Munich, Germany October 4 – 7, 2016 at Ludwig-Maximilians-University Annual Meeting of the German Annual Meeting of the – DPhG Pharmaceutical Society Sciences Pharmaceutical live interdisciplinarity We

DPhG Annual Meeting 2016 – Conference Book www.2016.dphg.de

ISBN 978-3-9816225-3-9 Annual Meeting of the – DPhG German Pharmaceutical Society Munich, Germany October 4 – 7, 2016 at Ludwig-Maximilians-University www.2016.dphg.de Conference Book

Pharmaceutical Sciences We live interdisciplinarity Annual Meeting of the German Pharmaceutical Society 2016 – DPhG Printed by:

REPRODUKT digital GmbH Stahlgruberring 20 81829 München

Munich, Germany, Oktober 2016 ISBN 978-3-9816225-3-9

Annual Meeting of the German Pharmaceutical Society – DPhG

Conference Book Pharmaceutical Sciences We live interdisciplinarity

Munich, Germany October 05 – 07, 2016 at Ludwig-Maximilians-University www.2016.dphg.de

Institutional Sponsors

Förderer der DPhG-Jahrestagung 2016

TABLE OF CONTENTS

CONFERENCE COMMITTEES ...... 2 WELCOME ADDRESS ...... 3 GENERAL INFORMATION ...... 4 LOCATIONS ...... 6 CONFERENCE PROGRAM OVERVIEW ...... 9 1 PLENARY LECTURES ...... 17 2 SCIENTIFIC LECTURES ...... 25 2.1 Materials for Drug Delivery ...... 26 2.2 Ion Channels in Health and Disease ...... 30 2.3 Progress in Drug Synthesis ...... 34 2.4 New Research, New Researchers I ...... 38 2.5 New Approaches in Gene and Stem Cell Therapy ...... 44 2.6 MS Based Drug Screening ...... 48 2.7 The Diversity in Pharmaceutical Biology ...... 51 2.8 The Quality of Therapeutic Proteins ...... 55 2.9 Anti-Infectives ...... 59 2.10 New Research, New Researchers II ...... 62 2.11 Clinical Pharmacy ...... 68 2.12 Advances in Drug Formulation and Biopharmaceutics ...... 72 2.13 Fighting Depression ...... 76 2.14 Interface Tumor/Inflammation ...... 79 2.15 Industrial Pharmacy ...... 83 3 POSTERS ...... 87 3.1 Analytics ...... 88 3.2 Inflammation ...... 97 3.3 Cancer/Inflammation ...... 105 3.4 Biotechnology/Protein Drugs ...... 115 3.5 Clinical Pharmacy ...... 118 3.6 Drug Design/Medicinal Chemistry ...... 123 3.7 GPCR/Ion Channels ...... 142 3.8 Natural Compounds/Chemical Biology ...... 145 3.9 Biopharmaceutics ...... 147 3.10 Pharmaceutical Technology and Biomaterials ...... 152 3.11 Pharmacology ...... 165 3.12 Regulatory Sciences/Industrial Drug Development ...... 169 3.13 Antiinfectives ...... 171 3.14 Neurological Disorders ...... 174 3.15 Drug Screening ...... 175 3.16 Other Topics ...... 177 AUTHOR INDEX ...... 182

DPhG Annual Meeting 2016 Conference Book • 1 CONFERENCE COMMITTEES

Scientific committee: Prof. Dr. Stefan Laufer Prof. Dr. Ulrich Jaehde Prof. Dr. Andreas Link Prof. Dr. Heyo Kroemer Dr. Olaf Queckenberg Prof. Dr. Irmgard Merfort Prof. Dr. Christoph Friedrich Prof. Dr. Klaus Mohr Prof. Dr. Peter Gmeiner Prof. Dr. Peter Ruth Prof. Dr. Jochen Klein Prof. Dr. Manfred Schubert-Zsilavecz Prof. Dr. Peter Langguth Prof. Dr. Andrea Sinz Prof. Dr. Kristina Friedland Prof. Dr. Holger Stark Prof. Dr. Angelika Vollmar Prof. Dr. Dieter Steinhilber Prof. Dr. Hermann Wätzig Prof. Dr. Werner Weitschies Prof. Dr. Thomas Efferth Prof. Dr. Gerhard Winter Prof. Dr. Ulrike Holzgrabe

Organization committee: Dr. Lars Allmendinger Dr. Ulrich Lächelt Prof. Dr. Martin Biel PD Dr. Johanna Liebl Prof. Dr. Franz Bracher Dr. Jörg Pabel Dr. Simone Braig Prof. Dr. Franz Paintner Laura Engelke Dr. Selma Speith-Kölbl Prof. Dr. Wolfgang Frieß Prof. Dr. Angelika Vollmar PD Dr. Dr. Christian Grimm Prof. Dr. Ernst Wagner Dr. Verena Hammelmann Prof. Dr. Christian Wahl-Schott Katharina Heimberger Prof. Dr. Klaus Wanner Dr. Sandra Hemmers Prof. Dr. Gerhard Winter Dr. Marco Keller

2 • DPhG Annual Meeting 2016 Conference Book WELCOME ADDRESS

As President of the Deutsche Pharmazeutische Gesellschaft (DPhG) and as congress Chairman of this meeting it is our pleasure to welcome you in Munich as attendees of our 2016 Annual Meeting.

Although our conference should be seen as a continuation of many successful annual meetings held by our society over the last years, we want to invite you to jointly move one step further with respect to emphasizing the interdisciplinarity of our profession and our pharmaceutical sciences. We have put that vision not only into our conference title but also let it come to life within our sessions and plenary lectures. You will notice that the themes touched in the 6 plenary lectures are all extended in one session linked to the subject, indicating that the structure of the meeting is not arbitrarily set but clearly laid out to certain focus topics. The poster session with its truly colorful variety of topics from all disciplines has long been a signature part of the meeting and we have added more time to allow all of us to see the posters without being distracted by too many parallel events. The number of posters we received fills the capacity of the available premises at the Chemistry and Pharmacy Campus in Großhadern with three sessions.

Another focus we, the President and the Chairman, share is the strong appreciation of the work of our young academic researchers. In particular with our German academic systems in mind, we know that the decision for an academic career is risky and seems often less rewarding in the early years compared to other careers. It is therefore of utmost importance to support those who pursue such an academic path. They carry the future of the pharmaceutical sciences on their shoulders. Consequently we have assigned two full prime time sessions, chaired by the President and the Vice- President, to young researchers and their work.

Many thanks go to the local organizers, the scientific chairs of the sessions, to the DPhG sections and scientific advisory board who all have worked together almost seamlessly to set the stage for this event. Besides the science and networking during the conference, we invite you to spend some time before or after in Munich and around in beautiful Upper Bavaria.

Our social event in the world famous Augustinerkeller with its typical Bavarian style may allow us all to relax with old and also new-found friends. At that point we want to express our gratitude to our General secretary Dr. Stein, who worked hard behind the scenes to ensure that the logistics of such a conference finally work well.

The conference book you hold in your hand carries all the necessary information on schedules, abstracts, poster-dates etc. and may also serve as a reference, later when you are back home.

We hope we have a fruitful meeting and a good time together

Stefan Laufer, President Gerhard Winter, Chairman

DPhG Annual Meeting 2016 Conference Book • 3 GENERAL INFORMATION

The Annual DPhG Meeting 2016 takes place at the faculty of chemistry and pharmacy (Butenandtstraße 5-13) in building F of the Ludwig-Maximilians-University Munich.

LANGUAGE The Conference language is English, no simultaneous translation will be provided.

INSTRUCTIONS FOR USING CONFERENCE WLAN If your institution is member of the “eduroam” community, you can use the wireless network “eduroam”. The configuration of your device should be the same as instructed by your home institution. Please use your account and the domain of your home institution. If your institution is not member of the “eduroam” community, you can obtain a guest account and a password at the Conference Office. WLAN: mwn-events USER-NAME: DPhGLMU2016

CONFERENCE OFFICE The Conference office is located at the Leipelt-Foyer in the Conference building F. Opening hours: Wednesday, October 5th, 2016: 9:30 – 19:00 Thursday, October 6th, 2016: 8:00 – 19:00 Friday, October 7th, 2016: 8:00 – 14:00

LIABILITY The organizers of the conference cannot be held responsible for any loss, theft, damage or injury to any person or property during the Conference, whatever the cause may be. The liability of persons and enterprises providing means of transportations or other services remains unaffected. Each congress participant and accompanying person takes part in all tours at his/her own risk.

4 • DPhG Annual Meeting 2016 Conference Book ABSTRACT AND POSTER NUMBERS Each abstract has a unique identifier, a letter-number combination. Letters refer to the conference topic, a contribution was assigned to (plenary lectures are identified by the letter “P”, scientific lectures by the letters “SL”, and poster presentations by the letters “POS”). Please note that in case of poster presentations the abstract number is identical with the poster number. Please refer to the author’s index on page 182 for direct access to specific abstracts. For all speakers: please hand over your presentation as a Power Point file at the conference desk until 8.30 am (for morning sessions) or 12.30 pm (for afternoon sessions).

POSTER SESSIONS Topics: Analytics, Inflammation, Cancer/Inflammation, Biotechnology/Protein Drugs, Clinical Pharmacy, Drug Design/Medicinal Chemistry, GPCR/Ion Channels, Natural Compounds/Chemical Biology, Biopharmaceutics, Pharmaceutical Technology and Biomaterials, Pharmacology, Regulatory Sciences/Industrial Drug Development, Antiinfectives, Neurological Disorders, Drug Screening, Other Topics. The posters were assigned to three poster sessions. Check the list below in which session you are presenting your poster. Presenting authors are requested to be present at their poster during the poster session.

Poster Session I: POS.47 – POS.70, POS.92 - POS.118, POS.158 – POS.170 and POS.223 – POS.237

Poster Session II: POS.26 – POS.45, POS.119 – POS.145, POS.171 – POS.199 and POS.217 – POS.219

Poster Session III: POS.1 – POS.23, POS.71 – POS.91, POS.146 – POS.157, POS.200 – POS.216 and POS.220 – POS.222

Poster session I Poster session II Poster session III Wednesday, Thursday, Friday, October 5th, 2016, October 6th, 2016, Session October 7th, 2016, 15:00 – 15:30 and 11:30 – 13:00 and 11:30 – 13:00 18:00 – 21:00 15:30 – 16:00 Wednesday, Thursday, Friday, Set-up October 5th, 2016, October 6th, 2016, October 7th, 2016, before 13:00 before 10:00 before 10:00 Wednesday, Thursday, Friday, Dismantling October 5th, 2016, October 6th, 2016, October 7th, 2016, after 21:00 after 18:30 after 15:00

CONFERENCE DINNER Separate registration necessary (special fee). Please refer to the Conference Office for registration and details. The Conference dinner will take place at “Augustinerkeller”, Arnulfstr. 52, 80335 Munich.

BADGES: Badges will be issued to all registered participants and enable access to all scientific sessions.

DPhG Annual Meeting 2016 Conference Book • 5

LOCATIONS

Ludwig-Maximilians-University, campus Großhadern: The Congress will take place at Ludwig-Maximilians-University Munich (Campus Großhadern), faculty of chemistry and pharmacy, Butenandtstr. 5-13, building F (ground floor and basement), 81377 Munich.

Arriving by plane and public transport: From the Munich airport take the suburban train S8 or S1 to Marienplatz. Change to the subway U6 in the direction of Klinikum Großhadern and get off at Großhadern. Take the rear extit and there take the left stairway. You are now on Würmtalstraße. Now you can walk along Würmtalstraße until the campus comes into view (large modern buildings) on the left (about 10 to 15 minutes walk). To arrive by bus from the subway station Großhadern, follow the sign bus 160 in the direction of Pasing or bus 268 in the direction of Gräfelfing. Exit the bus at Waldhüterstraße.

Arriving by train and public transport: Munich Central station (München Hauptbahnhof) is connected to all international long-distance routes. From there, take the subway U1 or U2 to Sendlinger Tor. Change to the subway U6 in the direction of Klinikum Großhadern and get off at Großhadern. Take the rear extit and there take the left stairway. You are now on Würmtalstraße. Now you can walk along Würmtalstraße until the campus comes into view (large modern buildings) on the left (about 10 to 15 minutes walk). To arrive by bus from the subway station Großhadern, follow the sign bus 160 in the direction of Pasing or bus 268 in the direction of Gräfelfing. Exit the bus at Waldhüterstraße.

Pas in g

Waldhüterstraße Großhadern

Hörsaalbereich

Figure 1: How to arrive to the Annual Meeting of the DPhG from the subway station Großhadern: Dashed line: arriving by foot / Continuous line: arriving by bus (picture from MVV, with kind permission)

6 • DPhG Annual Meeting 2016 Conference Book Arriving by car:

From the Nürnberg highway (A9): get onto the Mittlerer Ring B2R (direction Autobahn Lindau, A96), then onto the Lindau highway to the exit Blumenau, keep left onto Waldwiesenstraße until you reach the subway station Großhadern. From there, you will find several parking areas as described below.

From the Stuttgart highway (A8): from the end of the highway in Obermenzing turn off to Pasing, from Pasing drive in the direction of Gräfelfing, then turn left to Großhadern. From there, you will find several parking areas as described below.

From the Salzburg (A8) or Garmisch (A95) highways: drive onto the Mittlerer Ring B2R in the direction of Großhadern and Stuttgart. At Luise-Kiesselbach-Platz turn left towards Großhadern. Follow Waldfriedhofstraße, then Würmtalstraße until the subway station Großhadern. From there, you will find several parking areas as described below.

Parking areas: The only parking possibilities close to the conference building are located at Klinikum Großhadern. The daily parking fee is € 7,-. At the subway station Großhadern enter Sauerbruchstraße and follow the signs towards Klinikum Großhadern. Take the second street to your right hand side entering Marchioninistraße. Here you will find several parking areas.

If you are using the navigation system of your mobile phone, scan the following QR-codes. They will lead you to google maps, where the address of the parking areas and the congress building is already inserted.

Parking Building F, possibilities Butenandtstraße 11

(how to get to the (how to get to the parking areas) campus Großhadern)

Figure 2: Front view of the congress building F

Figure 3: Plan of the congress building F with its lecture halls

DPhG Annual Meeting 2016 Conference Book • 7

Augustiner-Keller (Conference Dinner, Thursday, 6th October, 19.30): Arnulfstraße 52, 80335 München

Arriving from Central Station: The subways U1, U2, U4 and U5 and suburban trains S1 – S8 are leading to Central Station. Take the northern exit towards Arnulfstraße and keep left. After 650 m you will find Augustiner-Keller on your right hand side.

Arriving from Hackerbrücke: The suburban trains S1 – S8 are leading to Hackerbrücke. Exit Hackerbrücke, enter Grassnerstraße and keep right. After 300 m you will find Augustiner-Keller on your left hand side.

If you are using the navigation system of your mobile phone, scan the following QR-code. It will lead you to google maps, where the address of Augustiner-Keller is already inserted.

Augustiner-Keller

(how to get to the Augustiner-Keller)

Vorsymposium der Fachgruppe Geschichte der Pharmazie (Ehrensaal des Deutschen Museums):

Arriving from Isartor:

All suburban trains in the direction of Ostbahnhof are leading to Isartor. There, follow the signs “Deutsches Museum” and exit the station onto Zweibrückenstraße. Follow Zweibrückenstraße until you reach to the river Isar. There turn right and follow Erhardtstraße. Take the first bridge to your left (Boschbrücke) and enter the main entrance of the Deutsches Museum. There will be a Meeting Point where a representative of “Fachgruppe der Geschichte der Pharmazie” will guide you to the lecture hall (Ehrensaal des Deutschen Museums).

8 • DPhG Annual Meeting 2016 Conference Book CONFERENCE PROGRAM OVERVIEW

CONFERENCE PROGRAM OVERVIEW

Pre-Meeting Program

Tuesday, October 4th Vorsymposium der Fachgruppe „Geschichte der Pharmazie“: Pharmazie in München Ort: Ehrensaal des Deutschen Museums, München Begrüßung durch den Vorsitzenden der FG Geschichte der Pharmazie 14:00 – Prof. Dr. Christoph Friedrich, Marburg 14:15 und des Generaldirektors des Deutschen Museums Prof. Dr. Wolfgang M. Heckl 14:15 – Zur Entwicklung des Hochschulfaches Pharmazie an der Universität München 15:00 Dr. Doris Winter, München 15:00 – Zur Entwicklung des Apothekenwesens in München 15:45 Dr. Gerhard Gensthaler, München 15:45 – Kaffeepause 16:15 Zur Geschichte der pharmazeutischen Industrie in München: Pharmazeutische 16:15 – Familienunternehmen – Know How, Flexibilität und eigene Marken 17:00 Dr. Ursula Lang, Seefeld 17:00 – Die pharmaziehistorische Sammlung des Deutschen Museums München 17:45 Dr. Florian Breitsameter, München

19:00 – Treffen Arbeitsgemeinschaft Katastrophenpharmazie 21:30 Ort: Campus Großhadern, Haus F, Butenandt-HS Notfall- und Katastrophenpharmazie - Die Arbeit des Apothekers in der internationalen 20:00 – Katastrophenhilfe 21:30 Dr. Carina Vetye-Maler, Buenos Aires

DPhG Annual Meeting 2016 Conference Book • 9 CONFERENCE PROGRAM OVERVIEW

Wednesday, October 5th Main Symposium (Congress language English) 10:30 – Sitzung Verband der Professoren an Pharmazeutischen Hochschulinstituten (Konferenz der 12:00 Fachbereiche Pharmazie), Leitung: Prof. Dr. Bernd Clement (Buchner-HS) 13:00 – Opening of the Annual DPhG Meeting 2016 (Liebig-HS) 13:30 Pharmaceutical Sciences, We live interdisciplinarity 13:30 – Plenary lecture 1, Achim Göpferich, The challenge of delivering biologics (Liebig-HS) P.1 14:15 14:15 – Plenary lecture 2, Jörg Striessnig, L-Type Ca2+ channels in brain disorders? New targets for old 15:00 drugs? (Liebig-HS) P.2 15:00 – Poster viewing (Cancer/Inflammation, Drug Design/Medicinal Chemistry I, Biopharmaceutics, Other 15:30 Topics) and coffee break SHORT TALKS (parallel sessions I) 15:30 – SL1 (Buchner-HS) SL2 (Butenandt-HS) SL3 (Willstätter-HS) 17:00 Materials for Drug Delivery Ion Channels in Health and Progress in Drug Synthesis Disease Chairs: W. Frieß, W. Weitschies Chairs: R. Lukowski, P. Ruth Chairs: P. Gmeiner, M. Heinrich 15:30 SL.01 15:30 SL.05 15:30 SL.09 Ferdinand Brandl: Thomas Wieland: Burghard König: Hydrogels for controlled release Phosphorylation of histidine Making and breaking of of protein therapeutics residues by nucleoside chemical bonds with light - diphosphate kinase B: A novel Visible light photocatalysis and mechanism to regulate ion photochromic molecular channel activity in disease switches 15:55 SL.02 15:55 SL.06 15:55 SL.10 Anne Seidlitz: Alexander Dietrich: Michael Müller: Evaluation of different polymers TRPC channels in lung function Diversity-oriented synthesis of for implants produced via fused and disease pharmaceuticals deposition modeling 16:20 SL.03 16:20 SL.07 16:20 SL.11 Peter Wich: Achim Schmidtko: Nicolas Blanchard: Biopolymers as multifunctional Pain control by calcium- Chemical synthesis of materials for nanoparticulate activated potassium channels mycolactone analogs - insights drug delivery into human mycobacterium ulcerans infection 16:40 SL.04 16:45 SL.08 16:45 SL.12 Gregor Fuhrmann: Christian Schmidt: Christian Ducho: Extracellular vesicles as smart Noradrenergic and serotonergic Inhibitors of the bacterial carriers for small molecule compounds to target narcoleptic translocase MraY as potential drugs episodes in a mouse model novel antibiotics

17:00 – Meetings der DPhG-Fachgruppen (in den Hörsälen) 18:00 Fachgruppe Fachgruppe Fachgruppe Fachgruppe Fachgruppe Fachgruppe Pharm./Med. Pharm. Pharma- Pharm. Klinische Industrie- Chemie Biologie kologie Technologie Pharmazie pharmazie P. Gmeiner A. Vollmar J. Klein P. Langguth K. Friedland C. Küster (Liebig) (Buchner) (Butenandt) (Wieland) (Willstätter) (Baeyer) 18:00 – Poster viewing (Cancer/Inflammation, Drug Design/Medicinal Chemistry I, Biopharmaceutics, Other 21:00 Topics) and welcome reception

10 • DPhG Annual Meeting 2016 Conference Book CONFERENCE PROGRAM OVERVIEW

Thursday, October 6th 9:00 – Plenary lecture 3, Eckhard Wolf, Genetically tailored pigs as organ donors and models for medical 9:45 research (Liebig-HS) P.3 SHORT TALKS (parallel sessions II) 10:00 – SL4 (Buchner-HS) SL5 (Butenandt-HS) SL6 (Willstätter-HS) 11:30 New Research, New Approaches in Gene and MS based Drug Screening New Researchers I Stem Cell Therapy Chairs: M. Lämmerhofer, Chairs: S. Laufer, A. Link Chairs: M. Biel, E. Wagner K. Wanner 10:00 SL.13 10:00 SL.19 10:00 SL.23 Julia Engert (Pharmaceutical Volker Busskamp: Gabriella Massolini: Technology): Evaluating microRNA-based Frontal affinity chromatography Application of a dried H1N1 therapies in stem cell-derived and mass spectrometry: a vaccine by epidermal powder retinal organoids perfect fit in drug discovery immunization in piglets using a novel pyrotechnically driven applicator elicits antigen-specific antibodies 10:15 SL.14 Alexander Titz (Med. Chem.): The virulence factor LecB varies in clinical isolates: consequences for ligand binding 10:25 SL.20 and drug discovery Caroline Le Guiner: Localized vs systemic gene therapy using rAAV vectors: 10:30 SL.15 achievements and remaining 10:30 SL.24 Christian Grimm challenges; the example of Johannes Ottl: (Pharmacology): Duchenne Muscular Dystrophy MS in pharmaceutical drug From mucolipidosis type IV to gene therapy discovery ebola: Insights into function and pharmacology of endolysosomal TRP channels 10:45 SL.16 10:50 SL.21 Astrid Kahnt (Biochemistry Hildegard Büning: and Pharmacology): From viruses to designer Molecular mechanisms of nanoparticles – tailoring adeno- lipoxin and resolvin biosynthesis associated viruses for gene 11:00 SL.17 therapy 11:00 SL.25 Andreas Koeberle Kai Scheffler: (Biochemistry): High resolution mass Insights from functional spectrometry of antibody drug lipidomics into the long-term conjugates using regulation of kinases by 11:10 SL.22 the orbitrap mass analyzer vitamin A Stylianos Michalakis: Gene therapy for human 11:15 SL.18 achromatopsia Finn Hansen (Med. Chem): α-Aminoxy peptides: from membranolytic anticancer foldamers to the first in class peptidomimetic Hsp90 C- terminal domain dimerization inhibitors

DPhG Annual Meeting 2016 Conference Book • 11 CONFERENCE PROGRAM OVERVIEW

11:30 – Poster viewing (Inflammation, Drug Design/Medicinal Chemistry II, Pharmaceutical Technology and 13:00 Biomaterials, Neurological Disorders) and lunch break 13:00 – Plenary lecture 4, Hans-Georg Rammensee, Impact of the immunogenic landscape of cancers on 13:45 immunotherapy (Liebig-HS) P.4 SHORT TALKS (parallel sessions III) 14:00 – SL7 (Buchner-HS) SL8 (Butenandt-HS) SL9 (Willstätter-HS) 15:30 The Diversity in The Quality of Therapeutic Anti-Infectives Pharmaceutical Biology Proteins Chairs: I. Merfort, T. Efferth Chair: H. Wätzig Chair: R. Hartmann 14:00 SL.26 14:00 SL.30 14:00 SL.34 Thomas Efferth: Martin Schiestl: Rolf Müller: Beyond malaria: The clinical Analytical tools in the biosimilar Innovative antibiotics from anticancer activity of artesunate development microorganisms: Some case studies 14:30 SL.27 14:30 SL.31 Rudolf Bauer: Christoph Scherübl: 14:35 SL.35 Application of plant Technical challenges for Shahriar Mobashery: metabolomics in herbal drug development and New antibiotics for the post- research manufacturing of antibiotic era biopharmaceuticals 15:00 SL.28 15:00 SL.32 Jennifer Herrmann: Ellen Köpf: Cystobactamids: Novel gyrase How does pH Affect interfacial inhibitors from myxobacteria antibody behaviour and 15:10 SL.36 that inhibit multi-resistant aggregation upon shaking? Ralph Holl: pathogens LpxC inhibitors – a novel class 15:15 SL.29 15:15 SL.33 of antibiotics Leonard Kaysser: Stefan Krimmer: Genome mining-guided drug Rational design of discovery: from proteasome to thermodynamic and kinetic protease inhibitors binding profiles by optimizing surface water networks coating protein bound ligands 15:30 – Poster viewing (Inflammation, Drug Design/Medicinal Chemistry II, Pharmaceutical Technology and 16:00 Biomaterials, Neurological Disorders) and coffee break

12 • DPhG Annual Meeting 2016 Conference Book CONFERENCE PROGRAM OVERVIEW

SHORT TALKS (parallel sessions IV) 16:00 – SL10 (Buchner-HS) SL11 (Butenandt-HS) SL12 (Willstätter-HS) 17:30 New Research, Clinical Pharmacy Advances in Drug Formulation New Researchers II Chair: K. Friedland, C. Wahl- and Biopharmaceutics Chairs: A. Link, S. Laufer Schott Chairs: P. Langguth, H. Rein 16:00 SL.37 16:00 SL.43 16:00 SL.47 Steffen Lüdeke Carsten Culmsee: Peter Kleinebudde: (Pharmaceutical Analytics): Evidence-based evaluation Thoughts on a manufacturing How polysaccharide system for OTC drugs classification system superstructure impacts hydrogel properties: A Raman optical activity study 16:15 SL.38 16:25 SL.44 16:25 SL.48 Dominique Lunter Dorothea Strobach: Christel Müller-Goymann: (Pharmaceutical Technology): Influence of over-the-counter Formulation development for Skin penetration analysis by drugs and prescription-only- topical treatment of tinea pedis confocal Raman microspectros- medication on male fertility and onychomycosis copy – potentials and pitfalls 16:30 SL.39 16:50 SL.45 16:50 SL.49 Sarah Hedtrich Hartmut Derendorf: Richard Hirsch: (Pharmacology): Evidence-based dose finding Improving the quality of split Influence of Th2 cytokines on using modeling and simulation tablets the cornified envelope, tight on earth and in space junction proteins and ß- defensins in filaggrin-deficient skin equivalents 16:45 SL.40 17:15 SL.46 17:10 SL.50 Karin von Schwarzenberg Sebastian Wicha: Jozef Al-Gousous: (Pharmaceutical Biology): The value of pharmacometrics Toward biopredictive dissolution Tumor selectivity of V-ATPase in development, optimisation for enteric coated dosage forms inhibition is based on and clinical use of anti-infective differential regulation of AMPK therapies 17:00 SL.41 Oliver Koch (Med. Chem.): Analysing the framework of protein ligand interactions: Ligand-sensing cores and privileged scaffolds

17:15 SL.42 Pierre Koch (Med. Chem.): A fluorescence polarization- based competition binding assay for detecting compounds interacting with inactive mitogen-activated protein kinases… 17:45 – Anke Krüger, Stefan Wulle: PubPharm - der Fachinformationsdienst Pharmazie: Präsentation der 18:30 neuen Rechercheplattform für die Pharmazie (POS.231) (Buchner-HS) 19:30 Conference dinner (Augustiner-Keller, Arnulfstr. 52, 80335 München)

DPhG Annual Meeting 2016 Conference Book • 13 CONFERENCE PROGRAM OVERVIEW

Friday, October 7th 9:00 – Plenary lecture 5, Florian Holsboer, Personalized therapy of depression – 9:45 the future has begun (Liebig-HS) P.5 SHORT TALKS (parallel sessions V) 10:00 – SL13 (Buchner-HS) SL14 (Butenandt-HS) SL15 (Willstätter-HS) 11:30 Fighting Depression Interface Tumor/ Industrial Pharmacy Inflammation Chairs: F. Paintner, K. Wanner Chairs: R. Fürst, O. Werz Chairs: O. Queckenberg, C. Olbrich 10:00 SL.51 10:00 SL.54 10:00 SL.58 Felix Hausch: Alexandra Kiemer: Hubertus Rehbaum: FKBP51 inhibitiors - a The mRNA binding protein Introduction of continuous pharmacological concept to p62/IGF2BP2 as a promoter of manufacturing from an enhance stress resilience metaflammation and engineering perspective hepatocellular carcinoma 10:30 SL.52 10:30 SL.55 10:20 SL.59 Theo Rein: Oliver Werz: Thomas De Beer: The stress protein FKBP51 Targeting monocytes and Model based PAT shapes antidepressant macrophages for intervention implementation in pharmacology with inflammation-related pharmaceutical manufacturing cancer processes 11:00 SL.53 11:00 SL.56 10:45 SL.60 Thomas Kirmeier: Tessa Lühmann: Armin Schweiger: Reengineering in the field of Clickable IL-4 cytokines to Modular, continuous API Psychopharmacology: Learning induce M2 macrophage production units by INVITE from successful models polarization

11:15 SL.57 11:10 SL.61 Dian-Jang Lee: Axel Zeitler: Oligoaminoamide-based siRNA Advancing process carriers for in vivo tumor understanding in film coating targeting and gene silencing by in-line terahertz pulsed imaging, optical coherence tomography and discrete element modelling 11:30 – Poster viewing (Analytics, Biotechnology/Protein Drugs, Clinical Pharmacy, GPCR/Ion Channels, 13:00 Natural Compounds/Chemical Biology, Pharmacology, Regulatory Sciences/Industrial Development, Antiinfectives, Drug Screening) and lunch break 13:00 – Plenary lecture 6, Dario Neri, Antibody-cytokine fusion proteins for the treatment of cancer and of 13:45 chronic inflammation: from the bench to Phase III clinical trials (Liebig-HS) P.6 14:00 – Award Session and Closing (Liebig-HS) 15:00 15:00 – Gemeinsame Sitzung von Repräsentanten der Deutschen Pharmazeutischen Gesellschaft und 18:00 des Verbandes der Professoren an Pharmazeutischen Hochschulinstituten (Konferenz der Fachbereiche Pharmazie), separate Einladung (Butenandt-HS)

14 • DPhG Annual Meeting 2016 Conference Book

Post-Meeting Program

Saturday, October 8th 15:00 – Tag der Offizinpharmazie: FG Allgemeinpharmazie der DPhG in Kooperation mit der Deutschen 18:30 Gesellschaft für Schmerzmedizin (DGS) und der Bayerischen Landesapothekerkammer (BLAK) Ort: Ludwig-Maximilians-Universität München, Fakultät für Chemie und Pharmazie, Butenandtstraße 5-13, Haus F (Buchner-Hörsaal) 15:00 Prof. Dr. Stefan Laufer, Präsident der DPhG Prof. Dr. Gerhard Winter, Congress Chairman Begrüßung und Einführung

15:15 Dr. med. Johannes Horlemann, Vizepräsident der Deutschen Gesellschaft für Schmerzmedizin, DGS Differentialtherapie und Beratung zu Opiaten

16:15 Prof. Dr. Theo Dingermann, Institut für Pharmazeutische Biologie, Goethe-Universität in Frankfurt am Main Cannabis – Die öffentliche Apotheke wird sich kümmern müssen

17:00 Pause mit Kaffee und Imbiss

17:30 Prof. Dr. Dieter Steinhilber, Institut für Pharmazeutische Chemie, Goethe-Universität Frankfurt am Main Prof. Dr. Theo Dingermann, Institut für Pharmazeutische Biologie, Goethe-Universität Frankfurt am Main Keith Richards und Osteoarthrose – wenn die Gelenke schmerzen

ANMELDUNG Eine Anmeldung zum Tag der Offizinpharmazie in Kooperation mit der Deutschen Gesellschaft für Schmerzmedizin ist nicht erforderlich. Die Veranstaltung ist kostenfrei. www.dphg.de/apo16

DPhG Annual Meeting 2016 Conference Book • 15

1 PLENARY LECTURES

DPhG Annual Meeting 2016 Conference Book • 17 PLENARY LECTURES

P.1 The Challenge of Delivering Biologics

Göpferich, A. Pharmaceutical Technology, Department of Chemistry and Pharmaceutical Sciences, University of Regensburg, Universitätsstrasse 31, 93040 Regensburg, Germany

Over recent years drug products of biological origin gained continuously of importance since they improved numerous therapies from cancer to ocular diseases such as age related macular degeneration. With respect to pharmaceutical applications proteins and nucleic acids are the most prominent examples for such drugs that became known as biologics. On the one hand they have a high therapeutic potential but on the other hand they also come with a number of caveats with respect to formulating them to medicinal products that stem from their structure and their physicochemical properties [1]: some of the molecules are of a size that does not allow for an unhindered distribution in the organism [2], they carry a multitude of electrical charges [3] which is a handicap for crossing biological barriers and they often tend to be of limited chemical and physical stability to mention just a few. To make them available for therapy, biologics have frequently been formulated as aqueous solutions for parenteral injection since it is in many cases the safest and simplest way of application. But even then a number of problems have to be solved. Aqueous solutions of antibodies for example often suffer from high viscosities due to their macromolecular character [4] and are frequently subject to aggregate formation [5] that have been reported to be immunogenic in some cases [6]. However, even though aqueous solutions are the most straightforward way to make biologics available for therapy, there are a number of scenarios in which the controlled release oft such compounds over extended periods of time could be highly advantageous. In such cases, the materials need to be processed to delivery systems, which is quite challenging. For proteins there are two general options that we can choose from. Biologics such as proteins can simply be embedded into matrix materials of hydrophobic character from which they can be released by degradation and erosion of the material in combination with diffusion. While this approach has definitely its merits, the interaction of proteins with the matrix material is frequently causing problems due to the presence of phase boundaries, degradation products as well as the changing physicochemical environment. Hydrogels are a promising alternative as carrier materials. Their advantage is a high water content that does not compromise protein stability as much as hydrophobic materials. In some cases, the interaction oft a polymer backbone with a biologic can even become part of the hydrogel structure such as in the case of antibodies that interact via electrostatic interactions with polymer chains [3]. However, also hydrogels have drawbacks. Loading proteins into their three-dimensional network can be difficult and is often solved by simply soaking hydrogel matrices in concentrated protein solutions. Also controlling the release of proteins can be sometimes challenging with respect to extensive premature release, the duration of release that is often too short and the release kinetics that can frequently not be tailored to the intended application. Nucleic acids finally ‘suffer’ from their highly negative charge density. We usually intend to overcome this drawback by formulating them with oppositely charged cationic materials to nanostructures that allow for cellular uptake via endocytotic processes. Nanoparticles offer a number of advantages for the delivery of biologics. However, they suffer from similar problems as the macromolecules they intend to deliver. Size and charge make it difficult to bring them to a target site in sufficient quantities. In many cases a poor target structure affinity and low target cell specificity seem to be problems that deserve more attention [7]. The talk will elucidate the need for delivering biologics beyond the injection of aqueous solutions. Concomitantly it will it will expand on some of the problems that have to be dealt with when formulating delivery systems for biologics. A special focus will, thereby, be on materials for protein delivery and on nanoparticles as well as hydrogels as carrier systems.

References: 1. Mitragotri, S., Burke, P. A. & Langer, R. Nat. Rev. Drug. Discov. 2014, 13: 655–672. 2. Schweizer, D., Serno, T. & Goepferich, A. Europ. J. Pharm. and Biopharm. 2014, 88: 291–309. 3. Schweizer, D. et al. Biomacromolecules 2013, 14: 75–83. 4. Yadav, S., et al. J. Pharm. Sci. 2010, 99: 1152–1168. 5. Mahler, H.-C., et al. J. Pharm. Sci. 2009, 98: 2909–2934. 6. Moussa, E. M. et al. J. Pharm. Sci. 2016, 105: 417–430. 7. Wilhelm, S. et al. Nat, Rev. Mat. 2016, 1: 1–12.

18 • DPhG Annual Meeting 2016 Conference Book PLENARY LECTURES

P.2 L-Type Ca2+ Channels in Brain Disorders? New Targets for Old Drugs?

Striessnig, J.1,2 ; Ortner, N. J.1,2 ; Pinggera, A.1,2 ; Negro, G.1,2 ; Mackenroth, L.3 ; Hoferm, N.1,2 ; Tuluc, P.1,2 ; Dougalis, A.4 ; Duda, J.4 ; Ciossek, T.5 ; Draheim, H. J.5 ; Liss, B.4 1 Pharmacology and Toxicology, Institute of Pharmacy and 2 Center for Molecular Biosciences, Univ. of Innsbruck, Innsbruck, Austria; 3 Institute for Clinical Genetics, Technical University, Dresden; 4 Institute of Applied Physiology, University of Ulm, Ulm, Germany; 5 Boehringer Ingelheim Pharma GmbH & Co KG, CNS Research, Biberach an der Riß, Germany.

Organic Ca2+-channel blockers ("Ca2+ antagonists", e.g. the dihydropyridines amlodipine, nimodipine, isradipine) are clinically used for first-line treatment of hypertension. They inhibit ion permeation through voltage-gated L- type Ca2+-channels (LTCCs, Cav1) in arterial smooth muscle and the heart. However, LTCCs are not only expressed in muscle cells of the cardiovascular system but are also key regulators of Ca2+-dependent signaling processes in many other electrically excitable cells, including neurons and (neuro-) endocrine cells (1-3 for review). Cav1.2 and Cav1.3 LTCC isoforms are expressed in the brain, both located postsynaptically at dendrites and the cell soma (1-3). They control neuronal excitability, synaptic morphology and couple synaptic activity to gene transcription. Through their distinct biophysical properties Cav1.2 and Cav1.3 contribute in different ways to various forms of learning, memory, emotional and drug-taking behaviours (1,3). No CNS side effects have been reported during antihypertensive therapy with brain-permeable LTCC blockers. However, recent evidence from human genetics strongly suggests an important role of enhanced LTCC function for neuropsychiatric disease risk (1). Timothy syndrome is a rare disease in which the mutation-induced increase in Cav1.2 channel activity (CACNA1C gene) causes not only long QT syndrome and other organ abnormalities but also autism (1). Genome-wide association studies revealed a strong association between intronic SNPs in CACNA1C and susceptibility for various psychiatric disorders, including bipolar disorder, schizophrenia and major depression. One of these SNPs (rs1006737) leads to increased Cav1.2 activity in fibroblast-derived induced neurons (3). We (4) and others (5) have recently discovered somatic mutations in the α1-subunit of Cav1.3 (CACNA1D) LTCCs which cause increased Cav1.3 activity and excess aldosterone production in aldosterone producing adenomas. When present germline, two of these mutations cause a severe congenital syndrome with primary aldosteronism, seizures and neurodevelopmental deficits at early age (PASNA, 5). Moreover, we identified very similar de novo missense mutations in the pore-forming 1-subunit of Cav1.3 channels in three unrelated patients with sporadic autism and intellectual disability (6). These mutations are all located in the channel's activation gate and cause pronounced gating changes leading to a strong gain of channel function. Together these data suggest that increased Cav1.2 and Cav1.3 channel activity can substantially contribute to neuropsychiatric disease risk in humans. This makes already existing brain-permeable LTCC inhibitors, such as isradipine or nimodipine, promising therapeutics in individuals carrying LTCC risk genes. We therefore investigated the sensitivity of Cav1.2 and Cav1.3 channels to the dihydropyridine LTCC blocker isradipine in HEK293 cells stably expressing these channels. IC50 values were obtained during activity patterns simulating either neuronal firing patterns or arterial smooth muscle - like depolarizations. We found that during neuronal-like activity isradipine is a significantly weaker inhibitor of Cav1.3 than of Cav1.2 and that inhibition of Cav1.3 is splice-variant dependent. Isradipine inhibited Cav1.2 channels during arterial smooth muscle – like depolarizations with an IC50 of 1.5 nM, which is 2-fold lower than for neuronal Cav1.2 and 4.5 - 11-fold lower than for neuronal Cav1.3 splice variants. Our data indicate that inhibition of neuronal Ca2+ channel with existing LTCC blockers could be a novel option for treatment of neuropsychiatric disease. However, chronic treatment may require high maintenance doses likely to cause hypotension.

Acknowledgments: Support: Austrian Science Fund (FWF) F44020; P27809; W1101-B12; DFG LI 1754/1 References: 1. Striessnig, J. et al., Wiley Interdiscip. Rev. Membr. Transp. Signal. 2014, 3 (2): 15–38. 2. Ortner, N.J. and Striessnig, J. Channels (Austin) 2016, 10 (1): 7-13. 3. Striessnig, J. et al. Curr. Mol. Pharmacol. 2015, 8 (2): 110-122. 4. Azizan et al. Nat. Genet. 2013, 45 (9): 1055-1060. 5. Scholl et al. Nat. Genet. 2013, 45 (9): 1050-1054. 6. Pinggera, A. et al. Biol. Psychiatry 2015, 77 (9): 816-822.

DPhG Annual Meeting 2016 Conference Book • 19 PLENARY LECTURES

P.3 Genetically tailored pigs as organ donors and models for medical research

Wolf, E.1,2,3; Klymiuk, N.2; Renner, S.2,3; Reichart, B.4 1 Gene Center, LMU Munich, Feodor-Lynen-Str. 25, D-81377 Munich, Germany 2 Center for Innovative Medical Models (CiMM), LMU Munich, D-85764 Oberschleißheim, Germany 3 German Center for Diabetes Research (DZD), D-85764 Oberschleißheim, Germany 4 Walter-Brendel-Center for Experimental Medicine, LMU Munich, D-81377 Munich, Germany

The number of donated human organs and tissues for patients with terminal organ failure falls far short of the need, a situation that threatens the life of many potential recipients, especially in Germany. Alternative techniques such as xenotransplantation (and stem cell therapies) are therefore urgently needed. The DFG-funded Transregional Collaborative Research Center 127 “Biology of Xenogeneic Cell, Tissue and Organ Transplantation – from Bench to Bedside” is a unique Consortium encompassing basic research on xenogeneic immune mechanisms, generation and evaluation of novel genetically (multi-)modified donor pigs, and preclinical porcine islet, heart valve, and heart transplantation studies. Hearts of triple-modified donor pigs lacking the major xenoantigen Gal-alpha1,3-Gal and expressing the human complement regulator hCD46 as well as human thrombomodulin [1] survived after heterotopic abdominal transplantation into baboons for up to 945 days [2], demonstrating that cardiac xenotransplantation may become a realistic option. Since T-cell mediated rejection is considered as the major barrier for long-term survival of pancreatic islet xenografts, we generated transgenic pigs expressing the T-cell co-stimulation blocking molecule LEA29Y under the control of the porcine INS promoter. After transplantation under the kidney capsule of diabetic NOD-SCID Il2rg-/- (NSG) mice, LEA29Y expressing porcine neonatal islet cell clusters (NICCs) restored glucose control and were – in contrast to wild-type NICCs – not rejected by transplanted human blood mononuclear cells. Only very low levels of LEA29Y were detectable in the circulation of mice grafted with transgenic islets, supporting the concept of local immune modulation by LEA29Y [3]. The LEA29 transgene was backcrossed to an Gal-alpha1,3-Gal deficient, hCD46 transgenic background to facilitate efficacy studies of multi-modified NICCs in diabetic nonhuman primates. Genetically modified pigs cannot only serve as tissue and organ donors, but also as tailored models of human diseases. For instance, genetically engineered pig models may help to bridge the gap between basic research and clinical studies in (pre)diabetic patients. Type 2 diabetic patients exhibit a reduced insulinotropic action of the incretin hormone glucose-dependent insulinotropic polypeptide (GIP). To mimic this disturbance in a large animal model, we generated transgenic pigs expressing a dominant-negative GIP receptor (GIPRdn) in the pancreatic islets. GIPRdn transgenic pigs exhibit a blunted insulinotropic action of GIP, a progressive deterioration of glucose control due to delayed and – at later stages – quantitatively reduced insulin secretion, and an impairment of physiological age-related expansion of beta-cell volume [4]. GIPRdn transgenic pigs thus provide a unique opportunity to screen for biomarker candidates during the pre-diabetic period [5] and to test therapeutic strategies targeting the glucagon-like peptide 1 (GLP1) receptor [6]. Missense mutations in the INS gene have been identified as common cause of permanent neonatal diabetes mellitus, also referred to as mutant INS gene- induced diabetes of youth (MIDY). We produced a transgenic MIDY pig line expressing INSC94Y. MIDY pigs show early-onset clinical diabetes mellitus, reduced body weight gain and beta-cell volume associated with a marked reduction of insulin secretory granules and severe dilation of the endoplasmic reticulum in the beta-cells [7]. MIDY pigs can be used for insulin treatment studies or for testing the efficacy of gene or cell therapies as well as islet transplantation. Secondary lesions of diabetes mellitus are another interesting area of research. We thus established the Munich MIDY pig biobank as a unique resource for studying systemic consequences of chronic hyperglycemia.

Acknowledgments: Funded by the Deutsche Forschungsgemeinschaft (TRR127: Biology of xenogeneic cell, tissue and organ transplantation – from bench to bedside) and by the German Center for Diabetes Research (DZD). References: 1. Wuensch, A. et al.: Transplantation 2014, 97(2): 138-47. 2. Mohiuddin, M.M. et al.: Nat Commun 2016, 7: 11138. 3. Klymiuk, N., Wolf-van Bürck, L. et al.: Diabetes 2012, 61(6): 1527-32. 4. Renner, S. et al.: Diabetes 2010, 59(5): 1228-38. 5. Renner, S. et al.: Diabetes 2012, 61(8): 2166-75. 6. Streckel, E. et al.: J Transl Med 2015, 13: 73.

20 • DPhG Annual Meeting 2016 Conference Book PLENARY LECTURES

P.4 Impact of the immunogenic landscape of cancers on immunotherapy

Rammensee, H.-G. Department of Immunology, University of Tuebingen, Auf der Morgenstelle 15, D-72076 Tuebingen, Germany

We have analyzed tumor samples of intermediate mutation load, in particular hepatocellular carcinomas, renal cell carcinomas, ovarian carcinomas and several leukemia types, by exome sequencing for somatic mutations and by mass spectrometry analysis for the identification of HLA ligands. Careful attention was given to HLA ligands possibly containing mutations; sequences containing nonsynonymous mutations on DNA level were subjected to prediction of peptides fitting to the relevant HLA class I molecules. High score predicted peptides were synthesized and used as references to identify the expected tumor peptide by mass spectrometry. In no instance we were able to find mutated peptides presented by tumor HLA molecules, although we had shown that the approach works with tumor cell lines. These findings are in accordance with the assumption that many human tumors expressing an intermediate or low number of somatic mutations do not express immunogenic mutated antigens visible for T cells. In contrast, in analysing the entire detectable landscape of HLA ligands on these tumor samples, consisting of 1000 through 5000 peptides per sample, we do find dozens to hundreds of peptides in germline sequence with apparently tumor specific expression, based on the absence of these peptides on adjacent autologous benign tissue and absence on a large number of normal tissue samples from all organs and tissue types available for analysis, all, of course, within the sensitivity limits of our technology. A fraction of these peptides is derived from gene products with tumor specific expression (like cancer testis antigens), another fraction, however, is derived from ubiquitously expressed proteins, pointing to tumor specific altered RNA or protein processing. The population of these tumor peptides is highly different between patients. Many of these tumor specific peptides are immunogenic, as tested by in vitro priming experiments with human T cells from healthy donors. We analyzed patients for the presence of antigen experienced T cells specific for these tumor peptides and not only found that such T cells exist in a fraction of patients but also that this fraction of patients shows a better overall survival. We conclude that 1.) Germline sequence HLA ligands with tumor specific expression should be efficient as targets for personalized antigen specific immunotherapy and 2.) Although the generally accepted view at present is that checkpoint inhibition only works via mutation specific T cells, we would not be surprised if in addition T cells against immunogenic germline sequence HLA ligands with individualized tumor specific expression will also be found if one looks for these.

DPhG Annual Meeting 2016 Conference Book • 21 PLENARY LECTURES

P.5 Personalized therapy of depression – the future has begun

Holsboer, F. HMNC Brain Health, Maximilianstraße 34, 80539 Munich, Germany

Biomedical progress has shown that diagnostic entities are not necessarily reflecting uniform disease mechanisms. Oncology heralded the birth of personalized medicine on a large scale demonstrating that specific target engagement is superior to a “one-size-fits” approach. Personalized treatment of depression is obviously more complex than in oncology as our knowledge how the disease evolves and how antidepressants act is limited. Studies of causes underlying depression focused on genetic risk, e.g. polymorphisms and environmental factors, e.g. stressors. The complexity is reflected by the absence of gene loci that confer vulnerability and the fact that stress is an unspecific risk factor for all psychiatric disorders. With that in mind, a gene test and biomarker-guided, personalized medicine was deemed as futile undertaking. Two examples will show that this perception is premature, if not wrong. The central master hormone, coordinating the whole range of adaptations needed to cope with stress is corticotropin-releasing hormone, CRH. If elevated over a longer period depression might precipitate, which resulted in pharmaceutical efforts to block the CRH/CRHR1 signaling pathway. CRHR1 antagonist failed to be superior to placebo in large clinical trials, which led industry to terminate CRHR1 programs. The negative results where unsurprising as only a fraction of patients has a CRH-based pathology. Research into identification, who might respond, has led to a gene test and to a sleep-EEG-based biomarker that predict CRHR1 response. While translation of these findings into clinical application will take time, another gene test demonstrates that personalized treatment of depression is already available: Antidepressants need to enter the brain but their passage is controlled by a transporter molecule P-glycoprotein (Pgp) that is encoded by the ABCB1-gene. ABCB1-gene variants determine if a given antidepressant that is a Pgp-substrate will be effective, or whether a higher dose is needed, or whether switching to an antidepressant that is not a Pgp-substrate, is appropriate. These examples demonstrate that personalized approaches in depression therapy are no longer utopian.

22 • DPhG Annual Meeting 2016 Conference Book PLENARY LECTURES

P.6 Antibody-cytokine fusion proteins for the treatment of cancer and of chronic inflammation: from the bench to Phase III clinical trials

Neri, D.1 1 Institute of Pharmaceutical Sciences, ETH Zürich, Vladimir-Prelog-Weg-4, CH-8093 Zürich (Switzerland)

Antibodies can be used to deliver bioactive molecules (drugs, cytokines, photosensitizers, radionuclides, etc.) to the tumor environment, thus sparing normal tissues. The antibody-based targeting of certain modified extracellular matrix (e.g. splice isoforms of fibronectin and of tenascin-C) is particularly attractive, as these antigens represent accessible, abundant and selective tumor-associated antigens [1-6].

In this lecture, I will present an overview of preclinical and clinical product development activities, performed by my group in collaboration with Philogen (www.philogen.com), for the development of antibody-cytokine fusion proteins (also termed “immunocytokines). A number of products, featuring IL2 or TNF as pro-inflammatory payloads, have moved to advanced clinical trials in oncology, while other immunocytokines (based on anti- inflammatory payloads) are currently being considered for the treatment of rheumatoid arthritis, inflammatory bowel disorders and other conditions.

Acknowledgment: The research activity of the group Neri is financed by the ETH Zürich, the Swiss National Science Foundation, the European Research Council (ERC Grant “Zauberkugel”) and the Swiss Federal Commission for Technology and Innovation (KTI). References: 1. Neri, D. & Bicknell, R.. Nature Rev. Cancer. 2005, 5, 436-446 2. Neri, D. & Supuran, C. Nature Rev. Drug Discov., 2011, 10, 767-777 3. Neri, D. & Pasche, N. Drug Discov. Today, 2012, 17, 583-590 4. Gutbrodt, K. et al. Science Transl. Med., 2013, 5, 201ra118 5. Hemmerle T. et al. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 12008-12012 6. Bootz, F. & Neri, D. Drug Discov. Today, 2016, 21, 180-189

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2 SCIENTIFIC LECTURES

DPhG Annual Meeting 2016 Conference Book • 25 SCIENTIFIC LECTURES

2.1 Materials for Drug Delivery

Chairs: W. Frieß, W. Weitschies

SL.01 Hydrogels for controlled release of protein therapeutics

Brandl, F. P. 1,2; Gregoritza, M.1 1 University of Regensburg, Department of Pharmaceutical Technology, 93040 Regensburg, Germany 2 Present address: BASF SE, 67056 Ludwigshafen, Germany

Hydrogels have many favorable properties that make them attractive for applications in controlled protein release [1,2]. For example, the preparation procedure is beneficial for preserving protein stability, since protein denaturating conditions (i.e., use of organic solvents, extreme temperature and/or pH values, high shear forces etc.) can be avoided. The mobility of incorporated proteins can be restricted by the polymer network; the hydrogel then forms a reservoir from which the protein is slowly released. Depending on the size of the protein and the average network mesh size, the release can be controlled by diffusion, swelling, degradation, or a combination of these mechanisms. And finally, in situ forming hydrogels can be administered by minimally invasive methods; these are injected in liquid form and turn into a viscoelastic drug depot at the site of application. However, despite the favorable properties of hydrogels, it is often challenging to guarantee the stability and availability of encapsulated proteins [1,2]. The stability of proteins depends on the method of drug loading, the polymer concentration, the type of the cross-linking reaction, the initiator concentration, and the use of stabilizing agents (e.g., radical scavengers). The availability of proteins is influenced by the method of drug loading, the type and molecular mass of the polymer, the polymer concentration, the cross-linking density, the hydrogel degradation rate, and the affinity of the payload for the carrier. While many cross-linking reactions can be used for the preparation of hydrogels (e.g., radical polymerization, Michael-type addition, “click” reactions etc.), only few reactions do not compromise the stability and availability of incorporated proteins. For example, the Diels–Alder reaction has been used for the preparation of hydrogels [3,4,5]. The reaction readily proceeds in water without catalyst or initiator; moreover, the cross-linking process is reversible which allows the preparation of degradable materials. However, side reactions with nucleophilic groups of proteins (e.g., cysteine or lysine residues) can affect their stability and availability [6]. In this talk, possible methods of protein loading and different principles of protein release will be discussed. Several cross-linking methods will be introduced that can be used for applications in controlled protein release. And last but not least, strategies will be presented to protect proteins during cross-linking.

References: 1. Lin, C.-C.; Anseth, K.S.: Pharm. Res. 2009, 26(3): 631–643. 2. Vermonden, T.; Censi, R.; Hennink, W.E.: Chem. Rev. 2012, 112(5): 2853–2888. 3. Kirchhof, S. et al.: J. Mater. Chem. B 2013, 1(37): 4855–4864. 4. Kirchhof, S. et al.: J. Mater. Chem. B 2015, 3(3): 449–457. 5. Kirchhof, S. et al.: Eur. J. Pharm. Biopharm. 2015, 96: 217–225. 6. Hammer, N. et al.: Macromol. Biosci. 2015, 15(3): 405–413.

26 • DPhG Annual Meeting 2016 Conference Book MATERIALS FOR DRUG DELIVERY

SL.02 Evaluation of different polymers for implants produced via fused deposition modeling

Kempin, W.; Bogdahn, M.; Weitschies, W.; Seidlitz, A. Biopharmaceutics and Pharmaceutical Technology, Institute of Pharmacy, EMA University of Greifswald, Felix-Hausdorff-Str. 3, 17487 Greifswald, Germany

3D printing of medicinal products and medical devices is a promising alternative to conventional dosage form manufacturing [1], especially when the therapeutic outcome can be improved by individually adapting the shape of the dosage form to the patients’ needs. This would be especially benefical when the placement of an implant in a natural passage or conduit is intended. Fused depositon modeling (FDM) is a 3D printing technique in which a polymer filament is introduced into the printhead, heated, extruded through the nozzle and deposited on the coordinates defined in the machine instructions (G-code). In order to print drug-loaded implants using this technique with commonly designed machines it is mandatory to produce filaments containing the drug and the polymer and if necessary further excipients. In this work we evaluated the formation of filaments with different polymers and the model drug quinine. The tested polymers included poly-L-lactide (PLLA), polycaprolactone (PCL), polydioxanone (PDO), polyhydroxybutyrate (PHB), poly(ethylene-vinyl acetate) (PEVA), ethyl acrylate methyl methacrylate copolymer (Eudragit® RS), and ethyl cellulose (EC under addition of triacetin). The polymers and quinine were mixed using a solvent casting technique at a quinine content of 5 % of total solids. The obtained films were formed into filaments using hot-melt extrusion using a self-constructed extruder (nozzle diameter 3 mm). Filaments that possessed the properties required for printing, e.g. homogeneity and acceptable consistency of diameter, were further tested regarding their printability using a Multirap M420 printer (nozzle diameter 0.5 or 0.35 mm). Model implants were printed as hollow cylinders with a designated height of 3 mm and an outer diameter of 4 or 5 mm. Model drug release of the successfully printed implants was assessed in an incubation setup with phosphate buffered saline solution (PBS) pH 7.4 over several weeks. Quinine content of the samples obtained was determined via fluorescence spectrometry. Filaments and model implants were successfully produced using PLLA, PCL, Eudragit® RS and EC. Implants based on PEVA were printed but showed visible shape variations and thus drug release was not investigated. The other polymers could not be formed into filaments either because no homogenous mixtures of the polymer and the model substance could be produced or since no suitable temperature was identified at which the blends were extrudable but not too liquid to be adequately deposited. Exemplaric images of a filament and model implant composed of PCL and quinine are given in figure 1. The optimum temperature at the noozle was very different for the different polymers ranging from 53 °C (PCL) to 164 °C (PLLA). Under addition of quinine to Eudragit® RS the process temperature for FDM was reduced compared to the pure polymer indicating a plasticizing effect of the model drug. Quinine release from the implants also showed marked differences depending on the type of polymer used. After 8 weeks, releases ranged from 3 % (Eudragit® RS) to 76 % (PCL) of total drug load. The addition of 10 % polyethyleneglycol as a pore forming agent to the model implant composed of PCL and quinine was able to moderately accelerate the release behaviour. FDM is a very promising technique for the preparation of drug- loaded implants adapted to patient anatomy. In this pilot study several polymers were identified that could be used to manufacture model implants containing the model drug quinine and yielded a wide array of release behaviours. More research is needed to identify suitable compositions and processing parameters for implant applications. These parameters are expected to change with every change of the compositon, e.g. changes in processing temperature caused by concentration-dependent plasicizing effects of the active pharmaceutical ingredients as well as excipients. Also it must be kept in mind that drug as well as polymer might (partially) degrade due to the high temperatures used for processing.

Acknowledgments: Financial support of the conducted research by the German federal ministry of education and research within “RESPONSE” is gratefully acknowledged. Furthermore, the authors thank L.-C. Koster and C. Franz for their assistance. References: 1. Lee Ventola, C.: P T. 2014, 39(10): 704–711.

DPhG Annual Meeting 2016 Conference Book • 27 SCIENTIFIC LECTURES

SL.03 Biopolymers as Multifunctional Materials for Nanoparticulate Drug Delivery

Wich, P. R. Johannes Gutenberg Universität-Mainz, Institut für Pharmazie und Biochemie, Staudingerweg 5, 55128 Mainz, Germany

Biopolymers, such as polysaccharides and proteins show a remarkable versatility as multifunctional materials for therapeutic applications. They can be easily modified with the toolkit of bioorganic chemistry and are particularly attractive because of their degradability and biocompatibility [1]. We present a chemical modified polysaccharide (acetal-modified dextran) that can be formulated into nano- und microparticles using a variety of common emulsion-based techniques (A). It is possible to transport proteins, DNA and RNA, as well as small hydrophobic drugs. Ac-DEX particles can release their encapsulated payload under mild acidic conditions including those found in sites of inflammation, tumor tissue, or endocytic vesicles. In addition, the degradation rate can be easily tuned within the time scale of relevant cellular processes. The low- toxicity and payload versatility makes Ac-DEX particles an ideal platform for a wide range of biotherapeutic delivery applications including immunotherapy[2] and gene delivery [3,4]. We also present a new universal approach for the preparation of a new class of protein-based nanoparticles for the delivery of therapeutic payloads (B). The lipophilic surface modification of proteins allows the use of solvent evaporation techniques for the formation of nanoparticles. Unlike previous approaches, we preserve the native structure of the proteins and our particles are stable without denaturation or crosslinking of the biopolymers. The material shows low toxicity at high concentrations and successfully delivers drugs, for example chemo- therapeutics to cancer cells [5,6].

References: 1. Wich, P. R.: Nachrichten aus der Chemie 2015, 63 (2): 128–138. 2. Broaders, K. E. et al.: Proc. Natl. Acad. Sci. USA, 2009, 106: 5497–5502. 3. Cohen, J. L. et al.: Bioconj. Chem. 2011, 13: 1902–1905. 4. Wich, P. R. et al.: Aust. J. Chem. 2012, 1: 15–19. 5. Radi, L. et al.: Med. Chem. Commun. 2016, Advance Article, DOI: 10.1039/c5md00475f. 6. Fach, M; Radi, L; Wich, P. R.: J. Am. Chem. Soc. 2016, Article ASAP, DOI: 10.1021/jacs.6b06243.

28 • DPhG Annual Meeting 2016 Conference Book MATERIALS FOR DRUG DELIVERY

SL.04 Extracellular vesicles as smart carriers for small molecule drugs

Fuhrmann, G.1,2; Serio, A.2; Stevens, M. M.2 1 Helmholtz Centre for Infection Research, Helmholtz Institute for Pharmaceutical Sciences Saarland, Saarland University, Campus Building E8.1, 66123 Saarbrücken, Germany 2 Imperial College London, Department of Materials, Department of Bioengineering, Prince Consort Road, SW7 2AZ London, United Kingdom

Extracellular vesicles (EVs) are cell-derived lipid membrane particles decorated with surface and membrane proteins [1]. EVs are nature’s way to deliver information as they transfer protein and nucleic acid based cargoes selectively to their target cell. Moreover, EVs feature a naturally derived composition; can potentially bypass complement activation and coagulation factors leading to reduced immunogenicity and increased stability in biological fluids. In addition, they often transit to their specific target cell rendering them promising candidates for drug delivery applications. In order to harness these properties, new ways of encapsulation of drugs into EVs need to be developed. Although EVs have been investigated to deliver RNA-based therapeutics [2], their use as carriers for small molecule drugs has not been studied in detail.

a b c

Figure 1. (a) Chemical structure of hydrophobic porphyrin. (b) Size and morphology of EVs analysed by nanoparticle tracking analysis and electron microscopy. (c) Cell uptake of EV and liposome loaded porphyrin, or free drug (1 μM) in MDA or HUVEC cells (*p<0.05, **p<0.01 vs. free drug, ANOVA, Tukey post-hoc test).

In this work, we discuss the potential of EVs as smart drug delivery systems [3]. EVs from various cell types (endothelial HUVEC, stem MSC and cancer MDA cells) were isolated and compared regarding size distribution and morphology. Subsequently, they were loaded with model compounds (porphyrins) of different hydrophobicities (Fig. 1a). Porphyrins are currently investigated as potent drugs for photodynamic therapy (i.e., light activatable cytotoxic drug) but their cellular uptake under physiological conditions is often poor. Here, we show successful loading of porphyrins into EVs using various active and passive encapsulation techniques and assessed their therapeutic efficiency. First, EVs from various cell types showed an average diameter of 171-197 nm (single population) as confirmed by TEM (Fig. 1b). Subsequently, EVs were loaded with hydrophobic porphyrin at high ratios and more efficiently than into standard liposomes. EV-mediated delivery of encapsulated porphyrin significantly increased its cellular uptake in cancer and endothelial cells. EVs from MDA and HUVEC cells were more efficient to bring the hydrophobic porphyrin into the cells compared to MSC EVs but all EVs induced a significantly better drug uptake in cancer cells compared with free or liposome encapsulated porphyrin which indicated their potential for drug delivery applications (Fig. 1c). Upon light illumination, cells that had previously taken up EV-encapsulated porphyrin had a significantly increased cumulative risk to undergo cell death compared to free drug and control cells. Our results indicate that EVs are promising carriers that are able to overcome physiological barriers and deliver drugs with high efficiency and at the cellular level.

Acknowledgments: This work was supported by the European Union (Marie Curie Intra-European Fellowship for GF) and the German Academic Exchange Service (Postdoctoral Fellowship for GF). References: 1. Fuhrmann, G.; Herrmann, I.K.; Stevens M.M.: Nano Today. 2015, 10(3): 397-409. 2. El Andaloussi, S. et al.: Nat. Rev. Drug Discov. 2013, 5: 347-357. 3. Fuhrmann, G. et al.: J. Control Release. 2015, 205: 35-44.

DPhG Annual Meeting 2016 Conference Book • 29 SCIENTIFIC LECTURES

2.2 Ion Channels in Health and Disease Chairs: R. Lukowski, P. Ruth

SL.05 Phosphorylation of Histidine Residues by Nucleoside Diphosphate Kinase B: A Novel Mechanism to Regulate Ion Channel Activity in Disease

Wieland, T.1; Zhou, X. B.2; Feng, Y. X.1; Ruth, P.3 1 Institute of Experimental and Clinical Pharmacology and Toxicology and 2 1st Medical Clinic, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 3 Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tübingen, Tübingen, Germany

Reversible phosphorylation of amino acid side chains in proteins is a frequently used mechanism in cellular signal transduction and alterations of such phosphorylation patterns are very common in cardiovascular diseases. They reflect changes in the activities of the protein kinases and phosphatases involving signaling pathways. Reversible histidine phosphorylation, a well-known regulatory signal in lower organisms, has been largely neglected as it has been generally assumed that histidine phosphorylation is of minor importance in vertebrates. During the last decade, it has become evident that the nucleoside diphosphate kinase isoform B (NDPK B), an ubiquitously expressed enzyme involved in nucleotide metabolism, and a highly specific phosphohistidine phosphatase (PHPT-1) form a regulatory histidine protein kinase /phosphatase system in mammals. At least two ion channels substrates of NDPK B are known, the intermediate conductance potassium channel SK4 and the Ca2+ conducting TRP channel family member, TRPV5. In both proteins the phosphorylation of a specific histidine residue regulates channel activity. As SK4 channel is involved in pathological VSMC proliferation, we assessed function and expression of SK4 channels in VSMC from injured mouse carotid arteries by patch-clamping and real-time PCR. ISK4 was detectable in VSMC from injured but not from uninjured arteries correlating with the occurrence of the proliferative phenotype. Direct application of NDPK-B to the membrane of inside-out patches increased ISK4 whereas NDPKB did not alter currents in VSMC obtained from injured vessels of SK4-deficient mice. The NDPK-B-induced increase in ISK4 was prevented by PHPT-1 indicating that ISK4 is regulated via histidine phosphorylation in proliferating VSMC: Moreover genetic NDPK-B ablation reduced ISK4 by 50% suggesting a constitutive activation of ISK4 in proliferating VSMC. In line, neointima formation after wire-injury of the carotid artery was substantially reduced in mice deficient in SK4 channels or NDPK-B. Our data therefore show that NDPK-B to SK4 signaling is required for neointima formation. Targeting this interaction via e.g. activation of PHPT-1 may thus provide clinically meaningful effects in vasculoproliferative diseases such as atherosclerosis and post angioplasty restenosis.

30 • DPhG Annual Meeting 2016 Conference Book ION CHANNELS IN HEALTH AND DISEASE

SL.06 TRPC channels in lung function and disease

Dietrich, A.1; Hofmann, K.1; Fiedler, S.1; Weber, J.1; Gudermann, T.1; Königshoff, M.2 1 Walther-Straub-Institute for Pharmacology and Toxicology, Member of the German Center for Lung Research (DZL), LM-University of Munich, Munich, Germany 2 Comprehensive Pneumology Center, Member of the German Center for Lung Research (DZL), Munich, Germany

Classical transient receptor potential (TRPC) channels are highly expressed in different lung tissues, but their physiological function was still elusive. By construction of TRPC-deficient mouse models and analysis of lung function in comparison to wild-type animals, essential roles of these channels were identified. Moreover, signal transduction cascades for TRPC activation and pathways induced by TRPC dependent cation influx, which are important for smooth muscle cell proliferation1 and contraction2 as well as endothelial barrier function3 were dissected by the same approach. Recently, we focused on another pathophysiological role in the lung. Pulmonary fibrosis (PF) is a progressive lung disease ultimately leading to death with no effective therapeutic options.4 Next to other unknown factors PF may be induced by drugs like bleomycin. One of the pathological features of PF is the formation of myofibroblast foci and the deposition of extracellular matrix (ECM). Many different cell types are suggested to transdifferentiate into these activated myofibroblasts e.g. alveolar epithelial cells type I and II, resident and peripheral fibroblasts. Although the mechanism of PF is not fully understood yet, the profibrotic transforming growth factor β (TGF-β) is considered to play a crucial role. TRPC6 is an unselective cation channel which might contribute to PF since it is known to play an important role in myofibroblast transdifferentiation and wound healing in cardiac and dermal fibroblasts.5 To study a potential role of TRPC6 in the development of PF we analyzed lung function, gene and protein expression in wild-type (WT) and TRPC6-deficient (Trpc6-/-) lungs utilizing a bleomycin-induced PF-model. WT mice died more frequently than TRPC6-deficient mice during the fibrotic phase. Moreover, collagen accumulation in bleomycin-treated Trpc6-/--lungs was indistinguishable to PBS- treated control animals, while treated WT mice showed increased collagen levels. To analyze TRPC6 function on a cellular level, we isolated primary murine lung fibroblasts (PMLFs) from mice of both genotypes and incubated them with TGF-β. Most interestingly, barrier function and contraction of a collagen matrix was significantly reduced in Trpc6-/--PMLFs in clear contrast to WT cells, which also showed increased TRPC6-expression after exposure to TGF-β. Therefore, defining TRPC6 function might help to identify pharmacological targets for new therapeutic options in PF.

References: 1. Malczyk, M. et al., American journal of respiratory and critical care medicine 188, 1451-1459, doi:10.1164/rccm.201307-1252OC (2013). 2. Weissmann, N. et al., Proc Natl Acad Sci U S A 103, 19093-19098, doi:0606728103 [pii]10.1073/pnas.0606728103 (2006). 3. Weissmann, N. et al., Nature communications 3, 649, doi:10.1038/ncomms1660 (2012). 4.King, T. E., Jr., Pardo, A. & Selman, M., Lancet 378, 1949-1961, doi:10.1016/S0140-6736(11)60052-4 (2011). 5. Davis, J., Burr, A. R., Davis, G. F., Birnbaumer, L. & Molkentin, J. D., Developmental cell 23, 705-715, doi:10.1016/j.devcel.2012.08.017 (2012).

DPhG Annual Meeting 2016 Conference Book • 31 SCIENTIFIC LECTURES

SL.07 Pain control by calcium-activated potassium channels

Schmidtko, A.1 1 Pharmakologisches Institut für Naturwissenschaftler, Goethe-Universität, Fachbereich Biochemie, Chemie und Pharmazie, 60438 Frankfurt am Main, Germany

Potassium (K+) channels are the most populous and widely distributed class of ion channels, governed by at least 77 genes in humans. Accumulating research has highlighted a prominent involvement of different K+ channel subunits in pain processing, and they are increasingly recognized as potential targets for treatment of pain. K+ channels are organized into voltage-gated, two-pore, inwardly rectifying, and Ca2+-activated (KCa) channels. Based on their single-channel conductance and sequence homology of transmembrane cores, KCa channels are further divided into KCa1.1, KCa2.1-3, KCa3.1, KCa4.1-2, and KCa5.1 subunits. Recent studies demonstrated a distinct expression pattern of KCa subunits in various cell populations of the nociceptive system. Moreover, behavioral studies in knockout mice and pharmacological modulation of KCa channels revealed that certain KCa subunits are directly gated to pain-relevant stimuli, whereas others are specifically activated during the processing of chronic inflammatory or neuropathic pain. Here I will present some of the pain-relevant functions of KCa channels with focus on KCa1.1, KCa3.1 and KCa4.1. These data suggest that targeting of KCa subunits with dominant roles in pain processing could yield novel analgesic treatments.

32 • DPhG Annual Meeting 2016 Conference Book ION CHANNELS IN HEALTH AND DISEASE

SL.08 Noradrenergic and serotonergic compounds to target narcoleptic episodes in a mouse model

Schmidt, C.; Leibiger, J.; Fendt, M.

Symptoms of narcolepsy are excessive daytime sleepiness, REM sleep disturbance, nighttime wakefulness and cataplexy. The latter is characterized by a sudden loss of muscle tone triggered by high emotions and is often accompanied with falls. In human, a deficiency of orexin/hypocretin neurons are the underlying basis of these symptoms. Beyond other drugs noradrenergic as well as serotonergic antidepressants are used to treat the cataplectic attacks. Using a mouse model of narcolepsy (orexin-deficient mice), we here compared the effects of the selective norepinephrine reuptake inhibitor reboxetine (doses 0.05 – 5.0 mg/kg) and the selective serotonin reuptake inhibitor escitalopram (doses 1.0 – 9.0 mg/kg) on narcoleptic episodes. Furthermore, the effects of noradrenergic ɑ1-receptor stimulation agonism and antagonism were evaluated. After treatment with reboxetine, escitalopram, prazosin or cirazoline the number and duration of narcoleptic episodes were measured and analyzed. Additionally, the effects of the compounds on locomotor activity were evaluated in an open field test. Our data demonstrated that reboxetine reduces narcoleptic episodes in orexin-deficient mice more effective than escitalopram. Prazosin and cirazoline had no significant effects on narcoleptic episodes. Treatment with cirazoline but no other drug reduced significantly the locomotor activity in orexin-deficient mice. The results of our study support the idea that selective noradrenergic reuptake inhibitors should be used for the treatment of narcolepsy.

DPhG Annual Meeting 2016 Conference Book • 33 SCIENTIFIC LECTURES

2.3 Progress in Drug Synthesis Chairs: P. Gmeiner, M. Heinrich

SL.09 Making and breaking of chemical bonds with light - Visible light photocatalysis and photochromic molecular switches

König, B. Fakultät für Chemie und Pharmazie, Universität Regensburg, D-93040 Regensburg, Germany

Light is a fascinating reagent for chemistry as it provides energy to drive reactions, but leaves no trace. In visible light photoredox catalysis [1] coloured redox active dyes, such as ruthenium(trisbipyridine) [2], eosin Y [3] or rhodamine 6G [4], convert the absorbed light into redox energy allowing photoinduced electron transfer reactions. We discuss recent examples using the method for metal free cross-coupling reactions [5] in organic synthesis and the late stage functionalization of complex molecules [6].

Light can also be used to control the conformation of bioactive molecules. Photo-switchable enzyme inhibitors for sirtuins illustrate this application [7].

Acknowledgement: We thank the Deutsche Forschungsgemeinschaft (GRK 1626 and GRK 1910) for financial support. References: 1. König, B. (Ed.) Chemical Photocatalysis (De Gruyter) 2013. 2. Ravelli, D.; Protti S.; Fagnoni, M. Chem. Rev. 2016, DOI: 10.1021/acs.chemrev.5b00662 3. Hari, D. P.; König, B. Chem. Commun. 2014, 50, 6688. 4. Ghosh, I.; König, B. Angew. Chem. Int. Ed. 2016, 55, 7676. 5. Ghosh, I.; Ghosh, T.; Bardagi, J. I.; König, B. Science 2014, 346, 725. 6. Brachet, E.; Ghosh, T.; Ghosh, I.; König, B. Chem. Sci. 2015, 6, 987 7. Falenczyk, C.; Schiedel, M.; Karaman, B.; Rumpf, T.; Kuzmanovic, N.; Grøtli, M.; Sippl, W.; Jung, M.; König, B. Chem. Sci. 2014, 5, 4794.

34 • DPhG Annual Meeting 2016 Conference Book PROGRESS IN DRUG SYNTHESIS

SL.10 Diversity-oriented synthesis of pharmaceuticals

Müller, M. Institute of Pharmaceutical Sciences, Albertstr. 25, University of Freiburg, 79104 Freiburg

Diversity-oriented strategies are widespread in biosynthesis, or it may be more appropriate to say that biosynthesis is the archetype of diversity-oriented synthesis. We are working towards the elucidation and application of diversity-oriented aspects of biosynthesis and biocatalysis, like stereoselective phenolic coupling, chorismate-derived microbial products [1] or the use of multi-purpose biocatalysts for C–C bond formation [2].

CO2H NH2

OH 2,3-trans-CHA

CO2H CO2H NH2 OH

O CO2H OH ADIC 2,3-trans-CHD CO2H OH

CO H 2 O CO H CO Shikimat 2 2 Biosynthese oder Isochorismat O CO2H Glucose O CO2H OH HO2C OH CO2H Chorismat SHCHC

O CO2H CO2H CO2H NH2 ADC OH OH NH OH 2 3,4-trans-CHD 3,4-tr ans-CHA

By using biosynthetic strategies we introduced novel biocatalytic and biomimetic syntheses of small bioactive molecules like HMG-CoA reductase inhibitors, antiviral compounds and antibiotics[3]. The long-term goal of this project is the identification of advantageous trajectories for the synthesis of putative new bioactives without the need for the synthesis of huge compound libraries.

References: 1. J. Bongaerts, S. Esser, V. Lorbach, et al., Angew. Chem. 2011, 123, 7927–7932. 2. M. Müller, Adv. Synth. Catal. 2012, 354, 3161–3174. 3. M. Müller, Current Opin. Biotechnol. 2004, 15, 591–598.

DPhG Annual Meeting 2016 Conference Book • 35 SCIENTIFIC LECTURES

SL.11 Chemical synthesis of mycolactone analogs - insights into human Mycobacterium ulcerans infection

Blanchard, N. Université de Strasbourg, CNRS, Laboratoire de Chimie Moléculaire, 25 rue Becquerel 67087 Strasbourg, France, E-Mail: [email protected]

Buruli ulcer is a necrotizing skin disease present in more than thirty countries in the world, located mainly in Western and Central Africa but also in Australia and now Japan [1]. To date no specific treatment of Buruli ulcer has been developed which correlates with the dramatic lack of understanding of the associated chemical and biological mechanisms. This infection is caused by Mycobacterium ulcerans that secretes a toxin called mycolactone. This macrolide is the first polyketide isolated from a human pathogen. For the past years, we have been involved in a research program at the frontier of chemical synthesis and immunology (in close collaboration with the Institut Pasteur of Paris for the latter part), aiming at better understanding the mode of action of these human toxins [2,3]. We have devised a modular synthetic approach of mycolactone-derived probes that contributed to the discovery of the first structure-activity relationship of these exotoxins. Based on this knowledge, structurally related fluorescent hybrids were prepared and used in confocal microscopy. Overall, these informations were crucial to the discovery and confirmation of the first proteic target of mycolatones, a long- standing goal in this research area [4].

Mycolactones analogs Me Me Key Biological Ques ons (with areas of structural altera ons) Me Answered by our work OH OH Me The first Structure‐Ac vity Rela onships O O Finding the cri cal mo fs for func onal Diverted H interac ons is decisive for the development Total of a therapeu c solu on Small building Me Me Synthesis blocks O (3 or 4 carbon The development of fluorescent hybrids for

atoms) O cellular localiza on Me A modular synthe c strategy based on click chemistry has been successful in this regard

Iden fica on of a proteic target of Me mycolactone HO OH The use of simplified deriva ves and HO fluorescent probes allowed the iden fica on of a proteic target of mycolactone for the Me first me

References: 1. Review: N. Blanchard et al., Nat. Prod. Rep. 2013, 30, 1527-1567. 2. (a) N. Blanchard et al., Chem. Eur. J. 2011, 17, 14413-14419; (b) N. Blanchard et al., J. Med. Chem. 2014, 57 7382–7395; (c) N. Blanchard et al., In Strategies and Tactics in Organic Synthesis, Harmata, M., Ed. Academic Press 2015; 11, pp 85-117. 3. For selected contributions from other groups, see: (a) C. A. Brown, V. K. Aggarwal, Chem. Eur. J. 2015, 21, 13900-13903; (b) K.-H. Altmann et al., Chem. Eur. J. 2011, 17, 13017-13031; (c) Y. Kishi, Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 6703-6708; (d) E.-i Negishi et al., Chem. Eur. J. 2011, 17, 4118-4130. 4. Demangel, C. et al., Science - Trans. Med. 2015, 7, 289ra285.

36 • DPhG Annual Meeting 2016 Conference Book PROGRESS IN DRUG SYNTHESIS

SL.12 Inhibitors of the bacterial translocase MraY as potential novel antibiotics

Ducho, C.1 1 Saarland University, Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Campus C2 3, 66123 Saarbrücken, Germany

Emerging resistances of bacterial strains, e.g. methicillin-resistant Staphylococcus aureus (MRSA), towards established antibiotics cause an urgent need for the development of novel antibacterial agents. One approach to achieve this goal is the systematic investigation of naturally occurring antibiotics with new or previously unexploited modes of action. Muraymycins (e.g. 1-4, see Figure) belong to the class of nucleoside-peptide antibiotics and were isolated from a Streptomyces sp. as a collection of 19 compounds [1]. They inhibit the bacterial membrane protein translocase I (MraY), a key enzyme in the intracellular part of peptidoglycan biosynthesis and therefore an attractive target for new antibacterial drugs [2,3].

We have developed a modular tripartite synthetic approach for the preparation of muraymycins and their analogues, e.g. 5'-deoxy muraymycin C4 5 (see Figure) [4-8]. This tripartite route and also an alternative dipartite route were employed for the synthesis of a series of muraymycin analogues, including 5'- and 6'-epimers [9]. Using a fluorescence-based in vitro assay [10], the inhibitory activity against MraY was determined, with IC50 values of several analogues being in the nM to low M range. These results as well as further studies on the properties of synthetic muraymycin analogues [11,12] will be presented.

Acknowledgements: Deutsche Forschungsgemeinschaft (DFG, SFB 803 "Functionality controlled by organization in and between membranes"), Fonds der Chemischen Industrie (FCI, Sachkostenzuschuss) for funding. References: 1. McDonald, L. A. et al.: J. Am. Chem. Soc. 2002, 124(35): 10260-10261. 2. Winn, M. et al.: Nat. Prod. Rep. 2010, 27(2): 279-304. 3. Wiegmann, D. et al.: Beilstein J. Org. Chem. 2016, 12: 769-795. 4. Spork, A. P. et al.: Tetrahedron: Asymmetry 2010, 21(7): 763-766. 5. Spork, A. P. et al.: J. Org. Chem. 2011, 76(24): 10083-10098. 6. Büschleb, M. et al.: Amino Acids 2012, 43(6): 2313-2328. 7. Ries, O. et al.: Beilstein J. Org. Chem. 2014, 10, 1135-1142. 8. Spork, A. P. et al.: Chem. Eur. J. 2014, 20(47): 15292-15297. 9. Spork, A. P.; Ducho C.: Synlett 2013, 24(3): 343-346. 10. Brandish, P. E. et al.: J. Biol. Chem. 1996, 271(13): 7609-7614. 11. Rodolisa, M. T. et al.: Chem. Commun. 2014, 50(86): 13023-13025. 12. Ries, O. et al.: Med. Chem. Commun. 2015, 6(5): 879-886.

DPhG Annual Meeting 2016 Conference Book • 37 SCIENTIFIC LECTURES

2.4 New Research, New Researchers I Chairs: S. Laufer, A. Link

SL.13 Application of a dried H1N1 vaccine by epidermal powder immunization in piglets using a novel pyrotechnically driven applicator elicits antigen-specific antibodies

Engert, J.1; Anamur, C.1; Engelke, L.1; Fellner, C.2; Lell, P.2; Henke, S.3; Stadler, J. 4; Zöls, S.4; Ritzmann, M.4; Winter, G.1 1 Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximilians-University Munich, Butenandtstr. 5, D-81377 Munich, Germany 2 PyroGlobe GmbH, Hauptstr. 15, D-85276 Hettenshausen, Germany 3 IIS Innovative Injektionssysteme GmbH, Lohmannstr. 2, D-56626 Andernach, Germany 4 Klinik für Schweine, Ludwig-Maximilians-University Munich, Sonnenstr. 16, D-85764 Oberschleißheim, Germany

Epidermal powder immunization (EPI) has become a promising emerging approach in the search for new, needle-free vaccination strategies [1]. The principle of EPI relies on the acceleration of vaccine particles to high speed which are administered into the skin, thereby avoiding the use of needles and syringes [2-4]. Ideally, vaccine particles are deposited in the epidermis which lacks blood vessels and nerve endings, thereby making the administration pain free and in consequence may improve patient compliance. In this work, we present the results of an in vivo study in piglets using a dried influenza model vaccine which was applied by EPI using a novel pyrotechnically driven applicator. A liquid influenza vaccine (Pandemrix®) was first concentrated by tangential flow filtration, then transformed into a dry powder by collapse freeze-drying and subsequently cryo-milled to obtain antigen-loaded sugar particles in the desired size range of 20 – 80 µm. The vaccine powder was attached to a membrane of a novel pyrotechnical applicator using oily components with or without adjuvants. Upon actuation of the applicator, particles were accelerated to high speed as determined by a high speed camera setup. Piglets were immunized twice using either the novel pyrotechnical applicator or classical intramuscular injection with the commercial product. Blood samples of the animals were collected directly before vaccine administration and at various time points during the study and analysed for antigen-specific H1N1 IgG antibodies by an enzyme- linked immunosorbent assay. Our study shows that the administration of a dry, cryo-milled powder vaccine into piglets using a novel pyrotechnically driven applicator is possible. The speed and impact of the particles is sufficient to breach the stratum corneum of piglet skin. Importantly, the administration of the vaccine powder with or without adjuvants leads to an increase in the H1N1-specific IgG antibody titre in vivo compared to a negative control containing no antigen. The study provides evidence that it is possible to combine the advantages of a storage stable dry vaccine with a pain free injection technique.

Acknowledgements: The authors would like to thank PD Dr. Wolfgang Rebel (WOREB-TOX Consulting, Neustadt, Germany) and Dr. Monir Majzoub-Altweck (LMU Munich, Germany) for assistance with sample analysis. This work was supported by a grant from the Federal Ministry for Education and Research Germany (BMBF), grant number FKZ 13N11315 – 13N11319. References: 1. Mitragotri S.: Nat. Rev. Immunol. 2005. 5(12): 905-916. 2. Chen D et al.: Expert. Rev. Vaccines 2002. 1(3): 265-276. 3. Chen D et al.: Cell Res. 2002. 12(2): 97-104. 4. Dean HJ et al.: Vaccine 2004. 23(5): 681-686.

38 • DPhG Annual Meeting 2016 Conference Book NEW RESEARCH, NEW RESEARCHERS I

SL.14 The virulence factor LecB varies in clinical isolates: consequences for ligand binding and drug discovery

Titz, A.1 1 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus E8.1, 66123 Saarbrücken, Germany

P. aeruginosa causes a substantial number of nosocomial infections and is the leading cause of death of cystic fibrosis patients. This Gram-negative bacterium is highly resistant against antibiotics and further protects itself by forming a biofilm. Moreover, a high genomic variability among clinical isolates complicates therapy. Its lectin LecB is a virulence factor and necessary for adhesion and biofilm formation. We analyzed the sequence of LecB variants in a library of clinical isolates and demonstrate that it can serve as a marker for strain family classification. LecB from the highly virulent model strain PA14 presents 13% sequence divergence with LecB from the well characterized PAO1 strain. These differences might result in differing ligand binding specificities and ultimately in reduced efficacy of drugs directed towards LecB. Despite several amino acid variations at the carbohydrate binding site, glycan array analysis showed a comparable binding pattern for both variants. A common high affinity ligand could be identified and after its chemoenzymatic synthesis verified in a competitive binding assay: an N-glycan presenting two blood group O epitopes (H-type 2 antigen). Molecular modeling of the complex suggests a bivalent interaction of the ligand with the LecB tetramer by bridging two separate binding sites. This binding rationalizes the strong avidity (35 nM) of LecBPA14 to this human fucosylated N-glycan. Biochemical evaluation of a panel of glycan ligands revealed that LecBPA14 demonstrated higher glycan affinity compared to LecBPAO1 including the extraordinarily potent affinity of 70 nM towards the monovalent human antigen Lewisa. The structural basis of this unusual high affinity ligand binding for lectins was rationalized by solving the protein crystal structures of LecBPA14 with several glycans.

References: 1. R. Sommer, S. Wagner, A. Varrot, C. M. Nycholat, A. Khaledi, S. Haussler, J. C. Paulson, A. Imberty, A. Titz, Chem. Sci., 2016, doi:10.1039/C6SC00696E

DPhG Annual Meeting 2016 Conference Book • 39 SCIENTIFIC LECTURES

SL.15 From mucolipidosis type IV to Ebola: Insights into function and pharmacology of endolysosomal TRP channels

Grimm, C.1; Chen, C. C.1; Chao, Y. K.1; Plesch, E.2; Keller, M.2; Bracher, F.2; Wahl-Schott, C.1, Biel, M.1 1 Department of Pharmacy - Center for Drug Research and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians- Universität, Butenandtstraße 5-13, 81377 München, Germany 2 Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität, Butenandtstraße 5-13, 81377 München, Germany

Endosomes and lysosomes are cell organelles involved in transport, breakdown and secretion of proteins, lipids, and other macromolecules. Dysfunction of the endolysosomal system can cause storage disorders such as mucolipidoses, sphingolipidoses, or neuronal ceroid lipofuscinoses, but is also implicated in the development of metabolic diseases, neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease, retinal and pigmentation disorders, infectious diseases and cancer. Endolysosomal cation channels are highly critical for the complex regulation of the multiple endolysosomal trafficking and transport processes including endo- and exocytotic events as well as the regulation of calcium, sodium, proton and other ionic concentrations within the endolysosomal vesicle lumen. With a combined approach comprising endolysosomal patch-clamp electrophysiology, imaging techniques, molecular biology techniques, and transgenic mouse models, we have recently contributed to the elucidation of the biophysical properties and physiological roles of some of the ion channels found in endosomes and lysosomes such as TRPML channels (also called mucolipins) and Two-pore channels (TPCs). Thus, we have generated (TRPML3, TPC2) or made use of (TRPML1, TPC1) knockout mouse models to study the pathophysiological consequences of the loss of these cation channels for the endolysosomal system. We found that loss of TPC2 makes mice more susceptible for the development of hypercholesterinemia and fatty liver hepatitis, we have explored the role of TPCs in viral infections such as Ebola virus infection and we have studied the role of TPCs for the migration of cancer cells and the formation of metastases. We have further developed small molecule tools to biophysically characterize channel properties and to explore options for a small molecule treatment of human patients carrying mutations in TRPML1 which cause the lysosomal storage disease mucolipidosis type IV. In summary, we provide novel insights into the function and pharmacology of different endolysosomal cation channels and we present novel technical approaches to characterize these proteins functionally such as selective early endosomal versus late endosomal/ lysosomal patch clamp techniques.

References: 1. Grimm, C. et al, Chemistry and Biology, 2010, 17(2): 135-148. 2. Cang, C. et al, Cell, 2013, 152: 778-790. 3. Grimm, C. et al, Nature Communications, 2014, 5: 4699. 4. Chen, C.-C. et al, Nature Communications, 2014, 5: 4681. 5. Sakurai, Y. et al, Science, 2015, 347: 995-998.

40 • DPhG Annual Meeting 2016 Conference Book NEW RESEARCH, NEW RESEARCHERS I

SL.16 Molecular mechanisms of lipoxin and resolvin biosynthesis

Lehmann, C.1; Cumbana, R.2; Ebert, R.2; Toewe, A.3; Angioni, C.3; Ferreirós, N.3; Geisslinger, G.1,3; Parnham, M. J.1; Steinhilber, D.2; Kahnt, A. S.2 1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology, Theodor Stern Kai 7, 60596 Frankfurt/Main, Germany 2 Goethe-University, Institute of Pharmaceutical Chemistry, ZAFES, Max-von-Laue-Str. 9, D-60438 Frankfurt/Main, Germany 3 Goethe University, Institute of Clinical Pharmacology, Pharmazentrum Frankfurt, ZAFES, Theodor Stern Kai 7, D-60590 Frankfurt/Main, Germany

The resolution of inflammation is an active process controlled by endogenous specialized pro-resolving lipid mediators (SPM) which are formed by various immune cells from polyunsaturated fatty acids during the course of an acute inflammatory response by the concerted action of different lipoxygenases, cyclooxygenase-2 and cytochrome P450 enzymes. Lipoxins and resolvins represent two members of this still growing family of SPM. However, the molecular mechanisms underlying cellular lipoxin and resolvin biosynthesis in humans are not completely understood. But this knowledge is of great importance for the development of pro-resolutionary pharmacotherapies as well as a better comprehension of potential resolution-toxic effects induced by already existing anti-inflammatory therapies. Therefore, we aimed to investigate lipoxin and D-type resolvin formation in primary immune cells such as neutrophils, macrophages, platelets and monocytic cell lines in detail. We found that various enzymes known to be involved in the biosynthesis of pro-inflammatory leukotrienes such as FLAP, cPLA2α, LTA4 hydrolase and LTC4 synthase also influence transcellular lipoxin and resolvin formation [1]. In addition, we studied differently polarized and stimulated macrophages as single cell model for lipoxin and resolvin biosynthesis. Here, polarisation and activation considerably rearranged the enzymatic layout of the cells leading to substantial changes in lipid mediator patterns.

References: 1. Lehmann, C. et al., FASEB J. 2015 (12):5029-43.

DPhG Annual Meeting 2016 Conference Book • 41 SCIENTIFIC LECTURES

SL.17 Insights from functional lipidomics into the long-term regulation of kinases by vitamin A

Pein, H.1; Voelkel, M.1; Schneider, F.1; Rossi, A.2; Koeberle, S. C.3; Loeser, K.1; Morrison, H.3; Sautebin, L.2; Werz, O.1; Koeberle, A.1 1 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, 07743 Jena, Germany 2 Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy 3 Leibniz Institute of Age Research - Fritz-Lipmann-Institute, 07745 Jena, Germany

Functional lipidomics combines comprehensive lipid profiling with target identification and lead discovery to reveal novel strategies for pharmacological intervention. Using this approach, we found that phosphatidylcholines with polyunsaturated fatty acids (PUFA-PC) oscillate during the cell cycle. PUFA-PC suppresses cell proliferation by inhibiting membrane binding and thus activation of the survival kinase Akt [1] and is apparently part of a feed- forward mechanism of apoptosis. By screening a library of nutrients, natural products and drugs for selective effects on the phospholipidome and lipid mediator production, we identified vitamin A as long-term regulator of Akt through modulation of PUFA-PC. The pleiotropic effects of retinol (vitamin A) on adult physiology and embryonic development are mediated through the active metabolite all-trans retinoic acid (RA). Bound to retinoic acid receptors (RARs), RA controls transcription but also fine-tunes the expression of RA target genes by activating kinases such as Akt. The mechanisms for long-term regulation of Akt by vitamin A are incompletely understood. We have shown by lipidomic profiling that retinol and RA deplete NIH-3T3 fibroblasts from phosphatidylcholines (PC) with polyunsaturated fatty acids, in particular linoleic acid (18:2), and concomitantly induce long-term Akt activation. Moreover, we found that the cellular ratio of 18:2-PC determines the activation state of Akt, and ascribed the effects of vitamin A on lipid composition and Akt signaling to retinoic acid X receptor (RXR) activation by using selective agonists. Administration of vitamin A to mice decreased 18:2-PC levels in brain (but not in other tissues) and in parallel enhanced basal Akt activation, which was attributed to astrocytes rather than to neurons in dissociated cortical cultures. Our study reveals how vitamin A is likely to regulate long- term Akt signaling: binding to nuclear receptors, modulating the membrane lipid composition and subsequently enhancing Akt membrane translocation and thus activation. We anticipate this cascade to be key for brain homeostasis in light of the well-established roles of vitamin A, polyunsaturated fatty acids as well as Akt for brain physiology.

References: 1. Koeberle, A. et al.: Proc. Natl. Acad. Sci. U. S. A. 2013, 110(7): 2546-2551.

42 • DPhG Annual Meeting 2016 Conference Book NEW RESEARCH, NEW RESEARCHERS I

SL.18 α-Aminoxy peptides: from membranolytic anticancer foldamers to the first in class peptidomimetic Hsp90 C-terminal domain dimerization inhibitors

Diedrich, D.1; Bhatia, S.2; Frieg, B.1; Stein, S.3; Bopp, B.4; Lang, F.2; Ernst, T.5; Rodrigues Moita, A. J.1; Rüther, A.6; Lüdeke, S.6; Kassack, M. U.1; Hochhaus, A.5; Borkhardt, A.2; Jose, J.4; Kurz, T.1; Gohlke, H.1; Hauer, J.2; Hansen, F. K.1 1 Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany. 2 Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich Heine University Düsseldorf, Medical Faculty, Düsseldorf, Germany. 3 Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Germany. 4 Institute for Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Münster, Germany. 5 Klinik für Innere Medizin II, Universitätsklinikum Jena, Erlanger Alle 101, 07747 Jena. 6 Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 25, Freiburg, Germany.

α-Aminoxy peptides are peptidomimetic foldamers with high proteolytic and conformational stability. We have recently developed an improved synthetic route to α-aminoxy peptides by the straightforward use of a combination of solution-phase and solid-phase supported methods.[1] In particular long-chained oligomers showed a remarkable anticancer activity against a panel of cancer cell lines.[1] Preliminary mode of action studies revealed that the anticancer activity of long-chained derivatives is at least in parts related to membranolysis whereas α-aminoxy hexapeptides did not possess membranolytic properties.[1] By X-ray crystallography, 2D NMR experiments, MD simulations, and CD spectroscopy we identified a 28-helical conformation as the preferred secondary structure of α-aminoxy peptides.[1] Molecular modeling studies revealed that this 28-helix can mimic the spatial arrangement of peptide side chains in α-helices.[1] Based on these results we hypothesized that α-aminoxy peptides can serve as α-helix mimetics and thus could be a suitable scaffold to inhibit protein-protein interactions (PPIs). To validate this hypothesis, we selected the C-terminal dimerization of the heat shock protein 90 (Hsp90) as a proof of concept PPI target. Hsp90 has been discovered as a promising target to combat cancer and several inhibitors targeting the ATP binding pocket in the N-terminal domain (NTD) of Hsp90 are currently under clinical investigation.[2] Unfortunately, all N-terminal Hsp90 inhibitors trigger a survival mechanism in cancer cells referred to as the heat shock response (HSR), which represents a major efficacy problem in clinical use. In contrast to Hsp90 NTD inhibitors, compounds that act at the C-terminal domain (CTD) of Hsp90 do not initiate the unfavorable HSR.[2] One novel approach to inhibit the CTD of Hsp90 is to target the Hsp90 CTD dimerization interface.[3,4] However, only a few peptidic CTD dimerization inhibitors have been described so far.[4] We now utilized the knowledge about the folding propensity of α-aminoxy peptides[1] and recently discovered hot spots at the Hsp90 CTD dimerization interface[3] to design and synthesize the first peptidomimetic Hsp90 CTD NO NO NO NO NO NO dimerization inhibitors. The hit compound 1 (Cbz- Phe- Phe- Ile- Ile- Leu- Leu-NH2), an α-aminoxy hexapeptide, exhibited promising anti-proliferative and cytotoxic activity in several human myeloid leukemic cell line models including imatinib resistant cell lines. The specificity of 1 to Hsp90 was determined by a Hsp90- dependent luciferase refolding assay and further confirmed by analysis of the downstream signalling. Furthermore, binding of 1 to the CTD of Hsp90 was demonstrated by MST measurements with the purified recombinant CTD of Hsp90. Most notably, in vivo proof of concept studies demonstrated the efficacy of 1 at 0.5 mg/kg in a K-562-Luciferase Xenograft tumor model. Compound 1 reduced the tumor burden significantly with respect to tumor weight (1: 0.2 ± 0.01g vs vehicle: 1.26 ± 0.44g (p = 0.04)). Immunoblot analysis of tumor samples derived from mice treated with 1 revealed the absence of HSR. Taken together, we have developed the first in class peptidomimetic Hsp90 CTD dimerization inhibitors.

References: 1. Diedrich, D. et al.: Chem. Eur. J. 2016: in press. 2. Wang, Y.; McAlpine, S. R.: Future Med. Chem. 2015, 7(2): 87–90. 3. Ciglia, E. et al.: PLOS ONE 2014, 9(4): e96031. 4. Bopp, B. et al.: BBA – Gen. Subjects 2016, 1860(6): 1043–1055.

DPhG Annual Meeting 2016 Conference Book • 43 SCIENTIFIC LECTURES

2.5 New Approaches in Gene and Stem Cell Therapy Chairs: M. Biel, E. Wagner

SL.19 Evaluating microRNA-based therapies in stem cell-derived retinal organoids

Busskamp, V. Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany

The outer segments of cones serve as light detectors for daylight color vision, and their dysfunction leads to several human blindness conditions. Here we show that the cone-specific disruption of the DGCR8 locus in adult mice led to the loss of miRNAs and the loss of outer segments, resulting in photoreceptors with significantly reduced light responses. However, the number of cones remained unchanged. The loss of the outer segments occurred gradually over one month, and during this time the genetic signature of cones decreased. Re- expression of the sensory-cell-specific miR-182 and miR-183 prevented the loss of outer segments. These miRNAs were also necessary and sufficient for the formation of inner segments, connecting cilia and short outer segments, as well as light responses in stem-cell-derived retinal cultures. Our results show that miR-182- and miR-183-regulated pathways are necessary for cone outer segment maintenance in vivo and functional outer segment formation in vitro.

44 • DPhG Annual Meeting 2016 Conference Book NEW APPROACHES IN GENE AND STEM CELL THERAPY

SL.20 Localized vs systemic gene therapy using rAAV vectors: achievements and remaining challenges; the example of Duchenne Muscular Dystrophy gene therapy

Le Guiner, C. Atlantic Gene Therapies, INSERM UMR 1089, Nantes, France

Among vector systems that allow efficient in vivo gene transfer, recombinant Adeno Associated Virus vectors (rAAV) hold great promise and are currently evaluated in multiple clinical trials for the treatment of inherited diseases. In particular, gene-therapy of muscle diseases rapidly gained attention because delivery of rAAV vectors of several serotypes results in very efficient transduction of skeletal muscles. Duchenne Muscular Dystrophy (DMD) is an example of a devastating muscle disorder without strongly effective treatment, which could benefit from the reconstitution of a deficient protein after rAAV-mediated gene transfer. DMD is a X-linked inherited muscle-wasting disease primarily affecting young boys with a prevalence of 1:5,000. The disease is caused by loss-of-function mutations in the gene encoding for the Dystrophin protein and is characterized by systemic, progressive, irreversible and severe loss of muscle function. Using a large network of laboratories with complementary skills, we demonstrated the efficacy of two different rAAV-based gene therapy strategies for DMD, in which the vector was injected in the GRMD (Golden Retriever Muscular Dystrophy) dog model, after one single locoregional intravenous injection in a forelimb. In GRMD dogs, such intravenous perfusion, in which the limb is transiently isolated from the general circulation by an atraumatic tourniquet, allowed diffuse transduction of the skeletal muscles of the limb, with significant normalisation of histological, NMR imaging and spectroscopy parameters and muscle strength, without deleterious immune responses. These results paved the way for a human trial of the upper-limb of non-ambulatory DMD patients. This locoregional approach was essential at a time when no one had been able to show therapeutic efficacy of rAAV vectors for muscular diseases beyond one isolated muscle. This proof of efficacy in a whole forelimb, as well as the significant increase in production capacity of rAAV vectors, recently opened the way to an injection in the whole body. The goal is to reach the entire muscles of the body including the heart and the diaphragm, and thus hope for a real treatment of systemic muscle diseases, like DMD. We then systemically administered one of our therapeutic rAAV vectors by a single intravascular injection in several GRMD dogs. Such injection led to long- term transduction of distant muscle groups and extended lifespan (up to 2 years) of the treated GRMD dogs. When using high doses of the therapeutic rAAV vector profound improvement of multiple clinical features was observed and no toxicity or deleterious immune consequences were observed. The specific development and the recent results of these translational projects will be presented, with a particular focus of the remaining challenges regarding rAAV vector delivery for muscle diseases.

DPhG Annual Meeting 2016 Conference Book • 45 SCIENTIFIC LECTURES

SL.21 From viruses to designer nanoparticles - tailoring adeno-associated viruses for gene therapy

Büning, H.1,2,3 1 Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 21, 50931 Cologne, Germany 2 German Center for Infection Research (DZIF), partner sites Bonn-Cologne and Hannover-Braunschweig-Berlin 3 Institute of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

Vectors based on the replication-defective parvovirus adeno-associated virus (AAV) have emerged as one of the leading gene transfer systems for in vivo gene therapy. As of yet, AAV vectors demonstrated an excellent safety profile and clinical success has been reported for a variety of disorders. The main focus of AAV’s clinical application is long-term gene transfer into post-mitotic tissues such as liver, muscle, brain or the eye. Furthermore, AAV vectors do show promise as tools for transient modification of proliferating cells and as templates in gene correction approaches, and have recently entered the area of vaccine development. However, since AAV vectors use common receptors for cell binding and cellular uptake, target as well as off- target cells are transduced when AAV vectors are applied in vivo. Besides increasing the vector dose needed to achieve therapeutic efficacy, transgene expression in off-target cells may result in undesired adverse events. Furthermore, locally as well as systemically applied AAV particles are transported via the blood to the liver, where they accumulate. Since the native tropism is mainly defined by the viral capsid, capsid engineering is exploited to overcome these drawbacks and optimize the AAV vector system for clinical use. Using AAV serotype 2 (AAV-2), the so far best characterized serotype, as basis, we have developed strategies to expand AAV's tropism by overcoming pre- and/or post entry barriers enabling thereby transduction of cell types refractory to transduction with natural serotypes. The same strategies are explored to re-directed AAV vectors towards a receptor of choice tackling the challenge of off-target transduction. As the capsid also represents the main target of the host immune system, we exploited capsid engineering for developing AAV vector variants that transduce their target cells despite the presence of neutralizing antibodies. Furthermore, we could demonstrate the potency of capsid engineering in augmenting immunogenicity of AAV vector-based vaccines.

46 • DPhG Annual Meeting 2016 Conference Book NEW APPROACHES IN GENE AND STEM CELL THERAPY

SL.22 Gene therapy for human achromatopsia

Michalakis, S. Department Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München

The past decade has seen major developments in the field of ocular gene therapy. The use of recombinant adeno-associated virus (rAAV) vectors has “jump started” the field and led to the development of very promising gene therapies for the treatment of previously untreatable genetic diseases of the eye like achromatopsia (ACHM). ACHM is an autosomal recessive vision disorder, which occurs approximately in one out of every 30,000 births. Patients with ACHM suffer from severely impaired daylight vision, characterized by poor visual acuity, photophobia, nystagmus (involuntary rapid eye movements), and lack of the ability to discriminate colors. Currently, six disease causing genes have been identified including the two genes encoding the cone-specific cyclic nucleotide gated channel subunits CNGA3 and CNGB3. Up to 80% of ACHM patients carry mutations in either CNGA3 or CNGB3. CNGB3 mutations are more prevalent in Europe and United States whereas CNGA3 is the most frequent disease gene in the Middle East and China. Within the collaborative research consrotium RD-CURE (http://www.rd-cure.de), which was established in 2012 between scientists and clinicians from the LMU Munich and the University Eye Hospital Tübingen we developed and evaluated a therapeutic strategy for ACHM based on rAAV vector-mediated CNGA3 gene supplementation. These activities led to the first human gene therapy trial for achromatopsia (https://clinicaltrials.gov; NCT02610582). In this presentation I will provide an overview on the preclinical development, toxicology and biodistribution studies and on the ongoing phase I/II clinical trial.

Acknowledgement: This work was supported by the Tistou and Charlotte Kerstan Foundation.

DPhG Annual Meeting 2016 Conference Book • 47 SCIENTIFIC LECTURES

2.6 MS Based Drug Screening Chairs: M. Lämmerhofer, K. Wanner

SL.23 Frontal affinity chromatography and mass spectrometry: a perfect fit in drug discovery

Massolini, G.; Calleri, E.; Temporini, C.; Brusotti, G. Dipartimento di Scienze del Farmaco, Viale Taramelli 12 Università degli Studi di Pavia, 27100 Pavia, Italy

Molecular interactions are the engine of biological processes, they determine the efficacy of drugs and therapeutics. Thus, considerable efforts have been devoted to manage the screening of a large number of compounds for ligands of biologically relevant targets in order to accelerated drug discovery processes. Frontal affinity chromatography (FAC) using immobilized receptor-based stationary phases represents a novel screening method among the affinity-based screening strategies to quickly prioritize compound hits for further biological testing and holds particular appeal due to its simplicity, wealth of approaches for coupling biomolecules, and modularity that supports the application of a range of detectors. In particular, FAC coupled to mass spectrometry (FAC-MS) has been shown to be a viable screening tool that has been y applied to a wide range of biological targets. Aside from the utility of the frontal analysis method to provide the opportunity to rank-order binding strengths in a single experiment as each compound has a unique m/z value, the technique provides precise and accurate Kd measurements on single ligands. In the presentation some examples of the power of FAC-MS approach [1-5], developed in our laboratory, using membrane and cytosolic/Nuclear receptors will be discussed together with the key parameters to be considered for the development of a reliable bioaffinity tool.

References: 1. Calleri E. et al : J. Med. Chem. 2010, 53 : 3489–3501 2. Calleri E et al. :Journal of Chrom. A, 2012, 1232 : 84– 92 3. Temporini C. et al. : Anal Bioanal Chem, 2013 405: 837–845 4. Temporini C. et al. Journal of Chrom. A, 2013, 1284 : 36– 43 5. de Moraes M.C. et al J Chrom A 1338 (2014)

48 • DPhG Annual Meeting 2016 Conference Book MS BASED DRUG SCREENING

SL.24 MS in pharmaceutical drug discovery

Ottl, J. Novartis Pharma NIBR, Basel, Switzerland

Application of biophysics/label-free detection technologies in drug discovery has gained more and more interest in the past years. This lecture will shed some light on the current state of the art use of biophysics suitable for higher throughput industrial lead/drug discovery applications. Key focus of the presentation is the utilization of mass spectrometry (MS) as biophysical method at early stages of the drug discovery process in miniaturized high throughput formats. In our group we are successfully applying MS-based screening to detect and qualify the binding of low molecular weight (LMW) lead candidates to their respective target proteins. In projects we mostly follow one of two typical application routes: the detection of the LMW cpd after being presented to the target protein or the detection of the protein/compound complex formed when reactive LMW cpds are exposed to target proteins. Case studies show how our application is impacting the early stages of drug discovery and how they are augmenting other technologies and approaches.

DPhG Annual Meeting 2016 Conference Book • 49 SCIENTIFIC LECTURES

SL.25 High Resolution Mass Spectrometry of Antibody Drug Conjugates Using the Orbitrap Mass Analyzer

Scheffler, K. Thermo Fisher Scientific, Dreieich, Germany

The complexity of modern therapeutic proteins presents a great analytical challenge. Most often a whole set of analytical techniques is used to characterize these proteins. With mass spectrometry alone several complementary methods are required to analyze protein drugs on the intact protein and on the peptide levels. Native mass spectrometry on the intact protein level allows also for the analysis of molecules which rely on non- covalent interactions to preserve critical structural features, such as antibody-drug conjugates (ADC). In addition the use of 100% aqueous buffers in native MS analysis produces lower charge states detected at higher m/z values compared to analysis under denaturing condition and thus improves mass separation of heterogeneous mixtures. Recent technical advancement on the benchtop Orbitrap mass spectrometry platform offers now complete characterization of the complex conjugates composed of small molecule drugs attached to antibodies on a single instrument. In this presentation data obtained from two different types of ADCs are presented and relevant analysis workflows as well as challenges are discussed.

50 • DPhG Annual Meeting 2016 Conference Book THE DIVERSITY IN PHARMACEUTICAL BIOLOGY

2.7 The Diversity in Pharmaceutical Biology Chairs: I. Merfort, T. Efferth

SL.26 Beyond malaria: The clinical anticancer activity of artesunate

Efferth, T. 1 Department of Pharmaceutical biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany. E-mail: [email protected]

More than a decade ago, we initiated a research program on the molecular pharmacology of phytochemicals derived from Chinese medicinal herbs. Bioactive plant extracts have been fractionated by chromatographic techniques. We isolated bioactive compounds and elucidated their chemical structures by nuclear magnetic resonance and mass spectrometry. A promising compounds was artemisinin from Artemisia annua L. and its semisynthetic compound artesunate. Artemisinin and artesunate are anti-malarial drugs. Our data indicated profound activity against cancer cells, but also against various viruses, Schistosoma, Trypanosoma, and even plant crown gall tumors. To elucidate the molecular mode of actions against cancer, we applied molecular biological and pharmacogenomic approaches in vitro and in vivo. Different signaling pathways were identified not only in cancer cells but also in cells infected with viruses, e.g. HCMV, HSV1 and others. To translate the experimental results in cell lines and animals to the bedside, we report on the compassionate use of artesunate in single cancer patients as well as on our efforts to initiate several clinical phase I/II trials in veterinary tumors as well as in human cervix or colorectal carcinoma. These pilot studies indeed indicate that artesunate is not only useful as antimalarial drug, but also exerts activity against cancer and viral diseases. Clinical results will also be presented that not only artesunate as semisynthetic chemical derivative of artemisinin, but also herbal extracts from Artemisia annua are active in veterinary and human tumor patients. Artesunate represents an illustrative example for the therapeutic potential of medicinal herbs and drugs derived from traditional Chinese medicine.

References: 1. Efferth et al.: Journal of Molecular Medicine 2002;80:233-42. 2. Efferth: Drug Resistance Updates 2005;8:85-97. 3. Efferth et al.: Molecular Cancer Therapeutics 2008;7:152-61. 4. Li et al.: Cancer Research 2008;68:4347-51. 5. Shapira et al.: Clinical Infectious Diseases 2008;46:1455-7. 6. Efferth et al.: Clinical Infectious Diseases 2008;47:804-11. 7. Krishna et al.: Ebiomedicine 2014;2:82-90. 8. Saeed et al.: Pharmacological Research 2016;110:216-226.

DPhG Annual Meeting 2016 Conference Book • 51 SCIENTIFIC LECTURES

SL.27 Application of plant metabolomics in herbal drug research

Bauer, R.1 1 Institute of Pharmaceutical Sciences, University of Graz, Universitaetsplatz 4, 8010 Graz, Austria

In recent years, plant metabolomics has increasingly been used for the identification of active constituents in medicinal plants. LC-HRMS based metabolomics and multivariate data analyses allow correlation of the chemical profiles of herbal extracts and their pharmacological activities, and the prediction of compounds which contribute to the activity of the extract. Within the FWF funded national research network (NFN) “Drugs from nature targeting inflammation”, the metabolic profile of ethanolic extracts from 36 Lonicera samples has been analyzed by UHPLC-ESI-HRMS using a Q ExactiveTM Hybrid Quadrupole Orbitrap-MS (Thermo Fisher). In parallel the extracts have been tested for inhibitory effects on NO production in RAW 264.7 macrophages, IL-8 expression in HUVECs, and NF-κB activation in HEK 293 cells, as well as their effect on the activation of PPAR-β/δ in HEK 293 cells. The LC-MS data were processed in an untargeted approach by MZmine 2 [1]. Peaks were identified and quantified by Lipid Data Analyzer [2]. The abundance of the peaks was linked to the pharmacological activity of the extracts using SIMCA 13 [3]. Correlations were modelled using orthogonal partial least squares-discriminant analysis (OPLS-DA) and the results were visualized by means of the S-plot. This led to the identification of compounds which were predicted to be most relevant for the in vitro anti-inflammatory activity in the particular bioassays [4]. The metabolic potential of the human microbiome also needs to be considered for the explanation of the activity of herbal extracts. Intestinal microbiota may metabolize herbal constituents and form active compounds, and herbal medicine may help to balance the gut ecosystem [5].Metabolomics can be used to compare herbal extracts before and after incubation and to identify new metabolites [6]. Quality of control of herbal medicines is currently mainly based on specifications of pharmacopoeias including assays on single marker compounds. More holistic methods, like metabolic profiling with multivariate data analysis may be applied in the future.

Acknowledgments: We gratefully acknowledge the funding provided by the Austrian Science Fund (FWF) for projects S107- B13 (NFN: Drugs from Nature Targeting Inflammation) and P26148 “High-Throughput Identification of Lipid Molecular Species in LC-MS/MS Data”, and the support by Dr. Kenneth Bendix Jensen, NAWI Graz Central Lab "Environmental, Plant & Microbial Metabolomics". References: 1. Pluskal, T. et al.: BMC Bioinformatics 2010, 11: 395. 2. Hartler, J. et al.: Bioinformatics 2011, 27: 572-577. 3. Whelehan, O.P. et al. : Chemometr. Intell. Lab. 2006, 84: 82–87. 4. Waltenberger, B. et al.: Monatshefte für Chemie. 2016,147: 479-491. 5. David, L.A. et al.: Nature 2014, 505: 559-563. 6. Selma, M.V. et al.: Food Funct. 2014, 5: 1779-1784.

52 • DPhG Annual Meeting 2016 Conference Book THE DIVERSITY IN PHARMACEUTICAL BIOLOGY

SL.28 Cystobactamids: Novel gyrase inhibitors from myxobacteria that inhibit multi-resistant pathogens

Herrmann, J.1,2; Müller, R.1,2 1 Helmholtz Centre for Infection Research (HZI), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarland University, Campus E8 1, 66123, Saarbrücken, Germany 2 German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig

Infectious diseases caused by bacterial pathogens remain a major health issue, not only restricted to developing countries. Especially antibiotics with activity against hard-to-treat and multidrug-resistant pathogens of the so- called ESKAPE panel (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are urgently needed. Cystobactamids are a group of newly discovered myxobacterial natural products isolated from Cystobacter spp. that show strong antibacterial activity against many Gram-negative and Gram-positive pathogens in the submicromolar range. Based on analysis of the biosynthesis gene cluster and self-resistance determinants in the natural producer organism, the molecular target of this compound class was identified. Cystobactamids target bacterial type IIa topoisomerases and their mechanism of inhibition resembles that of fluoroquinolone (FQ) antibiotics such as ciprofloxacin. However, only limited FQ cross-resistance was observed and we conclude that the cystobactamid binding site on gyrase might be overlapping but is not identical to that of ciprofloxacin. The basic structure of the cystobactamids, that consist of a central amino acid linker region flanked by substituted p-amino benzoic acid units, provides a new scaffold for the generation of innovative antibiotic drugs to combat infections caused by Gram-negative and Gram-positive pathogens. Intriguingly, in our search for alternative myxobacterial producer strains we discovered some new natural analogs with even improved antibacterial spectrum. This enabled an initial structure-activity- relationship (SAR) study that now aids to the rational design of second generation cystobactamids by means of chemical synthesis. Preclinical studies are currently being pursued with two frontrunner molecules with the overall aim to provide a first proof-of-concept for the in vivo efficacy of the cystobactamids.

References: 1. Baumann, S. et al.: Angew. Chem. Int. Ed. Engl. 2014, 53(52): 14605-14609.

DPhG Annual Meeting 2016 Conference Book • 53 SCIENTIFIC LECTURES

SL.29 Genome mining-guided drug discovery: from proteasome to protease inhibitors

Kaysser, L.1,2; Zettler, J. 1,2; Zubeil, F.3; Leipoldt, F. 1,2; Wolf, F.1,2: Schorn, M.4; Bauer, J.1,2; Bendel, T.1,2; Kulik, A.5; Dorrestein, P. C.6; Moore, B. S.4,6; Gross, H.1,2; Grond, S.3 1 Dept. Pharmaceutical Biology, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany 2 German Centre for Infection Research (DZIF), partner site Tübingen 3 Inst.. Organic Chemistry, University of Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany 4 Scripps Institution of Oceanography, UC San Diego, 9500 Gilman Drive, La Jolla CA, 92093-0204, USA 5 Interfaculty Institute of Microbiology and Infection Medicine Tübingen (IMIT), University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany 6 Skaggs School of Pharmacy, UC San Diego, 9500 Gilman Drive, La Jolla CA, 92093-0751, USA

Proteases and protease-like enzymes are ubiquitous in all aspects of life and have thus been established as therapeutic targets for various diseases such as cancer, hypertension, thrombosis, diabetes as well as viral and bacterial infections. Notably, a substantial number of pharmaceutical relevant protease inhibitors have been developed or at least been inspired by natural products. We recently identified the biosynthetic gene clusters of two bacterial epoxyketone proteasome inhibitors, eponemycin and epoxomicin.[1] The latter served as a lead compound for the development of carfilzomib (Kyprolis®) which has been approved by the European Commission for the treatment of multiple myeloma in 2015. Bioinformatic analysis and genetic engineering of the gene clusters as well as precursor feeding studies showed that a hybrid non-ribosomal peptide synthetase/polyketide synthase (NRPS/PKS) machinery and a conserved acyl-CoA dehydrogenase are responsible for the building of the characteristic epoxyketone warhead in these compounds.[2] Genome mining revealed a number of homologous gene clusters in various bacteria. Interestingly, one of these gene clusters encoded for the production of the hydroxamate metalloproteinase inhibitor matlystatin. Biosynthetic investigations of this molecule are ongoing and current results will be presented.

Acknowledgments: This work was funded by a Feodor Lynen Research Fellowship from the Aelxander von Humboldt-Foundation to L.K., a DFG Research Grant KA 3071/4-1 to L.K. and a PhD Stipend from the Ministry of Science Baden-Wuerttemberg to F.W. References: 1. Schorn, M. et al.: ACS Chem. Biol. 2014, 9(1): 301-309. 2. Zettler, J. et al.: Chembiochem 2016, 17(9): 792-798.

54 • DPhG Annual Meeting 2016 Conference Book THE QUALITY OF THERAPEUTIC PROTEINS

2.8 The Quality of Therapeutic Proteins Chair: H. Wätzig

SL.30 Analytical tools in the biosimilar development

Schiestl, M. 1 Sandoz GmbH, Biochemiestrasse 10, A-6250 Kundl

The evolvement in analytical tools over the last decades provided ever increasing insights in the nature of biopharmaceuticals. From an industry perspective, this evolvement facilitated the development of manufacturing processes. It allowed us to move from the former way of development where a ‘process defined the product’ to a nowadays more systematic, i.e. Quality by Design approach which gains better process and product understanding. This knowledge lead to better control strategies with an increased process robustness. It also facilitated the ability to change processes without altering the product quality.

The central role of analytics becomes especially striking when we look at biosimilars, which are products containing essentially the same active pharmaceutical ingredients as their reference products [1]. The focus in biosimilar development is the demonstration of similarity, which can be done best and in the most sensitive manner by analytical tools. Already the early manufacturing process development requires a wide panel of protein characterization methods for making a biosimilar candidate. Otherwise it is unlikely to achieve the normally very narrow development target which is defined by the measured variability of the reference product.

The presentation will show practical examples highlighting the role of analytics in biosimilar development.

References: 1. European Commission Consensus Information Document 2013 “What you need to know about Biosimilar Medicinal Products“ http://ec.europa.eu/DocsRoom/documents/8242/attachments/1/translations/en/renditions/native

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SL.31 Technical challenges for development and manufacturing of biopharmaceuticals

Scherübl, C.1 1 Boehringer Ingelheim Pharma GmbH & Co. KG, Biopharma Fill & Finish Germany, Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany

The characteristics of biopharmaceuticals pose several challenges for drug product development and manufacturing. The presentation summarizes the main challenges (e.g. aseptic filling, aggregation, light sensitivity and interaction with packaging material) and shows how they are addressed within routine drug product manufacturing.

56 • DPhG Annual Meeting 2016 Conference Book THE QUALITY OF THERAPEUTIC PROTEINS

SL.32 How does pH Affect Interfacial Antibody Behaviour and Aggregation upon Shaking?

Köpf, E.1; Schroeder, R.2; Brezesinski, G.3; Friess, W.1 1 Ludwig-Maximilians-Universitaet Muenchen, Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, 81377 Munich, Germany 2 AbbVie Deutschland GmbH & Co. KG, 67061, Ludwigshafen am Rhein, Germany 3 Max-Planck Institute of Colloids and Interfaces, Department of Interfaces, 14476 Potsdam, Germany

In the development of protein pharmaceuticals the high tendency to form aggregates requires special consideration. Specifically, the liquid-air interface is known to negatively impact protein stability and to trigger aggregate and particle formation. Although considerable effort has been put into the understanding of the mechanisms behind the link between the interfacial behaviour and the formation of particles is still poorly understood. In this study three antibodies (mAB1, mAB2, human IgG) were investigated for the impact of pH on their liquid-air interfacial protein film characteristics, as well as on protein-protein interactions and therefore protein stability in terms of aggregation. The formation of particles strongly depends not only on the protein itself, but also on pH [1]. Surface pressure measurements indicate a time- and concentration dependent adsorption [e.g. Πmax = 18.5 mN/m for IgG after 4h]. Equilibrium surface pressure as well as interfacial film compressibility investigated in a Langmuir trough is not considerably influenced by pH except for pH values of ≤ 2. This can be explained by pH-induced changes in secondary structure (Fig. 1). Moreover, with decreasing pH the numbers of particles formed upon shaking become less (Fig. 2).

Infrared Reflectance Absorbance (IRRA) spectra substantiate the presence of the IgG molecules at the interface by their amide bands. Both, adsorption and compression do not induce any conformational changes. Brewster Angle Microscopy (BAM) shows a continuous but heterogeneous protein distribution over the interface (Fig. 3) [2].

Submerse AFM of the interfacial film reveals inhomogeneities, similar to BAM results but at different scale, with areas of telescoped material which appear upon compression. Determination of B22 via DLS [3] points out that protein-protein interactions depend on the IgG itself and that B22 decreases with increasing pH values. Therefore, attractive forces between IgG molecules increase with increasing pH thereby causing increased numbers of particles after 48h shaking. Hence, the combinatorial use of the different methods described provides comprehensive insight into interfacial protein behaviour on the one hand, and on interaction parameters in solution on the other. Protein pharmaceuticals are exposed to liquid-air interfaces at many points during development, production and storage. Choosing an appropriate formulation pH is of crucial importance to improve protein stability against interface related stress.

References: 1. Jayaraman M., et al., Eur. J. Pharm. Biopharm. 2014, 87: 299–309 2. Rodrigueznino M., et al., Food Hydrocoll. 2005, 19 (3): 417–428 3. Menzen T. and Friess W., J. Pharm. Sci. 2014, 103 (2): 445–455

DPhG Annual Meeting 2016 Conference Book • 57 SCIENTIFIC LECTURES

SL.33 Rational Design of Thermodynamic and Kinetic Binding Profiles by Optimizing Surface Water Networks Coating Protein Bound Ligands

Krimmer, S. G.1; Cramer, J.1 ; Betz, M.1 ; Fridh, V.2 ; Karlsson, R.2 ; Heine, A.1 ; Klebe, G.1 1 Institute of Pharmaceutical Chemistry, University of Marburg, Marbacher Weg 6, 35032 Marburg, Germany 2 GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden

In any biological system, the binding reaction between an inhibitor and its target protein takes place in water. Therefore, water molecules have to be considered as additional binding partners besides protein and inhibitor. Nevertheless, the influence of water molecules on protein–ligand binding is still poorly understood. With the aim to study the thermodynamic influence of the rearrangement of water molecules around a newly formed protein- ligand complex, we analysed a series of eight congeneric thermolysin inhibitors exhibiting side-chains of increasing size and hydrophobicity (from methyl to a phenylethyl) by high-resolution crystallography and isothermal titration calorimetry [1]. Across the eight complexes, the crystal structures revealed water networks of different degrees of completeness adjacent to the protein-bound ligands. The observed structural differences correlated remarkably well with the differences observed between the thermodynamic signatures of complex formation. The establishment of a well-ordered, pronounced water network resulted in an increase in ∆H°, whereas the disruption of a water network resulted in an increase in –T∆S°. The inhibitor with the highest affinity exhibited a medium-sized hydrophobic side-chain stabilizing a pronounced water network, resulting in an highly favourable ∆H° overcompensating losses in –T∆S°. Based on these observations, we designed new inhibitors with the aim to further improved the water network stabilization and thereby boost affinity [2]. Prior to ligand synthesis, we validated the newly designed ligands by predicting the putative water networks by MD simulations. Afterwards, the new ligands were synthesized, and structurally and thermodynamically characterized. Furthermore, we also determined their binding kinetics by surface plasmon resonance. As a result, one of the new ligands showed the most pronounced water network, and, consequently, the highest affinity with an overall improvement of 1.5 orders of magnitude. Moreover, due to the pronounced water network caging the protein- bound ligand, a decreased dissociation rate constant was determined for this ligand.

References: 1. Krimmer, S.G. et al.: Chem. Med. Chem. 2014, 9, 833-846 2. Krimmer, S.G. et al.: submitted

58 • DPhG Annual Meeting 2016 Conference Book ANTI-INFECTIVES

2.9 Anti-Infectives Chair: R. Hartmann

SL.34 Innovative antibiotics from microorganisms: Some case studies

Müller, R.1 1 Helmholtz-Institutut für Pharmazeutische Forschung, Saarland

Natural products continue to be a major source for novel antibiotic lead structures often exhibiting new mode of action(s). In this presentation I will describe our efforts to identify innovative sources of antibiotics and the path from microbial extracts to promising lead structures for pharmaceutical development. As a first example for entirely new structures I will describe the recently identified cystobactamides as a new class of natural products with broad activity against gram-negative ESKAPE pathogens. The ESKAPE panel of bacteria represents the currently most difficult to treat causative and often multiresistant agents of nosocomial infections. The current status of our efforts to further develop and characterize the cystobactamid gyrase inhibitors in preclinical studies will be presented. As second example an update on our efforts to identify novel tuberculosis agents will be given. Despite modern antibiotics and the development of a curative regimen for this devastasting disease, tuberculosis remains a worldwide problem and the emergence of drug-resistant Mycobacterium tuberculosis has prioritised the need for new drugs. We show that new and optimised derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo. After identification of the griselimycin biosynthetic gene cluster we were able to clarify the biosynthesis of the biosynthetic precursor methyl-proline which is incorporated into the natural product at the site of metabolic lability. This finding opened up oportunities to improve the ratio of metabolically stable versus unstable griselimycin derivatives. Based on self-resistance studies in Streptomyces and genomic analyses of resistant mycobacteria, we found that griselimycins inhibit the DNA polymerase sliding clamp DnaN; these interactions were characterised by surface plasmon resonance and crystal structure analysis. Furthermore, we discovered that infrequent resistance to griselimycin is associated with highly unusual target amplification in mycobacteria. Our results demonstrate that griselimycin and its derivatives have high translational potential for tuberculosis, validate DnaN as an antimicrobial target and capture the process of antibiotic pressure- induced target amplification.

DPhG Annual Meeting 2016 Conference Book • 59 SCIENTIFIC LECTURES

SL.35 New Antibiotics for the Post-Antibiotic Era

Mobashery, S. Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 USA

-Lactam antibiotics are the most important antibiotics targetting the bacterial cell wall. However, their utility has been compormised due to broad resistance by bacteria to these antibiotics. Yet, their targets, penicillin-binding proteins (PBPs) still remain important targets for antibiotics. The importance of PBPs, especially the high- molcular-mass variants, is due to their critical functions in biosynhesis of bacterial cell wall. But equally important, these proteins decorate the surface of the plasma membrane, hence access by antibiotics is often less of a problem than is for the cytoplasmic targets. I will describe an in silico search for novel classes of antibiotics carried out with the X-ray structure of the PBP2a from methicillin-resistant Staphylococcus aureus (MRSA). MRSA is a global scourge, infections by which afflicate 100,000 individuals annually in the USA alone. A significant proportion of these cases leads to mortality. Resistance to -lactam antibiotics in these organisms is overencompassing, including penicillins, cephalosporins, carbapenems, among others. I will describe discovery of the oxadiazole and quinazolinone classes of antibacterials, which target PBPs in MRSA. The lead compounds were elaborated synthetically into a library of several hundred members, which were screened for antibacterial activity. Both classes of antibiotics target bacterial cell-wall biosynthesis, they exhibit favorable pharmacokinetic properties, they are efficacious in a rodent model of MRSA infection and they are orally bioavailable. Both classes of compounds hold great promise in addressing clinical needs in treating infections by MRSA.

60 • DPhG Annual Meeting 2016 Conference Book ANTI-INFECTIVES

SL.36 LpxC inhibitors – a novel class of antibiotics

Holl, R.1,2 1 Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany 2 Institut für Organische Chemie, Universität Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany

Due to the constantly increasing number of multidrug resistant bacteria, the successful treatment of bacterial infections with the currently available antibiotics is becoming more and more difficult. Therefore, novel antibacterial compounds addressing so far unexploited bacterial targets, thereby circumventing established mechanisms of resistance, are urgently required [1,2]. A promising strategy to combat infections caused by multidrug resistant Gram-negative bacteria is the inhibition of LpxC, a Zn2+-dependent deacetylase, which was validated as an antibacterial drug target. LpxC catalyzes the first irreversible step of lipid A biosynthesis in Gram-negative bacteria, the deacetylation of UDP-3-O-[(R)-3- hydroxymyristoyl]-N-acetylglucosamine. Lipid A, the hydrophobic membrane anchor of lipopolysaccharides in the outer membrane of Gram-negative bacteria, is essential for growth and viability of the majority of Gram-negative bacteria. As the inhibition of lipid A biosynthesis is lethal to these bacteria, LpxC inhibitors represent a promising new class of antibiotics [3]. The potent LpxC inhibitor CHIR-090, containing a hydroxamate moiety to chelate the catalytic Zn2+-ion and a hydrophobic region to mimic the fatty acyl chain of the enzyme’s natural substrate, was chosen as lead compound for the development of C-furanosidic and proline-derived LpxC inhibitors as well as of the respective open-chain derivatives [4,5]. To access the envisaged compounds, chiral-pool syntheses were elaborated. Disc diffusion assays against various clinically important Gram-negative bacteria were performed to reveal the antibacterial properties of the synthesized compounds. Additionally, their inhibitory activity of was determined in an LpxC enzyme assay.

References: 1. Projan, S., J. et al.: Curr. Opin. Microbiol. 2002, 5, 463-465. 2. Cooper, M. A. et al.: Nature 2011, 472, 32. 3. Kalinin, D. V. et al.: Curr. Top. Med. Chem. 2016, 16, 2379-2430. 4. Tangherlini, G. et al.: Bioorg. Med. Chem. 2016, 24, 1032-1044. 5. Müller, H. et al.: Eur. J. Med. Chem. 2016, 110, 340-375.

DPhG Annual Meeting 2016 Conference Book • 61 SCIENTIFIC LECTURES

2.10 New Research, New Researchers II Chairs: A. Link, S. Laufer

SL.37 How polysaccharide superstructure impacts hydrogel properties: A Raman optical activity study

Lüdeke, S.1 ; Rüther, A.;1 ; Forget, A.2; Roy, A.3; Carballo, C.3; Dukor, R. K.3; Nafie, L. A.3; Johannessen, C.4; Shastri, V. P.5 1 Inst. of Pharmaceutical Sciences, University of Freiburg, Albertstr. 25,79104 Freiburg, Germany 2 Future Industries Insitute, University of South Australia, MM Building, 5095 Mawson Lakes, Australia 3 BioTools, Inc., 17546 Beeline Hwy, Jupiter, FL, USA 4 Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium 5 Institute for Macromolecular Chemistry, University of Freiburg, Stefan-Meier-Str. 31, 79104 Freiburg, Germany

Hydrogels are cross-linked, water swollen polymers with numerous applications, particularly for biomedical and pharmaceutical purposes [1]. The choice of a hydrogel for a specific application is determined by its mechanical properties. Polysaccharide hydrogels are particularly advantageous, because they are non-toxic and biocompatible but achieving defined stochastic roughness and stiffness is often not trivial. We have shown that polysaccharide backbone carboxylation alters the chain organization of agarose-derived hydrogels, thereby leading to much softer hydrogels, which can be used to mimic the biomechanical properties of the extracellular matrix [2]. We applied Raman optical activity (ROA), a spectroscopic technique used previously to study polysaccharides [3], on gel samples of agarose and agarose with different degrees of carboxylation (28, 60, and 93%). The carboxylation-dependent spectra showed features that could be clearly attributed to higher order structure. By employing matrix least squares global fitting [4] we were able to identify two spectral species that interconvert into each other as a function of chemical modification. Comparison to ROA spectra from quantum chemical calculations allowed to assign them to double helical structure in agarose and β-strand-like conformation in the carboxylated derivatives. Our results suggest that 40% of the polysaccharide are already single stranded in native agarose and that carboxylation fully inhibits chain organization as double strands. This suggests that the rigidity and stiffness of agarose hydrogels depends on the presence of double helix structure.

References: 1. Peppas, N. A., et al.: Eur. J. Pharm. Biopharm 2000, 50(1): 27–46. 2. a) Forget, A., et al.: Proc. Natl. Acad. Sci. U. S. A. 2013, 110(32): 12887–12892. b) Forget, A., et al.: Macromol. Rapid Commun. 2015, 36(2): 196–203. 3. Bell, A. F., et al.: J. Raman Spectrosc. 1995, 26(12): 1071–1074. 4. Rüther, A., et al.: J. Phys. Chem. B 2014, 118(14): 3941–3949.

62 • DPhG Annual Meeting 2016 Conference Book NEW RESEARCH/NEW RESEARCHERS II

SL.38 Skin penetration analysis by confocal Raman microspectroscopy – potentials and pitfalls

Lunter, D.1 1 Department of Pharmaceutical Technology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany

In the course of the development of new dermal dosage forms, one major aspect that needs to be evaluated is the dermal absorption of the active from the dosage form. The use of confocal Raman microspectroscopy (CRM) to investigate this aspect has gained increasing attention over the past years. Among the potentials of CRM are the ability to perform penetration analysis in vivo as well as ex vivo with no or only little sample preparation. High spatial resolution and high chemical sensitivity enable label free detection. Two methods for CRM skin penetration analysis are currently used: depth profiling and 2D imaging. The former is the acquisition of spectra along a line perpendicular to the skin surface and subsequent calculation of the relative amount of the active across the depth of the skin that results in a penetration-depth-profile. The latter is the acquisition of spectra across a 2D image plane and subsequent calculation of the relative concentration of the active in each image point. It can be used to generate a color coded image of the distribution of the active within the scanned area. I investigated the feasibility of depth profiling and imaging to investigate the effect of penetration enhancers on the dermal penetration of a model active (procaine HCl) from a model formulation (hydrophilic gel) [1]. Both methods give similar results on the extent of penetration enhancement while the imaging approach is able to give more detailed information on the localization of the active with respect to the skin constituents. As CRM has not yet become a standard technique in dermo-pharmaceutical research consensus on optimal parameters for the conduct of skin penetration studies remains yet to be established. Factors like objective numerical aperture, pinhole size, laser wavelength and removal of the formulation from the skin surface strongly impact the results. Consequently are among the pitfalls: the use of inadequate microscope configuration (objectives and pinholes) and the insufficient removal of the formulation from the skin surface. I thus investigated the effect of the use different objectives and pinholes on the outcome of CRM depth profiling experiments. Regarding objective configuration, I found that simply by varying the objective and pinhole penetration depths- values varied between 10 and > 50 µm for the same sample. The effect of sample preparation was found to be even more pronounced. If the formulation was left on the skin or simply wiped off, the amounts of active which were erroneously detected in the skin were an order of magnitude higher than the actual penetrated amount [2]. To exclude such bias careful evaluation of the methods variables needs to be performed prior to any penetration study.

Acknowledgements: PD Dr. Martin Schenk is acknowledged for the donation of pig ears. This project was supported by the European Social Fund and by the Ministry of Science, Research and the Arts Baden-Wuerttemberg References: 1. Lunter D, Daniels R, J. Biomed. Opt., 2014, 19(12): 126015-126019. 2. Lunter D, Skin Pharma. Physiol., 2016, 29: 92–101.

DPhG Annual Meeting 2016 Conference Book • 63 SCIENTIFIC LECTURES

SL.39 Influence of Th2 cytokines on the cornified envelope, tight junction proteins and ß-defensins in filaggrin-deficient skin equivalents

Hönzke, S.1; Schäfer-Korting, M.1 ; Hedtrich, S.1 1 Institute of Pharmacy, Pharmacology & Toxicology, Königin-Luise-Straße 2+4, 14195 Berlin, Germany

Atopic dermatitis (AD) is a chronic inflammatory skin disease which is characterized by an impaired skin barrier function. In 2006, mutations in the filaggrin gene (FLG) were identified as a major predisposing factor for the manifestation of AD [1]. Aside from barrier deficiencies, AD is characterized by over-shooting Th2-mediated inflammatory processes and impaired innate immunity such as reduced expression of antimicrobial peptides (AMP) [2]. The Th2 cytokines IL-4 and IL-13 significantly contribute to the pathogenesis of AD, but their effects on the skin barrier and particularly the interdependencies with FLG deficiency are not yet fully understood. In this study, the influence of FLG knockdown on the expression of the AMP´s human β-defensin 1-3 and skin barrier proteins under normal and inflammatory conditions was evaluated. Histological examination revealed a thickening of the viable epidermis in the skin models following IL-4 and IL-13 treatment; FLG knockdown amplified this effect (FLG+ 89.5 ± 9.9 µm vs. FLG+/IL-4/13 128.8 ± 22.1 µm and FLG- 109.8 ± 10.2 µm vs. FLG-/IL-4/13 160.7 ± 30.8 µm). Additionally, supplementation of FLG- models with IL-4/IL-13 resulted in a major shift towards higher pH values (pH 6.37 ± 0.03) compared the normal skin models (pH 5.45 ± 0.05). Furthermore, we observed a compensatory 3-fold upregulation of involucrin and occludin in FLG- models, which was considerably disturbed by IL-4/13 exposure. Concordantly, these cytokines significantly reduced the expression of the skin barrier proteins FLG and involucrin in normal skin models. Most interestingly, for the first time we detected significantly (~ 5-fold) higher expression of HbD2 in FLG- models. This was particularly noteworthy because HbD2 is known to be upregulated through bacteria or inflammation but not by a genetic defect [2]. Interestingly, this up-regulation was markedly reduced under inflammatory conditions. In conclusion, these results indicate that defects in the epidermal barrier and cutaneous innate immune response are not primarily linked to filaggrin deficiency but are rather secondarily induced by Th2 inflammation [3].

Acknowledgments: Financial support by the Collaborative Research Center 1112 for the projects C02 is gratefully acknowledged. References: 1. Palmer, C.N., et al. Nat Genet, 2006. 38(4) 2. Kopfnagel, V., J. Harder, and T. Werfel. Curr Opin Allergy Clin Immunol, 2013. 13(5) 3. Hönzke, S., et al. J Invest Dermat, 2016. 136 (3)

64 • DPhG Annual Meeting 2016 Conference Book NEW RESEARCH/NEW RESEARCHERS II

SL.40 Tumor selectivity of V-ATPase inhibition is based on differential regulation of AMPK von Schwarzenberg, K.1; Menche, D.2; Müller, R.3; Vollmar, A. M.1 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich 2 Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Gerhard - Domagk-Str.1, 53121 Bonn, Germany 3 Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO 151150, Universitätscampus E8 1, 66123 Saarbrücken, Germany

Altered tumor metabolism is a hallmark of cancer directly linked to tumor progression. A key player involved in metabolic adaption is the AMP activated protein kinase-1 (AMPK) which is often deregulated in tumors but its role for tumor cell survival is controversial discussed. The recent finding that the vacuolar H+-ATPase (V-ATPase) is involved in AMPK activation during glucose deprivation promoted a role of the V-ATPase in controlling cellular metabolism and represents an interesting new option for targeting cancer. Here we show that AMPK is differentially regulated in tumor and non-tumor cells upon V-ATPase inhibition. The V-ATPase inhibitor archazolid lead to an increased phosphorylation as well as lysosomal localization of AMPK in non-tumor cells – in contrast to tumor cells where no regulation by archazolid was observed. Induction of AMPK by archazolid was shown to have a pro-survival role as silencing of AMPK in non-malignant cells decreased growth rate of the cells whereas induction of AMPK in tumor cells protected them from archazolid induced cytotoxicity. These effects where accompanied by a distinct metabolic regulation concerning ATP level, glucose uptake, ROS production and NADPH level in tumor and non-tumor cells. The pro-survival effects could be attributed to AMPK ability to maintain redox homeostasis by inhibiting ROS production and maintaining NADPH level. Therefore this work identifies a novel role for V-ATPase in metabolic regulation and presents the AMPK as a key protein in tumor-specific cytotoxicity of V-ATPase inhibition.

DPhG Annual Meeting 2016 Conference Book • 65 SCIENTIFIC LECTURES

SL.41 Analysing the framework of protein ligand interactions: Ligand-sensing cores and privileged scaffolds

Koch, O. TU Dortmund University, Faculty of Chemistry and Chemical Biology, Otto-Hahn-Straße 6, 44227 Dortmund, Germany

Binding site comparison is a widely established approach for the identification of similar ligand binding, which is typically based on the comparison of potential protein-ligand interaction patterns [1]. However, it was shown that the possible binding pocket space is limited [2] and that similar spatial arrangement of secondary structure elements around the ligand binding site (‘ligand-sensing cores’) can recognize similar scaffolds independent of the overall fold (see Figure 1) [3]. We are focusing on the development of different tools, to get a better insight into the underlying assumption and to analyse how to use this information for computational molecular design.

Fig. 1: Glycogen synthase kinase 3 and trypanothione synthetase show a similar ligand-sensing core and known inhibitors that share a similar paullone scaffold

A python based workflow was developed that utilise Scaffold Hunter [4] to analyse bioactivity databases for privileged scaffolds that belong to ligands binding to completely different targets. Using this tool, a scaffold binding to different targets was identified and used to find new inhibitors for one of the proteins (BRD4) with IC50s in the low micromolar range. Biochemical validation using two orthogonal assays and crystallisation studies proved the BRD4 binding of ligands similar to known ligands of the other protein target. So, a new relationship between BRD4 and another protein target was discovered. Overall, this example underlines the basic concept of privileged scaffolds and how to use this information in drug discovery workflows. An automated method to determine ligand-sensing cores of otherwise unrelated proteins was also developed. The current implementation allows the calculation of an all-against-all comparison of predicted binding site within all known protein structures (>100,000) on a current workstation within a reasonable time. In addition, for the identification of similar ligand-sensing cores of one selected binding site whole proteins can be used, neglecting the drawbacks of automated binding site identification methods. The final aim is a database with all known ligand- sensing cores that will hopefully be available soon for general use. The underlying approach and newly developed computational tools will be discussed in detail and promising results will be presented to demonstrate the usefulness.

References: 1. Ehrt C, Brinkjost T, Koch O., J. Med. Chem.. 2016, 59: 4121-4151. 2. Skolnick, J., et al., Bioorg. Med. Chem. Lett. 2015, 25:1163-1170. 3. Koch, O., Future Med. Chem. 2011, 3: 699-708 4. Klein, K., Koch, O., Kriege, N., Mutzel, P., Schäfer, T., Mol .Inf. 2013, 32: 964–975.

66 • DPhG Annual Meeting 2016 Conference Book NEW RESEARCH/NEW RESEARCHERS II

SL.42 A fluorescence polarization-based competition binding assay for detecting compounds interacting with inactive mitogen-activated protein kinases and development of covalent inhibitors of c-Jun N-terminal kinase 3

Koch, P.1 1 Eberhard Karls Universität Tübingen, Institute of Pharmaceutical Sciences, Department of Medicinal Chemistry, Auf der Morgenstelle 8, 72076 Tübingen, Germany.

The two mitogen-activated protein kinases (MAPK) c-Jun N-terminal kinase 3 (JNK3) and p38α MAPK have emerged as attractive drug targets due to their implication in several pathologic conditions such as neurodegenerative diseases and inflammation.[1-3] Biological evaluation of inhibitors for these two kinases is generally carried out through activity assays, although these methods are often expensive and time consuming. Fluorescence polarization (FP)-based competition binding assays were developed for both enzymes using probe 2 (JNK3: Kd = 3.0 nM; p38α MAPK: Kd = 5.7 nM) obtained by labelling of our potent dual JNK3/p38α MAPK inhibitor 1 (JNK3: IC50 = 24 nM; p38α: IC50 = 17 nM).[4] The assays were validated with known inhibitors of the two enzymes and results showed good correlation with data obtained from activity assays. The developed JNK3-FP assay represents the first example of an FP-based binding assay for this protein kinase and was recently employed to study the targeting of the gatekeeper of JNK3 with halogen bonds.[5] These features, together with the viability of both FP binding assays for the high throughput screening format, makes the assays suitable as fast and inexpensive pre-screening protocols for JNK3 and p38α MAPK inhibitors. In continuing efforts to enhance both, JNK3 selectivity and activity of our pyridinylimidazole-based kinase inhibitors, we successfully applied the approach of covalent targeting of JNK3 described by Zhang et al.[6] to our dual JNK3/p38α inhibitor 1 (Figure 1). It is crucial for the linker between the hinge-binding motif and the electrophilic warhead to comprise the optimal length and angle in order to orient the warhead ideally for the nucleophilic attack of the thiol to occur and simultaneously retain the original binding mode of the scaffold. Therefore, we synthesized a broad variety of linkers, altering the meta- and para-substitution pattern on both phenyl rings. Covalent JNK3 inhibitor 3 was finally identified as a potent JNK3 inhibitor showing an IC50-value in the low nanomolar range and displaying 735-fold selectivity against p38α MAPK (JNK3: IC50 = 2 nM; p38α: IC50 = 1,543 nM). Compound 3 is metabolic stable when exposed to human liver microsomes and displays a good selectivity profile in a screening against 410 kinases.

Fig.1: Pyridinylimidazole-based dual JNK3/p38α MAPK inhibitor 1 as a lead compound for development of FP-probe 2 and covalent JNK3 inhibitor 3.

References: 1. Manning, A. M.: Nat. Rev. Drug Discov., 2003, 2, 554-565. 2. Koch, P. et al.: J. Med. Chem. 2015, 58, 72-95 3. Cuenda, A.: Biochim. Biophys. Acta, 2007, 1773, 1358-1375 4. Ansideri, F. et al.: Anal. Biochem, 2016, 503, 28-40. 5. Lange, A. et al.: J. Am. Chem. Soc. 2015, 137, 14640-14652. 6. Zhang, T et al.: Chem. Biol. 2012, 19, 140-154.

DPhG Annual Meeting 2016 Conference Book • 67 SCIENTIFIC LECTURES

2.11 Clinical Pharmacy Chair: K. Friedland; C. Wahl-Schott

SL.43 Evidence-based evaluation system for OTC drugs

Achenbach, J.1; Culmsee, C.1 1 Institut für Pharmakologie und Klinische Pharmazie, Philipps-Universität Marburg, Karl-von-Frisch-Straße 1, 35032 Marburg

Background and Objectives: Methods of evidence-based medicine and pharmacy are becoming more and more important. Especially the field of self-medication has been neglected in terms of evidence-based principles so far. In community pharmacies there is often not enough time to perform literature searches on over-the- counter (OTC) drugs during daily practice. So far, a system that provides transparent, fast and evidence-based evaluations of OTC drugs to support counselling and scientifically-based information on self-medication is not available. The aim of this project is to develop an evaluation and information system for OTC drugs by using the indication migraine. This system has to provide a clear structure and easy orientation. Moreover, it has to present detailed data that are necessary to practice evidence-based and individual counselling. Methods and Results: In order to structure the evaluation system aspects of the “Analytic Hierarchy Process” (AHP) have been applied [1]. This method developed by Thomas L. Saaty is used to support decision problems by defining the problem or goal (e.g. an effective and safe migraine therapy) and by structuring the problem in a hierarchy (e.g. the single efficacy and safety criteria for different drugs used in this indication). Furthermore, the AHP provides the possibility to include individual patient preferences. A systematic procedure including systematic literature searches has been used in order to evaluate efficacy and safety criteria. Moreover, parts of the “Grading of Recommendation, Assessment, Development and Evaluation (GRADE)-system” have been integrated into the evaluation system. Thus, it is possible to incorporate quality ratings of the included studies and to provide a transparent and reproducible presentation of the data. Results of the evaluations for the different drugs are shown in structured and easily accessible “Summary-of- Findings-(SoF)-tables”. Conclusion: The system provides evidence-based data as well as transparent and reproducible results of the efficacy and safety evaluations of different drugs used in the self-medication of acute migraine headaches. This way it can support pharmacists to provide evidence-based counselling on OTC drugs.

References: 1. Saaty, T.L.: EJOR 1990, 48: 9–26.

68 • DPhG Annual Meeting 2016 Conference Book CLINICAL PHARMACY

SL.44 Influence of over-the-counter drugs and prescription-only-medication on male fertility

Strobach, D. University Hospital Munich, LMU

The number of couples seeking consultation for infertility problems has steadily increased over the past decades. It is assumed that male infertility concerns at least 50% of all affected couples. Male infertility is a multifactorial state. Among other risk factors, drugs can adversely affect male fertility and sexual function. But, epidemiological data on potential adverse drug reactions (ADRs) on male fertility and reproduction are sparse. In addition, information on these effects for a specific drug is often not easy accessible. Overall, the awareness of potential ADRs on male fertility and reproduction is often lacking. Drugs may impair male fertility by direct gonadotoxic effects, alteration of the hypothalamus-pituitary-gonadal (HPG) axis, impairment of ejaculation and erectile function, and libido. Both, over-the-counter drugs and prescription-only-medications can cause relevant effects. The presentation will include epidemiological data on drug use in men seeking fertility evaluation recently analysed by our group. Exemplarily, cases of an ongoing prospective study will be presented. Commonly used drug classes with potential negative impact on male fertility and sexual function will be discussed. In addition, the problem of information gathering on potential ADRs on male fertility and sexual function will be addressed.

DPhG Annual Meeting 2016 Conference Book • 69 SCIENTIFIC LECTURES

SL.45 Evidence-based Dose Finding Using Modeling and Simulation on Earth and in Space

Derendorf, H. University of Florida, Gainesville, United States

The cost of drug development has exploded in recent years and risen to a level that soon will no longer be affordable to society. The public expectation of drug safety and guaranteed therapeutic success has become unrealistic. As a result, the number of new drug approvals can be expected to go down in the near future, a trend that is already noticeable in drug classes with low market potential due to short term therapeutic use, e.g. antibiotics. One reason for the high cost of drug development are many unnecessary studies where the results could have been predicted with reasonable certainty. PK/PD modeling is a tool that can be used to collect and integrate all the available information about a drug candidate and its class in order to make rational decisions on studies that will decrease the uncertainty of the compound. It is based on quantitative data on drug exposure and response and particularly well suited to address the question of dose finding and optimization. In the drug development process, it bridges the complete cycle from discovery to clinical use. The advantage of this approach is to define objective go/no-go decision criteria for the development process rather than relying on subjective empirical decisions. There is no way that today all developing questions can be answered by experimental evidence, and modeling and simulation is a powerful alternative approach. The presentation will feature a number of recent examples from our group where we contributed to dose optimization and development of better medicines. The examples will include some recent work in collaboration with NASA to explore if doses of sleeping medications and antibiotics will need to be modified on the planned mars missions.

70 • DPhG Annual Meeting 2016 Conference Book CLINICAL PHARMACY

SL.46 The value of pharmacometrics in development, optimisation and clinical use of anti-infective therapies.

Wicha, S. G.1 1 Department of Pharmaceutical Biosciences, Uppsala Universitet, Husargatan 3, 75124 Uppsala, Sweden

Resistant infective organisms are on the rise and render our anti-infective armamentarium progressively ineffective. WHO has recently been alarmed by this development and prognoses worse-clinical outcomes world- wide due to a potentially upcoming back-transition into the pre-antibiotic era, and calls for leadership to stimulate the development of new anti-infective agents and foster the rationale use of existing drug entities [1]. The science of pharmacometrics represents an intersection between mathematics and statistics on the one hand, and pharmacy, medicine and physiology on the other hand aiming at quantitative characterisation of biological systems, such as bacteria or drug behaviour in humans by mathematical models. The presentation will outline three examples how pharmacometrics can help addressing the abovementioned WHO calls. The first example [2] will focus on a novel translational prediction approach in tuberculosis research, which can be utilised to streamline the development of new anti-tuberculosis drugs. In this approach, in vitro experiments can be utilised by means of mathematical modelling and simulation to predict animal dose fractionation studies as well as the result of phase IIb clinical trials and hence can support informed decision making for first-in-man dose selection and study design. The second example [3,4] outlines how combination regimens can be comprehensively evaluated by pharmacometric techniques to optimise for synergistic drug effects and avoid unfavourable combinatory effects such as antagonism or evolvement of bacterial resistance. Also, a comparison to conventional interaction assessment is made that can generate misleading conclusions. The third example [5] illustrates how the value of pharmacometric models and pharmacokinetic- pharmacodynamic relationships can be translated to the bedside to personalise anti-infective therapy by using a newly developed, open-access web-application (TDMx software, www.TDMx.eu, developed by the author). In summary, the presentation will display application fields of pharmacometrics from drug development to clinical treatment with anti-infective drugs, illustrating the diversity of clinical pharmacy research within the pharmaceutical sciences.

References: 1. World Health Organisation: Antimicrobial Resistance Global Report on Surveillance 2014. 2. Wicha, S.G. et al.: 25th European Congress on Clinical Microbiology and Infectious Diseases, Amsterdam, The Netherlands 2016. 3. Wicha, S.G. et al.: Pharm. Res 2015, 32(7): 2410-2418. 4. Wicha, S.G. et al.: 25th Population Approach Group Europe Meeting, Lisbon, Portugal 2016. 5. Wicha, S.G. et al.: Int. J. Antimicrob. Agents.2015, 45(4): 442-444.

DPhG Annual Meeting 2016 Conference Book • 71 SCIENTIFIC LECTURES

2.12 Advances in Drug Formulation and Biopharmaceutics Chairs: P. Langguth, H. Rein

SL.47 Thoughts on a Manufacturing Classification System

Kleinebudde, P. 1 Heinrich-Heine-University, Institute of Pharmaceutics and Biopharmaceutics, Universitätsstr. 1, 40225 Düsseldorf, Germany

The Biopharmaceutical Classification System (BCS) became popular during the last 20 years. It is based on the solubility and permeability of APIs. The BCS was modified several times and can be used for many purposes including regulatory issues. Taking the BCS characteristics into account, it is of interest to select an appropriate manufacturing technology for a certain API. For a new API it is of interest to select a manufacturing technology as early as possible. The question is, if there are any API properties, which can guide to choose an appropriate technology. For oral solid dosage forms an approach was made to compile first ideas for a Manufacturing Classificaton System (MCS) [1]. There is a clear link between the MCS and BCS as the reproducible production of a dosage form that does not impede dissolution and subsequent absorption is critical. Having a tablet product in mind four major routes for manufacturing were identified: (a) direct compression, (b) dry granulation, (c) wet granulation, (d) other technologies like spray drying or melt extrusion before tableting. The costs of production, the number of process steps and the stresses (shear, moisture, heating) applied to the API during production increase from (a) to (d). Some thoughts are provided for the construction of a MCS. The drug load in the dosage form is of major importance. At high drug load the behaviour during production is dominated by the API. It is more difficult to compensate for impaired properties like poor flowability or tabletability. The concept of percolation theory may be helpful to classify the drug loading based on percolation thresholds. The developability of an API is determined by several different properties like the dose, particle size, morphology, surface area, shape or density. Other parameters like flowability, segregation tendency, mechanical behaviour or surface adhesion are complicating the description. The first attempts to select a production route include a number of properties of the API. A major goal is to ideally select few properties based on first principles to construct a simple but useful MCS. The discussion is going on in the MCS group. Formulations on the market are analysed with respect to APi properties and production routes. Based on this, a simplified MCS based on two factors is proposed as an umbrella: Dose of the API in the proposed formulation and BCS class of that API i.e. BCS Class 1/3 vs BCS Class 2/4.

Acknowledgments: Michael Leane, Kendall Pitt, Gavin Reynolds and the MCS discussion group for lively and constructive discussions on the topic. References: 1. Leane, M. et al.: Pharm. Dev. Technol. 2015 20: 12-21.

72 • DPhG Annual Meeting 2016 Conference Book ADVANCES IN DRUG FORMULATION AND BIOPHARMACEUTICS

SL.48 Formulation development for topical treatment of tinea pedis and onychomycosis

Müller-Goymann, C.; Täuber, A. Institut Pharmazeutische Technologie TU Braunschweig, Mendelssohnstr. 1, 38106 Braunschweig, Germany

Superficial fungal skin infections (e.g., tinea pedis) belong to the most common infections worldwide. The most prevalent trigger is the dermatophyte fungus Trichophyton rubrum. Due to the fungus’ ability to feed on keratin, it is mostly located in the human nail plate and the horny layer of the skin, i.e., the stratum corneum (SC). The treatment is usually done with topical formulations containing active antifungal ingredients. Since the fungi may enter the nail plate via fissures and gaps, tinea pedis often goes along with fungal nail infections (onychomycosis) being considerably more difficult to treat. A convenient approach supporting the patient’s compliance would be a single formulation treating both diseases simultaneously. Hitherto, no such formulation is marketed due to the distinct barrier properties of nail and skin. The nail is considered as a hydrophilic gel membrane with an additional lipophilic pathway, whereas the skin represents a lipophilic partition membrane.

Formula( on* PET.foil* T. rubrum Polyamide*ring* Stratum*corneum*with*filter*as* backing*

Treatment

Infected stratum corneum model Successfully treated stratum corneum model

We developed such a simultaneous formulation from hydrophilic and lipophilic compounds and incorporated an antifungal active ingredient (API). Variations in composition led to different consistencies (liquid to semi-solid). The antifungal efficacy of the formulations was tested in a novel in vitro model, in which human SC was infected with T. rubrum. After 6 days of incubation, a variety of liquid formulations indicated complete fungal growth inhibition, whereas a marketed antifungal cream for the treatment of tinea pedis as a reference did not inhibit fungal growth completely. Moreover, a loosening of the tight microstructure of the SC was proven by DSC measurements aiding drug penetration and resulting in a better fungal growth inhibition. One-year stability studies at 30 °C proved API contents > 95 % (with one exception) and unchanged macroscopic appearance during storage. The semi-solid formulations did not show any phase separation phenomena, while some of the liquid formulations indicated reversible creaming. Combining these data with previously published results (1-6), in vitro antifungal efficacy against T. rubrum on infected SC as well as on artificial nail models was proven. In vitro permeation studies across SC and nail models indicated promising permeation behaviour for a variety of formulations. Therefore, we suggest a simultaneous antifungal therapy as a future therapy approach.

References: 1. Täuber A, Müller-Goymann CC: Mol. Pharm. 2014, 11(7):1991-1996. 2. Täuber A, Müller-Goymann CC: Int. J. Pharm. 2015, 489(1-2):73-82. 3. Täuber A, Müller-Goymann CC: Int. J. Pharm. 2015, 494(1):304-311. 4. Täuber A, Müller-Goymann CC: Akt. Dermatologie 2015, 41:1-7. 5. Täuber A, Müller-Goymann CC: Int. J. Pharm. 2016, 505(1-2):20-23. 6. Täuber A, Müller-Goymann CC: 2015 http://atlasofscience.org/one-for-two-one-medicine-against-two-diseases/

DPhG Annual Meeting 2016 Conference Book • 73 SCIENTIFIC LECTURES

SL.49 Improving the quality of split tablets

Hirsch, R.1 1 TH Köln – University of Applied Sciences, Claudiusstr. 1, 50678 Cologne, Germany

Tablets are split prior to ingestion for many reasons, varying from therapeutical need to purely economical considerations. The pharmaceutical quality of the split tablet – notabliy uniformity of content – will, however, decrease, even if stability and bioavailability (with their impact on efficacy and safety) are not affected. In order to minimise the mass variance of split tablets, Wenz Blister-Verpackungstechnik in cooperation with TH Köln is developing a device for the exact cutting of tablets, which will eventually enable pharmacies or facilities of medicinal or geriatric care to provide optimally split tablets to their customers. The development of the splitting device is accompanied by a finite element modeling of the breaking process in scored and unscored tablets. Critical parameters on breakability that should be considered during formulation development are crushing strength, porosity, and height of the tablets. Their impact on the variance introduced by the splitting process is investigated as is the influence of the plasticity and elasticity of the excipients. The concept of an effective particle number is introduced in order to characterise the homogeneity of the tableting mass. It can be used to predict the uniformity of content of the fragments from their mass variance. With this information the probability of acceptance of the relevant pharmacopoeial tests can be calculated for any formulation candidate.

Acknowledgements: The author wishes to thank the AiF Projekt GmbH (grant no. KF3275402US4) for financial support.

74 • DPhG Annual Meeting 2016 Conference Book ADVANCES IN DRUG FORMULATION AND BIOPHARMACEUTICS

SL.50 Toward biopredictive dissolution for enteric coated dosage forms

Al-Gousous, J.1.; Amidon, G. L.2; Langguth, P.1 1 Insititute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudinger Weg 5, 55099 Mainz, Germany. 2 Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan 48109, USA.

The currently established methodologies for in vitro release testing of enteric-coated dosage forms suffer from poor biopredictivity because of the too high buffer capacity of the employed buffer systems, which makes successful development of such products challenging [1]. Therefore, in this work, we undertook the development of a new dissolution testing method with improved biopredictivity for this type of dosage forms in the fasted state. Two commercially available enteric-coated aspirin products were used as model formulations: Aspirin Protect 300 mg (Bayer AG, Germany) , and Walgreens Aspirin 325 mg (LNK International, USA). The new method was developed by selecting the phosphate buffer resulting in a tablet disintegration performance that most closely matches that in a physiological bicarbonate buffer. The fact that the pH and the buffer molarity along the small intestine are far from being constant was also accounted for. This was done by introducing an algorithm where the pH and the buffer molarity of the medium were increased by adding a calculated amount of a concentrated Na2HPO4 solution to the dissolution vessel for products that release less than 75% of the drug within one hour of starting the test in buffer. Dissolution performance using the accordingly developed method was compared to that observed when using two well-established dissolution methods: the United States Pharmacopoeia (USP) method and blank Fasted State Simulated Intestinal Fluid (FaSSIF). The resulting dissolution profiles were convoluted using GastroPlus software to obtain predicted pharmacokinetic profiles. A pharmacokinetic study on 12 healthy human volunteers, in the fasted state, was performed to evaluate the predictions made by the different dissolution setups for the two aforementioned model formulations. The novel method provided the best prediction, by a relatively wide margin, for the difference between the lag times of the two tested formulations indicating its being able to predict the post-gastric emptying onset of drug release with reasonable accuracy. The post-lag time absorption rate predictions were evaluated using the Cmax/AUC0-24 ratio as an absorption rate metric. Regarding this metric’s predictions, the new method predicted the two products' performances relative to each other very accurately (only 1.05% prediction error in this regard). This prediction was superior to those made by the currently established methods (6.18% and 18.64% prediction errors for the USP and blank FaSSIF methods respectively). The reason behind the method’s success in this regard was found not to rest solely on its lower buffer capacity but also on its accounting for the changing pH and buffer molarity values faced by the dosage form as it traverses the small bowel. As for the new method’s predictions for the individual products’ values in absolute terms, they were borderline (22.58% maximum prediction error) but superior to those made by the currently established methods (maximum prediction errors of 41.67% for the USP method and 33.97% for the blank FaSSIF method). Therefore, the new method has been shown to be of improved biopredictivity compared to so far established methods, and so it could make successful development of enteric-coated products easier.

Acknowledgments: The German Academic Exchange Service (DAAD) is acknowledged for supporting Jozef Al-Gousous with a scholarship and the PHATI foundation for support. We also thank Dr. Brian Krieg Jung and Mr. Uwe for their help in setting up the bicarbonate system. We thank ACDIMA Biocenter (Amman, Jordan) for conducting the pharmacokinetic study Additional thanks goes to Dr. Michael Bolger for helpful discussion on GastroPlus. This work has been contributed to Innovative Medicines Joint Undertaking (www.imi.europa.eu) as a sideground. References: 1. Al-Gousous J, Langguth P. Dissolut. Tech. 2015, 22(3): 6-8.

DPhG Annual Meeting 2016 Conference Book • 75 SCIENTIFIC LECTURES

2.13 Fighting Depression Chairs: F. Paintner, K. Wanner SL.51 FKBP51 Inhibitiors - a Pharmacological Concept to Enhance Stress Resilience

Hausch, F.1 1 Technical University Darmstadt, Alarich-Weiss-Str. 4, 64287 Darmstadt, Germany

FKBP51, encoded by the FKBP5 gene, has attracted considerable attention due to its robust genetic and epigenetic association with numerous psychiatric disorders as well as stress-related endophenotypes. Several animal models confirmed FKBP51 as a key regulator of stress endocrinology. In addition, we and others could recently show that FKBP51 also contributes to chronic inflammatory pain and to diet-induced obesity. Drug discovery for FKBP51 has been hampered by the inability to pharmacologically differentiate against the highly homologous functional counter-player FKBP52 and all known FKBP ligands are unselective.[1] Here, we present the discovery of the first potent and highly selective inhibitors of FKBP51, SAFit1 and SAFit2.[2] This novel class of ligands achieves selectivity for FKBP51 by an induced-fit mechanism that is much less favorable for FKBP52. By using these ligands we demonstrate that selective inhibition of FKBP51 enhances neurite outgrowth in vitro and reduces glucocorticoid secretion, improves stress-coping, alleviates pain pathology and protectes from weight gain in vivo.[3] We thus propose FKBP51 inhibitors as a novel pharmacological concept to enhance resilience against overshooting stress that could be useful for the treatment of depression, obesity of chronic inflammatory pain.

Acknowlegements: This work was supported by the m4 Award 2011 from the Bayerische Staatsministerium für Wirtschaft, Infrastruktur, Verkehr und Technologie

References: 1. Pomplun et al., Angew. Chem. Int. Ed., 2015, 54, 345-8. 2. Gaali et al., Nat. Chem. Biol., 2015, 11, 33-37. 3. Maiaru et al., Sci. Transl. Med. 2016, 8, 325ra19

76 • DPhG Annual Meeting 2016 Conference Book FIGHTING DEPRESSION

SL.52 The stress protein FKBP51 shapes antidepressant pharmacology

Gassen, N. C.1; Stepan, J.2; Balsevich,G.2; Hartmann, J.2; Genewsky, A.2; Hafner, K.1; Schmidt, M. V.2; Eder, M.2; Rein, T.1 1 Max Planck Institute of Psychiatry, Department of Translational Research in Psychiatry, Kraepelinstr. 10, Munich 80804, Germany; 2 Max Planck Institute of Psychiatry, Department of stress neurobiology and neurogenetics, Kraepelinstr. 10, Munich 80804, Germany

FK506 binding protein (FKBP) 51 has been implicated in antidepressant response in several genetic studies. Initially, we had characterized FKBP51 as potent regulator of the glucocorticoid receptor and thereby also of the stress hormone axis [1]. Our recent research deciphered several novel FKBP51-directed molecular pathways that might explain the observed FKBP51 dependency of antidepressant action. These pathways include GSK3beta signalling, DNA methyltransferase 1 dependent epigenetic processes and autophagy [2-5]. We here present novel mechanistic actions of FKBP51 that characterize this remarkably multifunctional protein as scaffolder organizing a protein complex that regulates the stability of the autophagy driver Beclin1. This complex can be targeted by small molecules. The novel pharmacological treatment induces autophagy as indicated by several markers. In addition, it produces antidepressant-like behavioural effects in mice as well as antidepressant-like effects in synaptic function in slices and in vivo. Further experimental evidence suggests that the underlying mechanism involves autophagy-like molecular machineries for membrane fusion at synapses. Together, these results provide a novel route to autophagy, reveal a particular form of neuronal autophagy, and identify novel compounds for autophagic therapy in depression and several other diseases.

References: 1. Rein, T.: Bioessays 2016, in press [doi: 10.1002/bies.201600050]. 2. Gassen, N.C. et al.: PLoS Medicine 2014, 11(11):e1001755. doi: 10.1371. 3. Gassen, N.C. et al.: Autophagy 2015, 11(3): 578-580. 4. Gassen, N.C. et al.: Sci. Signal. 2015, 8(404):ra119. doi: 10.1126/scisignal.aac7695 5. Gassen, N.C. et al.: Mol. Psychiatry 2016, 21(2):277-89.

DPhG Annual Meeting 2016 Conference Book • 77 SCIENTIFIC LECTURES

SL.53 Reengineering in the field of Psychopharmacology: Learning from successful models

Kirmeier, T. Max Planck Institute of Psychiatry; HMNC Brain Health

Antidepressants are commonly used to treat depressive symptoms not only within major depressive disease (MDD) but also within many other psychiatric disorders. The remission rate of depressive symptoms within MDD upon antidepressant treatment has been reported to lie between 58 % and 67 %. Genetic variability is suspected to account significantly for individual response rates on antidepressants’ treatment. It is widely accepted that not a single genetic marker but the combination of various genetic markers account for the clinical effects upon antidepressant treatment. Despite numerous pharmacogenetic and pharmacogenomic attempts determining genetic markers for antidepressant response no breakthrough has been achieved. More recently, awareness emerged that in addition to genetic variation also the identity of direct drug target structures may be necessary to unravel pharmacogenetic effects upon drug treatment. The aim of this research program was to develop a chemical tool allowing for the identification of direct interaction partner of antidepressant. We were able to develop such a chemical tool (azidobupramine) characterized by two additional chemical groups, one for photoaffinity labelling and the other one for click chemistry. We could show that azidobupramine is characterized by equal affinities to the monoamine transporters as those found for clinically effective substances. Furthermore, it was possible to demonstrate that the intracellular distribution pattern of azidobupramine corresponds to that one of clinically active substances. Finally, we were able to select risk genes for pharmacogenomic analyses according to their probability to directly interact with antidepressants. According to our results, most promising risk genes for antidepressant response rates belong to protein families involved in provision of energy. With this study, we are the first using direct interaction partner of antidepressant as candidate genes for pharmacogenetic analysis. We think that our results may not only contribute to the discovery of yet unidentified mechanisms of MDD but also help to identify novel therapeutic options in the treatment of MDD.

78 • DPhG Annual Meeting 2016 Conference Book INTERFACE TUMOR/INFLAMMATION

2.14 Interface Tumor/Inflammation Chairs: R. Fürst, O. Werz

SL.54 The mRNA binding protein p62/IGF2BP2 as a promoter of metaflammation and hepatocellular carcinoma

Kiemer, A. K. 1 Saarland University, Department of Pharmacy, Pharmaceutical Biology, Campus C2 3, 66123 Saarbrücken

Hepatocellular carcinoma (HCC) represents the third-leading cause of cancer-related death worldwide. Hepatitis B and C infections and alcohol-related liver disease frequently lead to fibrosis and cirrhosis representing risk factors for the development of HCC. A substantial increase in non-alcoholic fatty liver disease (NAFLD) linked to the metabolic sydrome significantly contributes to the rising incidence of HCC (1). NAFLD represents an epidemic, which affects an estimated 10 to 20 million people in Germany. When metabolic disturbances are accompanied by inflammatory processes, i.e. when metaflammation occurs, the condition is classified as non- alcoholic steatohepatitis (NASH). NASH patients have an estimated annual risk to develop HCC of 2.6% (1). The IGF2 mRNA binding protein p62/IMP2-2 was identified in 1999 as an autoantigen in an HCC patient and was observed to exhibit an oncofetal expression pattern, i.e. the healthy adult liver expresses no p62. In order to investigate the role of elevated p62 we generated transgenic mice expressing p62 only in the liver. These animals developed steatosis and showed a highly elevated expression of the metabolic growth factor Igf2 (2). Igf2 proved to be causative for lipid deposition in p62 transgenic animals (3-4). The hepatic lipid composition in transgenic animals was characterized by a distinct increase of elongated (i.e. C18) fatty acids and of free cholesterol, both representing hallmarks of hepatic metaflammation (3;5-6). Interestingly, fatty acid elongation seems to be a characteristic feature for NASH and human NASH-associated HCC, while playing no role in virus-induced human HCC (7-8). In a NASH feeding model p62 amplified hepatic lipid accumulation and accelerated inflammation and fibrosis development (9). The actions of p62 as an inducer and promoter of metaflammation are directly linked to carcinogenic and tumor- promoting actions of p62: p62 induces the generation of reactive oxygen species and genomic instability (10). In a murine HCC model, p62 accelerates hepatocarcinogenesis; in human HCC, p62/IMP2 expression strongly correlates with markers of an aggressive tumor type. What is more, p62 facilitates chemoresistance in hepatoma cells by activating ERK as survival pathway (11). Taken together, p62 represents a pivotal inducer and promoter of hepatocarcinogenesis by amplifying metaflammation and therapy resistance. Antagonizing p62 actions might therefore represent an interesting therapeutic target for the prevention and treatment of NASH-associated HCC.

Acknowledgments: The project was funded, in part, by the Deutsche Krebshilfe (107751), the Else Kröner-Fresenius-Stiftung (2012_A250), and the BMBF (01KU1216F, Deutsches EPigenom Programm DEEP). Dr. Sonja M. Kessler was supported by an EASL Dame Sheila Sherlock Fellowship and a Bank Austria Visiting Scientists Programme Fellowship; Dr. Stephan Laggai obtained a post-graduate fellowship from Saarland University. Dr. Yvette Simon was awarded the 2014 Apotheker Jacob Award. References: 1. Malek, N.P. et al.: Dtsch Arztebl Int 2014; 111: 101-106 2. Tybl, E. et al.: J Hepatol 2011 54: 994-1001 3. Laggai, S. et al.: J Lipid Res 2014 55: 1087-1097 4. Kessler, S.M. et al.: Front Physiol 2016 7: 147 5. Laggai, S. et al.: World J Hepatol 2013 10: 558-567 6. Simon, Y. et al.: World J Gastroenterol 2014 20: 17839-17850 7. Kessler, S.M. et al.: Cancer Res 2014 74: 2903-2904 8. Kessler, S.M. et al.: Int J Mol Sci 2014 15: 5762-5773 9. Simon, Y. et al.: Gut 2014 63: 861-863 10. Kessler, S.M. et al.: Cell Death Dis 2015 6: e1894 11. Kessler, S.M. et al.: Am J Physiol - Gastroint Liver Physiol 2013 304: G328-G336

DPhG Annual Meeting 2016 Conference Book • 79 SCIENTIFIC LECTURES

SL.55 Targeting monocytes and macrophages for intervention with inflammation-related cancer

Werz, O. Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, 07743 Jena, Germany

Monocytes are peripheral blood leukocytes that can differentiate into various subsets of macrophages and dendritic cells. These cells play essential roles in innate immunity and protect the host against pathogenic microorganisms, but are also important for cancer immunosurveillance. Aberrant activation of monocytes or macropahges, however, may lead to numerous events (e.g. secretion of cytokines, growth factors, lipid mediators, and reactive oxygen species) that can promote inflammation-related disorders such as atherosclerosis or cancer. For example, when monocytes become activated they may infiltrate tumors and differentiate towards tumor-associated macrophages thereby promoting the persistence of an inflammatory milieu and thus, differentially contribute to various phases of the cancer process. In fact, pharmacological suppression of monocyte infiltration and differentiation into macrophages by trabectedin inhibited the production of interleukin-6 within the tumor microenvironment and is considered as clinically relevant approach for ovarian cancer therapy [1]. In this presentation, novel pharmacological concepts using natural products will be presented that target monocytes and different macrophage subsets (M1 and M2). Focus is placed on two types of natural compounds that had been described as anticancer agents with therapeutic potential: (i) the actin-targeting agent chondramide and (ii) the vacuolar-type ATPase inhibitor archazolid. Chondramide induces G-actin nucleation in macrophages and thereby predominantly depletes pro-tumoural M2 while promoting the tumour-suppressive M1 phenotype, associated with a greater likelihood of a protective anti-tumour immune response. Archazolid suppresses the secretion of pro-inflammatory cytokines and the formation of lipid mediators in primary monocytes related to cancer [2], and selectively increases tumor necrosis factor-α release from M1 but not from M2. Our results propose induction of G-actin nucleation and interference with v-ATPase as novel and promising pharmacological approaches for simultaneously targeting macrophage subtypes, in addition to cancer cells.

References: 1. Tang, X. et al., Immunology, 2015, 138:93-104 2. Scherer, O. et al., Biochem. Pharmacol., 2014, 91:490-500

80 • DPhG Annual Meeting 2016 Conference Book INTERFACE TUMOR/INFLAMMATION

SL.56 Clickable IL-4 cytokines to induce M2 macrophage polarization

Lühmann, T.1; Spieler, V.1; Werner, V.1; Fiebig, J.2; Müller, T. D.2; Meinel, L.1 1 Institute of Pharmacy and Food Chemistry, University of Würzburg, 97074, Germany 2 Lehrstuhl für Botanik I, University of Würzburg, 97070, Germany

Introduction: Regulation of macrophage (Mφ) polarization and phenotypic plasticity opens exciting new treatment strategies against inflammatory diseases, including impaired wound healing, rheumatoid arthritis or arteriosclerosis [1,2]. Although interleukin-4 (IL-4) effectively triggers M2-Mφ polarization thereby providing benefit for tissue repair and regeneration processes, its systemic use is constrained by dose-limiting toxicity. We addressed this demand by deploying genetic code expansion, thereby integrating unnatural amino acids (uAA) with either azide or alkyne functionalities into the IL-4 backbone at position 42 during protein synthesis in Escherichia coli (E. coli). These modifications enable controlled and site-specifc immobilisation of IL-4, aiming at sustained and localized activity. Methods: Human IL-4 and IL-4 muteins were expressed in E.coli BL21 (DE3) and were purified using cationic exchange chromatography Characterisation was performed by MALDI-MS, ESI-LC-MS/MS, SPR and RP-HPLC. Bioactivity of IL-4 and analogues was determined by proliferation of TF-1 cells and by using a secreted alkaline phosphatase (SEAP) STAT-6 reporter gene assay. NHS modified agarose particles were decorated with azide or dibenzooctyl functionalities for immobilisation of IL-4 either via copper catalyzed or copper free click chemistry [3,4]. Monocytes were isolated from human peripheral blood mononuclear cells obtained from blood buffy coats. Monocytes were differentiated into Mφ with M-CSF-1 (unpolarized, M0) and further treated with a combination of M-CSF-1 and IL-4 muteins or wild-type IL-4 for M2 polarization or with a cocktail of LPS and IFNγ for M1 polarization, respectively. Degree of Mφ polarization was assessed by RT-PCR for clicked agarose surfaces decorated with IL-4 and for respective controls (physio-adsorbed IL-4), respectively. Results and Discussion: Alkyne and azide functionalized IL-4 muteins were successfully expressed in the presence of 3 mM uAA and were purified by cationic exchange chromatography in an analogue manner to wild-type IL-4. The correct incorporation of the uAA at position 42 was confirmed by MALDI-MS and ESI-MS analysis and peptide mapping after trypsin digest. The soluble IL-4 muteins were as active as the wild-type protein in respect to TF-1 cell proliferation, SEAP stimulation and high affinity (IL-4Rα receptor) as well as low affinity receptor (IL-13Rα1 / common gamma chain) interaction. M2 polarization of Mφ as analysed by M2 marker gene upregulation was similarly in the presence of soluble IL-4 muteins compared to the wild type IL-4. Copper catalyzed (CuAAC) and copper free strain promoted (SPAAC) 1,3-dipolar azide alkyne cycloadditions were used to site-selectively anchor IL-4 to agarose surfaces. These surfaces had sustained IL-4 activity as demonstrated by TF-1 cell proliferation and M2 but not M1 polarization of M-CSF generated human Mφ. Site-directed anchoring of ‘clickable IL-4 muteins’ on surfaces by defined bioorthogonal chemistry can provide sustained immune modulating stimuli. The presented approach herein provides a blueprint for the engineering of cytokine-activated surfaces profiled for sustained and spatially controlled activity.

Acknowledgments: Support by DFG (grant ME 3920/3-1 ‘Macrophage plasticity deployed for efficient bone (re-) generation’) is gratefully acknowledged. References: 1. Mosser, D.M. and J.P. Edwards: Nat. Rev. Immunol. 2008, 8(12):958–69. 2. Chazaud, B: Immunobiology. 2014, 219:172–178. 3. Lühmann, T. et al: ACS Biomater. Sci. Eng. 2015, 1(9):740–746. 4. Gutmann, M. et al: ChemBioChem. 2016, 17: 866–875.

DPhG Annual Meeting 2016 Conference Book • 81 SCIENTIFIC LECTURES

SL.57 Oligoaminoamide-based siRNA carriers for in vivo tumor targeting and gene silencing

Lee, D. J.1,2; Kessel, E.1,2; He, D.1,2; Klein, P. M.1; Lächelt, U.1,2; Wagner, E.1,2 1 Department of Pharmacy & Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 Munich, Germany 2 Nanosystems Initiative Munich, Schellingstraße 4, 80799 Munich, Germany

Successful applications of RNAi-based cancer therapy depend upon efficient intracellular delivery of siRNA and effective knockdown of targeted transcripts. To achieve this, oligoaminoamide-based polycationic oligomers, which can form polyplexes with anionic siRNA by electrostatic interaction, have shown great potential as siRNA carrier [1]. However, delivery of siRNA with specificity to the tumor site remains a major limitation [2]. In two different approaches, we have synthesized a series of sequence-defined oligomers which include a cationic oligoaminoamide core, cysteines (as bioreversible disulfide-forming units), and polyethylene glycol chain (for shielding surface charges) coupled to a terminal ligand to target folate receptor (FR)-overexpressing tumors [1-3]. First, to enhance the targeted antitumor effect, the antifolate drug methotrexate (MTX) was employed as both targeting ligand for FR-mediated uptake and as an anticancer agent as it is toxic to the target cells by blocking de novo thymidylate and purine synthesis [4]. MTX-conjugated polyplexes are spherical nanoparticles with a hydrodynamic diameter of 6.5 nm (Fig. A). Treatments with these polyplexes containing EG5 siRNA in FR- expressing tumor cells triggered knockdown of EG5 gene and caused augmented cytotoxicity. Intratumoral administration of the MTX-based polyplexes showed significantly enhanced tumoral retention (168 h) compared to the non-targeted polyplexes (48 h), and mediated the longer survival time (>70 days) than untreated controls (24 days) in tumor-bearing mice (Fig. B) [5]. Second, to increase the polyplex stability for systemic delivery, we optimized the physicochemical properties of polyplexes by combinatorial optimization of PEGylated folate- conjugated oligomer (for FR targeting and shielding of surface charges) and 3-arm thiol-oligomer (for size modification and particle stability) (Fig. C). For uni-directional fast coupling between the two groups of oligomers, we activated the cysteine thiol groups of one of the oligomers with 5,5’-dithio-bis(2-nitrobenzoic acid) to achieve a fast chemical linkage through disulfide formation with the free thiol groups of the other oligomer. These targeted combinatorial polyplexes (TCPs) are homogeneous spherical particles with favorable size and strong siRNA binding activity. TCPs were internalized into cells by FR-mediated endocytosis (Fig. D & E; arrowheads: FR), triggered significant eGFP-luciferase marker gene silencing, and transfection with antitumoral EG5 siRNA suppressed cell proliferation in FR-expressing tumor cells. Moreover, the most promising formulation TCP1 after i.v. administration in tumor-bearing mice exhibited siRNA delivery into the tumor, reducing the EG5 gene expression by 46% at mRNA level (Fig. F) [6]. Therefore, we developed highly functionalized and defined siRNA carrier systems, which could be a potential strategy for RNAi-based cancer therapeutics.

Acknowledgments: The study was supported by DFG Excellence Cluster Nanosystems Initiative Munich (NIM). References: 1. Lächelt, U., Wagner, E.: Chem. Rev. 2015, 115 (19), 11043-78. 2. Lee, D.J. et al.: Methods Mol. Biol. 2015, 1206:15-27. 3. Dohmen, C. et al.: ACS Nano. 2012, 6(6): 5198-208. 4. Lächelt, U. et al.: Mol. Pharm. 2014, 11(8): 2631-9. 5. Lee, D.J. et al.: Biomaterials. 2016, 77, 98-110. 6. Lee, D.J. et al.: J. Control. Release. 2016, DOI: 10.1016/j.jconrel.2016.06.011.

82 • DPhG Annual Meeting 2016 Conference Book INDUSTRIAL PHARMACY

2.15 Industrial Pharmacy Chairs: Q. Queckenberg, F. Kramer SL.58 Introduction of Continuous Manufacturing from an Engineering Perspective

Rehbaum, H.1 1 Dr. Rehbaum Consulting, Berlin 10117, Germany

Driven by initiatives of national regulatory authorities, pharmaceutical companies and international societies, continuous manufacturing for secondary manufacturing of solid dosage forms has gained increasing interest in the pharmaceutical industry. Early adopters were among both pharmaceutical manufacturers and equipment suppliers, resulting in different approaches as either single unit operations or turnkey solutions for continuous manufacturing. However, continuous manufacturing implies a change of the mindset for all participating parties. This leads to different expectations and understandings, especially when pharmaceutical companies are approaching equipment suppliers to jointly evaluate technologies and continuous processes. In his presentation, Dr. Rehbaum will report about hands-on experience and examples from his work as technology consultant for the pharmaceutical industry, targeting especially pharmaceutical companies currently in preparation to engage into continuous manufacturing.

DPhG Annual Meeting 2016 Conference Book • 83 SCIENTIFIC LECTURES

SL.59 Model based PAT implementation in pharmaceutical manufacturing processes

De Beer, T.1 1 Ghent University, Department of Pharmaceutical Analysis, Laboratory of Pharmaceutical Process Analytics & Technology (LPPAT), Ottergemsesteenweg 460, B-9000 Ghent, Belgium

Process Analytical Technology (PAT) refers to a toolbox used to ensure that quality is built into products herewith improving process understanding, increasing efficiency, and decreasing costs. PAT is getting more and more adopted by the pharmaceutical industry, as stimulated by the regulatory authorities. The PAT toolbox contains process analyzers, multivariate analysis tools, modelling and simulation tools, process control tools, and continuous improvement/knowledge management/information technology systems. The integration and implementation of these tools is complex, and has resulted in uncertainty with respect to both regulation and validation. The paucity of staff knowledgeable in this area may complicate adoption. This presentation will especially focus – by means of cases studies - on the challenges related to the correct implementation of process sensors (e.g., interfacing of measurement probes) in pharmaceutical process environments allowing the obtain representative and meaningful real-time measurement. Case studies will demonstrate how model based analysis (e.g., based on computational fluid dynamics) may contribute to optimal process sensor implementation.

84 • DPhG Annual Meeting 2016 Conference Book INDUSTRIAL PHARMACY

SL.60 Modular, Continuous API Production Units by INVITE

Schweiger, A. INVITE GmbH, CHEMPARK Bldg. W32, D-51368 Leverkusen, Germany

Modular continuous production plants are a promising approach to face the challenges of volatile markets, shorter product lifecycles and diversification of the product range. Recent public funded projects successfully demonstrated the technical and economic potential of small scale modularly built production plants [1]. Small scale continuous production addresses various business sectors with different boundary conditions like specialties, fine chemicals and pharmaceuticals manufacturing. Through modularization, a flexible infrastructure is provided in order to achieve versatile production plants. This is required to be competitive with currently applied batch technology. The flexibility within the modular concept is achieved by modularization on different technical and information layers. Furthermore methods such as standardization, process intensification and scalability reinforce the continuous modular plant concept. The improved efficiency by transformable multi-purpose plants is strength of the modular concept, as well as the ability of capacity expansion by numbering-up the modular equipment. Additionally, the modularization allows accelerating the engineering by reusing know-how which leads to faster development times [2]. To enable the reuse of engineering information a systematic modularization approach is pursued at INVITE. This approach divides a continuous process into Process Equipment Design modules (PED). A PED represents at least one unit operation including the peripheral components. The PEDs ensure the documentation during engineering and plant lifecycle and contain for example typical engineering documents such as P&ID as well as safety and reliability assessments. Stored in a data base, the created PEDs can be reused over several projects. The physical module is then built following technical and geometrical guidelines to provide the functional modularization. This allows the reuse, replace and combination of single modules. Each module is designed as an autonomous module, decoupled from the overall system, which allows independent replacement, cleaning or maintenance. If a standalone or decentralized production is desirable, the modules can be integrated into a Process Equipment Container (PEC). These containers provide a fully integrated infrastructure to build up a mobile production environment. During the F³ Factory project a modular and flexible continuous production of an active pharmaceutical ingredient was demonstrated at the INVITE research centre. A multi-step synthetic batch process was partly transferred to the modular continuous infrastructure. This led to significant reduction in processing steps, reaction time and solvents used. Additionally, a reduction in design and installation costs as well as apparatus costs could be achieved [1]. The subsequent research project CONSENS (Integrated Control and Sensing) is now focussing to advance the continuous production of high-value products by introducing novel online sensing equipment and closed loop control of the key product parameters. For example, an online concentration measurement by Medium Resolution Nuclear Magnetic Resonance Spectroscopy (MR-NMR) will be integrated into the modular plant concept. Especially with regard to the pharmaceutical manufacturing, continuous modular plants can enable an efficient manufacturing with high quality. In spite of some regulatory uncertainties, the FDA even encourages the development towards continuous manufacturing [3], so that small scale modular plants can be seen as a suitable strategy of the future pharma production.

References: 1. F³ Factory: Flexible, Fast and Future Production Processe - Final Report. http://www.f3factory.com/scripts/pages/en/newsevents/F3_Factory_final_report_to_EC.pdf (accessed July 20, 2016) 2. Bramsiepe, C.; Schembecker, G.: CIT 2012, 84 (5), 581-587 3. Chatterjee, S.: FDA Perspective on Continuous Manufacturing, IFPAC Annual Meeting, January 2012, 4. Kessler, S.M. et al.: Front Physiol 2016 7: 147 5. http://www.fda.gov/downloads/AboutFDA/CentersOffices/OfficeofMedicalProductsandTobacco/CDER/UCM341197.pdf (accessed July 20, 2016)

DPhG Annual Meeting 2016 Conference Book • 85 SCIENTIFIC LECTURES

SL.61 Advancing process understanding in film coating by in-line terahertz pulsed imaging, optical coherence tomography and discrete element modelling

Lin, H.1; Dong, Y.2; Markl, D.3; Pei, C.4; Williams, B. M.5; Zheng, Y.5; Shen, Y. C. 2; Elliott, J.A.5; Zeitler, J. A.3 1 Department of Engineering, Lancaster University, Lancaster LA1 4YW, UK 2 Department of Electrical Engineering and Electronics, University of Liverpool, Liverpool L69 3GJ, UK 3 Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB2 3RA, UK 4 Department of Materials Science and Metallurgy, University of Cambridge, Cambridge CB3 0FS, UK 5 Department of Eye and Vision Science, University of Liverpool, Liverpool LT7 8TX, UK

We have recently developed the measurement technologies to acquire coating thickness measurements simultaneously in-line using two independent sensing modalities: terahertz pulsed imaging (TPI) and optical coherence tomography (OCT). Both techniques are sufficiently fast to resolve the coating thickness of individual pharmaceutical tablets in situ during the film coating operation and both techniques are direct structural imaging techniques that do not require multivariate calibration. The TPI sensor is suitable to measure films of > 50 μm and can penetrate thick layers even in the presence of pigments over a wide range of excipients. Due to the long wavelength of terahertz radiation it is not affected by scattering due to dust within the coater. In contrast, OCT can resolve coating layers as thin as 10 μm and is capable of measuring the intra-tablet coating uniformity as well as the inter-tablet coating thickness distribution within the coating pan. However, the OCT technique is less robust when it comes to the compatibility with excipients, dust and potentially the maximum coating thickness that can be resolved. Using a custom built laboratory scale perforated pan coating unit the coating thickness measurements were acquired independently by the TPI and OCT sensors throughout a film coating run. Results of the in-line TPI and OCT measurements were compared against one another and validated with off-line TPI and weight gain measurements. Compared to other process analytical technology sensors the TPI/OCT sensors can resolve the inter-tablet thickness distribution based on sampling a significant fraction of the populations of tablets in the process. By combining two complementary sensing modalities it was possible to seamlessly monitor the coating process over the range of film thickness of 20 to > 250 μm. To complement the in-line measurements and to further challenge the validity of the measurement data we have developed discrete element models (DEM) of the film coating operation at exactly the same process scale as the experimental work. Using the DEM simulations we were able to numerically evaluate the mixing of tablets in the coater and the mass transfer of the coating onto the tablet cores. The DEM results allowed us to check whether the chosen sensor locations and measurement geometry are suitable to measure a representative sample of the total tablet population in the coater and what process parameters have the most pronounced effect on the coating quality.

Acknowlegements: We would like to acknowledge financial support from the U.K. Engineering and Physical Sciences Research Council (EP/L019787/1 and EP/L019922/1). The authors acknowledge BASF for providing the materials used in this study, Colorcon Ltd. (Dartford, UK) for coating process recommendations, Huettlin GmbH (Bosch Packaging Technology, Schopfheim, Germany) for advice on the coating unit design and the staff of the electronics and mechanical workshops at the Department of Chemical Engineering and Biotechnology, University of Cambridge for building the lab scale coater.

86 • DPhG Annual Meeting 2016 Conference Book

3 POSTERS

DPhG Annual Meeting 2016 Conference Book • 87 POSTERS

The Interactions of various heparinoids (unfractionated and a low 3.1 Analytics molecular weight heparin), as well as pentosan polysulfate sodium (PPS), with human and bovine albumins (BSA and HSA) have been

studied. The experiments were performed at 23°C and 37°C. POS.1 Both BSA and HSA interact more strongly with PPS than with Investigating the Interaction of an adhesion protein unfractionated and low molecular weight heparins. For PPS, P-selectin with heparinoids using affinity capillary interactions can already be observed at low mg/L concentrations (3mg/L), and saturation is already obtained at approximately 20 mg/L. electrophoretic and computational methods Unfractionated heparin showed almost no interactions with BSA at 23°C, but weak interactions at 37°C at higher heparin concentrations. 1 1 1 1 Mozafari, M. ; Balasupraminiam, S. ; El Deeb, S. ; Wätzig, H. The peak shapes also changed at higher concentrations at 37°. This 1 Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstrasse 55, 38106 Braunschweig, Germany was the most substantial difference due to the temperature. In most cases the binding data were similar at both temperatures. However, P-selectin is a transmembrane protein located in the granules of temperature-dependant changes in the peak shapes have been platelets and the Weibel-Palade bodies of endothelial cells. P-selectin observed for heparin and PPS. imparts rolling of leukocytes on activated endothelial cells as well as Furthermore, HSA showed a characteristic splitting in two peaks interaction of platelets with leukocytes after binding to its nature ligand especially after interacting with PPS, which is probably attributable to called PSGL-1 [1,2]. This binding is dependent on the presence of the formation of two species or conformational change of HSA after calcium ions [3,4]. Studies have indicated that plasma P-selectin levels interacting with PPS. are higher in disorders associated with arterial thrombosis [5,6]. Hence The successive experiments and the presentation of the it is important to investigate the binding affinities of this protein to electropherograms, ordered by increasing heparinoid concentrations, potential inhibitors. seems to be very powerful to understand peak splitting phenomena and Therefore, heparin and pentosan polysulfate sodium (PPS) were differences between heparinoids and their albumin interactions. Marked investigated for their binding affinity to P-selectin. PPS is a highly differences have been found. sulfated semi synthetic polysaccharide which has shown to exhibit numerous pharmacological activities [7,8]. Acknowledgments: We gratefully acknowledge bene pharmaChem for providing the PPS substances and the financial support, Furthermore we thank PolymicroTM Technologies for For the purpose of binding studies, a fast and precise affinity capillary providing the capillaries used in this study. electrophoresis method has been developed and applied for the References: investigation of the interactions between P-selectin and heparinoids in 1. Rezaei-Tavirani, M. et al.: J. Biochem. Molec. Biol. 2006, 39 (5): 530-536. the presence and absence of calcium ions. The normalized mobility 2. Fanali, G. et al.: Molec. Aspects of Med. 2012, 33: 209–290. ratios (∆R/Rf) were used to evaluate the binding affinities [9,10]. 3. Gelamo, E.L. et al.: Biochim. Biophys. Acta. 2002, 1594: 84-99. It was found that P-selectin more strongly interacts with heparinoids in 4. Redweik, S. et al.: Electrophoresis. 2013, 34 (12): 1812–1819. the presence of calcium ions. The half-maximal concentration of 5. Alhazmi, H. A. et al.: J. Pharm. Biomed.l Anal. 2015, 107:311–317. heparinoids to affect P-selectin mobility was estimated to be 3 mg/L. 6. Mozafari, M. et al.: Electrophoresis. 2015, 36: 2665–2669.

Acknowledgments: We gratefully acknowledge bene pharmaChem for providing the PPS substances and the financial support, Furthermore we thank PolymicroTM Technologies for providing the capillaries used in this study.

References: POS.3 1. Danese, S. et al.: Digest. Liver Dis. 2005, 37 (11): 811–818. Determination of the Enantiomeric Purity of Nipecotic Acid 2. Lorant, D. E. et al.: J. Clin. Invest. 1993, 92 (2): 559–570. as 1-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl) Derivatives 3. Barondes, S. H. et al.: Trends Biochem. Sci. 1988, 13 (12): 480–482. 4. Ernst, B. et al.: Nat. Rev. Drug Discov. 2009, 8 (8): 661–677. Schmidt, S. K.1; Höfner, G.1; Wanner, K. T.1 5. Merten, M. et al.: Z. Kardiol. 2004, 93 (11): 855–863. 1 Department für Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität 6. Zhu, H. et al.: Med. Chem. Commun. 2013, 4 (7): 1066–1072. München, Butenandtstr. 5-13, 81377 München, Deutschland 7. Abdel-Haq, H., Bossu, E.: J. Chromatogr. A. 2012, 1257: 125–130. 8. Dürüst, N., Meyerhoff, M. E.: Anal. Chim. Acta. 2001, 432 (2): 253–260. Nipecotic acid is an important precursor for the synthesis of novel 9. Redweik, S. et al.: Electrophoresis. 2013, 34 (12): 1812–1819. γ 10. Alhazmi, H. A. et al.: J. Pharm. Biomed. Anal. 2015, 107: 311–317. enantiopure inhibitors of -aminobutyric acid transporters (GATs) [1,2], the inhibitory activity of which is frequently found to differ quite substantially. For example, the (S)-enantiomer DDPM-1457 is known to be more potent at the GABA transporter subtype mGAT4 than its (R)- enantiomer [(S): pIC50 = 5.87 ± 0.08; (R): pIC50 = 4.33 ± 0.05] [3,4] POS.2 (Figure 1). With the data of pharmacological testing being dependent on Using affinity capillary electrophoresis to investigate the the enantiopurity of the studied samples, the knowledge of the precise concentration-dependant binding of heparinoids to enantiopurity of nipecotic acid serving as starting material for the albumins synthesis of new GAT inhibitors is of fundamental importance. Therefore, an HPLC method for the determination of the enantiopurity Mozafari, M.1; El Deeb, S.1; Wätzig, H.1 of nipecotic acid (piperidine-3-carboxylic acid) was established. 1 Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstrasse 55, Derivatization of nipecotic acid prior to chromatography appeared 38106 Brunswick, Germany. promising to guarantee high wavelengths for UV-Vis detection ensuring a selective way of determination of the individual enantiomers of the Serum albumins are the most studied proteins over the years. They are analyte and as a consequence thereof the determination of high ee predominant protein component in blood plasma. Albumins are values. With 4-fluoro-7-nitrobenzo[c][1,2,5]oxadiazole, a derivatization important for transport of a variety of endogenous and exogenous reagent was found fulfilling this requirement. It enabled efficient substances in blood. They have many physiological functions; for separation of both enantiomers of nipecotic acid as 1-(7- instance, they bind a diversity of hydrophobic ligands such as fatty [c][1,2,5]oxadiazol-4-yl) derivatives on a ChiralPak ID-3 stationary acids and steroids; but also small molecules and some metal ions bind phase (Daicel, Illkirch, France) and reliable UV-Vis quantification at 490 to albumins [1-3]. nm (Figure 1). The established HPLC based method has been validated Binding the drugs to serum albumins is one of the most important regarding specificity, linearity, quantification limit (QL), accuracy, and pharmacokinetic determinants. This might have an impact on the precision. In addition, by spiking of highly enantiopure samples of lifetime of a drug in plasma [1-3]. nipecotic acid derivatives with small amounts of racemic mixture, the Hence, it is important to find out how strong the drugs interact with newly developed method has been shown to even enable the detection albumins. of slight changes of ee values and to be well suited for the accurate For this reason, an affinity capillary electrophoretic method has been determination of very high enantiomeric excesses. applied due to its numerous benefits such as short analysis duration and usage of small amounts of samples [4-6]. The evaluation of binding affinities was carried out using normalized mobility ratios (∆R/Rf) [4-6].

88 • DPhG Annual Meeting 2016 Conference Book ANALYTICS

O 7. SANCO Guideline SANCO/12571/2013 OH O MeO N F OH N N O N N O N NO POS.5 MeO 2 NO2 Determination of plasma protein binding for ephedrine and OMe stereoisomers DDPM-1457 4-fluoro-7-nitrobenzo[c][1,2,5] 1-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl) oxadiazole nipecotic acid Volpp, M.1; Holzgrabe, U.1 1 Fig. 1: mGAT4 inhibitor DDPM-1457, derivatization reagent 4-fluoro-7-nitrobenzo[c]- University of Würzburg, Institute for Pharmacy and Food Chemistry, 97074 Würzburg, [1,2,5]oxadiazole and 1-(7-nitrobenzo[c][1,2,5]oxadiazo-4-yl)nipecotic acid. Germany

References: (-)-Ephedrine and its diastereomer (+)-pseudoephedrine, both natural 1. Herein, the nomenclature for the murine GABA transporters (mGAT1-mGAT4) is used. occurring alkaloids from Ephedra species, have been used in therapy 2. Krogsgaard-Larsen, P. et al.: Epilepsy Research 1987, 1(2): 77–93. for centuries [1] and are regaining popularity in the last few years with 3. Kragler A., Höfner G., Wanner K. T.: Eur. J. Med. Chem. 2008, 43: 2404–2411. new over-the-counter combination drugs containing pseudoephedrine. 4. Pabel, J. et al.: Chem. Med. Chem. 2012, 7(7): 1245–1255. None the less there is scarcely any information on their plasma protein binding in literature. The substances’ plasma protein binding is an important parameter from a pharmacokinetic and pharmacodynamic point of view and can play a POS.4 crucial role in therapy. A few publications determined a binding constant Determination of pesticide exposure of Podarcis muralis by for either ephedrine or pseudoephedrine to bovine or human serum a micro-QuEChERS approach and GC-MS/MS albumin (BSA or HSA). A drawback of all publications was the application of different methods, like capillary electrophoresis [2], Stöckelhuber, M.1; Müller, C.1; Mingo, V.2; Lötters, S.2; Wagner, N.2, microdialysis [3] or ultracentrifugation [4], for their experiments, Bracher, F.1 resulting in a wide range of the extend of albumin binding. All of these 1 Department für Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität studies weren’t conducted under physiological conditions, impeding any München, Butenandtstraße 5-13, 81377 München, Germany kind of direct comparison. 2 Department für Biogeographie, Universität Trier, Universitätsring 15, 54296 Trier, Germany These circumstances lead us to conduct different experiments, determining the plasma protein binding for Pesticides are widely applied in a variety of different ways during the (-)-ephedrine, (+)-pseudoephedrine and their diastereomers. Since it production of foods to control the growth of weeds and fungi or to has been shown that the affinity to albumin, the main transport protein prevent crop damage by insects, mites, rodents and other pests [1]. in plasma, can vary for the drug’s different stereoisomers, e.g. Although many species within the European Union are strictly protected propranolol [5], we included all stereoisomers of those two alkaloids in in all member states due to the Habitats Directive, detrimental effects of order to find out whether their binding to albumin is enantioselective. pesticide use are possible within [2] and especially outside protected The methods used to determine protein binding were continuous and areas [3]. This is in particular remarkable as negative effects, which discontinuous ultrafiltration in order to have orthogonal methods to may lead to a regional diversity loss, have already been identified in verify our findings. The experiments were conducted in phosphate laboratory and mesocosm studies. In reptiles, as one of the various buffered saline, pH 7.4 with human serum, bovine or human serum threatened vertebrate groups, ongoing worldwide population declines albumin, and ultrafiltration filters (MWCO 10kDa) made of regenerated are recognized. The causes for these declines are highly assorted, and cellulose. To separate and quantify the enantiomers simultaneously, the it is believed that among all factors, habitat loss and degradation is the samples obtained by discontinuous ultrafiltration were analysed using major factor in industrialized countries, followed by use of capillary electrophoresis and a chiral selector. Results of different agrochemicals in their habitats. Although effects of pesticides on methods will be discussed. reptiles have been reviewed to some degree, and different studies have shown evidence of potential strong effects on reptile wildlife, there is still References: a great lack of data. Especially, toxicity data concerning squamates is 1. Chen, K.K.; Kao, C.H.: J. Am. Pharm. Assoc. 1926, 15(8):625-639. scarce, and data on effects of pesticides in species' natural habitats 2. Ye, N. et al.: J. Chromatogr. Sci. 2007, 45(5):246-250. even more so [3]. To evaluate the exposure risk of the lizard Podarcis 3. Yang, R. et al.: Luminescence 2011, 26(5):374-379. muralis the residue unit dose of its prey animals at different times after 4. Till, A.E.;Benet, L.Z.: J. Pharmacol. Exp. Ther. 1979, 211(3):555-560. exposition has to be determined. 5. Albani, F. et al.: Br. J. Clin. Pharmacol. 1984, 18(2):244-246. In the last few years a new multiresidue analytical method for the determination of pesticides, the QuEChERS concept (Quick, Easy, Effective, Cheap, Rugged and Save) has become very popular in the analysis of pesticides in fruits and vegetables [4,5]. QuEChERS POS.6 methodology (used for extraction and clean-up steps) has been applied with success on diverse food matrices [6]. However, the prey animals of Chiral separation of phenethylamines by capillary Podarcis muralis (mostly insects and spiders) possess another tissue electrophoresis using tetrabutylammonium chloride as composition than plant material with the result that the method had to background electrolyte additive be adapted to the current matrix. Therefore different cleaning procedures and extraction steps have been tested to obtain satisfying Wahl, J.1; Holzgrabe, U.1 recoveries and precision. 1 University of Würzburg, Institute of Pharmacy and Food Chemistry, Am Hubland, 97074 Würzburg, Germany Solving the problem with the minimal available tissue material (100 – 1000 mg), a so called micro-QuEChERS approach was developed and The enantiomers of a chiral drug can have different pharmacodynamic, combined with the powerful gas chromatography triple quadrupole pharmacokinetic and toxicological properties [1-3]. Thus, especially new system (GC-MS/MS). This combination allows the simultaneous drugs are launched as pure enantiomers. However, the isomeric purity analysis of pesticides in animal tissue with minute amounts of sample. has to be proven. Capillary electrophoresis (CE) is often used for the The method was fully validated according to SANCO/12571/2013 [7]. chiral separation of drugs and pharmaceutical products. Finally, the samples of the prey animals have been prepared with the Phenethylamines, like ephedrine, pseudoephedrine, methylephedrine new micro-QuEChERS approach and analyzed with GC-MS/MS to and norephedrine are active components extracted from ephedrae assess the oral exposure risk of Podarcis muralis. herba [4]. They are used as ingredients of cold medicines and abused as starting material for the synthesis of methamphetamine [5]. References: In the past few years a great interest was drawn towards ionic liquids 1. Boes, E. et al.: Procedia Chemistry 2015, 16:229 – 236. 2. Wagner, N. et al. Biol. Conserv. 2015, 191: 667-673. (ILs) in analytical separation techniques. Ionic liquids are defined as 3. Mingo V., Lötters S., Wagner N.: Environ. Pollut. 2016, 215:164-169. (semi-)organic salts with a melting point below 100 °C. One special 4. Müller, C.; Bracher, F.; Plößl, F.: Chromatographia 2011, 73:807-811. advantage of ILs is that they can be designed as requested. It is 5. Anastassiades, M. et al.: J. AOAC Int. 2003, 86 (2):412-431. possible to modify the melting point, the viscosity, the water-solubility/- 6. Castillo, M.; González, C.; Miralles, A.: Anal. Bioanal. Chem. 2011, 400 (5):1315–1328.

DPhG Annual Meeting 2016 Conference Book • 89 POSTERS miscibility and the electrochemical behavior by altering the combination stationary phases also showed individual characteristics under achiral of cations and anions. Ionic and/or proton donor-acceptor interactions RP- and HILIC-test conditions due to the influence of secondary between analyte and IL are possible interactions facilitating new kinds interactions emerging from silica surface, polymeric film and of separation mechanisms. In capillary electrophoresis ILs can be used endcapping. as main electrolyte, as electrolyte additive and for dynamic coating of Its multi-purpose usability and its compatibility with modern mass the capillary surface. ILs possess many properties making them spectrometers such as QTOF and Ion Trap instruments make these excellent additives in CE background electrolytes (BGE). The most new chiral stationary phases a promising tool for various applications, important property is the charge of the dissolved ions in BGE enabling especially in pharmaceutical analytics. the cations to interact with deprotonated silanol groups on the capillary surface and thereby modifying the electroosmotic flow. References: The enantioseparation of four phenethylamines (ephedrine, 1. Zimmermann, A. et al.: J. Chromatogr. A 2016, 1436: 73-83. pseudoephedrine, methylephedrine, norephedrine) was investigated by adding different concentrations of tetrabutylammonium chloride (TBAC) to a phosphate buffer containing β-cyclodextrin as chiral selector [6]. By increasing the concentration of TBAC an enhancement of the chiral POS.8 resolution was found. Due to an adsorption of the IL cations to the capillary surface, an increase of migration times was observed by Characterization and Application of Stable-bond Reversed- increasing the concentration of TBAC in the BGE. To ensure phase/Weak Anion-exchange Mixed-mode Silica in reproducible migration times different rinsing procedures (acidic, basic, Pharmaceutical Research organic) were examined. The best results have been observed for a basic rinsing step using electric voltage instead of pressure. Bäurer, S.1; Zimmermann, A.1; Horak, J.1; Lämmerhofer, M.1 To further optimize the chiral separation method, phosphate buffers in a 1 Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany range from 50 to 100 mM and from pH 2.0 to pH 3.0 have been tested.

Furthermore the concentration of TBAC was varied from 0 - 200 mol/L Nowadays, the use of High Performance Liquid Chromatography and different electrical voltages and capillary temperatures have been (HPLC) coupled with several methods of detection is the state of the art investigated. For three compounds (ephedrine, pseudoephedrine, in instrumental analysis for quality control of pharmaceutical drugs as methylephedrine) a baseline resolution was achieved. The best well as in research of biomolecules, drug development and synthesis enantioseparation for all analytes was found using a 75 mM phosphate control. For chemically differing analyte mixtures, as well as for mixtures buffer pH 2.5 containing 0.125 mol/L TBAC. of very similar polar and ionic compounds, complementary methods to

the well known and often used reversed phase (RP) chromatography References: 1. Bialer, M.: Adv. Drug Deliver. Rev. 2012, 64: 887-895. become more and more necessary. Mixed Mode (MM) chromatography 2. Duggan, K. C. et al.: Nat. Chem. Biol. 2011, 7: 803-809. combines multiple complementary interaction principles due to the 3. Blaschke, G. et al.: Arznei.-Forsch. 1979, 29: 1640-1642. multiple interaction sites of the immobilized selectors. Reversed 4. Liu, Y. M.; Sheu, S. J.: J. Chromatogr. 1992, 600: 370-372. phase/weak anion exchangers (RP/WAX) stationary phases have 5. Mikuma, T. et al.: Forensic Sci. Int. 2015, 249C: 59-65. already demonstrated to be nicely suitable for the analysis of drugs, 6. Holzgrabe, U.; Wahl, J.: Capillary Electrophoresis - Methods and Protocols 2016, article in metabolites and peptides [1, 2]. The stability of the covalently bonded press. selectors often turns out to be problematic due to their hydrolysis and

detachment from the silica surface, also known as column bleeding. On

the one hand, in the case of coupling with very sensitive detection methods, the ageing process of the modified silica is detected and will POS.7 limit the use of the separation material in combination with this detection mode. On the other hand, the decreasing selector surface coverage Development of quinine-derived chiral stationary phases causes an alteration of retention times and can affect adversely the for hyphenated enantioselective liquid chromatography- separation. Hence, a more stable immobilization strategy has been mass spectrometry developed which uses a multiple crosslinked poly(3- mercaptopropyl)methylsiloxane layer on the silica surface for Woiwode, U.1; Zimmermann, A.1; Sievers-Engler, A.1; Lämmerhofer, M.1 immobilization of the selector via thiol-ene click chemistry. Recently, it 1 Institute of Pharmaceutical Sciences, Pharmaceutical (Bio)Analysis, University of Tübingen, has been proven to be greatly MS compatible [3]. The RP/WAX Auf der Morgenstelle 8, 72076 Tübingen, Germany stationary phases obtained via polysiloxane layer coating have been State-of-the-art analytical HPLC and UHPLC for research and quality characterized according to their MM character and anion exchange control is more and more combined with mass spectrometry (MS) to capacity for evaluation of the influence of this stabilization strategy. provide comprehensive analytical results within a minimum of time. Further investigations utilizing this promising stabilized RP/WAX silica Despite the vast amount of data obtainable by MS, MS/MS and MSn, were done to overcome challenging separation problems, which were respectively, there is virtually no possibility to discriminate enantiomers previously successfully solved by comparable brush-type RP/WAX by this way of detection alone. Accordingly, separation on the LC-stage stationary phases. is required to access chiral compounds analytically, especially those of pharmaceutical relevance. References: 1. Hinterwirth, H. et al.: J. Sep. Sci. 2010, 33(21): 3273-82. In this attempt the cinchona alkaloid quinine was used as a backbone to 2. Zimmermann, A. et al.: J. Chromatogr. A. 2014, 1354: 43-55. produce chiral selector molecules by formation of C-9-carbamates with 3. Zimmermann, A. et al.: J. Chromatogr. A. 2016, 1436: 73-83. aliphatic or aromatic residues and immobilized to silica supports. However, selector bleeding from the column has a pronounced influence on ionization efficiency in positive MS mode when comparing, for example, to alkyl silanols cleaved from RP-phases. To obtain highly stable chiral stationary phases, a new immobilization strategy using a POS.9 siloxane polymer as carrier of several selector molecules was applied. Development of a bioreactor based on papain immobilized In contrast to commonly used direct immobilization of the selector via a on gold nanoparticles for efficient protein digestion single thioether bridge there is a higher stability of the polymer-type phase due to redundancy of linkages between the polymer and the Liu, S.1; Höldrich, M.1; Lämmerhofer, M.1 actual surface of the support [1]. The chiral stationary phases were 1 University of Tübingen, Institute of Pharmaceutical Sciences, Auf der Morgenstelle 8, further modified by a strongly acidic endcapping. Free thiol-groups on 72076 Tübingen, Germany the polymeric film were oxidized to form sulfonic acid residues. This caused secondary repulsive interactions for acidic compounds and Immobilized enzyme on nanostructure materials has gathered rendered milder elution conditions in polar organic elution mode, while increasing attention in recent years. Compared with free enzyme in maintaining separation performance. LC-MS compatible conditions solution, immobilized enzyme has many outstanding qualities such as including reduced mobile phase ion strength and lower flow rate were high catalytic efficiency, flexible control of reaction, easy removal after successfully implemented and gave consistent chiral separation results reaction, no contamination for product and repetitive usage. Due to the for sub-nanogram sample amounts within short cycle times. The new significant physical properties of the gold nanoparticle (GNP), it

90 • DPhG Annual Meeting 2016 Conference Book ANALYTICS becomes a popular substrate which can provide a large surface-to- Acknowledgements: We thank HiperScan GmbH, Dresden, Germany for providing the NIR volume ratio for the immobilization of enzyme. In our study, surface spectrometer and calibration software. modified GNPs were achieved via layer-by-layer (LBL) process with References: alternative cationic polyallylamine and anionic poly(acrylic acid). 1. Blanco, M. et al.: Analyst 1998, 123(8): 135R–150R. 2. Reich, G.: Adv. Drug Deliv. Rev. 2005, 57(8): 1109–43 Afterwards, papain was immobilized on the GNPs via the covalent 3. Jamrógiewicz, M.: JPBA 2012, 66: 1–10. amide coupling between the amino groups on papain and the terminal 4. Roggo, Y. et al.: JPBA 2007, 44(3): 683–700. carboxylic groups of the GNPs by using EDC and sulfo-NHS. The 5. Silva, M. et al.: Talanta 2012, 89: 342–51. resultant papain-functionalized gold nanoparticles were characterized 6. Lê, L.M. et al.: Talanta 2014, 119(36): 361–6. by surface plasmon resonance (SPR) band, dynamic light scattering 7. European Medicines Agency: Guideline on the use of near infrared spectroscopy by the (DLS) and zeta potential.[1] A new technology Resonant Mass pharmaceutical industry and the data requirements for new submissions and variations 2014 Measurement (RMM) was applied for determining the average number (URL:http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2014/06/W C500167967.pdf) of papain immobilized on per GNP by precise measurement of the single nanoparticle mass in the range of femtogram to attogram.[2] The activity of the immobilized enzyme was estimated by determination of the Michaelis-Menten parameter (Km, Vmax and Kcat) with the standard chromogenic substrate Nα-Benzoyl-DL-arginine-4-nitroanilide POS.11 hydrochloride (BApNA).[3] Further, the standard protein solution was 2D protein analysis – a part of the SynFoBiA project at the digested by the gold nanobiocatalyst to the peptides, and further Center of Pharmaceutical Engineering analysed by using UHPLC-ESI-QTof-MS/MS, which demonstrated the applicability of the bioreactor based on papain functionalized GNPs. Maul, K. J.1,2; Hahne, T.1; Wätzig, H.1,2 The immobilized papain not only has higher activity recovery and better 1 TU Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany stability, but also can be easily isolated from the reaction medium by 2 PVZ – Center of Pharmaceutical Engineering, Franz Liszt Straße 35a, 38106 Braunschweig, easy centrifugation steps for reusage. Germany

Acknowledgments: We acknowledge Dr. Markus Epe, Malvern GmbH (Herrenberg, Germany). Biopharmaceuticals become more and more important in the therapy of References: different illnesses. Thus it is important to develop new and cheap ways 1. Hinterwirth, H. et al.: Anal. Chem. 2013, 85: 8376-8384. to produce them. Like all active pharmaceutical ingredients the quality 2. Nejadnik, M. R.; Jiskoot, W.: J. Pharm. Sci. 2015, 104: 698-704. has to be very high. Proteins are, in contrast to most small molecules, 3. Hinterwirth, H.; Linder,W.; Lämmerhofer, M.: Anal. Chim. Acta. 2012, 733: 90-97. not that stable and may also form aggregates. To ensure the required quality a precise and fast analysis is mandatory, especially to detected unwanted impurities. For this purpose a 2D separation with a combination of LC and CE was POS.10 developed. In the first dimension a strong anion exchanger (SAX) column was used. Fractions were collected each 80 sec. These were Application of near infrared spectroscopy for content then analyzed with capillary gel electrophoresis (CGE). determination in semi-solid pharmaceutical formulations To determine the suitability of this method standard protein samples were prepared. They contain Myoglobin, β-Lactoglobulin, Ovalbumin, Schlegel, L.1; Schubert-Zsilavecz, M.1,2; Abdel-Tawab, M.1 BSA and a monoclonal Antibody. Six samples were prepared. Each 1 Zentrallaboratorium Deutscher Apotheker (Central laboratory of German pharmacists), was then analyzed with SAX five times and 23 fractions were collected Carl-Mannich-Str. 20, 65760 Eschborn, Germany 2 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, each time. Afterwards each collection was analyzed with CGE. 60438 Frankfurt, Germany The 2D-separation technique was applied to a real sample. This sample was produced within the Center of Pharmaceutical Engineering (PVZ) Over the last decades near infrared spectroscopy (NIRS) has become a project “New synthesis and formulation of poorly soluble API’s and valuable tool in quality control and is widely used in the pharmaceutical sensible biopharmaceuticals” (SynFoBiA: Neuartige Synthese- und industry for identification and qualification purposes of active Formulierungsverfahren für schwerlösliche Arzneistoffe und pharmaceutical ingredients (APIs) and excipients. It has also gained in empfindliche Biopharmazeutika). The PVZ combines pharmaceutics, importance in the course of the FDA PAT initiative for determination of chemical engineering, bioengineering and micro engineering, which is a critical process parameters such as moisture content and blend unique German collaboration placed at the TU Braunschweig. Institutes uniformity [1-3]. from the TU Clausthal and the Leibniz Universität Hanover also Use of NIRS for API quantification has been described for solid participate. formulations including tablets, capsules or powder mixtures [4] and also for fluids [4-6], while little data for the application to semi-solid Acknowledgements: We thank Polymicro for providing the capillaries used in the CGE and our formulations [4] is available. cooperation partners which provided the real samples. Since affordable NIR spectrometer with limited wavelength range are increasingly used in pharmacies and, furthermore, there is a rising need for economic and practical methods in quality control of extemporaneous mixtures the question arises whether those NIR spectrometer are suitable for API quantification in creams and POS.12 ointments. Characterization of Adsorption Phenomena of Antibody Therefore, we have evaluated the applicability of NIRS on preparations Conjugates with different frequently used APIs in hydrophilic and lipophilic creams as well as in water-free lipid systems. Duerr, C.1; Seifert, I.1; Friess, W.1 Due to the fact that NIRS is not suitable for analysis of very small drug 1 Ludwig-Maximilians-Universität München, Department of Pharmacy, Pharmaceutical amounts, APIs were chosen that are used in dermatologically effective Technology and Biopharmaceutics, Butenandtstr. 5, 81377 Munich, Germany concentrations above 1 % such as salicylic acid, urea, erythromycin and Introduction: Antibody-drug conjugates are becoming a vital tool in metronidazole. effective cancer treatment. Due to the added hydrophobic payload, We studied six different formulations each containing one of the antibody-drug conjugates may show different characteristics than the aforementioned drugs using NIR transflectance measurements and naïve mAb [1]. Since the amount of protein lost or unfolded due to developed quantitative models for content determination of common adsorption to container, processing material or the air-liquid interface is concentrations. To generate appropriate fitting methods with low a critical factor [2], the adsorption to glass and the air-liquid interface prediction errors concerning RMSEP and SEP as well as with was analyzed for a model antibody conjugate and the naïve mAb. acceptable coefficients of determination we used partial least squares Moreover, the mechanical stress stability of the samples was regression (PLS) and multiple spectral pretreatments. Finally, we investigated in this study. successfully validated one method based on the criteria of the EMA Materials and Methods: A therapeutic mAb (2 mg/mL) in 10 mM Guideline [7]. phosphate, 145 mM NaCl PBS at pH 7.2, was mixed with The results show that the application of NIRS for API quantification in (NHS)-Fluorescein in DMF. The same therapeutic mAb in 100 mM semi-solid preparations is possible in general, but accuracy and NaHCO3 buffer at pH 9.0 was mixed with Eosin-ITC in DMSO [3]. The variance depend on the active ingredient and its concentration range.

DPhG Annual Meeting 2016 Conference Book • 91 POSTERS products were purified and buffer exchanged to 10 mM phosphate, Rh(I)(NHC)(COD)Cl triggered antiproliferative effects against MCF-7 145 mM NaCl PBS pH 7.2 or 6.5. 0.5 mg/mL samples were analyzed on (human breast adenocarcinoma) and HT-29 (colon carcinoma) cells ServalytTM PrecotesTM gels pH 6-9 and stained with Serva Blue W to and inhibited the enzyme thioredoxin reductase (TrxR),[1,2] which is determine the isoelectric point. The adsorption behavior of 2 mg/mL overexpressed in tumor cells. It was confirmed, that the complexes with samples was analyzed on a SiO2 surfaced quartz crystal chip using a iodide (X = I) exhibited higher biological activity as their chloro- QCM apparatus qCell T, resembling a glass surface. Colloidal stability substituted (X = Cl) equivalents.[2] Rhodium-complexes were also was measured using a Zetasizer APS at protein concentrations proven to interact with the DNA.[3,4] between 0.2 and 10 mg/mL and calculated according to [4].To Scheme 1 shows the newly synthesized rhodium-based complexes, determine the surface pressure at the air-liquid interface, the samples which were characterized by 1H-NMR, 13C-NMR, elemental analysis were analyzed using a Teflon multiwellplate on a MicroTroughX. A and mass spectrometry. In our ongoing studies we evaluate the mechanical stress study was performed to analyze the continuous chemical and biological properties of the new complexes with a focus formation of a new air-liquid interface by shaking 0.2 mg/mL samples at on variations in the X ligand. The current results will be presented on 500 rpm for 24 hours. The samples were analyzed for visual the poster. appearance, turbidity at 350 nm (Nanodrop 2000) and subvisible particles (PAMAS SVSS-25). Results and Discussion: The degree of labelling was 6 for both model mAb conjugates. The calculated hydrophobicity is 2.55 (NHS-Fluorescein) and 4.52 (Eosin-ITC) resp., indicating that hydrophobic molecules were attached to the mAb and that the Eosin- conjugates are more hydrophobic than the Fluorescein-conjugates. The isoelectric points were decreased for the conjugates (5.3-7.4) compared to the naïve mAb (7.6-8.0). Consequently, at the pH values tested, the mAb is slightly net positively charged, whereas the mAb conjugates are Fig. 1: Rhodium(I)-NHC-complexes negatively charged or neutral. In QCM measurements, the mAb Acknowledgments: The financial support of the Center of Pharmaceutical Engineering (PVZ) is conjugates showed a smaller frequency shift and decreased total gratefully acknowledged. adsorbed and irreversible adsorbed masses at pH 7.2, as the charge References: interaction between the net neutrally charged mAb conjugates and the 1. L. Oehninger, L.N. Küster, C. Schmidt et al.: Chem. Eur. J., 2013, 19: 17871 – 17880. negatively charged chip surface is decreased compared to the slightly 2. L. Oehninger, S. Spreckelmeyer, P. Holenya et al.: J. Med. Chem., 2015, 58: 9591−9600. positively charged naïve mAb. The surface pressure was not increased 3. J. R. McConnell, D. P. Rananaware, D. M. Ramsey et al.: Bioorg. Med. Chem. Lett., 2013, for the mAb conjugates compared to the naïve mAb, showing that the 23: 2527–2531. adsorption to the air-liquid interface is not changed with conjugation. 4. C. L. Kielkopf, K. E. Erkkila, B. P. Hudson et al.: Nature Struct. Biol., 2000, 7: 117-121. Colloidal stability displayed net attraction for the mAb conjugates and net repulsion for the mAb, indicating that the mAb conjugates are more prone to aggregation. After the mechanical stress, the visual appearance of all samples remained unchanged. The turbidity showed a more pronounced increase for the conjugates. Moreover, the number POS.14/SL.17 of subvisible particles was significantly increased for the conjugates Insights from functional lipidomics into the long-term compared to the naïve mAb, illustrating that aggregates have formed. regulation of kinases by vitamin A Differences between the two mAb conjugates or an effect of the pH value could not be identified. Thus, the antibody conjugates are less Pein, H.1; Voelkel, M.1; Schneider, F.1; Rossi, A.2; Koeberle, S. C.3; resistant to mechanical stress. The formation of particles is not due to Loeser, K.1; Morrison, H.3; Sautebin, L.2; Werz, O.1; Koeberle, A. 1 an increased adsorption to the air-liquid interface or to the glass 1 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, 07743 Jena, Germany surface, but because of net attractive interactions of the mAb conjugate 2 Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy molecules. 3 Leibniz Institute of Age Research - Fritz-Lipmann-Institute, 07745 Jena, Germany Conclusion: The conjugation of negatively charged hydrophobic molecules to the mAb has slightly decreased protein adsorption to glass For Abstract see Short Lectures SL.17 imitating SiO2 chips. Adsorption to the air-liquid interface was not increased for the conjugates compared to the naïve mAb. However, the mAb conjugates were more sensitive to shaking stress and showed net attractive interaction. POS.15 Acknowledgments: Coriolis Pharma, Aftahy, K., Quraeschi, M. Gradual depolymerization of fucoidan from Fucus References: vesiculosus L. and its effect on the pharmacological profile 1. Wakankar, A.A. et al., Bioconj Chem. 2010, 21(9): 1588-1595. 2. Dixit, N., Maloney, K.M., Kalonia, D.S., Int. J. Pharm. 2011, 412: 201-27. Lahrsen, E.1; Schoenfeld, A.1; Alban, S.1 3. Molecular Probes by Life Technologies, Amine-Reactive Probes, MAN001774, 2013, 1 Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstrasse 76, Revision 2.0. 24118 Kiel, Germany 4. Menzen, T.; Friess, W.; J. Pharm. Sci. 2014.103(2): 445-455. The fucose-containing sulfated polysaccharides (syn. “fucoidans”) from brown algae exhibit a wide range of bioactivities and are therefore considered promising candidates for health-supporting and medicinal applications [1]. The past three decades, research on isolation, POS.13 molecular characterization, and screening of in vitro and in vivo Investigation of the Possible Anticancer Properties of the pharmacological activities has significantly increased. Regarding in vivo Rhodium(I) N-Heterocyclic Carbene Complexes application, the usually high-molecular mass of native fucoidans may, however, be associated with unfavourable biopharmaceutical properties Wölker, J.1,2; Ott, I.1,2 and possibly undesired effects so that it seems reasonable to develop 1 Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, fucoidan derivatives with reduced size. Beethovenstrasse 55, D-38106 Braunschweig, Germany 2 Center of Pharmaceutical Engineering (PVZ), Technische Universität Braunschweig, The aim of the presented study was to establish a suitable Franz-Liszt Strasse 35a, D-38106 Braunschweig, Germany depolymerization method for fucoidans without concomitant desulfation and to examine the activity profile of the resulting depolymerized N-heterocyclic Carbene moieties, especially the “Arduengo-type” metal fractions. For this, fucoidan extracted from Fucus vesiculosus L. with an complexes, have recently started to play an important role as new weight-averaged molecular weight (Mw) of 38.2 kDa, a degree of potential anticancer agents. Although the most extensively investigated sulfation (DS) of 0.63 and a fucose content of 83.1 % (mol/mol), was derivatives of this class of compounds contain gold(I) and silver(I) metal used [2]. ions, series of rhodium(I) N-heterocyclic carbene (NHC) complexes with The two most suitable methods turned out to be hydrothermal 1,5-cyclooctadiene (COD) and a halido ligand (X, Fig. 1), have also depolymerisation at 120 °C and degradation with hydrogen peroxide for shown potential as antitumoral agents.[1,2] The complexes of the type different durations. In this way, 19 fractions with Mw down to 10.3 and

92 • DPhG Annual Meeting 2016 Conference Book ANALYTICS

4.9 kDa, respectively, were obtained. Chemical characterization assays for the determination of aldosterone and renin concentrations in revealed no changes of their chemical composition and no elimination small sample volumes of serum and plasma [1,2]. of sulfate groups. The impact of degradation on the pharmacological Methods: Based on commercial available enzyme-linked profile was examined by the following in vitro activities: (1) elastase immunosorbent assay (ELISA) kits for the determination of aldosterone activity inhibition, (2) inhibition of the classical pathway-activated or renin concentrations, assays for the determination in small sample haemolytic complement activity, (3) inhibition of the classical pathway- volumes were improved. For aldosterone, a competitive assay was activated C5a generation, (4) potentiation of the complement factor C1s used, while the renin assay used was based on a sandwich inhibition by the serine protease inhibitor C1 inhibitor (C1inh) as well as immunoassay [3,4]. The validation plan covers the following the undesired effects (5) activation of Factor XII, and (6) anticoagulant parameters: calibration curve, accuracy, precision, analytical run activity in the activated partial thromboplastin time (APTT) assay. The procedures, stability, matrix effect, dilutional linearity, parallelism, activities of the native fucoidan and its fractions were compared either reproducibility and incurred sample reanalysis, processed sample by calculating the IC50, e.g. elastase inhibition assay, or by other stability, determination of hemolyzed blood samples. characteristic values, i.e. concentration for doubling coagulation time Results: Two enzyme-linked immunosorbent assays for the (DC). Table 1 presents the activity loss of four exemplary fractions determination of aldosterone or renin concentrations in small sample compared to native fucoidan. volumes (40 µl) were developed. Regarding both assays, the parameters calibration curve, accuracy, precision, analytical run procedures, reproducibility and incurred sample reanalysis, processed Fuc Mw 38.2 Fuc Mw 34.4 Fuc Mw 20.6 Fuc Mw 15.9 Fuc Mw 10.3 sample stability and determination of hemolyzed blood samples were IC50 0.48 0.53 0.61 1.18 4.27 successfully validated. All validation runs comply with current Elastase inhibition (µg/mL) Activity 0.0 % 8.5 % 20.0 % 59.1 % 88.7 % bioanalytical guidelines of EMA and FDA [1,2]. For aldosterone, the loss1 IC Complement 50 assay ranges from 31.3 pg/ml to 1000.0°pg/ml, while the renin assay (µg/mL) 4.50 4.73 8.67 20.30 81.4 inhibition Activity 0.0 % 4.9 % 48.1 % 77.8 % 94.5 % ranges from 4.0 pg/ml to 128.0 pg/ml. On the basis of six independent (haemolysis) loss1 assay runs, within-run as well as between-run accuracy and precision Complement IC50 (µg/mL) 34.5 40.4 45.3 63.6 118.3 were demonstrated. For both assays, five concentration levels were inhibition Activity 0.0 % 14.5 % 23.8 % 45.7 % 70.8 % (C5a generation) used for the evaluation of between-run accuracy and precision. loss1 Regarding accuracy, the relative error of the different concentration %2 24.84 25.75 26.28 20.75 9.87 C1inh potentiation Activity 0.0 % 0.0 % 0.0 % 16.5 % 60.3 % levels ranged from -3.8 % to -0.8 % (aldosterone) and from -3.1 % to loss1 +3.0°% (renin). Precision, reported as coefficient of variation, ranged %3 25.46 22.38 12.28 13.24 9.32 from 4.6 % to 8.5 % (aldosterone) and from 4.7°% to 10.7 % (renin). Factor XII activation Activity 0.0 % 12.1 % 51.8 % 48.0 % 63.4 % loss1 Regarding both assays, hemolyzed samples showed no effect on accurate sample determination within the calibration range of the DC (µg/mL) 16.70 19.47 18.63 32.14 76.00 Anticoagulant activity Activity 0.0 % 14.2 % 10.4 % 48.0 % 78.0 % assays. loss1 Conclusion: The developed enzyme-linked immunosorbent assays allow a precise and accurate determination according to current Table 1: Mw dependent activities of native fucoidan and degraded fucoidan fractions 1 calculated in relation to the native fucoidan, 2 sample conc. 6.25 µg/mL 3 in relation to bioanalytical guidelines of EMA and FDA. This reliable determination in Pathromtin SL® at sample conc. 25.0 µg/mL very small sample volumes provides the basis for sophisticated investigations in children. All the activities decreased with decreasing Mw, but both the extent and Acknowledgements: The research leading to these results has received funding from the the molecular weight range of activity loss and thus the overall shape of European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement the molecular weight dependent activity loss was different for any n°602295 (LENA). individual activity. For example, whereas the inhibitory effect on the References: haemolytic complement activity was lost at Mw of 10.3 kDa, the C1inh 1. EMA. Guideline on Bioanalytical Method Validation. EMA, Committee for Medicinal Products potentiation was only reduced at Mw < 20 kDa and at most by 60 %. for Human Use, London, UK 2011. In conclusion, the activities of the fucoidan from Fucus vesiculosus l. 2. US FDA. Guidance for Industry: Bioanalytical Method Validation. (Draft Guidance) US Department of Health and Human Services, US FDA, Center for Drug Evaluation and Research, proved to be dependent on the Mw. However, the impact of the Mw on the various activities turned out to differ resulting in modified Rockville, MD, USA 2013. pharmacological profiles of the fucoidan fractions. 3. Aldosterone ELISA Kit (Ref. EIA-5298), DRG Instruments GmbH, Germany 4. Renin ELISA Kit (Ref. EIA-5125), DRG Instruments GmbH, Germany

References: 1. Ruocco, N. et al.: Molecules 2016, 21(5): pii: E551. 2. Schneider, T. et al.: Glycobiology 2015, 25(8): 812–824. POS.17

Serum and cerebrospinal fluid concentrations of vancomycin in neurosurgical critically ill patients with POS.16 central nervous system infections Development and validation of enzyme-linked immunosorbent assays for the determination of Blassmann, U.1; Roehr, A. C.2; Frey, O. R.2; Vetter-Kerkhoff, C.1; Thon, aldosterone and renin concentrations in small sample N.3; Briegel, J.4; Huge, V.4 volumes - a paediatric-tailored approach 1 Pharmacy, University Hospital of Munich, Marchioninistr. 15, 81377 Munich, Germany 2 Pharmacy, Hospital of Heidenheim, Schlosshaustr. 100, 89522 Heidenheim, Germany 3 Neurosurgery, University Hospital of Munich, Marchioninistr. 15, 81377 Munich, Germany Schaefer, J.1; Burckhardt, B. B.1; Tins, J.1; Bartel, A.; Läer, S.1 4 Anesthesiology, University Hospital of Munich, Marchioninistr. 15, 81377 Munich, Germany 1 Institute of Clinical Pharmacy and Pharmacotherapy, Heinrich-Heine-University Duesseldorf, Universitaetsstr. 1, 40225 Duesseldorf, Germany Background: Combination therapy with meropenem and vancomycin is recommended for hospital-acquired central nervous system (CNS) Background: The LENA (Labeling of Enalapril from Neonates up to infections [1]. The limited penetration of vancomycin into the Adolescents) project investigates a novel age-appropriate enalapril cerebrospinal fluid (CSF) is well known. However, only limited data exist formulation to improve the pharmacotherapy of heart failure in young on the disposition of vancomycin in critically ill patients with CNS children. To gain a better understanding of the underlying disease and infections and non-inflamed meninges [2]. The aim of this study was to to evaluate the impact of the angiotensin-converting enzyme inhibitor describe concentrations of vancomycin in serum and CSF in critically ill enalapril in the diseased population, pharmacokinetic and humoral patients with external CSF drainage and proven or suspected CNS parameters require systematic investigation. As blood volume in infections. children is limited, validated bioanalytical assays for small sample Material/methods: This was an observational pharmacokinetic (PK) volumes are required. study in neurosurgical critically ill patients with proven or suspected Objective: Development and validation according to current CNS infection receiving vancomycin. Serial blood and CSF samples are international EMA (European Medicines Agency) and FDA (Food and taken and analysed by using an in vitro chemiluminescent micro particle Drug Administration) guidelines of paediatric-tailored bioanalytical immunoassay (ARCHITECT iVancomycin assay, Abbott; measuring range: 0.24 mg/l – 100.00 mg/l). Pharmacokinetic parameters are

DPhG Annual Meeting 2016 Conference Book • 93 POSTERS

analysed by a one compartment model. The primary References: pharmacokinetic/pharmacodynamic targets are the area under the 1. World Health Organization, Antimicrobial Resistance: Global Report on Surveillance, 2014, concentration curve (AUC) divided by the minimum inhibitory http://www.who.int/drugresistance/documents/surveillancereport/en/ concentration (MIC) value of 400 in serum and concentrations above 2. Rasko, D.A.; Sperandio, V.: Nat. Rev. Drug Discovery. 2010, 9: 117-128. 3. Aloush, V. et al.: Antimicrob. Agents Chemother. 2006, 50: 43-38. the MIC of suspected pathogens throughout the entire dosing interval in 4. Wretlind, B.; Pavlovskis, O.R.: Rev. Infect. Dis. 1983, 5(5): 998-1004. CSF (100 % T>MIC). According to EUCAST 67 % of staphylococci 5. Kamath, S. et al.: Mol. Microbiol. 1998, 30(5): 933-941. display an MIC of 1 mg/l, 13 % display an MIC of ≤ 0.5 mg/l. Variables are described with median values [interquartile range]. Results: Ten patients (mean age 54, mean weight 73) were enrolled. A total of 110 serum samples and 106 CSF samples were analysed. The median of peak and trough concentration in serum was 24.97 [19.96– POS.19 29.86] mg/l and 8.66 [6.60-10.99] mg/l, respectively. The median MicroScale thermophoretic investigation of deferiprone AUC24 in serum was 394.77 [337.93 – 450.89] mg/l. The median of interaction with selected biometals corresponding peak and trough concentration in CSF was 1.60 [0.24- 2.11] mg/l and 1.12 [0.24-2.50] mg/l, respectively. In CSF, 31 % of the Asmari, M.1; Kleusch, C.2; Michalcova, L.; El Deeb, S.1 samples remained below the detection limit. Assuming an MIC of 0.5 1 Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany mg/l all patients achieved an AUC/MIC value >400. Assuming an MIC 2 NanoTemper Technologies GmbH, Flössergasse 4, 81369 Munich, Germany of 1 mg/l 50 % of all sampling days achieved AUC/MIC > 400. In CSF, 3 Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 56 % of all concentrations reached 1 mg/l and 32 % of all berno 62500, Czech Republic concentrations reached 2 mg/l. Conclusions: Vancomycin demonstrated adequate CSF MicroScale Thermophoresis (MST) method has been developed and concentrations for high susceptible staphylococci/methicillinresistant applied for investigating the interaction of deferiprone (iron-chelator) staphylococcus aureus. With the high inter-individual PK variability with Cu2+, Zn2+, Ni2+, Mn2+, Co2+, Mg2+, Ca2+ and Fe3+.The experiments observed, therapeutic drug monitoring in CSF might be an option to were performed on Monolith NT. LabelFree MicroScale Thermophoresis optimize vancomycin dosing in critically ill patients with CNS infections. system. Pretest scanning indicated good fluorescence intensity of Higher serum level targets or switching to alternative antibiotics should deferiprone proper for label free experiments. Different concentrations be considered, if CSF concentrations are lacking. of the intended metals in the range of 0.048 - 500.0 µM were titrated against 100 µM fixed concentration of deferiprone dissolved in 0.1 M Acknowledgements: Stiftung Patient und Klinische Pharmazie, Lesmüllerstiftung Tris buffer at pH 7.4. MST measurements were performed in standard References: capillaries at 50% Excitation-power and 20% MST-power. The results 1. Tunkel, A.R. et al.: Clin. Infect. dis. 2004; 39(9): 1267-1284. indicated significant interactions of deferiprone with Cu2+, Zn2+, Ni2+, 2. Nau, R. et al.: Clin. Microb. Rev. 2010; 23(4): 858-83. Co2+ and Fe3+. The data fitted to Hill equation curves with Hill

coefficients of 1.5, 3.2, 1.5, 1.6, and 1.8 for Cu2+, Zn2+, Ni2+, Co2+ and

Fe3+, respectively, thus indicating more than 1:1 stoichiometry. EC50

values for the binding of Cu2+, Zn2+, Ni2+, Co2+ and Fe3+ to deferiprone POS.18 where calculated to be 38.1, 39.5, 101.1, 51.1 and 20.6 µM, Development of Novel Inhibitors Targeting Elastase (LasB) respectively. No binding of deferiprone with Mg2+, Ca2+ and Mn2+ were from Pseudomonas aeruginosa observed. The technique shows a fast and simple approach to study the binding of deferiprone to different biometals.

Kany, A. M.1; Sikandar, A.2; De Mello Martins, A. G. G.1; Eberhard, J.1;

Hamed, M.1; Haupenthal, J.1; Köhnke, J.2; Hartmann, R. W. 1,3 1 Helmholtz Institute for Pharmaceutical Research Saarland, Department of Drug Design and Optimization, Campus E8.1, 66123 Saarbrücken, Germany 2 Helmholtz Institute for Pharmaceutical Research Saarland, Department of Structural Biology of POS.20 Biosynthetic Enzymes, Campus E8.1, 66123 Saarbrücken, Germany MS Uptake Assays for the Serotonin Transporter (rSERT) 3 Saarland University, Pharmaceutical and Medicinal Chemistry, Campus E8.1, 66123 Saarbrücken, Germany Währa, M.1; Ackermann, T.1; Höfner, G.1; Wanner, K. T.1 1 Department Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität The increasing appearance of bacteria that are resistant to commonly München, Butenandtstr. 5-13, D-81377 Munich, Germany used antibiotics poses a threat to public health and makes the development of novel antibiotics an urgent necessity [1]. Targeting Uptake experiments are the fundamental assay type for bacterial virulence factors is a new approach in the field of antibacterial characterization of substrate transport and evaluation of inhibitor drug development. Virulence factors are known to play a pivotal role profiles at transport proteins. Most of the assays commonly used for this during the infection process of pathogenic bacteria [2]. The harmful zinc purpose are based on substrates labeled by radioisotopes or metalloprotease elastase (LasB) is such a virulence factor, secreted by fluorophores. The last years showed a steady development of uptake Pseudomonas aeruginosa. This pathogen is responsible for severe lung assays based on unlabeled substrates quantified by LC-MS/MS. This infections, especially in immunocompromised and cystic fibrosis concept e.g. named MS Uptake Assays has been applied to different patients[3]. LasB enables the bacteria to colonize a niche in the host, to transporters like the GABA transporters (hGAT1, hGAT2, hGAT3 and evade the host immune response and to obtain nutrition from the BGT1) [1] as well as the multidrug and toxin extrusion transporters 1 infected cells [4]. Furthermore, it is involved in the formation of P. and 2 (MATE 1 and MATE2) and the organic cation transporter 2 aeruginosa biofilm by proteolytic activation of nucleoside diphosphate (OCT2) [2]. In the present study, this strategy should be used to set up kinase (NDK) [5]. MS based transport assays for the Serotonin Transporter (SERT) Consequently, this protease represents a prime target for novel employing 1-methyl-4-phenylpyridinium (MPP+) as substrate. inhibitors which attenuate the aforementioned virulence mechanisms. In A sensitive LC-ESI-MS/MS quantification method for MPP+ (m/z 170 → order to identify novel active and selective LasB inhibitors, we 128) using five times deuterated MPP+ (d5-MPP+, m/z 175 → 133) as performed a fluorescence-based functional screening of a focused internal standard was developed and validated according to the FDA protease inhibitor library as well as of a number of fragments. Inhibition guideline for bioanalytical methods. Applying this quantification method, was crosschecked by additional LC-MS-based analysis of the MS Uptake Assays for rSERT (stably expressed in HEK293 TREX fluorescence assay to detect compounds whose apparent inhibition was cells) were established following the procedure shown in the scheme only due to quenching of fluorescence. below. We discovered a sulfonic acid derivative and a series of mercaptoacetamides as inhibitors of LasB. Treatment of PA14 wild type cultures with the sulfonic acid led to a reduction of biofilm volume. Using cell based and in vivo assays, the biological effects of LasB inhibition by our hits will be further investigated. Cocrystallization of these compounds with LasB is currently ongoing and will serve as a basis for chemical optimization regarding potency and selectivity against human matrix-metalloproteases (MMPs).

94 • DPhG Annual Meeting 2016 Conference Book ANALYTICS

FT-IR Analytics

100 TGA 31P MAS-NMR

80 Mass [%] Transmission [%] Transmission control control Monophosphonic Acid on ZnO Triphosphonic Acid on ZnO Diphosphonic Acid on ZnO Diphosphonic Acid on ZnO 60 Triphosphonic Acid on ZnO Monophosphonic Acid on ZnO

3850 3300 2750 2200 1650 1100 550 100 200 300 400 500 600 700 Wavenumber [cm-1] Sample Temperature [°C]

EDX XRD 60000

50000

40000

30000 counts 20000 10000

+ 0 Using this procedure, the KM-value of MPP for rSERT could be 0 500 1000 1500 2000 2 Theta [°] determined in saturation experiments. The obtained result is in good accordance with those of radio uptake assays. Finally, inhibitory Acknowledgements: We kindly like to thank A. Hensel from the Institute of Physical Chemistry potencies for a series of reference compounds were evaluated with the at the University of Hamburg for the analysis of the samples via TEM, EDX and XRD, Dr. Young Joo Lee from the Institute of Inorganic Chemistry at the University of Hamburg for conducting established MS Uptake Assays. The results derived therefrom correlate the solid-state NMR investigations and Dr. Silvia Gross from the Dipartimento di Scienze well with the affinities found in binding assays for hSERT. [3] Chimiche at the Università degli Studi di Padova, Italy for the synthesis of the ZnS and ZnO nanoparticles. References: References: 1. Schmitt, S., Höfner, G., Wanner K.T.: Anal. Chem. 2014, 86(15): 7575-7583. 1. Darouiche R. O., N.: Engl. J. Med. 2004, 350(14): 1422-1429. 2. Vath M et al.: J. Chromatogr. B. 2014, 967: 211-218. 2. Costerton J. W., Stewart P. S., Greenberg E. P.: Science 1999, 284(5418): 1318-1322. 3. Grimm, S.H., Höfner, G., Wanner, K.T.: Chem. Med. Chem. 2015, 10(6):1027-1039. 3. Banerjee I., Pangule R. C., Kane R. S.: Adv. Mater. 2011, 23(6): 690-718. 4. Lejars M., Margaillan A., Bressy C.: Chem. Rev. 2012, 112(8): 4347-4390. 5. Sisson A. L., Haag R.: Soft Matter 2010, 6(20): 4968-4975. 6. Khalil, F. et al.: Colloids Surf. B 2014, 117(2014): 185-192. 7. Shao Q.; Jiang S.: Adv. Mater. 2015, 27(1): 15-26. POS.21 8. Pujari S. P. et al.: Angew. Chem. 2014, 126(25): 6438-6474. Multimeric phosphonic acids as anchor molecules to generate stable antifouling surfaces on metals

Klitsche, F.1; Maison, W.1 POS.22 1 University of Hamburg, Institute of Pharmacy, Pharmaceutical and Medicinal Chemistry, Bundesstraße 45, 20146 Hamburg, Germany Total phenolic and tannins determination: A modification of Ph. Eur. 2.8.14 suitable for high-throughput screenings The stable modification of surfaces becomes increasingly important especially in the fields of clinical hygiene and implant medicine [1, 2]. Wiesneth, S.1; Heilmann, J.1; Jürgenliemk, G.1 1 Institute of Pharmaceutical Biology, University of Regensburg, Universitätsstraße 31, Bacterial infections during hospitalization and implantation cause high D-93053 Regensburg, Germany costs for the health care systems and have often serious consequences for the patients. In this context, strategies to prevent bacterial To get an overview of the natural compounds’ composition of an herbal attachment to surfaces the so-called antifouling are attractive to drug or extract thereof, very often convention methods are used to decrease microbial loading. There are two main approaches used to estimate the concentration of major groups of secondary plant generate antifouling surfaces: on the one hand an active approach ingredients. A principle mostly being used for phenolic compounds is employing surface-bound antibiotics, cations or proteases and on the the Folin-Ciocalteu’s reagent method described in the European other hand a repelling strategy with biopassive coatings based on Pharmacopeia method 2.8.14 (Ph. Eur.) [1] as not only the total polyethylene glycol (PEG), polyglycerols or zwitterions which inhibit phenolic content can be determined by this method, but also the tannin concentration, irrespective of the type of tannins (hydrolyzable or protein adsorption to the surface [3-7]. These effector moieties need to condensed tannins) [1]. Unfortunately, it is hardly possible to handle a be strongly bound to the surface via appropriate anchor molecules [8]. large number of samples in an appropriate time using this complex Herein we report the binding properties of different adamantane-based method. To work more efficiently, the aim of the present study was to phosphonic acids. These anchors can be immobilized on clinically simplify this procedure. relevant metal surfaces such as titanium and zirconium. We report a By changing the wavelength [2], the amounts of reagents [3], proof-of-concept study on different metal nanoparticles as model minituarizing the setting to microtiter scale and changing the time systems for larger metal surfaces. The obtained coated nanoparticles interval in between pipetting the assay and the measurement, the Ph. are characterized via different analytical methods (e.g. solid-state 31P Eur. method was optimized for a larger number of experiments. MAS-NMR, EDX, XRD, TGA and FT-IR-spectroscopy). Moreover the Calibration curves and time kinetics with different phenolic compounds stability of the coated particles towards different pH-values and time (catechol, gallic acid, (+)-catechin, tannic acid, salicylic acid, ferulic periods is examined. acid) were determined in addition to the molar extinction coefficient by extrapolation of each calibration curve and thus, to examine possible correlations between the reaction’s stoichiometry and the absorption. Also a complete data set of ascorbic acid, a non phenolic compound, was generated in order to check its response to this assay. The method was validated concerning its repeatability, robustness, linearity and reproducibility. Using this method, at least 120 samples can be handled per day by one person for the estimation of the total phenolic as well as tannin content.

Acknowledgments: Many thanks are due to M. Jünger for performing the experiments concerning this project (Institute of Pharmaceutical Biology, University of Regensburg). References: 1. 2.8.14: Gerbstoffe in pflanzlichen Drogen. Europäisches Arzneibuch 8. Ausgabe (Deutscher Apotheker Verlag) 2015; 383. 2. Glasl, H.: DAZ 1983, 123, 1979–1987. 3. Li, H. et al.: Food Chemistry 2007, 102, 771–776.

DPhG Annual Meeting 2016 Conference Book • 95 POSTERS

POS.23 The molybdoenzyme mARC detoxifies trimethylamine N- oxide, a risk factor for cardiovascular disease

Schneider, J.1; Girreser, U.1; Havemeyer, A.1; Tyl-Bielicka, A.2; Pysniak, K.2; Ramotowska, E.2; Mikula, M.2; Clement, B.1 1 Christian-Albrechts-University Kiel, Department of Pharmaceutical and Medicinal Chemistry, Gutenbergstraße 76, 24118 Kiel, Germany 2 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics, Wilhelma Konrada Roentgena 5, 02-781 Warsaw, Poland

Cardiovascular disease (CVD) is the leading cause of morbidity worldwide. Therefore, it is of the utmost importance to learn more about its genesis. Within this context, trimethylamine N-oxide (TMAO), the physiological metabolite of dietary phosphatidylcholine, is in the center of interest. As the metabolic profile of TMAO in plasma correlates with the risk for CVD due to a pro-atherosclerotic mechanism [1], any process that leads to decreased TMAO plasma levels could possibly reduce the risk for CVD. TMAO is an oxidation product of hepatic flavin monooxygenase (FMO), thus its reduction to the precursor trimethylamine (TMA) is an obvious option to diminish its plasma concentration. So far, no enzyme catalyzing this reaction was identified. The recently in our lab discovered mitochondrial amidoxime reducing component mARC, the fourth molybdenum containing enzyme in mammals [2], could potentially catalyze this reduction. Along with the heme-containing cytochrome b5 (CYB5) and its flavin-containing cytochrome b5 reductase (CYB5R), mARC forms an N-reductive enzyme system which is able to reduce a variety of N-hydroxylated compounds. The human genome encodes for two mARC proteins, hmARC1 and hmARC2. So far, the physiological role of mARC remains mainly unknown [2-3]. Biotransformation assays including the reconstituted recombinant N- reductive enzyme system and TMAO as substrate can easily reveal if a reduction to the metabolite TMA is of significance. The volatile character as well as the lack of a chromophore of TMA poses particular challenges for this task. Hence, we developed an LC/MS/MS-method involving a dedicated and reliable sample preparation comprising the derivatization of TMA to a non-volatile compound by quaternization of the amine. With this newly developed analytical tool we investigated the in vitro formation of TMA through the reconstituted N-reductive enzyme system. We found that hmARC1 but not hmARC2 reduces TMAO. Moreover, we show that murine liver homogenates of wild type mice and mARC2(-/-) knock-out mice reduce TMAO without significant difference in specific activity. These data suggest that only one mARC isoform participates in the detoxification of TMAO. Our results prove that mARC reduces TMAO, thus represents the counterpart to FMO and plays a role in the prevention of CVD. Furthermore, these findings also propose a physiological function of the molybdoenzyme mARC.

Acknowledgements: We thank A. Meier1 for preparing the murine liver homogenates. References: 1. Wang Z. et al., Nature 2011, 472(7341): 57-63. 2. Havemeyer A. et al., J. Biol. Chem. 2006, 281(46): 34796-34802. 3. Gruenewald S. et al., J. Med. Chem. 2008, 51(24): 8173-8177.

POS.24/SL.37 How polysaccharide superstructure impacts hydrogel properties: A Raman optical activity study

Lüdeke, S.1; Rüther, A.1; Forget, A.2; Roy, A.3; Carballo, C.3; Dukor, R. K.3; Nafie, L. A.3; Johannessen, C.4; Shastri, V. P.5 1 Inst. of Pharmaceutical Sciences, University of Freiburg, Albertstr. 25,79104 Freiburg, Germany 2 Future Industries Insitute, University of South Australia, MM Building, 5095 Mawson Lakes, Australia 3 BioTools, Inc., 17546 Beeline Hwy, Jupiter, FL, USA 4 Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium 5 Institute for Macromolecular Chemistry, University of Freiburg, Stefan-Meier-Str. 31, 79104 Freiburg, Germany

For Abstract see Short Lectures SL.37

96 • DPhG Annual Meeting 2016 Conference Book INFLAMMATION

chemoattractant for leukocytes produced by endothelial cells, was 3.2 Inflammation concentration-dependently down-regulated by C81 (qPCR). Furthermore, neither C81 treatment nor BMP2K silencing led to the

reduction of TNFα-induced IκBα degradation (Western blot) or p65 POS.25/SL.16 translocation into the nucleus (microscopy). Interestingly, silencing of BMP2K resulted in a marked decrease of TNFα-induced COX-2 Molecular mechanisms of lipoxin and resolvin biosynthesis expression (Western blot). Our study provides first insights into the anti-inflammatory and anti- 1 2 2 3 3 Lehmann, C. ; Cumbana, R. ; Ebert, R. ; Toewe, A. ; Angioni, C. ; angiogenic potential of the carbazole derivative C81 in vitro. Since the 3 1,3 1 2 Ferreirós, N. ; Geisslinger, G. ; Parnham, M. J. ; Steinhilber, D. ; inhibition of BMP2K seems to be responsible only for some actions of 2 Kahnt, A. S. C81, we will investigate the role of AAK1 in these processes. The 1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology, Theodor Stern Kai 7, 60596 Frankfurt/Main, precise role of BMP2K/AAK1 in angiogenic and inflammatory Germany endothelial processes as well as the involved pathways during 2 Goethe-University, Institute of Pharmaceutical Chemistry, ZAFES, Max-von-Laue-Str. 9, BMP2K/AAK1 silencing and C81 treatment will be further elucidated. D-60438 Frankfurt/Main, Germany 3 Goethe University, Institute of Clinical Pharmacology, Pharmazentrum Frankfurt, ZAFES, Theodor Stern Kai 7, D-60590 Frankfurt/Main, Germany

For Abstract see Short Lectures SL.16

POS.27 Vioprolide A interferes with pro-inflammatory processes in human endothelial cells

POS.26 Luong, B.1; Bischoff, I.1; Müller, R.2; Fürst, R.1 First insights into the action of the carbazole derivative 1 Institute of Pharmaceutical Biology, Biocenter, Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany C81 and the role of its primary target BMP2K in the 2 Helmholtz Institute for Pharmaceutical Research Saarland, Department of Microbial Natural activated vascular endothelium Products and Department of Pharmaceutical Biotechnology, Saarland University, Campus E8.1, 66123 Saarbrücken, Germany Bischoff, I.1; Dai, B.2; Strödke, B.3; Knapp, S.4,5; Bracher, F.3; Fürst, R.1 1 Institute of Pharmaceutical Biology, Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt Myxobacteria have been shown to be a rich source of potentially useful, am Main, Germany bioactive secondary metabolites [1,2]. A myxobacterial library of 259 2 Pharmaceutical Biology, Center for Drug Research, University of Munich, Butenandtstr. 5-13, purified compounds was screened for their effects on the TNFα-induced 81377 Munich, Germany 3 Department of Pharmacy - Center for Drug Research, University of Munich, Butenandtstr. 5- ICAM-1 expression and on the serum-induced migration and 13, 81377 Munich, Germany proliferation. One of the compounds identified was vioprolide A, a cyclic 4 Institute for Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, 60438 peptide obtained from the myxobacterium Cystobacter violaceus [3]. Frankfurt am Main, Germany 5 Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus, Headington, Aim of this study was to characterize the effects of vioprolide A on the Oxford OX3 7BN, United Kingdom tumor necrosis factor α (TNFα)-activated endothelium and to gain first insights into the underlying mode of action. Chronic inflammatory diseases, such as psoriasis or rheumatoid First, we investigated the impact of vioprolide A on the viability of arthritis, are characterized by constant leukocyte infiltration and ongoing primary human umbilical vein endothelial cells (HUVECs). angiogenesis in the inflamed tissue. As current anti-inflammatory Concentrations up to 10 nM did not significantly influence the metabolic pharmacotherapy is not always satisfying, there is a great need for the activity (CTB assay), the apoptosis rate (sub-diploid DNA content) or discovery of new drug leads and targets. The synthetic carbazole the LDH release. The combined treatment with 10 nM Vio A and derivative C81 acts as a kinase inhibitor. Results of a thermal shift 10 ng/ml TNFα slightly decreased the metabolic activity by about 11 % assay revealed that C81 shows by far the highest binding affinity to the without influencing the apoptosis rate. BMP-2-inducible kinase (BMP2K/BIKE) and the adaptor-associated Proinflammatory cytokines such as TNFα trigger the recruitment of kinase 1 (AAK1). Both kinases belong to the Numb-associated kinase leukocytes by an increased surface expression of leukocyte and (NAK) family, which has been linked to various biological functions, endothelial cell adhesion molecules [4]. Vioprolide A (10 nM) was able such as osteoblast differentiation or receptor-mediated endocytosis. to strongly reduce the TNFα-induced leukocyte adhesion (THP-1, Since the vascular endothelium crucially regulates inflammatory Jurkat, PBMC). Flow cytometry analysis revealed that 30 minutes processes, we hypothesized that these kinases might play a preincubation with 10 nM vioprolide A diminished the TNFα-induced pathophysiological role in the inflammation-activated endothelium. The surface expression of the endothelial cell adhesion molecules ICAM-1, functional role of both kinases has not been characterized in the VCAM-1 and E-selectin by more than 50 %. Quantitative real-time PCR endothelium so far. Therefore, we aimed to analyze the showed that vioprolide A also strongly downregulated the TNFα- pharmacological potential of C81 and, as a starting point, to investigate induced mRNA levels of all three adhesion molecules by about 80-90 the role of BMP2K in angiogenic and inflammatory processes in the %. The TNFα-induced expression of these cell adhesion molecules is vascular endothelium. mainly regulated by the transcription factor NFκB [4]. Therefore, we Initial experiments show that only high concentrations of C81 affected analysed the NFκB promotor activity using a dual luciferase reporter the viability of human umbilical vein endothelial cells (HUVECs) after 24 gene assay. TNFα stimulated the NFκB promotor activity and hours of treatment (IC50: 171 μM). Longer incubation periods (72 h) pretreatment with 10 nM vioprolide A led to a fulminant decrease of this reduced the proliferation of a human microvascular endothelial cell line induction by about 90 %. (HMEC-1) with an IC50 of 7 μM. C81 treatment (10 μM) resulted in a To get a deeper insight into the TNFα-related pathways that are reduced migratory capacity of HMEC-1. A tube formation assay on influenced by vioprolide A, we performed a transcriptome analysis using Matrigel® demonstrated that C81 significantly impaired the formation of the MACE (massive analysis of cDNA ends) and RNA-sequencing capillary-like structures in a concentration-dependent manner. In technique. We found 395 and 557 genes that were upregulated by addition, C81 (3 μM) reduced the formation of VEGF-induced sprouts in TNFα in HUVECs after 6 and 16 hours, respectively. From these genes, HUVEC spheroids. Interestingly, the analysis (Western blot) of signaling 133 (6 hours) and 239 genes (16 hours) were downregulated by molecules in HUVECs that play a crucial role in cell proliferation (e.g. preincubation with vioprolide A for 30 minutes. Within these genes, we ERK, Akt) revealed that these pathways are not influenced, neither by identified further NFκB targets such as the chemokines CX3CL1 and C81 treatment nor by knock-down of BMP2K (RNAi). In regard to monocyte chemoattractant protein 1 (MCP-1) or genes related to gene inflammatory processes, C81 treatment and BMP2K silencing of expression such as CBP/p300 activating peptide and interferon HUVECs decreased the adhesion of THP-1 cells, a monocytic cell line, regulatory factor 1 (IRF1). onto the activated endothelial cells. As the interaction of leukocytes is Taken together, vioprolide A inhibits the TNFα-induced leukocyte mainly mediated by cell adhesion molecules (CAMs), the effect of C81 adhesion and surface expression of ICAM-1, VCAM-1 and E-selectin or BMP2K silencing on their expression was analyzed (flow cytometry, probably via deactivation of the NFκB signalling pathway. Vioprolide A qPCR). While the expression of CAMs was strongly decreased after may interfere with NFκB-regulating molecules, such as CBP/p300 or C81 treatment, the knock-down of BMP2K did not markedly affect their IRF-1, to exert these effects. The in vivo relevance of these findings as expression. Physiologically, leukocytes are recruited to the site of well as the underlying mechanisms are currently studied. inflammation by chemotactic cues. The expression of CX3CL1, a

DPhG Annual Meeting 2016 Conference Book • 97 POSTERS

Acknowledgments: This work was supported by the German Research Foundation (DFG, FOR STW 5 is a standardized multi-component herbal preparation consisting 1406, FU 691/9-2). of hydro-alcoholic extracts of bitter candytuft, lemon balm, chamomile, References: caraway, peppermint, angelica, milk thistle, celandine, and licorice. It 1. Reichenbach H.: J. Ind. Microbiol. Biotechnol. 2001, 27(3): 149-156. has been used effectively in functional dyspepsia [1] and irritable bowel 2. Weissmann K.J., Müller R.: Nat. Prod. Rep. 2010, 27(9): 1276-1295. 3. Schummer D. et al.: Liebigs Ann./Recl.1996, 6:971-978. syndrome [2] and showed efficacy in experimental dextran sodium 4. Newton K., Dixit V.M.: Cold Spring Harb. Perspect. Biol. 2012, 4(3). sulfate induced colitis as a model for ulcerative colitis [3]. The present study was conducted to investigate its potential usefulness in 2, 4, 6- trinitrobenzene sulfonic acid (TNBS) induced colitis as a model of Crohn’s disease. Colitis was induced by instilling TNBS in the colon of male Wistar rats POS.28 under light ether anesthesia. In a prophylactic setting, STW 5 was given Anti-inflammatory and cytoprotective effects of Hypericum orally one week before induction of colitis and continued for three days extract STW 3-VI onwards. After 24 h rats were sacrificed. In the curative setting, STW 5 was given orally 48 h after colitis induction daily for one week and the Schwendler, A.1; Abdel-Aziz, H.2; Kelber, O.3; Hüther, J.1; Bonaterra, G. rats sacrificed 24 h later. Mucosal colonic damage was assessed A.1; Cordes, A.1; Kinscherf, R.1 macroscopically. Sulfasalazine was used as a reference drug. Colon 1 Dept. of Medical Cell Biology, Philipps-University Marburg, Marburg, Robert-Koch-Str. 8, homogenates and serum samples were used to assess levels of 35032 Marburg, Germany inflammatory and oxidative stress parameters. Immuno-histochemical 2 Medicial Affairs, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany staining of colon sections was used to assess calprotectin and IL-17A 3 Innovation & Development, Phytomedicines Supply and Development Center, Bayer levels, reported to be implicated indicators for IBD in man. Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, TNBS colitis led to severe ulcerative damage, inhibition of reduced Germany glutathione and a rise in myeloperoxidase in colon homogenates.

Relevant cytokines TNFα, IL-1β, ICAM-1 were elevated as well as LT- Glutamate toxicity and inflammation play an integral role in a variety of B4 and PGE2. Immuno-histochemical examination showed a rise in disorders as depression. Additionally, innumerable evidence indicates calprotectin and IL-17A. Pre-treatment with STW 5 prevented such that adaptation to chronic stress involves response from both, the effects in a similar manner to sulfasalazine. In the curative setting, STW neuroendocrine and immune systems. Depression has also been linked 5 tended to normalize changes in reduced glutathione and to an inflammatory response since proinflammatory cytokines like myeloperoxidase and in ulcerative indices induced by TNBS. Present interleukin 6 (IL-6) and tumor necrosis factor (TNF) are increased in findings provide good supportive evidence for the potential usefulness depressed patients. In this context, the combined antioxidant and anti- of STW 5 in Crohn’s disease. inflammatory properties [1] of St. John's wort (Hypericum perforatum L.) extract are suggested to contribute to the antidepressant effects [2] by References: normalization of an overactive hypothalamic-pituitary-adrenal axis [3]. 1. Schmulson, M. J.: Nat Clin Pract Gastroenterol Hepatol 2008; 5: 136-137 Thus, the aim of our investigations was to determine the effects of STW 2. Madisch, A. et al.: Aliment Pharmacol Ther 2004; 19: 271‐279 3-VI on protection of differentiated mouse hippocampal HT22 cells from 3. Wadie, W. et al.: Int J Colorectal Dis 2012; 27: 1445‐1453 the cytotoxic effects of glutamate or NMDA and the possible anti- inflammatory properties on LPS-activated macrophages (MΦ). Differentiated HT22 cells were pre-treated with STW 3-VI to investigate the protective effects against glutamate or NMDA cytotoxicity. The anti- inflammatory properties of STW 3-VI were evaluated by quantification of POS.30 the TNF release on LPS activated PMA-differentiated THP 1 MΦ using Development of smart cell-free and cell-based assay ELISA assay and the mRNA expression of TNF and IL-6 by qRT-PCR. systems for investigation of leukotriene C4 synthase Glutamate or NMDA (0.1mM) induced 30% cytotoxicity in HT22 cells. (LTC4S) activity and evaluation of inhibitors Pre-incubation (24h) with 10μg/ml of STW 3-VI improved the viability by 30% compared to the control. Pre-treatment (48h) of LPS activated MΦ Liening, S.1; Scriba, G. K.1; Rummler, S.2; Weinigel, C.2; Kleinschmidt, with STW 3-VI (60, 80 or 100 μg/ml) induced a significant lowering T. K.3; Haeggström, J. Z.3; Werz, O.1; Garscha, U.1 (54%, 64% and 53%) of TNF release. QRT-PCR revealed that 48 h pre- 1 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller- treatment with 60 or 80 μg/ml STW 3-VI inhibited the mRNA expression University Jena, Germany, 2 Institute of Transfusion Medicine, Jena University Hospital, Jena, Germany, of IL-6 and TNF respectively by LPS-activated MΦ. 3 Division of Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska In conclusion, STW 3-VI protects hippocampal cells from glutamate or Institutet, Stockholm, Sweden NMDA induced cytotoxicity and activates the anti-inflammatory defense by inhibition of the cytokine production by MΦ. These effects are in Cysteinyl-leukotrienes (cys-LT) are powerful pro-inflammatory accordance with the therapeutic use of STW3-VI in depression. mediators that cause bronchoconstriction in anaphylaxis and asthma. They are formed by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) Acknowledgements: The study received financial support from Steigerwald Arzneimittelwerk yielding the unstable leukotriene A4 (LTA4) that is subsequently GmbH conjugated with glutathione (GSH) by LTC4 synthase (LTC4S), an References: integral membrane protein that belongs to the superfamily of 1. Breyer, A et al.: Phytomedicine 2007, 14: 250-255 membrane-associated proteins in eicosanoid and glutathione 2. Denke, A et al.: Drug Res 2000, 50 (5): 415-419 metabolism (MAPEG), to form LTC4. The tripeptide side chain of LTC4 3. Gastpar, M; Singer, A; Zeller, K.: Pharmacopsychiatry 2006, 39: 66-75 4. Grundmann, O; Kelber, O; Butterweck, V.: Planta Medica 2006, 72: 1366-1371 is then cleaved in two successive steps to form LTD4 and LTE4. These cys-LTs are recognized by GPCRs. Cys-LT receptor antagonists as well as LTC4S inhibitors have been developed, but only the former have reached the market. One reason might be the high structural homology of LTC4S to related enzymes of the MAPEG family. One the other hand POS.29 a convenient test systems to identify potential LTC4S inhibitors was Mode of action of a phytomedicine, STW 5, in an missing due to instability of exogenously added LTA4 as substrate. experimental model of Crohn´s disease The main purpose of our work was to establish cell-free and cell-based assay systems based on in situ-generated LTA4 that allow studying Khayyal, M. T.1; Wadie, W.1; Abdallah, D.1; Schneider, M.2; Efferth, T.2; LTC4S activity and investigating LTC4S inhibitors. Co-incubations of Kelber, O.3; Abdel-Aziz, H.4 microsomes from HEK293 cells stably expressing LTC4S together with 1 Department of Pharmacology, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, isolated 5-LOX efficiently converted exogenous AA to LTC4 (~1.3 11562 Cairo, Egypt µg/200 µg protein). Stimulation of HEK293 cells co-expressing 5-LOX 2 Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes and LTC4S with Ca-ionophore A23187 and 20 µM AA leads to a strong Gutenberg University, Staudinger-Weg 5, 55128 Mainz, Germany 3 Medicial Affairs, Phytomedicines Supply and Development Center, Bayer Consumer Health, LTC4 formation (~ 250 ng/106 cells). MK-886 consistently inhibited LTC4 Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany formation in the assay types (IC50 = 3.2 and 3.1 µM, respectively) and 4 Innovation & Development, Phytomedicines Supply and Development Center, Bayer we successfully confirmed TK04a as potent LTC4S inhibitor in these Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany assay systems (IC50 = 17 and 300 nM, respectively). Further, we

98 • DPhG Annual Meeting 2016 Conference Book INFLAMMATION

demonstrated transcellular LTC4 biosynthesis between human “FY-cork” [1]. We examined the influence of these residues on 5-LOX neutrophils or 5-LOX-expressing HEK293 cells that produce LTA4 from activity, substrate access, membrane binding, and interaction with AA and HEK293 cells expressing LTC4S that transform LTA4 to LTC4. FLAP in intact HEK293 cells expressing 5-LOX in the absence and Summarizing, we established cell-free and cell-based HEK 293 systems presence of FLAP [2]. Uncapping the 5-LOX active site by mutation of for evaluating LTC4S inhibitors. Our assay approaches are F177 and/or Y181 to less bulkier alanine (5-LOX-F177A, 5-LOX-Y181A, advantageous as the substrate LTA4 is generated in situ and are 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX suitable for studying enzymatic functionality of LTC4S including site- membrane association in A23187-stimulated cells. Additionally, for 5- directed mutations. LOX-F177A and 5-LOX-F177/Y181A, the formation of 5-LOX products was dramatically reduced relative to 5-LOX-wild-type (wt). Strikingly, co- expression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE by these same 5-LOX mutants (≈ 60- 70% 5-LOX-wt levels) but not of LTA4 hydrolysis products. Substitution POS.31 of Y181 by less bulkier residues as phenylalanine or alanine, allows Thermodynamic properties of leukotriene A4 hydrolase dioxygenation at carbon 5 and generated 5-H(p)ETE at levels inhibitors comparable to 5-LOX-wt but mainly prevented LT formation. Again, co- expression of FLAP partially restored LTA4 hydrolysis product formation Wittmann, S.1; Kalinowsky, L.1; Kramer, J. S.1; Blöcher, R.1; Knapp, S.1; by 5-LOX-Y181A. Steinhilber, D.1; Pogoryelov, D.2; Proschak, E.1; Heering, J.3,* Together, the data suggest that amino acids (F177 and Y181) 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Street 9, obstructing access to the active site are essential for membrane 60438 Frankfurt, Germany 2 Institute of Biochemistry, Goethe University Frankfurt, Max-von-Laue Street 9, association. Additionally, protein-lipid (5-LOX/membrane) as well as 60438 Frankfurt, Germany protein-protein (5-LOX/FLAP) interaction promote LT formation in the 3 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group cell. Translational Med-icine and Pharmacology TMP, Theodor-Stern-Kai 7, D-60596 Frankfurt am Main, Germany * Author to whom all correspondence should be addressed. E-mail: Acknowledgments: Financial support was provided by Deutsche Forschungsgemeinschaft [email protected] (DFG) within the SFB1127: Chemical Mediators in complex Biosystems. References: The leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme, 1. Gilbert, N. et al. Science 2011, 331: 217-219 containing a peptidase and a hydrolase activity [1]. The hydrolase 2. Gerstmeier, J.; Garscha, U. et al. FASEB J. 2016, 30(5):1892-900 activity is responsible for the conversion of leukotriene A4 to pro- inflammatory leukotriene B4 Both activities having opposing functions regulating inflammatory processes [2,3]. The hydrolase activity is responsible for the conversion of leukotriene A4 to pro-inflammatory POS.33 leukotriene B4, and hence, selective inhibitors of the hydrolase activity are of high pharmacological interest [4]. The size of the binding pocket Particulate matter (PM2.5) from biomass combustion alters complicates the development of potent inhibitors. We therefore worked the methylation profile of genes related to cancer out the thermodynamic properties of five known LTA4H inhibitors. The apo structure was solved by X-ray crystallography and utilizing an in Heßelbach, K.1; Kim, G.-J.2; Flemming, S.2; Dornhof, R.1; Häupl, T.3; silico method we determined the position of stabilized water molecules. Günther, S.2; Merfort, I.1; Humar, M.1 1 Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs University of From the occupancy of different regions within the binding pocket we Freiburg, 79104 Freiburg, Germany predicted an individual profile for the selected inhibitors. A variety of 2 Pharmaceutical Bioinformatics, Albert-Ludwigs University of Freiburg, 79104 Freiburg, biochemical and biophysical methods was then applied to evaluate the Germany 3 Department of Rheumatology and Clinical Immunology, Charité University Hospital Berlin, derived mapping of the binding pocket, which in the future could 10117 Berlin, Germany facilitate inhibitor development. Biomass combustion is increasingly used as a renewable, CO2 neutral References: alternative energy source. However, biomass combustion significantly 1. Haeggström, J. Z. et al, (1990) Leukotriene A4 hydrolase: an epoxide hydrolase with contributes to indoor air pollution and emissions by biomass generated peptidase activity. Biochem. Biophys. Res. Commun. 173, 431–7. 2. Rao, N. L. et al, (2007) Anti-inflammatory activity of a potent, selective leukotriene A4 PM might be regionally comparable or even higher than traffic related hydrolase inhibitor in comparison with the 5-lipoxygenase inhibitor zileuton. J. Pharmacol. Exp. emissions. Nowadays, it is generally known that anthropogenic PM in Ther. 321, 1154–60. ambient air is a major health hazard [1] resulting in a variety of 3. Wells, J. M. et al, (2014) An aberrant leukotriene A4 hydrolase-proline-glycine-proline diseases, including chronic obstructive pulmonary disease (COPD), pathway in the pathogenesis of chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care acute respiratory infections, fibrosis, and lung cancer [2, 3]. Here, Med. 190, 51–61. particles less than 2.5 μm in diameter (PM2.5) are considered to be 4. Stsiapanava, A. et al, (2014) Binding of Pro-Gly-Pro at the active site of leukotriene A4 most harmful, as they penetrate deeply into the lungs and adversely hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor. Proc. Natl. Acad. Sci. U. S. A. 111, 4227–32. affect bronchioles or alveoli. However, it is still not completely understood how particles emitted during biomass combustion affect human health. Recent reports indicate that the occurrence of these diseases is closely connected to epigenetic aberrations, such as alterations in CpG residue methylation, histone modifications and POS.32 changes in micro(mi)RNA expression [4]. 5-Lipoxygenase-activating protein (FLAP) rescues activity Here, we analyzed the effect of PM2.5 using human epithelial bronchial of 5-lipoxygenase mutations that delay nuclear membrane alveolar cells (BEAS-2B) as a model on the genome-wide methylation association and disrupt product formation of CpG nucleotides by an Illumina Methylation450K BeadChip array and linked the results with the impact on the transcriptome by an Affymetrix

Human Genome U133 Plus 2.0 Array. We filtered 155 genes which Garscha, U.1; Gerstmeier, J.1; Newcomer, M. E.2; Romp, E.1; Werz, O.1 1 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller- were either hyper- or hypomethylated and simultaneously University, 07743, Jena, Germany transcriptionally differently regulated. From these genes, 66 were 2 Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana related to lung diseases, especially to lung cancer. Our results give first insights that epigenetic aberrations induced by chronic exposure to PM Leukotrienes (LT) are pro-inflammatory lipid mediators that are formed may be involved in the development of various lung diseases. from arachidonic acid (AA) via the 5-lipoxygenase (5-LOX) pathway and are pronounced in asthma, allergy and cardiovascular diseases. AA is Acknowledgments: The presented work is part of an interdisciplinary EU-funded research converted by 5-LOX first to 5(S)-hydroperoxyeicosatetraenoic acid (5- project (see http://www.biocombust.eu), supported in part through the Interreg IV Program “Oberrhein” (project C35 BIOCOMBUST). HPETE) and subsequently to LTA4. In cellulo, 5-LOX receives its substrate from the membrane-embedded 5-LOX-activating protein References: (FLAP) for product formation, and inhibition of FLAP or genetic knock- 1. Lelieveld J. et al.: Nature. 2015, 525(7569): 367-71. down blocks LT formation. Beside the absolute necessity of FLAP for 5- 2. Anderson JO., Thundiyil JG., Stolbach A.: J Med Toxicol. 2012, 8(2): 166-75. 3. Raaschou-Nielsen O. et al.: Lancet Oncol. 2013, 14(9): 813-22. LOX activity in the cell, the crystal structure of 5-LOX revealed an active 4. Heerboth S. et al.: Genet Epigenet. 2014, 6: 9-19. site that is concealed by two residues, F177 and Y181 referred to as

DPhG Annual Meeting 2016 Conference Book • 99 POSTERS

POS.34 POS.35 Eculizumab, the only complement inhibitor in the clinic is 6‐Arylamino‐3,4‐dihydroisoquinolin‐1(2H)‐ones as new not always efficient: mechanistic evidence for incomplete pharmacophores for linear hinge binders inducing the inhibition under eculizumab and how the problem can be glycine-flip fixed Praefke, B. A.1; Laufer, S. A.1 Harder, M. J.1; Simmet, T.1; Schmidt, C. Q.1; 1 University of Tuebingen, Faculty of Science, Pharmaceutical and Medicinal Chemistry, Auf der Morgenstelle 8, 72076 Tuebingen, Germany 1 Ulm University, Institute of Pharmacology of Natural Products & Clinical Pharmacology, Ulm, Germany Various diseases can be linked to dysregulated protein kinase activity Eculizumab inhibits the terminal and lytic pathway of complement by [1]. Achieving selectivity with ATP-competitive kinase inhibitors is blocking the activation of the complement protein C5. The terminal difficult, due to highly conserved ATP-binding sites. We previously pathway (TP) of the complement cascade generates the anaphylatoxin developed highly potent and selective carbonyl based linear C5a and the plasma membrane penetrating membrane attack complex hingebinding p38α MAPK inhibitors inducing a so called “glycine-flip”, a and, thus, holds the most inflammatory potential of the whole rotation of Gly110 in the hinge region, resulting in the formation of two complement cascade. Treatment with eculizumab shows remarkable hydrogen bonds from the carbonyl oxygen to the amide-NH of Gly110 clinical benefits in the complement mediated diseases atypical and Met109 [2,3]. As only 46 of the known 518 protein kinases bear a haemolytic uraemic syndrome (aHUS) and paroxysmal nocturnal glycine in the equivalent position, targeting the glycine-flip represents a haemoglobinuria (PNH). However, several reports demonstrate that way to increase kinome selectivity [4]. activation of C5 is not always completely suppressed in patients even in presence of excess amounts of eculizumab. This suggests that residual C5 activity may limit the drug’s therapeutic benefit under certain conditions. Therefore, we challenge the prevailing dogma that C5 inhibitory agents, like the therapeutic antibody eculizumab, completely inhibit the TP and set out to elucidate the underlying molecular mechanism responsible for the observed residual TP activity in presence of C5 inhibitors. We show in several experiments ex vivo that C5 inhibition by eculizumab and other C5 inhibitory agents is generally susceptible to incomplete suppression of the TP. By using biophysical and several cell based Derived from our dibenzosuberone, dibenzoxepinone and studies we demonstrate that it is the surface density of the deposited benzosuberone scaffolds we developed a new hinge binding motif by complement opsonin C3b that directs the level of this residual lytic replacing the ketone by an amide group. We expect an increase in complement activity. For example, we show in a clinical relevant model electron density of the amide oxygen to strengthen the bidentate of autoimmune mediated haemolysis that residual lytic activity under C5 hydrogen bonds to the flipped glycine and the adjacent amino acid. The inhibition occurs on human erythrocytes after forceful complement arylamino moiety was decorated with various substituents in order to activation via alloantibodies directed against blood group antigens. The cover a broad range of electronic and lipophilic properties to explore the level of such residual C5 activity directly correlated with the hydrophobic region I of kinases possessing a glycine in the hinge strength/titer of the complement activating alloantibodies. This indicates region. that the therapeutic benefit of eculizumab is especially impaired in clinical situations that are characterised by a particular forceful References: activation of complement which actually would require a particular 1. G. Manning et al.: Science. 2002, 298(5600), 1912-1934. efficient suppression of the complement system. 2. S. C. Koeberle et al.: J. Med. Chem. 2012, 55(12), 5868-5877. We found two potential solutions how this clinical problem of residual 3. B. Baur et al., J. Med. Chem. 2013, 56(21), 8561-8578. C5 activity, e.g. after ischemia reperfusion injury or during antibody 4. K. E. Martz et al., J. Med. Chem. 2012, 55(17), 7862-7874. mediated transplant rejection, may be alleviated in the future. In ex vivo studies on patient material we demonstrate that inhibition of the TP by simultaneous employment of two different C5 inhibitors can completely block the lytic complement activity even after very forceful complement POS.36 activation. Alternatively, a biotherapeutic fusion molecule derived from novel protein-engineering approaches of several natural complement Chemical Tuning of Anthranilamides Towards Selective inhibitors also proved to be extremely potent in suppressing PPARδ Agonism inflammatory TP activities after powerful initiation of the complement cascade. Heitel, P.1; Proschak, E.1; Schubert-Zsilavecz, M.1; Merk, D.1 1 In conclusion, with our study we advance the mechanistic Goethe University of Frankfurt, Max-von-Laue-Straße 9, 60438 Frankfurt, Germany understanding of complement C5 activation by demonstrating that high cell surface densities of C3b opsonins can override the blocking effects of different C5 inhibitors. We demonstrate that such inflammatory, residual TP activity under C5 inhibition can be circumvented by simultaneously employing two orthogonal C5 inhibitors or by using a novel, engineered fusion protein that efficiently blocks all three complement activation pathways. Thus, our study not only provides the rational explanation for the clinically observed phenomenon of residual TP activity under eculizumab therapy, which has significant implications for anti-C5 therapy in general, but also indicates potential future Peroxisome proliferator-activated receptors (PPARs) are members of therapeutic avenues that allow efficient blockage of complement even the nuclear receptor family that function as ligand-activated transcription after very forceful activation of this innate immune cascade. factors [1]. PPAR activation by endogenous ligands - fatty acids and eicosanoids - leads to the expression of various genes involved in proliferation of liver peroxisomes, metabolic regulation of lipid and glucose homeostasis, as well as inflammation [2-5]. In mammals, three subtypes have been identified which differ in expression and physiological function. Whereas PPARα and PPARγ agonists have been extensively studied because of hypolipidemic and antidiabetic properties, the physiological role of PPARδ (also referred to as PPARβ) remained unknown for a long time. By now, it has been figured out that PPARδ is ubiquitously expressed and plays a pivotal role in fatty acid oxidation in key metabolic tissues such as skeletal

100 • DPhG Annual Meeting 2016 Conference Book INFLAMMATION muscle [6]. Besides, PPARδ activation exhibits anti-inflammatory effects To exploit the concept of dual FXR/sEH modulation for NASH treatment and hence gained interest as therapeutic target. However, in contrast to we developed dual agents with partial FXR agonistic and sEH inhibitory PPARα and PPARγ, no PPARδ ligand has been approved as drug so potency. Initially, we merged known pharmacophores[9,10] for both far. Although first clinical trials with PPARδ agonist GW501516 targets to generate our lead compound that exhibited moderate FXR demonstrated promising results such as decreased plasma triglyceride activation and sEH inhibition at 50 µM in vitro. By systematic levels, elevated HDL levels and enhanced insulin sensitivity in obese exploration of the structure-activity relationship (SAR) of the compound patients [7-8], GW501516 promoted the growth of intestinal adenomas class on both targets, we optimized the potency for partial FXR [9]. activation and sEH inhibition to low nanomolar values and finally used In initial studies, we have already shown that anthranilamides are this knowledge to generate compounds with the desired dual activity promising candidates to overcome the need for selective PPARδ and well-balanced nanomolar potency. The most promising compounds agonists [10]. In the process, compound 1 proved to be selective over were intensively trialed in vitro for FXR target gene induction, selectivity, PPARα and PPARγ, showing a low micromolar EC50 value on PPARδ. metabolic stability and toxicity. The compounds revealed a favorable Starting from computational docking of 1 into the PPARδ ligand binding profile and pilot in vivo data is encouraging. domain (LBD), we investigated the acidic head group, substitution of the In summary, we report the first class of dual FXR agonists/sEH aromatic moieties, as well as introduction of heteroaromatic systems to inhibitors and based on favorable in vitro and in vivo properties, further exploit the interaction between the ligand and the binding pocket. In this exploration of the concept and the compound class is warranted. structure-activity relationship (SAR) study, we chemically optimize the potency of anthranilamide 1 in several cycles to come up with a References: selective, nanomolar PPARδ agonist, which is tested in a PPAR-Gal4 1. Rinella, M.: JAMA 2015, 313(22): 2263–73. transactivation assay for each subtype. Further research including in 2. Gawrieh, S.; Chalasani, N.: Semin. Liver Dis. 2015, 35(3): 338–48. vivo investigations will reveal whether this compound class is suited as 3. Ratziu, V. et al.: Gastroenterology 2016, 150(5): 1147–1159. 4. Neuschwander-Tetri, B. et al.: Lancet 2014, 385(9972): 956–65. novel strategy for treatment of metabolic syndrome. 5. Zhang, Z; Dales, N.; Winther, M.: J. Med. Chem. 2014, 57(12): 5039–56. 6. Liu, Y. et al.: PLoS One 2012, 7(6): e39165. Acknowledgements: Financial support by the Else Kröner-Fresenius-Stiftung, Translational 7. He, J. et al.: J. Diabetes 2016, 8(3): 305–13. Research Innovation - Pharma (TRIP) is gratefully acknowledged. 8. Arab, J. et al.: Hepatology 2016, doi:10.1002/hep.28709. References: 9. Merk, D. et al.: J. Med. Chem. 2014, 57(19): 8035–55. 1. Gronemeyer, H.; Gustafsson, J.-Å.; Laudet, V.: Nat. Rev. Drug Discov. 2004, 3(11): 950–964. 10. Blöcher, R. et al.: J. Med. Chem. 2016, 59(1): 61–81. 2. Forman, B. M.; Chen, J.; Evans, R. M.: Proc. Natl. Acad. Sci. 1997, 94(9): 4312–4317. 3. Xu, H. E. et al.: Mol. Cell 1999, 3(3): 397–403. 4. Keller, H. et al.: Proc. Natl. Acad. Sci. U.S.A. 1993, 90(6): 2160–2164. 5. Kliewer, S. A. et al.: Proc. Natl. Acad. Sci. 1997, 94(9): 4318–4323. 6. Neels, J. G.; Grimaldi, P. A.: Physiol. Rev. 2014, 94(3): 795–858. POS.38 7. Sprecher, D. L. et al.: Arterioscler. Thromb. Vasc. Biol. 2007, 27(2): 359–365. 8. Risérus, U. et al.: Diabetes 2008, 57(2): 332–339. 1-HeteroaryIpropan-2-one inhibitors of cytosolic 9. Gupta, R. A. et al.: Nat. Med. 2004, 10(3): 245–247. phospholipase A2with improved metabolic stability 10. Merk, D. et al.: Bioorg. Med. Chem. 2015, 23(3): 499–514. Althaus, J.; Hake, T.; Subeska, A.; Hanekamp, W.; Fabian, J.; Lehr, M. Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstr. 48, 48149 Münster, Germany

POS.37 Cytosolic phospholipase A2 (cPLA2) catalyzes the first step in the Facing non-alcoholic steatohepatitis with multi-target biosynthesis of pro-inflammatory lipid mediators such as agents prostaglandins, leukotrienes, and platelet activating factor (PAF) by cleaving membrane phospholipids in arachidonic acid and lyso- Schmidt, J.1; Rotter, M.1; Weiser, T.1; Wittmann, S.1; Kaiser, A.1; Weizel, phospholipids. Therefore, inhibition of cPLA2 is considered to be an L.1; Proschak, E.1; Merk, D.1 attractive target for the design of new anti-inflammatory drugs. 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, Recently, we have found that the indole-5-carboxylic acid derivative 1 is 60438 Frankfurt; [email protected] a potent inhibitor of cPLA2 in cell free systems as well as in intact

cells.1 However, the compound only showed a low bioavailability in mice Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic after peroral administration. One reason for this behaviour appears to steatohepatitis (NASH) arising from western diet and lifestyle evolve as be its excessive in vivo metabolism. Primarily, the reactive ketone group serious health burden with alarming incidence.[1] NAFLD and NASH of the propan-2-one scaffold is reduced to an alcohol and the carboxylic are characterized by accumulation of fat in liver subsequently causing acid moiety of the indole ring system is converted into an ester inflammation and fibrosis and are strongly associated with the metabolic glucuronide. In the present study we tried to increase the metabolic syndrome.[1] Although the high prevalence of NAFLD and NASH stability of this kind of compounds, e.g. by introduction of methyl elicited intensive research for novel treatment options there is still no substituents into the α-position of the reactive ketone functionality and satisfying pharmacological therapy.[2] Several molecular targets have by transformation of the aromatic carboxylic acid group to an aliphatic been identified as potentially suitable for NAFLD/NASH treatment. one, respectively.2 The effects of these structural variations on cPLA2α Promising clinical data has been reported for elafibranor[3], an agonist inhibitory potency as well as on phase I and phase II metabolic stability of the peroxisome proliferator-activated receptors (PPAR) α and δ as in rat liver homogenate are described. well as for obeticholic acid[4] which activates the farnesoid X receptor

(FXR). Additionally, the inhibition of a number of enzymes including stearyl-CoA desaturase 1 (SCD1)[5] and soluble epoxide hydrolase (sEH)[6,7] proved effective in treating NASH in vivo. In light of the multifactorial nature of NASH, modulation of more than one target might provide a superior therapeutic effect. Especially, combination of FXR activation that revealed anti-steatotic and anti- fibrotic effects in clinical trials with inhibition of sEH generating anti- inflammatory effects promises synergistic activity. The nuclear receptor References: 1. Ludwig, J. et al.: J. Med. Chem. 2006, 49: 2611-2620. FXR acts as intracellular bile acid sensor and liver protector. Its 2. Schwarzkopf, J. et al.: Med. Chem. Res. 2014, 23: 5250-5262. activation has various beneficial metabolic effects and via induction of small heterodimer partner (SHP) as well as sterol regulatory element binding protein 1c (SREBP1c) reduces liver fat content.[8] sEH is an enzyme of the arachidonic acid cascade located in the CYP pathway and catalyzes the degradation of anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). Therefore, sEH inhibition hinders EET degradation and has anti-inflammatory properties.[7]

DPhG Annual Meeting 2016 Conference Book • 101 POSTERS

POS.39 POS.40 Tuning Selectivity: Novel Covalent-Reversible JAK3- Enhancement of binding interactions between hydrophobic Inhibitors with High Isoform Specifity Targeting a Nitrile region I and deep pocket-p38 MAP Kinase inhibitors with Induced Arginine Pocket outstanding potencies

Forster, M.1; Chaikuad, A.2,4; Bauer, S. M.1; Holstein, J.3; Gehringer, Wentsch, H. K.1; Walter, N. M.1; Mayer-Wrangowski, S. C.2; Rauh, D.2; M.1,5; Pfaffenrot, E.1; Ghoreschi, K.3; Knapp, S.2,4; Laufer, S. A.1 Laufer, S. A.1 1 Department of Medicinal Chemistry, Eberhard-Karls-University Tuebingen, 1 Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Auf der Morgenstelle 8, 72076 Tuebingen, Germany Eberhard Karls Universitaet Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany 2 Nuffield Department of Clinical Medicine, Structural Genomics Consortium and Target 2 Faculty of Chemistry-Chemical and Biology, Technische Universitaet Dortmund, Discovery Institute, University of Oxford, Old Road Campus Research Building, Roosevelt Otto-Hahn-Strasse 6, 44227 Dortmund, Germany Drive, Oxford, OX3 7DQ, United Kingdom 3 Department of Dermatology, University Medical Center, Eberhard-Karls-University Tuebingen, Liebermeisterstr. 25, 72076 Tuebingen, Germany The fundamental role of p38 mitogen-activated protein kinases (MAP 4 Present address: Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University kinases) in the biosynthesis of proinflammatory cytokines like IL-1β and and Buchmann Institute for Molecular Life Sciences, Max-von-Laue-Strasse 9, TNFα underlines their importance as therapeutic targets for the D-60438 Frankfurt am Main, Germany 5 Present address: Department of Chemistry and Applied Biosciences, treatment of (auto)inflammatory diseases [1], cancer [2] and Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zurich, neurodegenerative diseases [3]. Although, a plethora of p38α MAP Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland kinase inhibitors arising from manifold structural classes have been developed over the last decades, no inhibitor has launched to the In the last decade, Janus kinases (JAKs) have evolved to targets of market yet. Thus, there is still an urgent need for promising clinical high interest in the development of anti-inflammatory and oncologic candidates with improved in vivo efficacies and reduced side effects [4]. agents. This family of cytosolic tyrosine kinases, consisting of four For p38 MAP kinase inhibitors the major challenges like outstanding isoforms (JAK1, JAK2, JAK3 and TYK2), has a variety of crucial potency, due to low ATP competitiveness, combined with excellent functions in many important signal pathways. While the other isoforms selectivity have already been solved [5, 6]. are ubiquitously expressed in different tissues, JAK3 is solely expressed in cells of the lymphoid lineage. Therefore, it plays a major role in the development of immunocompetent cells like T-cell or natural killer cells. Because of this isolated role, selective inhibition of JAK3 is supposed to be a promising strategy to achieve immunosuppression with less adverse effects [1]. However, the sufficiency of specific JAK3 inhibition is heavily debated, since it always is co-localized with JAK1 at the transmembranic γc-cytokine receptors [2,3]. To finally resolve this enigma, highly JAK3-selective chemical probes are required.

Fig. 1: Binding mode of compound 1 in p38α MAP kinase (PDB code: 3UVQ)

Our lead compound 1 (figure 1) showed an excellent IC50 value (IC50 = 1 nM) with respect to p38 MAP kinase, but unfortunately only a moderate inhibitory activity in a human whole blood TNFα release assay (IC50 = 280 nM). One reason might be a short target residence time due to the high intracellular ATP concentration and a competitive binding mechanism. Based on the X-ray structure (PDB code: 3UVQ) [6] we synthesized compounds which form the same interactions to the The current gold standard to investigate JAK dependent signalling is enzyme and can moreover enhance the interactions between Tofacitinib. Unless it has an excellent kinome wide selectivity, it suffers hydrophobic region I and the deep pocket of the enzyme. Both are valid from a poor selectivity within the JAK-family, being a potent inhibitor of strategies to overcome the shortcomings mentioned above. all four isoforms [4]. Starting from a tricyclic analogue of Tofacitinib with Furthermore, we pursued the strategy of parallel synthesis of a a simplified cyclohexyl side chain, we were able to develop a new class dibenzooxepinone and a dibenzosuberone scaffold to compare their of highly potent and selective JAK3 inhibitors. By targeting a JAK3 affinity to the enzyme and their activity in whole blood tests as well as to specific cysteine residue near the ATP binding pocket following a avoid potential metabolic disadvantages of the dibenzosuberone covalent-reversible approach, our inhibitors demonstrate both, an scaffold. Finally, we synthesized a variety of dibenzepinones following outstanding isoform specifity (400-, 2700- and 3600-fold JAK3 over the intentional binding mode and were able to improve the inhibitory JAK1, JAK2 and TYK2, respectively) as well as a high kinome wide potency on the isolated enzyme down to the picomolar range and selectivity. We were able to prove the covalent-reversible binding mode achieved IC50 values in whole blood system in the low double-digit of our compounds with protein Xray crystallography and simultaneously nanomolar range. discovered a novel binding pocket. This cavity is unique to our structures and is induced by interactions of the nitrile substituent of the References: inhibitor with arginine residues of the enzyme. The high potency and 1. Player, M. R. Curr Top Med Chem, 2009, 9, 598. selectivity of this inhibitor class are also successfully carried over to 2. Tsai, C. J.; Nussinov, R. Semin Cancer Biol, 2013, 23, 235-42. cellular models resulting in a selective inhibition of JAK3 dependent 3. Anton, R. et al.: PLoS One, 2014, 9, e95641.54. signalling in functional T-cells. Therefore, these compounds are suitable 4. Zhang, J. et al.: T. in Pharm.Science 2007, 28, 286-295. to serve as molecular probes to elucidate the role of selective JAK3 5. Baur, B. et al.: J Med Chem, 2013, 56, 8561-8578. inhibition. 6. Fischer, S. et al.: J Med Chem, 2013, 56, 241-53.

References: 1. Ghoreschi, K., Laurence, A., O’Shea, J.J.: Nat. Immunol. 2009, 4(10): 356-360 2. Haan, C. et al.: Chem. Biol. 2011, 3(18): 314-323 3. Thorarensen, A. et al.: ACS Chem. Biol. 2014, 9(7): 1552–1558 4. Thoma, G., Drückes, P., Zerwes, H.G.: Bioorg. Med. Chem. Lett. 2014, 19(24): 4617-4621

102 • DPhG Annual Meeting 2016 Conference Book INFLAMMATION

POS.41 POS.42 In-situ forming gel devices as local depot therapeutic for Transferrin-Polyethylenimine Nanoparticles for T Cell rheumatoid arthritis targeted siRNA Delivery as Novel Anti-inflammatory Asthma Therapy Mohammadi, M.1; Li, Y.2; Abebe, D.3; Xie, Y.; Kandil, R.; Kraus, T.; Gomez-Lopez, N.; Fujiwara, T.; Merkel, O. M. Kandil, R.1; Xie, Y.2; Kim, N. H.2; Nadithe, V.2; Thakur, A.2,3; Lum, L. 1 Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, G.2,3; Bassett, D. J. P.2; Merkel, O. M.1,2,3 Ludwig-Maximilian University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany 1 Department of Pharmacy, Pharmaceutical Technology and Biopharmacy, Ludwig-Maximilians- 2 Department of Pharmaceutical Sciences, Wayne State University, 259 Mack Ave, Detroit, Universität München, 81377 München MI 48201, USA 2 Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48201 3 Department of Chemistry, The University of Memphis, Memphis, TN 38152, USA 3 Karmanos Cancer Institute, Detroit, MI 48201 4 Department of Obstetrics and Gynecology & Immunology and Microbiology, School of Medicine, Wayne State University, CS Mott Center for Human Growth and Development, 275 E. Hancock, Detroit, Michigan 48201 Asthma is a major public health problem as the disease affects 235 5 Department of Oncology, Molecular Therapeutics Program, Wayne State University, million people worldwide, and in a considerable portion of patients it is 4100 John R St, Detroit, MI 48201, USA still not sufficiently controlled. Therefore, novel efficient anti- For new treatment options in rheumatoid arthritis (RA) folic acid (FA) inflammatory therapies with minimum side effects are urgently needed. coupled three layered micelles (3LM) were developed to encapsulate The disorder is characterized by infiltration of immune cells, including T nucleic acids. Their application as locally implantable, targeted, helper 2 cells (Th2), a type of activated T cells (ATC), in the lung, macrophage-specific RNA interference (RNAi)-based delivery system causing chronic inflammation of the airways. Various underlying could therefore revolutionize RA therapy [1, 2]. cascades are orchestrated via the secretion of Th2 cytokines, such as 3LM were formed from triblock copolymers of PLLA-PEI-PLLA and IL-4, IL-5, and IL-13. [1] All the more, than downregulation of these PLLA-PEG-PLLA in a three-step procedure [3]. Their structure and DNA single cytokines, the therapeutic interference with decisive transcription entrapment in the core were determined by staining DNA with silver factors involved in this disease process, such as GATA-3, is a nitrate and TEM detection [4]. Hydrodynamic diameters and zeta promising approach to early-on undermine pathologic pathways in potentials were measured by dynamic light scattering and laser Doppler asthma. [2] Since RNA interference was shown to induce transient and anemometry. DNA release in neutral and acidic pH was detected by reversible knockdown [3], it offers an auspicious therapeutic base for modified SYBR Gold assays [3]. Fluorescein labeled folic acid was used the silencing of clinically relevant genes. However, the lack of efficient for flow cytometric detection of the expression of functional folic biocompatible siRNA carrier systems to overcome extra- and receptor β in LPS-activated and resting macrophages. For targeting of intracellular barriers still impedes the clinical translation. activated macrophages, folic acid (FA) was attached to the PEG-chain To achieve the particularly challenging transfection of T lymphocytes, of a PLLA-PEG diblock affording PLLA-PEG-FA. 3LM were formed with we designed a novel nano-sized ATC targeted delivery system 75%PLLA-PEG-FA and 25%FA-PEG-PLLA in the outer polymer shell composed of Transferrin-N-Succinimidyl-3-(2-Pyridyldithio)-Propionate- for optimal targeting conditions. After RAW264.7 and primary Polyethylenimine (Tf-SPDP-PEI). By utilizing the physiological iron macrophages were activated with LPS [5] or left resting they were transport molecule Transferrin as a ligand, we both achieve cell treated with targeted and non-targeted 3LM loaded with fluorescently internalization and active targeting, as its endocytosis-mediating labeled DNA to optimize the uptake rate or GFP-Plasmid for receptor is overexpressed by activated but not by naïve T cells. The investigating transgene expression, quantified by flow cytometry. incorporated disulfide bond is stable extracellularly, but can easily be Thermoresponsive and injectable hydrogels as depot formulation were cleaved in the endosome, releasing the particles inside of the cells. formed by stereocomplexing 3LM which contain PLLA-PEG-PLLA in the Following the successful synthesis of the Tf-SPDP-PEI conjugate, size outer core with PDLA-PEG-PDLA [6]. To examine the stability and DNA and zeta potential of conjugate-siRNA polyplexes were measured by release of the hydrogels under physiologic and inflamed conditions, dynamic light scattering (DLS) and condensation efficiency was hydrogels were exposed to different conditions. determined by SYBR Gold assay. Particle uptake and siRNA delivery The core-corona structure and efficient DNA entrapment in the core were examined using flow cytometry and knockdown of GAPDH, a were confirmed by TEM. The sizes were found to be less than 200 nm, universal housekeeping gene, was quantified by real-time PCR. and the encapsulation efficiency of DNA was optimized based on the Prepared siRNA polyplexes featured favorable sizes of less than 200 ratio of the PEI block in PLLA-PEI-PLLA per DNA [3]. 3LM were stable nm, slightly negative zeta potentials and comparable siRNA at neutral pH but released DNA in an acidic environment [3]. FR- condensation rates with particles composed of non-modified PEI. The overexpressing activated cells, as successfully identified by designed conjugate was proven to successfully deliver siRNA to both internalization of FA-fluorescein, showed significantly higher uptake of human primary ATCs and murine T cells in a murine asthma model targeted 3LM and GFP expression in vitro and ex vivo than resting without causing considerable adverse effects in the latter. Furthermore, macrophages. Stereocomplexes of 3LM form hydrogels above their significant reduction of GAPDH expression was demonstrated in vitro phase transition temperature. In the physiologic environment almost no after delivery of respective siRNA in conjugate nanoparticles to human DNA was released and in an acidic environment most DNA was ATCs compared with PEI alone. encapsulated in 3LM while the mass of the gel degraded. In conclusion, our biocompatible targeted delivery system holds great Our findings confirm that FA-3LM are taken up by activated promise to be an innovative therapeutic option to improve asthma macrophages via folate receptor mediated endocytosis and thus could control in the future. become a promising delivery system for receptor-mediated drug or Acknowledgements: ERC Starting Grant (StG-2014-LS7-637830), NIH Boost Grant and Wayne gene delivery and novel therapy of rheumatoid arthritis in an in situ State Start-Up Grant to Olivia Merkel forming gel formulation. References: 1. Ray, A. and L. Cohn: J. Clin. Inves. 1999, 104(8): 985-93. References: 2. Sel, S. et al.: J. Allergy. Clin. Immunol. 2008, 121(4): 910-916 e5. 1. WHO, Chronic diseases and health promotion in: Chronic rheumatic conditions, 2015 , 3. Kole, R. et al.: Nat. Rev. Drug. Discov. 2012, 11(2): 125-40. Geneva. 2. Gordon S.,Taylor P.R: Nat Rev Immunol, 2005 5: 953-964. 3. Abebe D.G. et al.: Macromolecular Bioscience, 2015 1828 - 1836. 4. Zheng M. et al.: ACS Nano, 2012 6: 9447-9454. 5. Funk J.L. et al.: Atherosclerosis, 1993 98: 67-82. 6. Abebe D.G., Fujiwara T.: Biomacromolecules, 2012 13: 1828-1836. POS.43/SL.39 Influence of Th2 Cytokines on the Cornified Envelope, Tight Junction Proteins and ß-Defensins in Filaggrin- Deficient Skin Equivalents

Hönzke, S.1; Schäfer-Korting, M.1; Hedtrich, S.1 1 Institute of Pharmacy, Pharmacology & Toxicology, Königin-Luise-Straße 2+4, 14195 Berlin, Germany For abstract see Short Lecture SL.39

DPhG Annual Meeting 2016 Conference Book • 103 POSTERS

POS.44 A new class of selective and potent inhibitors of human delta 24-dehydrocholesterol reductase

Müller, C.1; Hemmers, S.1; Schreiber, F.1; Körner, A.2; Mirakaj, V.2; Giera, M.3; Bracher, F.1 1 Department für Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 München, Germany 2 Universitätsklinikum Tübingen, Eberhards Karls Universität Tübingen, Waldhörnlestraße 22, 72072 Tübingen, Germany 3 Leiden University Medical Center, Center for Proteomics and Metabolomics, Albinusdreef 2, 2300 RC Leiden, The Netherlands

In the 1960’s the pharma company William S. Merrell Co marketed triparanol (Mer-29) as a hypolipidemic drug inhibiting the enzyme Δ24-dehydrocholesterol reductase (24-DHCR). Inhibition of this enzyme leads to reduction of cholesterol levels and accumulation of desmosterol, the substrate of 24-DHCR ultimately leading to cholesterol. However, harmful side effects led to a withdrawal of the drug authorisation for triparanol [1]. Besides this early setback in the use of 24-DHCR inhibitors, several promising applications for such enzyme inhibitors have recently been claimed [2,3]. Spann et al. [2] observed in a murine atherosclerosis model that foam cells, evolving from transforming macrophages, surprisingly presented an intrinsically anti- instead of pro-inflammatory phenotype. The phenotype was attributed to the intra-cellular accumulation of desmosterol. Other studies showed that 24-DHCR inhibitors could play a crucial role in HCV infection [3]. These findings led us to focus our research on the development of potent and selective 24-DHCR inhibitors [4,5]. We used a whole cell assay in combination with targeted GC-MS based sterol pattern analysis for the characterization of target enzyme and selectivity system for the analysis of the obtained sterol pattern under inhibitor treatment. Furthermore, IC50 values reflecting inhibition of total cholesterol biosynthesis were established. Here we present lathosterol side chain esters as novel non-toxic, selective and potent 24-DHCR inhibitors with IC50 values in the sub- nanomolar range. Furthermore, we demonstrate that the well-known and established 24-DHCR inhibitors are not selective, and significantly less potent. The newly developed inhibitors are highly useful tools for further studies in the field of 24-DHCR inhibition.

References: 1. Rozman, D., Monstory, K.: Pharmacol. Ther. 2010, 127(1):19-40. 2. Spann, N.J. et al.: Cell 2012, 151(1):138-152. 3. Takano, T. et al.: J. Hepatol. 2011, 55(3):512-521. 4. Giera, M., Plössl, F., Bracher, F.: Steroids 2007, 73(8):633-642. 5. Müller, C. et al.: Steroids 2013, 78(5):483-493.

104 • DPhG Annual Meeting 2016 Conference Book CANCER/INFLAMMATION

The endo-ß-D-glucuronidase heparanase cleaves heparan sulfate (HS) 3.3 Cancer/Inflammation chains which are part of so called heparan sulfate proteoglycans (HSPG). HSPGs consist of a protein core with attached linear HS- chains and comprise versatile functions regarding the integration of the POS.46/SL.40 individual cell in and communication with its environment. HSPGs are able to bind macromolecules within the extracellular matrix (ECM), e.g. Tumor selectivity of V-ATPase inhibition is based on collagen and fibronectin and have adhesive functions during migration. differential regulation of AMPK In addition, HS-residues form a scaffold for diverse active moieties and growth factors, such as vascular endothelial growth factor (VEGF). [1] von Schwarzenberg, K.1; Menche, D.2; Müller, R.3; Vollmar, A. M.1 HS cleavage by heparanase enzymatic activity releases these factors 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich and brings them to action. Therefore, tumor cells make use of 2 Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Gerhard - Domagk-Str.1, 53121 Bonn, Germany heparanase activity during metastatic progression inducing an ECM- 3 Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection remodelling for transmigration purpose. Consequently, heparanase Research and Department of Pharmaceutical Biotechnology, Saarland University, PO 151150, expression and activity is considered as a bad prognostic factor in Universitätscampus E8 1, 66123 Saarbrücken, Germany cancer. [2] In addition to the active heparanase, the enzymatically inactive For abstract see Short Lecture SL.40 precursor form called “latent heparanase” displays activities

independent of HS-cleavage, but obviously related to HS binding.

These activities include the induction of multiple signalling events,

fostering integrin binding [2] or an increased secretion of VEGF by POS.47 tumor cells [3], which plays a key role in establishing the early New Inhibitors of Golgi alpha-mannosidase II metastatic niche. Aims of the study: The molecular mechanism by which VEGF-induction

is realized by latent heparanase is actually unknown and forms the Irsheid, L.1; Borek, C.1; Guilherme dos Santos, M.1; Weickert, A.2; pivotal question of our current studies. VEGF release in tumors has Seibel, J.2; Engels, B.2; Stauber, R.3; Schirmeister, T.1 1 Germany Institute of Pharmacy and Biochemistry, University of Mainz, Germany been related to thrombin activity and thrombin receptor overexpression 2 Institute of Physical and Theoretical Chemistry, University of Würzburg, Germany [4], however, this pathway has not been associated with heparanase, 3 Molecular and Cellular Oncology, University Medicine Center Mainz, Germany and a “heparanase receptor” is unknown. Results: Western blot analysis revealed that there is a substantial Golgi α- mannosidase II (GMII) plays a crucial role in the N- expression of thrombin receptors in our MV3 human melanoma cell glycosylation pathway. In various tumor cell lines, the distribution of the model system. Therefore we followed the effects of latent heparanase N-linked sugars on the cell surface is modified and correlates with the and the stimulation of thrombin receptors by TRAP-6 (thrombin receptor progression of tumor metastasis(1). GMII therefore is a molecular target activating peptide 6) on VEGF-expression by analyzing the culture for anticancer agents and its inhibition has shown to induce tumor supernatants after defined incubation periods via VEGF-ELISA. Both repression(2). GMII, a member of the family 38 glycoside hydrolases, interventions led to a significant increase in the VEGF level and hence cleaves two mannose units (α-(1,3) and α-(1,6)) of the intermediate join latent HPSE and thrombin receptor activation in giving an GlcNAcMan5(GlcNAc)2.The active site of the enzyme consists of two angiogenic stimulus. aspartate residues and a zinc cation. GMII acts as a retaining Besides this view on functional consequences of latent heparanase, an glycosidase and cleaves the sugars in a two-step-SN2-mechanism innovative biosensor technique allowed us to get insight into the resulting in a covalent glycosyl-enzyme complex. The mechanism intracellular signalling, applied for the first time in the heparanase preserves the configuration of the anomeric C-atom(3,4). Several natural research. The sensor device measures the dynamic mass redistribution product-based or synthetic inhibitors have been investigated. However, in the cytoplasm resulting from a cell (receptor) activation. Thus, a the clinical use of the known potent inhibitor swainsonine is restricted ligation of receptors in the cell membrane and a triggered due to the side effects resulting from inhibition of the closely related rearrangement of mass as a downstream effect can be recorded in a lysosomal α-mannosidases(5). We performed two virtual screenings with real time sensorgram. We applied TRAP-6 in different concentrations a library of about five million purchasable compounds to identify new and obtained a typical, in part complex concentration dependent DMR- chemotypes inhibiting the GMII. The docking studies were carried out response curve. The application of TRAP-6 together with a thrombin on the known active site and on a potential allosteric binding site receptor antagonist led to a decrease in DMR-response and verified the identified by molecular modeling. The most promising candidates were obtained signals to be specifically evoked by thrombin receptor purchased and subjected to enzyme inhibition assays yielding several activation. Interestingly latent heparanase also generated a weakly active compounds. The aim of our work is to derivatize the most concentration dependent DMR-curve and therefore latent heparanase active compounds in order to increase the inhibitory effect and elucidate downstream effects can be monitored and analysed using this method. the structure-activity relationships. Furthermore we synthesize Co-incubation with thrombin receptor antagonist did not influence the derivatives based on a central D-mannose core as promising lead DMR-signal in a consistent manner, and therefore a direct impact of structures for a selective, covalent GMII-inhibition. latent heparanase on thrombin receptors seems up to now not to be obvious. The elucidation of a potential heparanase / receptor link References: leading to a VEGF release remains the matter of further investigations. 1. Fujita, T. et al.: Org. Lett. 2004, 6 (5): 827-830. In light of ongoing clinical trials using heparanase inhibitors in cancer

2. Van den Elsen, Y. M. H.; Kuntz, D. A.; Rose,D. R.: The EMBO Journal 2001, 20 (12): 3008- and considering the established VEGF-blocking strategies in clinics, an 3017. 3. Petersen, L. et al.: J. Am. Chem.Soc. 2010, 132: 8291-8300. interrelation between both appears promising regarding the elucidation 4. Zhong, W. et al.: J. Am. Chem.Soc. 2008, 130: 8975-8983. of involved mechanistic patterns and might reveal new targets for anti- 5. Cheng, T.-J. R. et al: Chem. Asian J. 2013, 8: 2600-2604. angiogenic therapeutic options.

References: 1. Vlodavsky, I. et al.: Nat. Med. 1999, 5(7): 793–802. 2. Gerber U. et al.: Semin. Thromb. Hemost. 2015, 41(2): 244-254. POS.48 3. Zetser, A. et al.: Cancer Res. 2006, 66(3): 1455–1463. 4. Dorsam R. T., Gutkind J. S.: Nat. Rev. Cancer 2007, 7(2): 79-94. Latent Heparanase – Impact on angiogenic mediators within the metastatic niche

Hoß, S. G.1; Grundmann, M.2; Ilan, N.3; Vlodavsky, I.3; Bendas, G.1 1 University of Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry II, An der Immenburg 4, 53121 Bonn, Germany 2 University of Bonn, Institute for Pharmaceutical Biology, Section for Molecular-, Cellular- and Pharmacobiology, Nussallee 6, 53115 Bonn, Germany 3 Technion-Israel Institute of Technology, Vascular and Cancer Biology Research Center Rappaport Faculty of Medicine and Research Institute, P.O. Box 9649, 31096 Haifa, Israel

DPhG Annual Meeting 2016 Conference Book • 105 POSTERS

POS.49 POS.50 Fucoidans inhibit inflammation and tumor metastasis Integrin activation leads to increased chemoresistance supporting processes mediated by IL-8 or C5a against cisplatin, doxorubicin and mitoxantrone in breast cancer cell lines MCF-7 and MDA-MB-231 Liewert, I.1; Ehrig, K.1; Alban, S.1 1 Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstraße 76, Baltes, F.; Piva, M. B. R.; Schlesinger, M.; Bendas, G. 24118 Kiel, Germany University of Bonn, Pharmaceutical Institute, An der Immenburg 4, 53121 Bonn, Germany

Heparins are known to exhibit anti-inflammatory and antimetastatic Background: The development of resistance against activities, however their application in inflammatory diseases and chemotherapeutics is the major obstacle in the clinical treatment of cancer is limited due their immanent bleeding risk. Fucose-containing tumor diseases. The molecular mechanisms of chemoresistance are sulfated polysaccharides (syn. “fucoidans”) isolated from brown algae, versatile and in many cases of multifactorial origin. The contact which are currently considered promising candidates for health- formation of tumor cells with extracellular matrices has been shown to supporting and medicinal applications [1], showed anti-inflammatory contribute to a lower sensitivity for chemotherapy. Thus, this interaction and antimetastatic activity in vivo as well, but considerably reduced between cells and their microenvironment has been indicated as anticoagulant effects [2,3]. However, there is only limited knowledge “environment mediated drug resistance” (EM-DR) that can further be about their mode of actions and structure-dependent effects. Attractive divided into “cell adhesion mediated drug resistance” (CAM-DR) and targets for both anti-inflammatory and antitumor agents are the “soluble factor mediated drug resistance” (SFM-DR) depending on the anaphylatoxin C5a and interleukin 8 (IL-8) which are both closely linked molecules involved [1]. to inflammatory processes and tumor progression [4,5,6,7,8]. Integrins are heterodimeric transmembrane receptors that take an The aim of the current study was to investigate the affinity of two important role in communication between cells and their structurally distinct fucoidans from Saccharina latissima (S.l.-SP) and microenvironment. Consequently, their contribution to CAM-DR is highly Fucus vesiculosus (F.v.-SP) [9,10], as well as heparin (UFH) as probable but has not explicitly been shown for breast cancer cells. reference, to IL-8 and C5a and their effects on downstream processes Aim/objectives: The aim of this study was to investigate and quantify initiated in polymorphonuclear neutrophils (PMN) as cell type critically the effect of EM-DR in breast cancer cell lines MCF-7 and MDA-MB- involved in many inflammatory diseases. 231 depending on different integrin activation levels. In a competitive SPC-ELISA [11], both fucoidans, but not UFH, Methods: Sensitivity against cytostatic drug was measured by MTT displayed high affinity to IL-8 as well as C5a and S.l.-SP bound about 4 assay. Cells were seeded in triplicates, inoculated with either cisplatin, times stronger than F.v.-SP to both stimuli. doxorubicin or mitoxantrone and incubated for 72 h. The resulting dose- The impact of this binding on the PMN activation was examined by effect curves were used to get EC50 as a parameter for cell sensitivity. means of two intracellular signalling processes, i.e. phosphorylation of The assay was performed at least threefold for each treatment. the protein kinases Erk1 and Erk2 and intracellular calcium release. By treating the cells additionally with integrin influencing agents, the According to Western Blot analyses, both fucoidans inhibited the Erk receptors’ effect was measured. phosphorylation, whereby the inhibition was more pronounced in IL-8- Integrins were activated by cell ligation on extracellular matrices such than in C5a-stimulated PMN. Further, both fucoidans concentration- as collagen and fibronectin. Furthermore, manganese(II)-cations were dependently reduced the strong intracellular calcium release induced by added to allosterically activate integrins by binding to their MIDAS IL-8 as well as C5a, whereas UFH showed only moderate effects. structure [2]. Contrary to the binding assay, F.v.-SP was significantly more active In order to inhibit integrins we used low molecular weight heparins (e.g. than S.l.-SP. tinzaparin [3]) or a monoclonal integrin antibody (natalizumab). To As typical reaction of activated PMN, the IL-8- and C5a-induced PMN interfere with the intracellular integrin signaling pathway, FAK inhibitor chemotaxis was assessed using modified Boyden chambers. Similar to 1,2,4,5-benzenetetramine tetrahydrochloride or PI3K/mTOR inhibitor the Erk phosphorylation, chemotaxis to IL-8 was effectively inhibited by BEZ235 was applied. Changes in the expression of extracellular and both fucoidans and up to 30 % by UFH, whereas migration towards C5a intracellular molecules were investigated by Western Blot and FACS. was only reduced to about 50 % by both fucoidans and not at all by Results: The integrin activation by ECM ligation declines the sensitivity heparin. Strikingly, S.l.-SP and F.v.-SP displayed distinct shapes of the against the cytostatic drugs cisplatin, doxorubicin or mitoxantrone in concentration-dependent curves for inhibition of chemotaxis towards IL- both MCF-7 and MDA-MB-231 cells. Apart from binding to extracellular 8 with more pronounced inhibition by S.l.-SP at <10 µg/ml and a matrices, the allosteric activation by manganese(II)-cations displayed stronger effect of F.v.-SP at higher concentrations. the highest resistance increasing effect. Since both, blockade of integrin In conclusion, both examined fucoidans showed to interfere with IL-8- binding and interference with integrin downstream signaling enhance and C5a-induced stimulation of PMN by binding to these activators. sensitivity, a clear functional involvement of cellular integrins in They proved to be considerably more active than heparin, however, resistance formation can be suggested. However, differences in the their activity profiles differed. This suggests that further mechanisms cellular response to the different cytostatics and their relation to may be involved in their inhibitory effects on PMN. integrins became evident suggesting more complex mechanisms than a simple integrin-resistance way. Acknowledgements: We thank Cornelia Rodde for her excellent technical assistance. Conclusions: The data suggest that there is a molecular resistance References: mechanism based on integrin activation in breast cancer cell lines MCF- 1. Ruocco, N. et al.: Molecules 2016, 21(5): 2. Fitton, J., Stringer, D., Karpiniec, S.: Mar Drugs 2015, 13(9): 5920–5946. 7 and MDA-MB-231 that exceeds simple physical adhesion processes. 3. Kwak, J.-Y.: Mar Drugs 2014, 12(2): 851–870. This sheds a new light on integrins as potential targets to overcome 4. Manthey, H. et al.: The International Journal of Biochemistry & Cell Biology 2009, 41(11): tumor cells’ resistance to cytostatic treatments. 2114–2117. 5. Guo, R.-F., Ward, P.: Annu Rev Immunol 2005, 23: 821–852. References: 6. Darling, V. et al.: Expert Rev Clin Immunol 2015, 11(2): 255–263. 1. Meads, M. B.; Gatenby, R. A.; Dalton, W. S.: Nat Rev Cancer. 2009, 9(9): 665–74. 7. Harada, A. et al.: J Leukoc Biol 1994, 56(5): 559–564. 2. Chen, J.; Salas, A.; Springer, T.A.: Nat Struct Biol. 2003, 10(12): 995–1001. 8. Campbell, L., Maxwell, P., Waugh, D.: Pharmaceuticals (Basel) 2013, 6(8): 929–959. 3. Schlesinger, M. et al.: Thromb Haemost. 2009, 102(5): 816–22. 9. Ehrig, K., Alban, S.: Mar Drugs 2014, 13(1): 76–101. 10. Schneider, T. et al.: Glycobiology 2015, 25(8): 812–824. 11. Alban, S., Gastpar, R.: Journal of Biomolecular Screening 2001, 6(6): 393–400.

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2 Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf, POS.51 Universitätsstr. 1, D-40225 Düsseldorf 3 Department für Chemie, Institut für Biochemie, Universität zu Köln, Zülpicher Straße 47, The anti-metastatic properties of the tubulin-binding agent D-50674 Köln pretubulysin could be based on the trapping of tumor cells to the endothelium The inhibition of critical protein-protein interactions (PPIs) has become increasingly important in drug discovery. Although, targeting PPIs is Schwenk, R.1; Stehning, T.1; Bischoff, I.1; Ullrich, A.2; Kazmaier, U.2; considered as difficult, more and more success stories demonstrate the Fürst, R.1 feasibility of this approach. The lack of assays to investigate the 1 Institute of Pharmaceutical Biology, Biocenter, Goethe-University, 60438 Frankfurt, Germany inhibition of PPIs leads to the necessity of new methods to overcome 2 Institute of Organic Chemistry, Saarland University, 66123 Saarbrücken, Germany this hurdle. A novel method based on the Autodisplay technology facilitates to Tubulin-binding agents are the most widely used anti-cancer drugs. Due screen for small molecules that inhibit PPIs in vitro. As PPIs can be to the side effects and the development of resistances, the discovery of mainly divided into two classes, heteromeric and homomeric interaction, new agents is still of importance [1]. Recently, pretubulysin (PT), a two different targets were chosen to develop a flow cytometric naturally occurring precursor of the myxobacterial compound tubulysin, screening assay based on the Autodisplay technology: the was identified as a novel tubulin-binding compound [2]. Within the DFG heterotetrameric kinase CK2 [1] and the homodimeric chaperone Hsp90 research group FOR 1406, PT was characterized as an anti-tumoral, [2]. anti-angiogenic and vascular-disrupting compound [3,4,5]. Moreover, As proof of principle, Autodisplay and subsequent flow cytometric PT was also found to inhibit the formation of metastases in vivo [6]. Aim analysis were used to demonstrate the inhibition of CK2/CK2 of the present study was to gain insights into the mechanisms interaction. In this case, purified CK2 was labelled with a fluorophore underlying this anti-metastatic effect by investigating the influence of PT and binding to CK2 displayed on the surface of E. coli cells was on the interaction of endothelial and tumor cells in vitro. examined via flow cytometry. High cellular fluorescence intensity Treatment of primary human endothelial cells (HUVECs) with PT strongly increased the adhesion of breast cancer cells (MDA-MB-231) indicated binding of CK2 to CK2. Addition of a cyclic peptide derived onto HUVECs, but limited their transmigration through the endothelium from the C-terminal CK2 segment, known to inhibit this interaction, led (Transwell assay). Based on this data, the gene expression of to lowered fluorescence intensity, indicating inhibition of the presumably involved adhesion molecules was determined by qRT-PCR: CK2CK2 interaction [3]. ICAM-1, VCAM-1, E-selectin, N-cadherin, and galectin-3. Moreover, the For inhibition of a homomeric interaction, the homodimer Hsp90 was chemokine system CXCL-12/CXCR4 was analyzed. We found that the displayed on the surface of E. coli. As only dimerized Hsp90 is able to mRNA level of endothelial N-cadherin was upregulated by PT. While bind client proteins such as p53, the next step was to proof dimerization the total protein expression of N-cadherin was enhanced in PT-treated of Hsp90 on the surface of E. coli. For this purpose, purified and HUVECs, its surface expression was only marginally influenced by PT fluorophore labelled p53 was added to cells displaying Hsp90 followed (Western blot, flow cytometry). In line with this, blocking endothelial N- by flow cytometric analysis. The addition of labelled p53 to these cells cadherin by a neutralizing antibody revealed that this protein is not led to an increased cellular fluorescence intensity, indicating binding of involved in PT-evoked tumor cell adhesion. Interestingly, PT strongly labelled p53 to surface displayed and dimerized Hsp90. In augmented the mRNA expression of CXCL12 in HUVECs (qRT-PCR), consequence inhibition of dimerization should lead to reduced cellular whereas its protein expression and endothelial secretion was only fluorescence intensity. Addition of peptides derived from the C-terminal slightly enhanced by PT (Western blot, ELISA). However, the secretion dimerization interface of Hsp90 was used to inhibit the homomeric of CXCL12 (and cytokine secretion in general) is of no importance for interaction. Flow cytometric analysis revealed blocking of the the PT-evoked tumor cell adhesion, since there was no difference in dimerization of Hsp90 with these peptides with IC50 values in the low tumor cell adhesion when culture medium was changed or not before micromolar range [4]. With this new method we were able to screen and tumor cells were added. Moreover, an autocrine action of CXCL12 identify a new class of peptidomimetic compounds that inhibit could be excluded, since inhibition of the CXCL12 receptor CXCR4 on dimerization of Hsp90 with IC50 values in the low µM range. endothelial cells with plerixafor did not influence cancer cell adhesion. The novel screening method based on the Autodisplay technology By microscopic analyses, we found that PT treatment causes transient facilitates screening of inhibitors for both, heteromeric and homomeric gaps in the HUVEC monolayer, where tumor cells prefer to adhere. interactions, as shown for CK2 and Hsp90. It is simple to adapt for the Since β1-integrins on the tumor cells could mediate interactions screening of PPI inhibitors for other targets, making this technique a between cancer cells and extracellular matrix proteins in the gaps (e.g. valuable addition to the still limited arsenal of ex vivo assays to collagen), their influence in cell adhesion and transmigration assays investigate the inhibition of PPIs. was examined. Both the PT-evoked increase in cell adhesion and the decrease in transmigration was completely abolished when 1-integrins References: β 1. Niefind, K. et al.: EMBO J. 2001 , 20, 5320-31. were blocked on MDAs by a neutralizing antibody. 2. Wegele, H. et al.: Rev. Phyiol. Biochem. Pharmacol. 2004, 151, 1-44. These results indicate that the anti-metastatic action of pretubulysin 3. Raaf, J. et al.: (2013) ACS Chem. Biol. 2013, 8, 901-907. might be based on the trapping of tumor cells on the endothelium. 4. Bopp, B.; Ciglia, E. et al.: BBA – Gen. Subjects 2016, 1860(6): 1043–1055. Whether this effect is also relevant in vivo, will be analyzed in future studies using intravital microscopy.

Acknowledgments: This work was supported by the German Research Foundation (DFG, FOR 1406, FU 691/9-2). POS.53 References: Identification of clinically relevant patient factors on the 1. Dumontet, C.; Jordan, M. A.: Nat. Rev. Drug Disc., 2010, 9(10): 790-803 pharmacokinetics of tamoxifen based on study data of two 2. Ullrich, A. et al.: Angew. Chem. Int. Ed. Engl., 2009, 48(24): 4422-4425 3. Herrmann, J. et al.: PLoS ONE, 2012, 7(5): e37416 clinical trials: Exploratory data analysis 4. Rath, S. et al.: Br. J. Pharmacol., 2012, 167(5): 1048-1061 5. Kretzschmann, V. K. et al.: Arterioscler. Thromb. Vasc. Biol., 2014, 34(2): 294-303 Richter, T.1; Klopp-Schulze, L.1; Lintermans, A.2; Van Asten, K.2; 6. Braig, S. et al.: Cell Death Dis., 2014, 5: e1001 Jongen, L.2; Blomme, C.3; Poppe, A.3; Neven, P.2,3; Joerger, M.4; Kloft, C.1 1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universität Berlin, Germany, 2 KU Leuven - University of Leuven, University Hospitals Leuven, Department of Oncology, Leuven, Belgium POS.52 3 KU Leuven - University of Leuven, University Hospitals Leuven, Department of Gynecology A novel screening method to identify inhibitors of PPIs and obstetrics, Leuven, Belgium 4 Dept. of Medical Oncology & Hematology, Cantonal Hospital St. Gallen, Switzerland based on the Autodisplay technology Background and objective: Breast cancer is one of the 10 most 1 2 2 3 3 Bopp, B. ; Ciglia, E. ; Hansen, F. K. ; Neundorf, I. ; Hochscherf, J. ; frequent causes of death in Germany with 22 deaths per 100 000 2 3 2 1 Kurz, T. ; Niefind, K. ; Gohlke, H. ; Jose, J. inhabitants (2.1% of all inhabitants) in 2014. Tamoxifen (TAM), a 1 Westfälische Wilhelms-Universität Münster, PharmaCampus, Institut für Pharmazeutische und Medizinische Chemie, Corrensstraße 48, D-48149 Münster selective estrogen-receptor modulator (SERM), is used for adjuvant, neoadjuvant and metastatic setting with a dose of 20 mg daily. A high

DPhG Annual Meeting 2016 Conference Book • 107 POSTERS inter-individual variability in the pharmacokinetics (PK) of TAM and its hybrid ligand 1c decreased proliferation and migration. In mice, the metabolites has been observed1. Potent metabolites of TAM with a high hybrid ligand 1d was uterine protective whereas tamoxifen alone or receptor affinity are (Z)-endoxifen (ENDX) and (Z)-4-hydroxytamoxifen melatonin and tamoxifen (unlinked) stimulated uterine tissue similar to (100-fold higher than TAM), predominantly metabolised by CYP2D6.2 the effects of 17-estradiol alone. Moreover, 1d was shown to bind with The current investigation aims to identify influential patient factors which equal affinity to estrogen receptors and melatonin MT1 receptors similar could explain the high inter-individual variability found in the PK of TAM to tamoxifen or melatonin alone; however, at concentrations greater and its metabolites and guide towards TAM dose individualisation. than 1M, 1d increased melatonin receptor and estrogen receptor Material and Methods: The two multi-centre studies included 391 binding sites suggesting a dual but unique mechanism of action. Here, adult, female patients, treated with 20 mg once daily. In both studies we describe a new synthetic approach toward compounds 1a-e and in only patients with postmenopausal status and estrogen receptor- vitro anti-cancer action of the hybrid ligand 1c with respect to cancer positive (ER+) cancer were eligible. The primary objective of CYPTAM 2 cell proliferation, viability, and invasion. The findings provide a new (NCT00965939, npatients = 247, treatment: neoadjuvant or metastatic) promising perspective for the treatment of breast cancer. was to assess the relation between ENDX plasma concentrations and CH radiological treatment response to tamoxifen based on the RECIST O 3 n criteria 1.0 3, whereas the primary objective of CYPTAM 3 N N (CH ) MeO H 2 n (NCT00966043, npatients = 144, treatment: adjuvant) was to assess a 1a 2 change in endometrial thickness. In both studies, SNP genotyping of O 1b 3 N 1c 4 H various cytochrome P450 enzymes involved in TAM metabolism, such 1d 5 as CYP2D6, was performed. Besides that, they differed in the number 1e 6 H C of blood samples taken for PK analysis (129 samples in CYPTAM 3 and 3 441 samples in CYPTAM 2). An exploratory statistical and graphical data analysis was conducted in R (Version 3.2.4): To assess similarities Acknowledgments: Würzburg University, Prof. Dr. Ulrike Holzgrabe and differences in the two studies, continuous and categorical References: covariates, such as age, BMI and genotype, were statistically 1. Witt-Enderby, P.A. et al. US Patent 8785201. 2014 compared. For this purpose, study populations were stratified based on categorical covariates (e.g. treatment setting) into subgroups to identify potential patterns. Results: The patient characteristics between the subgroups of the studies, defined as “setting: adjuvant”, “setting: neoadjuvant” and POS.55 “setting: metastasised”, differed considerably. Approximately 75% of the Photoactivation of Pt(IV)-anticancer complexes coupled to patients in CYPTAM 2 suffered from a metastasised adenocarcinoma upconverting nanoparticles with a median age of 68 years (range: 50 – 88 years) and a median BMI 2 2 of 26.8 kg/m (range:16.6 – 50.7 kg/m ); the other 25% of the patients Perfahl, S.1; Natile, M.2; Natile, G.3; Bednarski, P. J.1 of CYPTAM 2 study were treated in a neoadjuvant setting with a 1 Institute of Pharmacy, University of Greifswald, 17487 Greifswald, Germany median age of 82 years (range: 49 – 96 years) and median BMI of 2 CNR- IENI, Department of Chemical Sciences, University of Padua, 35131 Padua, Italy 3 26.3 kg/m2 (range: 18.4 – 42.4 kg/m2). Patients of the CYPTAM 3 study, Department of Chemistry, University of Bari, 70125 Bari, Italy with a median age of 60 years (range: 41 – 85 years) and median BMI Photoactivated chemotherapy (PACH) has been attracting attention as of 26.1 kg/m2 (range: 18.4 – 39.2 kg/m2) were treated in an adjuvant a potential novel therapy of cancer. In particular, Pt(IV) complexes have setting. been utilized in this approach.[1,2] However, the need for relatively High inter-patient variability was observed for ENDX concentrations: In short wavelength light for activation of transition metal complexes to the CYPTAM 3 study the median concentration was 11.8 ng/mL (range: cytotoxic species limits wider applications because only superficial 0.77 - 31.8 ng/mL) after 3 - 6 months of treatment; in the CYPTAM 2 tumors can be treated due to the short penetration distances of light into study the median concentrations were similar with 12.4 ng/mL (range: tissues. By converting two or more longer wavelength photons into one 0.70 – 35.1 ng/mL) and 11.5 ng/mL (range: 2.20 – 30.5 ng/mL) in the photon of shorter wavelength, upconverting nanoparticles (UCNP) metastasised and neoadjuvant setting after 2 months of treatment, promise to help overcome this problem. Thus, light of longer respectively. wavelength, such as near infrared (NIR) radiation, could penetrate Conclusions: deeper into tumor tissues, become upconverted by nanoparticles to Overall, a high inter-individual variability in TAM ( 16-fold) and ENDX shorter wavelength, and selectively activate a nearby metal complex to concentrations ( 50-fold) was observed across the two studies. a reactive anticancer drug. In this poster, we present two strategies to Interestingly, ENDX concentrations showed similar distributions develop photoactivatable diiodido-Pt(IV) diamines coupled in various (median, range) in the different treatment settings. The results of this ways to UCNPs (Figure). Diiodido-Pt(IV) diamines were the first Pt exploratory data analysis will be used to assist a subsequent covariate complexes described that can be photoactivated to cytotoxic species[3], analysis utilising a population modelling approach. The PK model thus making them interesting candidates for UCNP assisted PACH. Our considering the clinically relevant covariates shall support individual results show that NIR is able to facilitate the release of reactive Pt treatment decisions. species from both forms of the modified UCNPs, which platinate calf

thymus DNA. Moreover, NIR potentiates the cytotoxic activity of References: 1. Schroth, W. et al.: J. Clin. Oncol., 2007, 25(33): 5187-5193. UCNPs in cell culture.[4] 2. Mürdter, T. E. et al.: Clin. Pharm. Ther., 2011, 89(5): 708-717. 3. Eisenhauer, E. A. et al.: Eur. J. Cancer., 2009, 45(2): 228-247.

POS.54 Melatonin-Tamoxifen Hybrid Ligand as Potent Agent against Breast Cancer

Zlotos, D. P.1; Marzouk, M. A.1; Hasan, M.2; Darveau, T.2; Guarinini, L.2; Browne, E.2; Witt-Enderby, P. A.2 1 The German University in Cairo, Dept. of Pharmaceutical Chemistry, New Cairo City, 11835 Cairo, Egypt 3 Duquesne University, Division of Pharmaceutical Sciences, School of Pharmacy, 421 Mellon Hall, Pittsburgh, PA, 15282, USA

Melatonin-tamoxifen hybrid ligands 1a-e have been recently reported to be promising new agents in the prevention and treatment of breast cancer.1 In particular, in vitro studies using MCF-7 (ER+/PR+), MDA- MB-231 (triple negative) and MMCs (HER2+/ER-/PR-) cancer cells the

108 • DPhG Annual Meeting 2016 Conference Book CANCER/INFLAMMATION

Acknowledgements:This work was supported by European Research Council (BioIncmed Histone deacetylase inhibitors (HDACis) belong to an emerging class of 247450) and the Italian Ministero dell’Università (PON01_01078 and FIRB RINAME RBAP114AMK). anticancer compounds which cause growth arrest and apoptosis of several tumor cells.[1] It is increasingly recognized that the combination References: 1. Bednarski, P. J.; Mackay, F. S.; Sadler, P. J.: Anti-Cancer Agents Med.Chem. 2007, 7: 75-93. of HDACis with established anticancer drugs (e.g. cisplatin) provides 2. Farrer, N. J.; Salassa, L.; Sadler P. J.: Dalton Trans 2009: 10690-10701. synergistic effects in the treatment of hematological and solid tumors, 3. Kratochwil, N. A. et al.:J. Med. Chem. 1996, 39: 2499-2507. probably generated through HDACi-mediated increased accessibility of 4. Perfahl, S. et al.: Mol. Pharmaceut. (in press). DNA.[2,3] Starting from LMK235, a HDACi with HDAC4 and 5 preference[4], we reasoned that the enlargement of the cap group and the connecting unit should lead to a novel type of HDACi with HDAC6 preference (Figure 1). The lead structure LMK214 (N-Hydroxy-6-((3-(quinolin-3- POS.56 yl)ureido)oxy)hexanamide) of the novel compound library showed Lung Cancer: EGFR Inhibitors with Low Nanomolar Activity potent inhibition of HDAC6 and no inhibition of HDAC4 and 8 up to a against a Therapy-Resistant L858R/T790M/C797S Mutant concentration of 10 µM. Based on these preliminary results we performed a docking study in order to understand the selectivity profile Günther, M.1; Juchum, M.1; Kelter, G.2; Fiebig, H.2; Laufer, S. A.1 of compound LMK214 and to design improved analogues. Here we 1 University of Tuebingen, Faculty of Science, Pharmaceutical and Medicinal Chemistry, describe the synthesis of new potent hydroxamate-based HDAC6 Auf der Morgenstelle 8, 72076 Tuebingen, Germany 2 Cell Biology & Compound Screening Oncotest GmbH, Am Flughafen 12 – 14, 79108 Freiburg, preferential inhibitors with a novel alkoxyurea connecting unit linker Germany region. A microwave-assisted protocol allowed the systematic variation of the cap moiety. The biological evaluation of the target compounds The treatment of non-small-cell lung cancer (NSCLC) with epidermal included cellular HDAC and MTT assays on Cal27 (human tongue growth factor receptor (EGFR) inhibitors is made challenging by squamous carcinoma cell line) and A2780 (human ovarian cancer cell acquired resistance caused by somatic mutations [1]. Third generation line) as well as their cisplatin resistant sublines. Some compounds EGFR inhibitors (WZ4002, Osimertinib) have been designed to showed improved effects on inhibition of cellular HDACs in a whole-cell overcome resistance, mediated by the T790M mutation, through HDAC assay compared to vorinostat. Based on their antiproliferative covalent binding to the Cys797 residue of the enzyme. These inhibitors effects and HDAC inhibition, the three most potent compounds were are effective against most clinically relevant EGFR mutations, however selected for isoform profiling against human HDAC1, HDAC6 and their high dependence on this particular interaction means that HDAC8. The most promising compound demonstrated excellent additional mutation of Cys797 results in poor inhibitory activity, which selectivity over HDAC8 (SI: 550), moderate selectivity over HDAC1 (SI: leads to tumor relapse in initially responding patients [2,3]. 15) and highly improved HDAC6 inhibition of 2.8 nM. Furthermore, drug combination studies with cisplatin revealed for all three selected F compounds a markedly enhancement of cisplatin-induced cytotoxicity and a synergistic antitumor effect with CI < 0.9 in the cell lines Cal27 R N 1 N and Cal27 CisR. These effects were more pronounced for the cisplatin R2 resistant subline Cal27 CisR. N OH N H H N N R HN HN 3

Based on a selectivity screening of a highly potent reversible p38 Fig. 1.: Strategy and target compounds inhibitor [4], we identified EGFR inhibition as an off-target effect of this compound. High potency, as well as moderate physicochemical Acknowledgments: J. Senger and M. Jung thank the Deutsche Forschungsgemeinschaft (DFG) properties and cellular activity against p38, led us to pick this compound for funding. (Ju295/9-2 within SPP1463, Ju295/10-2 within CRU201; Ju295/13-1). as a lead structure for further improvements in terms of mutant EGFR References: inhibition. With this concept, we have successfully developed highly 1. Witt, O. et al.: Cancer Lett. 2009, 277(1): 8–21. potent reversible and irreversible T790M EGFR inhibitors that showed 2. Ong, P.-S. et al.: Int. J. Oncol. 2012, 40(5): 1705–1713. 3. Stiborova, M. et al.: Curr Med Chem. 2012, 19(25): 4218–38. picomolar IC50-values in an enzyme assay and down to 18 nM GI50 in a 4. Marek, L. et al.: J. Med. Chem. 2013, 56(2): 427–436. double mutant (L858R/T790M) cellular assay. In contrast to classic third generation EGFR inhibitors, some of these compound showed high inhibitory activity in the low nanomolar range against the therapy- resistant L858R/T790M/C797S EGFR triple mutant [5]. POS.58/SL.18 α-Aminoxy peptides: from membranolytic anticancer References: 1. Juchum, M. et al.: Drug Resist. Updat. 2015, 20: 12-18. foldamers to the first in class peptidomimetic Hsp90 2. Piotrowska, Z. et al.: Cancer Discov. 2015, 5: 713-722. C-terminal domain dimerization inhibitors 3. Thress, K.S. et al.: Nat. Med. 2015, 21: 560-562. 4. Selig, R. et al.: J. Med. Chem. 2012, 55: 8429-8439. Diedrich, D.1; Bhatia, S.2; Frieg, B.1; Stein, S.3; Bopp, B.4; Lang, F.2; 5. Günther, M. et al.: Angew. Chem. Int. Ed., 2016, in press, doi:10.1002/anie.201603736 Ernst, T.5; Rodrigues Moita, A. J.1; Rüther, A.6; Lüdeke, S.6; Kassack,

M. U.1; Hochhaus, A.5; Borkhardt, A.2; Jose, J.4; Kurz, T.1; Gohlke, H.1;

Hauer, J.2; Hansen, F. K.1 1 Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany. POS.57 2 Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich Heine Alkoxyurea-based histone deacetylase inhibitors increase University Düsseldorf, Medical Faculty, Düsseldorf, Germany. 3 Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, cisplatin chemosensitivity Germany. 4 Institute for Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms- 1 1 1 1 University, Münster, Germany. Stenzel, K. ; Hamacher, A. ; Hansen, F. K. ; Gertzen, C. G. W. ; Leven, 5 Klinik für Innere Medizin II, Universitätsklinikum Jena, Erlanger Alle 101, 07747 Jena. M.1; Marek, L.1; Senger, J.3; Marek, M.4; Romier, C.4; Jung, M.3; Gohlke, 6 Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 25, Freiburg, H.1; Kassack, M. U.1; Kurz, T.1 Germany. 1 Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität, Universitätsstr. 1, 40225 Düsseldorf, Germany. For abstract see Short Lecture SL.18 2 Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104 Freiburg, Germany. 3 IGBMC, Université de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch Cedex, France.

DPhG Annual Meeting 2016 Conference Book • 109 POSTERS

POS.59 POS.60 A fluorescent oxaliplatin analogue sheds light on the Targeting cell membrane characteristics by soraphen A: mechanism of action of the drug and the mechanisms of A novel therapeutic option to fight cancer resistance Stoiber, K.1,4; Winzi, M.2; Pernpeintner, C.3,4; Koeberle, A. 5; Ulrich, M.1; Kalayda, G. V.1; Bosman, I.1; Gollos, S.2; Kullmann, M.1; Hamacher, A.3; Werz, O.5; Müller, R.6; Zahler, S.1; Lohmüller, T.3,4; Guck, J.2; Feldmann, Sarin, N.1; Galanski, M.4; Kassack, M. U.3; Müller, C. E.2 J.3,4; Vollmar, A. M.1,4; Braig, S.1 1 Department of Clinical Pharmacy, Institute of Pharmacy, University of Bonn, 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich, An der Immenburg 4, 53121 Bonn, Germany Munich, Germany. 2 Department of Pharmaceutical Chemistry I, Institute of Pharmacy, University of Bonn, 2 Biotechnology Center, Technische Universität Dresden, Tatzberg 47/49, 01307, Dresden, An der Immenburg 4, 53121 Bonn, Germany Germany 3 Institute of Pharmaceutical and Medicinal Chemistry, University of Düsseldorf, 3 Photonics and Optoelectronics Group, Department of Physics and Center for Nanoscience, Universitätsstr. 1, 40225 Düsseldorf, Germany Ludwig-Maximilians-University of Munich, Amalienstr. 54, 80799, Munich, Germany. 4 Institute of Inorganic Chemistry, University of Vienna, Währinger Strasse 42, 1090 Vienna, 4 Nanosystems Initiative Munich (NIM), Schellingstraße 4, 80799, Munich, Germany. Austria 5 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller- University Jena, Philosophenweg 14, 07743 Jena, Germany. 6 Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Oxaliplatin is a third-generation platinum-based anticancer drug Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical distinguished by its activity in colorectal cancer, which is intrinsically Biotechnology, Saarland University, Campus E8.1, 66123 Saarbrücken, Germany. resistant to the other platinum chemotherapeutics cisplatin and carboplatin. The development of acquired chemoresistance in the Albeit cellular membranes exert important functions in signaling, course of treatment with oxaliplatin is, however, common. The transport and recycling processes, targeting membrane characteristics mechanism of action of cisplatin and the mechanisms of cancer cell by using pharmacologically attractive compounds in anti-cancer therapy resistance to this drug have been extensively studied. In contrast, only has not been addressed so far. little is known about oxaliplatin. This study focused on the investigation Here we employed the acetyl-CoA carboxylase inhibitor soraphen A as of the cellular uptake and intracellular transport of oxaliplatin using an a tool to study the effects of a disturbed phospholipid homeostasis on analogue featuring a fluorescent tag. membrane characteristics and its functional consequences on cancer We have successfully synthesized and fully characterized an oxaliplatin progression. By using state-of-the-art biophysical methodologies such derivative CFDA-oxPt bearing a carboxyfluorescein-diacetate in the as real-time deformability cytometry, optical tweezers measurements of position 4 of the cyclohexane ring. In the sensitive human ileocecal giant plasma membrane vesicles and fluorescence recovery after colorectal adenocarcinoma cell line HCT-8 and in oxaliplatin-resistant photobleaching analysis we could demonstrate that the membrane variant HCT-8ox, the cytotoxicity of CFDA-oxPt was lower compared to deformability and fluidity of cancer cells are disturbed after soraphen A the parent drug as revealed using an MTT-based assay (oxPt, HCT-8: treatment. Sophisticated imaging techniques such as proximity ligation EC50 = 4.87 µM; oxPt, HCT-8ox: EC50 = 92.4 µM; CFDA-oxPt, HCT-8: assays revealed that soraphen A abrogates recycling processes and EC50 = 26.5 µM; CFDA-oxPt, HCT-8ox: EC50 = 865 µM). modulates the dimerization of growth factor receptors located in the Nevertheless, oxaliplatin-resistant HCT-8ox cells were found cross- membrane. Consequently, HER2, Src and EGFR receptor signaling resistant to CFDA-oxPt. The platinum-free label CFDA was not toxic up cascades are inhibited leading to diminished breast tumor growth in to 1 mM in both cell lines. These results indicate that CFDA-oxPt vitro and in vivo. represents a suitable model to investigate the mechanism of action of Thus, soraphen A might represent a promising lead substance for anti- oxaliplatin and mechanisms of resistance. cancer agents interfering with the so far undescribed field of membrane The study of the cellular processing of CFDA-oxPt using confocal laser properties. scanning microscopy revealed extensive accumulation of the drug in the plasma membrane of the sensitive cells without any nuclear accumulation visible. In oxaliplatin-resistant cells, CFDA-oxPt localized to the plasma membrane and vesicular structures. In order to further investigate the differences between the sensitive and POS.61 oxaliplatin-resistant cells, cellular accumulation of oxaliplatin was Targeting cholesterol metabolism in hepatocellular studied in more detail. Passive diffusion was found an important carcinoma – V-ATPase inhibition as a novel therapeutic component of oxaliplatin uptake in both cell lines. Inhibition of copper option transporter 1 (CTR1) by CuSO4 led to a significant reduction of oxaliplatin accumulation in HCT-8 and HCT-8ox cells as measured by Bartel, K.1; Winzi, M.2; Ulrich, M.1; Koeberle, A.3; Menche, D.4; Werz, atomic absorption spectrometry. Atropine, a potent inhibitor of organic O.3; Müller, R.5; Guck, J.2; Vollmar, A. M.1; von Schwarzenberg, K.1 cation transporter 1 (OCT1) significantly decreased oxaliplatin 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich 2 Biotechnology Center, Technische Universität Dresden accumulation but only in the sensitive cell line. Inhibition of organic 3 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich Schiller cation transporter 2 (OCT2) with cimetidine significantly reduced University Jena oxaliplatin accumulation in sensitive and resistant cells. Decynium-22, 4 Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn 5 Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection an inhibitor of organic cation transporter 3 (OCT3) had no effect on Research and Department of Pharmaceutical Biotechnology, Saarland University oxaliplatin accumulation in both cell lines. Plasma membrane accumulation of CFDA-Pt is being further investigated using co- Hepatocellular carcinoma (HCC) is still a major cause of cancer-related localization experiments between the oxaliplatin analogue and the death worldwide, however therapy options are limited, leaving a transport proteins mentioned above. pressing need for new therapies. Regarding HCC, cellular lipid and CTR1 is abundantly present in both cell lines but its expression is not cholesterol metabolism is gaining interest, as high cholesterol levels are significantly different between sensitive and resistant cells as revealed beneficial for proliferation tumor progression [1,2]. Cholesterol is by qRT-PCR and Western Blot. Also the expression of organic cation essential for membrane-related mitogenic signaling and increased cell transporters is similar in both cell lines. OCT2 is expressed to a softness of cancer cells, which increases malignancy and invasive marginal extent in HCT-8 and HCT-8ox cells. potential. Despite intensive research , targeting cholesterol metabolism Thus, the oxaliplatin derivative with a fluorescent tag (CFDA-oxPt) has effectively still remains challenging. We provide evidence, that the revealed plasma membrane accumulation in both cell lines and vacuolar ATPase (V-ATPase) inhibitor archazolid (arch), which was additional vesicular accumulation in oxaliplatin-resistant cells. The latter recently shown to interfere with cholesterol metabolism, has a major may indicate the mechanism of oxaliplatin resistance. CTR1 and OCT1 impact on the mechanical phenotype of HCC. Archazolids restrictes are likely involved in oxaliplatin accumulation in the sensitive cells but cellular access to free chol caused by lysosomal trapping through V- only CTR1 appears to mediate active uptake in resistant cells. The ATPase inhibition. Thereby it increases cell stiffness and membrane contribution of OCT2 appears to be marginal. polarity selectively in HCC, while leaving hepatocytes unaffected and as a consequence inhibits membrane-related Ras signaling leading to Acknowledgements: The authors acknowledge the financial support by the Deutsche Forschungsgemeinschaft. impaired proliferation in vitro and in vivo. Hence, V-ATPase inhibition represents a novel, central link between cell biophysical properties and

proliferative signaling in malignant HCC cells, providing the basis for an

attractive and innovative strategy against HCC.

110 • DPhG Annual Meeting 2016 Conference Book CANCER/INFLAMMATION

gene expression are responsible for a specific effect on migration of tumor cells, will be subject of future investigations.

Acknowledgments: This work was supported by the Deutsche Forschungsgemeinschaft (FOR 1406) References: 1. Karmann, L. et al.: Angew. Chem. Int. Ed. Engl. 2015, 54(15): 4502-7.

POS.63

Fig.1: Proposed mechanism of action of archazolid in HCC. (A) Under physiological Effects of the actin binding compound Miuraenamide A on conditions low-density lipoprotein (LDL) binds to its receptor (LDL-R) and is internalized. In the mechanosensitive gene expression in endothelial cells acidic environment of the lysosome, provided by the V-ATPase LDL dissociates from the receptor and is cleaved. Free cholesterol is released and integrated into membranes. Ras is a 1 2 3 1 membrane-bound small GTPase mainly activated in cholesterol-enriched membrane Gegenfurtner, F. A. ; Müller, R. ; Kazmaier, U. ; Vollmar, A. M. ; Zahler, microdomains. Ras in turn promotes proliferation and survival. (B) Archazolid inhibits the V- S.1 ATPase and thereby acidification of the endo-lysosome leading to cholesterol accumulation. 1 Ludwig-Maximilians-University Munich, Department of Pharmacy, Institute of Pharmaceutical Cholesterol is depleted from the membrane and cholesterol-enriched microdomains are Biology, Germany, Email: [email protected] disrupted, changing membrane properties. As a counsequence Ras activation and downstream 2 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) and Institute of signalling is disrupted, leading to reduced proliferation. Pharmaceutical Biotechnology, Saarland University Campus, 66123 Saarbrücken, Germany 3 Institute for Organic Chemistry, Saarland University, 66041 Saarbrücken, Germany Acknowledgements:This work was supported by the DFG research funds 1406 SCHW 1781/1-1 and AV 376/18-1 and by HTCR, a non-profit foundation under German civil law, which facilitates Apart from its classic structural and transport-related tasks in the cell, research with human tissue by providing an ethical and legal framework for prospective sample the actin cytoskeleton recently emerges as an important regulatory collection and the Alexander von Humboldt foundation (Alexander von Humboldt Professorship to JG). element of gene expression in a variety of mammalian cells. For example, it has been reported that nuclear actin participates in References: 1. Gorin, A.; Gabitova, L; Astsaturov, I.: Current opinion in pharmacology 2012, 12: 710-716, chromatin remodeling and directly binds to RNA Polymerases I, II and doi:10.1016/j.coph.2012.06.011. III. Furthermore, actin is involved in the regulation of mechanosensitive 2. Fages, A. et al.: BMC medicine 2015, 13: 242, doi:10.1186/s12916-015-0462-9. transcription factors such as MRTF-A and the Hippo-YAP axis[1]. Regarding the formation of new blood vessels, this is particularly important since the actin cytoskeleton undergoes constant structural remodeling in response to changing parameters of the cellular microenvironment. Consequently, both MRTF and YAP are being POS.62 discussed as potential key players in angiogenesis[2,3]. Migration Inhibition by Actin Binding Compounds Beyond By using the myxobacterial actin-binding compound Miuraenamide A as Simple Actin Polymerization a chemical tool, we examine the functional role of nuclear actin and the consequences on gene expression in endothelial cells with a focus on Moser, C.1; Kazmaier, U.2; Zahler, S.1; Vollmar, A. M.1 the mechanosensitive transcription factors MRTF and YAP. We show 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilian University, that Miuraenamide A is a potent inducer of MRTF-A nuclear Butenandtstraße 5-13, 81377 Munich, Germany translocation and subsequent SRF target gene expression in HUVEC. 2 Institute for Organic Chemistry, Saarland University, 66041 Saarbrücken, Germany In contrast, both, the Hippo-YAP axis, which shares a variety of target An astonishing number of natural compounds interfere with the genes with the SRF-MRTF pathway, and basal transcriptional activity cytoskeleton of mammalian cells. Compounds targeting the remain largely unaffected by our compound. On a structural level, we microtubules like vinca-alkaloids or taxanes, are extensively studied demonstrate that stimulation with Miuraenamide A causes pronounced and used for cancer therapy. nuclear deformation accompanied by the formation of nucleolar actin In contrast, knowledge about pharmacological properties of actin aggregates and an increase in heterochromatic regions. binding drugs is poor and drugs interfering with actin are far from In summary, we characterize Miuraenamide A as an activator of clinical use. On the other hand, the role of actin cytoskeleton in cancer mechanosensitive signaling pathways with a marked preference for the related processes such as migration and invasion as well as apoptosis MRTF-SRF axis in endothelial cells. We furthermore find that and proliferation of tumor cells is well accepted. Unfavourable Miuraenamide A provokes the remodeling of chromatin and nuclear cytotoxicity or other limitations such as the supply of respective natural actin structures, suggesting a regulatory influence of actin on gene compounds might be a reason, which, however, could be overcome by expression beyond the modulation of MRTF or YAP, which will be the synthetically accessible natural compounds. One promising option subject of further investigations. along this line might be the use of Miuraenamide (Miu), a cyclic Acknowledgments:This work was funded by the DFG depsipeptide isolated from a halophilic myxobacterium, previously synthesized by the Kazmaier group [1] and shown to induce actin References: 1. Hendzel; M. J.: Curr Opin Cell Biol 2014, 28: 84-9. hyperpolymerization and disruption of the cytoskeleton (CSK). 2. Franco, C. A. et al.: Development 2013. Our working hypothesis was that actin binding agents are able to evoke 3. Choi, H. J. et al.: Nat. Commun. 2015. distinct pharmacological effects beyond an obvious interruption of the actin CSK, when administered in subtoxic concentrations over time. We proposed that a subtoxic concentration of Miu leads to inhibition of migration of invasive tumor cells, such as ovarian cancer cell, driven by changes in transcriptional activity. In fact, first experiments showed an POS.64/SL.04 inhibition of cell migration after 72h Miu treatment, at a subtoxic Extracellular vesicles as smart carriers for small molecule concentration, without obvious disruption of the actin CSK. Interestingly, drugs this inhibition was independent of cancer cell adhesion, spreading and motility. Of note, migration was only inhibited in the setting of a Fuhrmann, G.1,2; Serio, A.2; Stevens, M. M.2 chemotactic Boyden chamber assay, where the cells have to squeeze 1 Helmholtz Centre for Infection Research, Helmholtz Institute for Pharmaceutical Sciences through narrow pores, but not in mechanical deformation independent Saarland, Saarland University, Campus Building E8.1, 66123 Saarbrücken, Germany 2D scratch assay. Moreover the examination of the transcription level of 2 Imperial College London, Department of Materials, Department of Bioengineering, Miu treated ovarian cancer cells indicated significantly regulated genes, Prince Consort Road, SW7 2AZ London, United Kingdom which are supposed to be important in migration, e.g., structural proteins and integrins. For abstract see Short Lecture SL.04 In sum we have evidence that actin binding agents such as Miu are able to induce distinct changes in transcriptional activity, when employed in concentrations which do not evoke an instant and pronounced effect on actin polymerisation. Whether these changes in

DPhG Annual Meeting 2016 Conference Book • 111 POSTERS

However, PEIs of lower molecular weight and hence higher POS.65 biocompatibility have not been explored so far. Protein-based nanoparticles for drug delivery applications We synthesized a series of three novel tyrosine-modified 2 kDa, 5 kDa and 10 kDa branched PEIs (termed “PxY” with x = molecular weight of Fach, M.1; Radi, L.1; Wich P. R.1 parent PEI), and for comparison the 25 kDa branched tyrosine-modified 1 Johannes Gutenberg Universität-Mainz, Institut für Pharmazie und Biochemie, PEI, via an amide linkage. All tyrosine-modified PEIs were initially Staudingerweg 5, 55128 Mainz, Germany characterized for optimal siRNA complexation conditions, complex size

and zeta potential. Biological activities were first determined in reporter Modern drug delivery systems for therapeutic applications are very cell lines stably expressing luciferase or eGFP. All PEIs benefitted from similar in size and shape to biological nanostructures with comparable the tyrosine modification, with knockdown efficacies of ~ 80 % for the functions, like exosomes or viruses. Polymer-based particles are in P5Y, P10Y, P25Y and 50 % even for P2Y. Importantly, the tyrosine particular interesting as drug, vaccine and gene delivery systems modification strongly enhanced the knockdown efficacies and required because they can be easily chemically modified [1] but they often lack lower amounts of the polymer as compared to the parent PEIs. Another biocompatibility and degradability. Alternatives are nature’s biopolymers critical aspect regarding PEI/siRNA complexes is their colloidal that are readily accessible, can be obtained in high quantity, are instability in buffers and the presence of serum. Notably, incubation of structurally well defined and trigger in most cases only a low reaction of the tyrosine-PEI/siRNA complexes in the presence of different serum the immune system. [2] concentrations revealed the absence of particle aggregation. In Especially proteins have interesting properties as structurally well- addition, we also identified suitable conditions, e.g. freezing and defined biopolymers for the formation of nanoparticles. They have lyophilisation, for long-term storage without loss of biological activity. several advantages over synthetic polymers, including biodegradability, Beyond reporter cell lines, the use of our tyrosine-modified PEI non-antigenicity, stability, binding capacity and toxicity. Because of derivatives was extended towards the therapeutically more relevant these unique properties, protein-based nanocarriers are promising siRNA-mediated targeting of the endogenous anti-apoptotic oncogene candidates for the delivery of drugs and genetic material. [3] survivin in different carcinoma cell lines. Transfection experiments led to Here, we present a new approach for the preparation of protein-based a marked >60% reduction of cell proliferation over negative control nanoparticles for the delivery of hydrophobic therapeutics. We are transfected cells upon siSurvivin delivery mediated by the 5 kDa to creating a protein-polymer conjugate that can be used in solvent 25 kDa modified PEIs. This was based on a >70% survivin knockdown, evaporation methods for the formation of stable nanoparticles. We as determined by RT-qPCR. The profound survivin knockdown thus obtain empty and drug-loaded nanoparticles with a size of around 100 also demonstrated the biological efficacy of tyrosine-PEI/siRNA nm that are stable in different physiological environments. The complexes in targeting endogenous, (patho-) physiologically produced nanoparticles are non-toxic but do not effect of the activity of overexpressed target genes. the encapsulated drug. Additionally, we transferred the method of NP The therapeutic application of non-viral RNAi in vivo requires the preparation to various proteins in different sizes showing that this delivery of intact siRNAs into the target tissue. Initially, the P10Y was method can be universally applied to any protein of choice. [4,5] selected and comprehensively characterized in a pre-clinical in vivo This novel method for nanoparticle preparation extends the range of study in tumour xenograft-bearing mice. The intact delivery of biocompatible materials beyond current known synthetic polymers and P10Y/siRNA complexes into various tissues and the xenograft tumours polysaccharides and potentially opens up new strategies for therapeutic upon systemic (intraperitoneal or intravenous) injection was applications. demonstrated in a radioactive biodistribution assay. Toxicity studies demonstrated that the repetitive administration of P10Y/siRNA caused no adverse effects such as hepatotoxicity, immunostimulation, alterations in the immunophenotype or weight loss. Finally, the therapeutic potential of the P10Y-mediated delivery of siRNAs was analysed in a melanoma xenograft tumour mouse model. The systemic application of P10Y/siRNA complexes targeting survivin demonstrated profound inhibition of tumour growth and down-regulation of the target oncogene. References: Taken together, we demonstrate that the simple modification of PEI with 1. Sapsford, K. E. et al.: Chem. Rev. 2013 113(3), 1904-2074. 2. Zolnik, B. S. et al.: Endocrinology 2010 151(2), 458-465. the aromatic amino acid L-tyrosine strongly enhances the biological 3. Elzoghby, A. O. et al.: J. Controlled Release 2012 157(1), 38-49. activity of low molecular weight PEIs in vitro compared to their parent 4. Radi, L. et al.: Med. Chem. Commun. 2016, Advance Article, DOI: 10.1039/c5md00475f. PEIs. The modification prevents serum-induced particle aggregation as 5. Fach, M; Radi, L; Wich, P. R.: J. Am. Chem. Soc. 2016, Article ASAP, DOI: one major critical factor for in vivo applications. The first in vivo study 10.1021/jacs.6b06243. with the 10 kDa derivative (P10Y) demonstrates the high biocompatibility in mice, the efficient delivery of intact siRNA and the applicability of P10Y/siRNA complexes as RNAi therapeutic.

Acknowledgements: This work was in part supported by grants from the Saxonian Ministry of POS.66 Science and Art (SMWK) and the Deutsche Krebshilfe (grant number 11616) to A.A., and by the L-tyrosine-modification of low molecular weight PEIs German Ministry of Education and Research (BMBF, grant # 1315883). increases siRNA knockdown efficacies in vitro and in vivo References: 1. Creusat, G.; Zuber, G.: Chembiochem : a European journal of chemical biology 2008, 9: 2787-2789. 1 2 2 1 Ewe, A. ; Przybylski, S. ; Burkhardt, J. ; Aigner, A. 2. Ewe, A. et al.: J Control Release 2016, 230:13-25. 1 Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology; Leipzig 3. Ewe, A. et al.: Drug Deliv Transl Res 2016 [Epub ahead of print] University, Härtelstrasse 16-18, D-04107 Leipzig, Germany 2 Fraunhofer Institute for Cell Therapy and Immunology (IZI), Perlickstrasse 1, D-04103 Leipzig, Germany

The non-viral delivery of small RNA molecules like siRNAs for specific POS.67 gene silencing still poses a major hurdle for their use in vivo. Their Inhibition of Cdk5 – a novel strategy to address cancer successful therapeutic application is thus dependent on the development of save and efficient delivery strategies. Polyethylenimines stem cells (PEIs) are cationic polymers that are well established for the delivery of 1 1 1 2 2 nucleic acids, but major concerns have been raised with regard to their Mandl, M. M. ; Zhang, S. ; Ulrich, M. ; Schmoeckel, E. ; Mayr, D. ; 1 1 limited efficacy and inherent toxicity. On the other hand, the presence of Vollmar, A. M. ; Liebl, J. 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich reactive amines in PEI molecules allows for chemical modifications, and (LMU), Munich, Germany thus offers new opportunities for more efficient and safer delivery 2 Institute of Pathology, Ludwig-Maximilians-University of Munich (LMU), Munich, Germany vectors. One promising strategy is based on the introduction of amino acids. Recently, amino acid modifications of the more toxic 25 kDa Cancer stem cells (CSCs) represent a major problem in tumor therapy branched PEI have shown improvement for nucleic acid delivery. as they account for chemoresistance, tumor recurrence and metastasis. However, strategies to address CSCs are limited. In this context, we

112 • DPhG Annual Meeting 2016 Conference Book CANCER/INFLAMMATION hypothesize the serine/threonine kinase cyclin dependent kinase 5 (Cdk5) as a particular interesting and promising player to explore. Cdk5 POS.69 exerts pivotal functions in the neuronal system and is associated with Inhibition of Cdk5 – a novel strategy to improve Sorafenib neurodegenerative diseases. During the last years, important non- response in HCC therapy neuronal functions of Cdk5 have been elucidated and recent studies indicate Cdk5 as a promising target for anti-cancer therapy. Although Ardelt, M. A.1; Fröhlich, T.2; Lehr, T.3; Wojtyniak, J.-G.3; Arnold, G. J.2; Cdk5 has recently been associated with epithelial-mesenchymal Vollmar, A. M.1; Liebl, J.1 transition (EMT), a role of Cdk5 in CSCs has not been described yet. In 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians University, 81377 Munich, Germany fact, here we suggest a function of Cdk5 in CSCs by showing that 2 Laboratory for Functional Genome Analysis, LAFUGA, Gene Centre, LMU, 81377 Munich, knockdown and pharmacological inhibition of Cdk5 impaired Germany tumorsphere formation and reduced tumor establishment in vivo. 3 Clinical Pharmacy, Saarland University, 66123 Saarbrücken, Germany Conversely, Cdk5 overexpression promoted tumorsphere formation which was in line with increased expression of Cdk5 in human breast Introduction: For the treatment of advanced hepatocellular carcinoma cancer tissues as shown by staining of a human TMA. In order to (HCC) Sorafenib is the only approved systemic therapy. Unfortunately understand how Cdk5 inhibition affects tumorsphere formation, we patients only gain a survival benefit of about 3 months and therefore identify a role of Cdk5 in detachment induced cell death: Cdk5 inhibition there is a great need for novel systemic treatment option or induced apoptosis in tumorspheres by stabilizing the transcription factor combinational treatments.[1] Recently we identified cyclin dependent Foxo1 which results in increased levels of the pro-apoptotic protein Bim. kinase 5 (Cdk5), an atypical kinase, as a novel target in HCC therapy: In summary, our study elucidates a Cdk5-Foxo1-Bim pathway in cell We found that Cdk5 was frequently overexpressed in HCC and death in tumorspheres and suggests Cdk5 as a potential target to rendered HCC cells sensitive for the treatment with DNA damaging address CSCs. agents by regulating DNA damage response.[2] The aim of this study was to investigate whether Cdk5 inhibition could also sensitize HCC cells for Sorafenib treatment and thereby enhance the tumour growth inhibition and to elucidate the corresponding mechanism. Methods: To evaluate the impact of Cdk5 inhibition on Sorafenib POS.68 treatment we used the pharmacological Cdk5 inhibitors roscovitine and Disturbing mitochondrial proteostasis by inhibition of Clp dinaciclib as well as genetic downregulation of Cdk5 via shRNA in complex as an anti-cancer strategy different cell based assays and mouse xenograft models. For the investigation of Cdk5 function and signalling in the context of Sorafenib Mandl, M. M.1; Gronauer, T.2; Fetzer, C.2; Stahl, M.2; Vollmar, A. M.1; treatment we used a proteomics screen of whole cell lysates of Sieber, S. A.2; Liebl, J.1 Sorafenib treated HCC cells with or without Cdk5 knockdown. 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-Universität München Results: In fact, the combination of Sorafenib with a functional ablation (LMU), Butenandtstraße 5-13, Munich, D-81377, Germany of Cdk5 synergistically inhibited the proliferation and clonogenic survival 2 Center for Integrated Protein Science at the Department of Chemistry, Technische Universität München, Lichtenbergstraße 4, Garching, D-85747, Germany of HCC cells in vitro. This synergism could also be confirmed in a xenograft mouse model, where a combinational therapy resulted in a The mitochondrial matrix protease Clp is implicated in ageing and significantly reduced tumour growth rate. Moreover, we could show that disease. By regulating the mitochondrial proteostasis and the Cdk5 inhibition not only reduced overall migration but also prevented mitochondrial unfolded protein response (UPRmt) Clp is a key player for the induced cancer cell migration caused by sub therapeutic the maintenance of mitochondrial function. Although it has been found concentrations of Sorafenib. The proteomics screen revealed on the lately that Clp subunit P (ClpP) is upregulated in a majority of Acute one hand, that Cdk5 knockdown cells seem to have deregulated Myeloid Leukemia cells and its activity has been linked to cisplatin metabolic activity and on the other hand that the epidermal growth resistance, our knowledge of the function of Clp complex in cancer is factor receptor (EGFR) is connected to Cdk5. Our further investigations still limited. Neither general mechanisms of action of its inhibition, nor could show that Cdk5 is involved in EGFR signalling and trafficking and the signaling from mitochondria to nucleus and substrates in prevents the activation of the PI3K/Akt pathway, which was shown to be mammalian cells, especially in cancer, have been investigated yet. caused by compensatory activation of EGFR.[3] According, the aim of the present study was to characterize the function Conclusion: The pharmacological inhibition of Cdk5 with drugs, which of Clp as a potential new target for anti-cancer therapy. First, we found are already clinically used in the treatment of other diseases, offers a ClpP and ClpX subunits to be expressed in various cancer cell lines new concomitant therapy option for patients treated with Sorafenib. The including myeloid and lymphatic leukemia, breast and liver derived combination has the benefit of not only inhibiting tumour cell growth to a cancer cell lines. To evaluate its therapeutic relevance, we used novel greater extent but also impeding tumour cell motility compared to Clp complex inhibitors, performed profound pharmacological testing in Sorafenib single treatment. To conclude, our work provides evidence for several functional cell-based assays and found that Clp inhibition Cdk5 inhibition as a new promising strategy to improve the therapeutic affected cell viability of leukemia, breast and liver cancer cells. effect of Sorafenib in HCC. Additionally, as an early event, ATP production was decreased pointing to a direct effect on mitochondria. Furthermore, Clp inhibition led to an Acknowledgements: Financial support by DFG (VO376/17-1) is gratefully acknowledged. induction of apoptosis and exerted chemosensitizing effects in leukemia References: cells towards chemotherapeutics including Imatinib and Etoposid. In 1. Llovet, J. M. et al.: N Engl J Med 2008, 358: 378-390. 2. Ehrlich, S. M. et al.: J Hepatol 2015, 63: 102-113. order to validate Clp as a therapy target and to characterize the 3. Blivet-Van Eggelpoel, M. J. et al.: J Hepatol 2012, 57: 108-115. mechanism of action of the inhibitors, we observed the induction of mitochondrial ROS production and common hallmarks of the intrinsic apoptosis mechanism, like the loss of mitochondrial membrane potential, PARP cleavage or Caspase activation. Clp inhibition was also found to be effective in patient-derived leukemia cells. Currently, we are POS.70 focusing on the general mode of action of Clp inhibition, such as, for Characterization of Sorafenib Resistance and Rebound instance, the impact on the mitochondrial proteome using stable isotope Growth in Hepatocellular Carcinoma Cells labeling with amino acids in cell culture (SILAC). In addition, identification of peptides released from mitochondria under stress Meßner, M.1; Ardelt, M. A.1; Meyer, K.2; Fröhlich, T.2; Vollmar, A. M.1; conditions and Clp inhibition using a mass spectrometry-based Liebl, J.1 mt approach should expand knowledge about UPR signaling and specific 1 Department of Pharmacy, Pharmaceutical Biology, LMU, Butenandtstr. 5-13, 81377 Munich, Clp substrates. Moreover, the establishment of an in vivo model should Germany further promote Clp inhibition as a promising strategy in cancer therapy 2 Laboratory for Functional Genome Analysis, LAFUGA, Gene Centre, LMU, Feodor-Lynen-Straße 25, 81377 Munich, Germany and may even improve treatment of resistant cancer cells. Introduction: Sorafenib represents the current standard of care for patients with advanced stage hepatocellular carcinoma (HCC) based on two recent phase III trials that reveal an overall survival benefit of three months compared to placebo [1]. Nevertheless, its use is hampered by the occurrence of drug resistance. Further, up to 80% of patients

DPhG Annual Meeting 2016 Conference Book • 113 POSTERS treated with sorafenib suffer from side effects necessitating dose reduction, short-term “drug holidays” or permanent treatment termination [2]. In order to improve sorafenib therapy, the aim of the study was to elucidate the molecular basis for this drug resistance in general. In addition we studied the effect of sorafenib withdrawal as rebound phenomena have been reported in the context of anti- angiogenic therapy abrogation. Methods: To investigate the molecular mechanisms involved in sorafenib resistance we developed a sorafenib resistant human liver cell line in which we studied morphology, protein expression as well as proliferative and migratory potential under sorafenib exposure and withdrawal conditions. The effects of sustained sorafenib exposure were additionally investigated on protein level by means of a mass spectrometry-based proteomics analysis. Results: The sorafenib resistant human liver cells changed their morphology to elongated spindle-shaped cells, lost E-Cadherin and showed high expression of N-Cadherin and Vimentin, indicating epithelial-to-mesenchymal transition but yet the migratory potential was decreased. Further they showed increased apoptosis, a G1-phase cell cycle arrest and evasive PI3K (phosphatidylinositol 3-kinase)/Akt pathway activation. Proteomics screen revealed on the one hand a strong induction of the translational machinery and on the other hand reduced fatty acid metabolic processes. Remarkably, following withdrawal of sorafenib, the resistant cells showed rebound growth, a phenomenon also found in patients. As broad cross-resistance is presumed to occure, this cell model was used to investigate sensitivity towards a variety of chemotherapeutics and pathway inhibitors in order to elucidate an alternative treatment or combination therapy. Conclusion: Our work demonstrated that several mechanisms are involved in the acquired resistance to sorafenib, such as crosstalks involving the PI3K/Akt pathway and epithelial-to-mesenchymal transition. However, the exact mechanisms accounting for sorafenib resistance remains unclear and further investigations are needed to improve our understanding and help to find effective strategies for overcoming sorafenib resistance in HCC. Importantly, this study revealed that halting antiangiogenic therapy with sorafenib could lead to rapid tumor growth rebounds, influencing clinical practice with regard to dosing schedules and presurgical intervention strategies.

References: 1. Llovet, J. M. et al.: N Engl J Med. 2008, 359: 378-390. 2. Abou-Alfa, G. K. et al.: J Clin Onkol. 2006, 24: 4293-4300.

114 • DPhG Annual Meeting 2016 Conference Book BIOTECHNOLOGY/PROTEIN DRUGS

analysed showing good physical and conformational stability [5]. Since 3.4 Biotechnology/Protein lipids are biodegradable and biocompatible [6] they represent a promising delivery platform for AMD treatment. Material and Methods: Triglycerides Dynasan® D118 and H12 were Drugs kindly donated by Cremer Oleo (Hamburg, Germany). Hydroxy-β- cyclodextrine (HP-β-CD) was a gift of Wacker Chemie AG (Burghausen, Germany). Ranibizumab (Lucentis®) is a fab-fragment with a molecular POS.71 weight of about 49 kDa. Ranibizumab was formulated with HP-β-CD at NADPH Cofactor regeneration on the cell surface: an a 1:1 ratio [m/m] before lyophilization. SLIs were prepared by twin- important step to pharmaceutical applications of surface screw extrusion as described before [6] using a ZE-5 extruder from displayed phase I P450 enzymes Three-Tec (Seon, Switzerland). A mixture of H12, D118 and 10% protein lyophilizate was fed to the extruder and extrusion was Schüürmann, J.; Lindhorst, F.; Jose, J. performed at 35°C. Implants had a semicircle shaped form which is Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westfälische Wilhelms- suitable for intravitreal use and contained 1.65mg protein per implant. Universität Münster, Corrensstraße 48, D 48149 Münster, Germany For in-vitro release studies, implants were placed into 2.0 ml centrifugation tubes and incubated at 37°C in a horizontal shaker The use of purified enzymes for preparative applications is still held (40rpm) in PBS pH 7.4. The release medium was completely back by costly production and purification processes, stability problems exchanged at predetermined time points and protein concentration was and reusability issues. In this regard, whole cell biocatalysts offer analyzed UV-metrically at 280 nm. Monomer content was determined significant advantages. Theses catalysts however are restricted to cell via SE-HPLC using a Waters 2695 Separations Module with a Waters permeable substrates and products. One solution to circumvent mass 2487 Dual λ Absorbance Detector (Waters, Milford, USA). The flow rate transfer problems and possible cross reactions with intracellular was adjusted to 0.5ml/min and 50µl were injected onto a TSKgel enzymes is the presentation of enzymes on the cell surface. G3000SWXL size exclusion column (300mm x 7.8mm; Tosoh Bioscience, Our group was recently able to display human cytochrome P450 phase Tokyo, Japan). The running buffer consisted of 50mM sodium I enzymes CYP1A2 and CYP3A4 in an active form on the cell surface of phosphate with 300mM NaCl and 0.05% NaN3 and was adjusted to pH E. coli using the Autodisplay technique [1,2]. Therefore the passenger 7.0. domain of a native autotransporter was replaced by the peptide or Results: A sustained release of Ranibizumab was achieved over protein of choice, which was subsequently presented at the cell surface approximately 110 days. The release profile is characterized a triphasic [3]. A current project focusses on the display of CYP102A1 from release behaviour without an initial burst. An initial phase for the first Bacillus megaterium for pharmaceutical applications. Surface exposure two weeks where 40% of the incorporated protein was released of the enzyme was confirmed by protease accessibility tests, flow- followed by a linear phase ranging from week 2 to week 14 releasing cytometry analysis and activity measurements. CYP102A1, also known 50% of encapsulated protein. During the last release phase, lasting as BM3 is one of the best studied cytochrome P450s to date and can from week 14 to 18, only small amounts of protein were released be engineered to accept many pharmaceutical relevant substances [4]. (approx. 3%). In total, nearly 90% of incorporated protein was released. In a proof of principle study we analyzed a small library of substances The monomer content of the released protein fractions was measured with the known R47L F87V L188Q triple mutant of CYP102A1 displayed over 18 weeks showing no loss of monomer compared to Ranibizumab at the cell surface of E. coli. bulk material showing that the fab-fragment is physically stable within However, the project also elucidated a major challenge for the use of the lipidic matrix over time. the technology in pharmaceutical applications. Bringing an enzyme to Conclusion: We successfully demonstrated that twin-screw extruded the cell surface blocks the access to intracellular cofactors, which are SLIs are a suitable technology platform for the long-term release of a too expensive to be added stoichiometrically. In an approach to therapeutically relevant protein providing a sustained release of 14 conserve the advantages of a surface displayed catalyst, we used weeks in linear manner. Good physical stability of the fab-fragment Autodisplay as well to present various dehydrogenases on the surface indicates sufficient protein stability within the lipidic matrix. Therefore, of E.coli cells for the regeneration of the essential cofactor NADPH. this approach is a promising platform for intravitreal delivery of anti- VEGF drugs for AMD treatment. References: 1. Quehl, P et al. Microb Cell Fact 2016, 15:1-15 References: 2. Schumacher S and Jose J J Biotechnol 2012, 161(2):113-120 1. Wong, W.L. et al.: Lancet Glob. Health 2014, 2: e106-e116 3. Schüürmann, J et al. Appl Microbiol Biotechnol 2014, 98:8031-8046 2. Lim, L.S. et al.:Lancet 2012, 379: 1728 – 1738 4. Whitehouse C, Bell S and Wong L. Chem Soc Rev 2012, 41:1218-1260 3. Lovett, M.L. et al.: Eur. J. Pharm. Biopharm.2015, 95: 271-278 4. Vollrath, M. et al.: Poster 10th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology, Glasgow, Scotland, UK, 2016 5. Vollrath, M. et al.: Poster AAPS Annual Meeting and Exposition, Orlando, FL, USA, 2015 6. Sax, G. et al.: J. Control. Release, 2012, 2: 195–202 POS.72 Long-term release of Ranibizumab from lipid based implants for intravitreal treatment of age-related macular degeneration (AMD) POS.73

Protein powder suspensions based on perfluorodecalin Engert, J.1; Vollrath, M.; Winter, G.1 1 Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig- Maximilians-University, Butenandtstrasse 5-13, 81377 Munich, Germany Marschall, C.1; Yasin, A.2; Witt, M.2; Friess, W.1 1 LMU Munich, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Butenandtstr.5, 81377, Munich, Germany Introduction: Age-related macular degeneration (AMD) is an eye 2 Novaliq GmbH, Im Neuenheimer Feld 515, 69120 Heidelberg,Germany disease characterized by a loss of the central vision causing visual impairment and blindness [1]. The main characteristic of the more Introduction: severe neovascular form of AMD (wet AMD) is an abnormal blood Currently there is a growing demand for highly concentrated protein vessel formation leading to the presence of blood in the back of the eye formulations for subcutaneous injection. However, conventional region [2]. According to the limitations of the repeated intravitreal aqueous formulations have different challenges such as increased injections of anti-VEGFs, the development of insertable devices viscosity and potentially decreased protein stability. Protein powder providing long-term release of anti-VEGF drugs is in the focus of current suspensions in non-aqueous vehicles represent a promising alternative. research [3]. Herein the protein is present in a solid state offering the chance of In our study, solid lipid implants (SLIs) were investigated for the higher protein stability compared to an aqueous solution. Furthermore sustained release of the recombinant monoclonal antibody fragment the suspensions may provide lower viscosity compared to the ® Ranibizumab (Lucentis ) for intraocular delivery. It has already been respective aqueous solution depending on the protein-protein- demonstrated that SLIs are a suitable platform for long-term protein interaction in water [1]. The choice of potential suspension vehicles is release since a sustained release of an IgG1 over 190 days was limited as traditional non-aqueous liquids such oils show an inherent described [4]. Additionally, protein stability of the released fractions was high viscosity [2]. A potential alternative is perfluorodecalin. Previous

DPhG Annual Meeting 2016 Conference Book • 115 POSTERS investigations by Knepp et al. showed high protein stability of 2, 4, 5]. So far, the material of choice for prefilled syringe barrels has suspended lyophilizates in this vehicle over one year at elevated been glass [2, 4]. These glass syringes need silicone oil as a lubricant temperatures comparable to the same lyophilizate stored at -80°C [3]. for a smoot injection. However, silicone oil can contribute to the particle Materials and methods: count of the solution. Either it sheds into the solution or can foster A lab scale spray-drier was used to produce lysozyme powder, protein aggregation. Particles, especially silicone oil droplets with formulated with trehalose as a stabilizer, at a high protein to stabilizer protein attached, in general compromise the safety and efficacy of the ratio. Process parameters were chosen according to Schuele et al. [4]. drug [1, 4, 5]. Therefore, silicon oil free, alternative materials for prefilled Feed solutions for spray-drying contained different total solid contents. syringes are necessary. A suitable material can be cyclic olefin polymer The obtained powders were analysed by differential scanning (COP). This material in combination with special coated stoppers works calorimetry, laser diffraction and scanning electron microscopy. well without silicone oil for lubrication [5]. COP syringes possess a Suspensions were prepared in perfluorodecalin at different powder major drawback, namely providing a lower gas barrier than glass [4]. contents up to 500 mg/ml using a dispersing device. The rheological This might limit their usage for the storage of biopharmaceuticals, properties of the suspensions were investigated using a cone-plate because many of those are sensitive to oxidation [3]. rheometer. However, oxygen barrier properties can be modified. One option is the Results: usage of multilayer syringes with a layer incorporated displaying high Spray-drying showed good powder yields (>80%). Increasing the total oxygen barrier properties. Yet, these systems are expensive and not yet solid content in the feed solution led to larger particle diameters up to available in high numbers. A cheap solution would be a tight aluminum 30 µm. The powder particles showed a typical spherical morphology. bag as secondary packaging which would also protect from light. The prepared suspensions showed a shear-thinning behaviour and the Another cost-effective option would be an oxygen impermeable label, viscosities were remarkably low even at high powder concentrations. as a label is necessary anyway. Even without additional measures The true density of the prepared powder was lower than the stability could be achieved, if the effect of oxygen permeation on the perfluorodecalin density leading to flotation of the powder. Suspensions particular protein is negligible. showed good redispersion behaviour as well as good suspension The suitability of such novel COP based syringes as a long term stability during short term storage at room temperature and elevated packaging material for biopharmaceuticals is evaluated systematically temperature. in this study. The study focuses especially on the issue of oxygen Conclusion: permeation and its consequences. To this end, two model proteins of Overall the suspensions benefit from perfluorodecalin’s exceptional high therapeutic relevance were investigated. Further, the study physical properties such as its low viscosity resulting in low suspension comprises the low cost options of an aluminum bag which is filled with viscosity and good redispersibility even at higher powder nitrogen and a tight label. Finally we benchmarked the COP syringes concentrations. Further work will show long term suspension stability of against commonly available glass syringes. the prepared protein powder suspensions in perfluorodecalin. Also The data of the ongoing study suggest a superiority of the COP other, more relevant proteins with lower protein stability, such as syringes regarding particle count. MicroFlow Imaging reveals clearly the monoclonal antibodies, will be formulated as suspensions in order to impact of silicone oil from glass syringes on the particle burden of the confirm the advantages of protein powder suspensions and to solution. Depending on the syringe setup, differences regarding investigate long-term protein stability. chemical stability of the proteins are visible. Ion exchange and reversed phase chromatography show the highest content of oxidized protein for References: COP syringes without any alterations. However, the storage of COP 1. M. a Miller, J. D. Engstrom, B. S. Ludher, and K. P. Johnston, “NIH Public Access,” vol. 26, syringes in nitrogen filled aluminum bags or with tight labels leads to no. 2, pp. 1067–1074, 2011. lower amounts of oxidized species than in glass syringes. The impact of 2. Y.-F. M. Mayumi Bowen, Nick Armstrong, “Investigating High-Concentration Monoclonal a higher oxygen permeability of the COP barrel in comparison to glass Antibody Powder Suspension in Nonaqueous Suspension Vehicles for Subcutaneous Injection,” J. Pharm. Sci., vol. 101, no. 12, pp. 4433 – 4443, 2012. syringes can easily be overcome by low cost modifications. 3. A. M. et al. V. M. Knepp, “Stability of nonaqueous suspension formulations of plasma derived factor IX and recombinant human alpha interferon at elevated temperatures,” Pharm. Res., vol. Acknowledgments: The authors thank West Pharmaceutical Services Deutschland GmbH & Co KG, Eschweiler, Germany and Gerresheimer Bünde GmbH, Bünde, Germany for the supply of 15, no. 7, pp. 1090–1095, 1998. syringe material. 4. S. Schüle, T. Schulz-Fademrecht, P. Garidel, K. Bechtold-Peters, and W. Frieß, “Stabilization of IgG1 in spray-dried powders for inhalation,” Eur. J. Pharm. Biopharm., vol. 69, no. 3, pp. References: 793–807, 2008. 1. Depaz, R.A., et al.: J Pharm Sci 2014, 103: 1384-1393. 2. Krayukhina, E., et al.: J Pharm Sci 2015, 104: 527-535. 3. Manning, M.C., et al.: Pharm Res 27, 2010, 544-575. 4. Nakamura, K., et al.: PDA J Pharm Sci Technol 2015: 69, 88-95. 5. Yoshino, K., et al.: J Pharm Sci 2014, 103: 1520-1528. POS.74/SL.32

How does pH Affect Interfacial Antibody Behaviour and Aggregation upon Shaking? POS.76 Koepf, E.1; Schroeder, R.2; Brezesinski, G.3; Frieß, W.1 Is bedside filtration an effective tool to diminish risks 1 Ludwig-Maximilians-Universitaet, Department of Pharmacy, Pharmaceutical Technology & associated with protein aggregates? Biopharmaceutics, 81377 Munich, Germany 2 AbbVie Deutschland GmbH & Co. KG, 67061 Ludwigshafen am Rhein, Germany 3 Max-Planck Institute of Colloids and Interfaces, Department of Interfaces, 14476 Potsdam, Werner, B. P.1; Winter, G.1 Germany 1 Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University, Butenandtstr. 5, 81377 Munich, Germany, For abstract see Short Lecture SL.32 [email protected], phone: +49 89 2180 77035

In the last decade much progress has been made in formulating stable

and easy to handle protein drug products despite the complex structure

of proteins and their chemical and physical instability [2, 3]. POS.75 Nevertheless, the chance remains that protein aggregates are formed Suitability testing of novel polymer syringes for long term during transportation, storage or handling. Generally, these protein particles could endanger the safety and efficacy of the protein drug [2]. storage of biopharmaceuticals Hence, these aggregates may present a hazard especially for

immunosuppressed and intensive care patients [1]. The potential of Werner, B. P.1; Winter, G.1 1 Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, protein aggregates to induce immune responses is known since the Ludwig-Maximilians-University, Butenandtstr. 5, 81377 Munich, Germany, 1950s and is nowadays broadly accepted [4, 7]. For several proteins, [email protected], phone: +49 89 2180 77035 like adalimumab, insulin or human growth hormone the formation of antidrug antibodies is observed [8]. The consequences of this antidrug Prefilled syringes receive more and more interest for the administration antibody formation can be negligible and minor but also very severe, of biopharmaceuticals, because of an easy and quick drug like the neutralization of an endogenous protein [5]. Today, the FDA administration in combination with an increased patient compliance [1, and other regulatory bodies have increased the pressure on drug

116 • DPhG Annual Meeting 2016 Conference Book BIOTECHNOLOGY/PROTEIN DRUGS manufacturers which now have to supply comprehensive precursor protein (APP) was implemented. The kinetics of matriptase-2 documentation regarding particle burden of protein solutions. inhibition by Kunitz-type inhibitors were analyzed and the intracellular An easy and practicable approach to protect patients from any risks downstream effects of matriptase-2 inhibition were investigated by associated with protein aggregates could be bedside filtration. In our applying a set of different assays using human liver cell systems. Co- unique and comprehensive survey, we identified more than 50 products immunoprecipitation revealed the formation of matriptase-2/inhibitor of more than 300 marketed products which are already filtered during complexes. Western blotting and reporter gene assays were employed preparation and administration (Figure 1) [8]. Considering these findings to analyze the impact on matriptase-2-mediated hemojuvelin cleavage and the positive benefits associated with bedside filtration, like reduced and hepcidin expression. Binding modes of the matriptase-2/Kunitz-type occurrence of thrombi [6] or sepsis, we like to suggest contemplating a inhibitor complexes were suggested by molecular modeling. broader application of final bedside filtration. In conclusion, we have characterized Kunitz-type inhibitors as powerful, slow-binding inhibitors of matriptase-2 with HAI-2 being the most effective inhibitor. Thus, HAI-2 provides an excellent framework for the development of a drug inhibiting matriptase-2 as a therapeutic agent in iron overload diseases.

References: 1. Finberg, K. E. et al.: Nat. Genet. 2008, 40(5): 569-571. 2. Du, X. et al.: Science 2008, 320(5879): 1088-1092. 3. Silvestri, L. et al.: Cell. Metab. 2008, 8(6): 502-511.

Fig. 1: Already 15.9% of all protein drug products listed in the Rote Liste® are filtered during POS.78/SL.56 preparation or administration of the drug. Adapted from Werner, B.P. and Winter, G. [8]. Clickable IL-4 cytokines to induce M2 macrophage

We critically assessed several practical aspects arising from a broader polarization application of final bedside filtration. First, ejection of a high 1 1 1 2 2 concentrated protein solution is still possible after filter attachment, Lühmann, T. ; Spieler, V. ; Werner, V. ; Fiebig, J. ; Mueller, T. D. ; whereas the filter has only a minor impact on the ejection force. Meinel, L.1 1 Institute of Pharmacy and Food Chemistry, University of Würzburg, 97074, Germany Second, the so far investigated filters do not contribute to the particle 2 Lehrstuhl für Botanik I, University of Würzburg, 97070, Germany burden of the solution, but are highly capable in reducing the amount of protein particles. Further, protein adsorption or structural changes of the For abstract see Short Lecture SL.56 protein caused by filtration are not observed. Overall, our data shows that bedside filtration during protein drug preparation and administration can be a powerful and effective tool to guard patients from potentially negative effects associated with protein aggregates. POS.79/SL.03 References: 1. Bethune, K., et al.: Nutrition 2001, 17: 403-408. Biopolymers as Multifunctional Materials for 2. Kessler, M., et al.: Dial Transplant 2006, 21: v9-v12. Nanoparticulate Drug Delivery 3. Liu, L., Randolph, T.W., Carpenter, J.F.: J Pharm Sci. 2012, 101(8): 2952-9. 4. Mahler, H.C., et al.: J Pharm Sci 2009, 98 (9): 2909-2934. Wich, P. R. 5. Rosenberg, A. AAPS J. 2006, 8 (3): E501-E507. Johannes Gutenberg Universität-Mainz, Institut für Pharmazie und Biochemie, 6. Schellekens, H. Best Pract Res Clin Haematol 2005, 18: 473-480. Staudingerweg 5, 55128 Mainz, Germany 7. van Lingen, R.A., et al.: Acta Paediatr 2004, 93 (5): 658-662. 8. Wang, W., et al.: Int J Pharm 2012, 431: 1-11. For abstract see Short Lecture SL.03 9. Werner, B.P., Winter, G.: Int J Pharm 2015, 496: 250-267.

POS.77 Kunitz-type inhibitors targeting matriptase-2: Promising therapeutics for iron overload diseases

Beckmann, A.-M.1; Maurer, E.1; Mangold, M.1; Furtmann, N.1, 2; Bajorath, J.2; Walter, J.3; Becker-Pauly, C.4; Gütschow, M.1; Stirnberg, M.1 1 Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 2 Department of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and Medicinal Chemistry, University of Bonn, Dahlmannstr. 2, 53113 Bonn, Germany 3 Department of Neurology, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany 4 Institute of Biochemistry, Unit for Degradomics of the Protease Web, Christian Albrechts University, Rudolf-Höber-Str. 1, 24118 Kiel, Germany

The type II transmembrane serine protease (TTSP) matriptase-2 (gene name: TMPRSS6) is a critical stimulator of iron absorption by negatively regulating hepcidin, the key hormone of iron homeostasis [1,2]. By cleaving the BMP co-receptor hemojuvelin matriptase-2 suppresses BMP/SMAD signalling thereby downregulating the expression of hepcidin [3]. Consequently, this protease attracted much attention as a target to treat primary and secondary iron overload diseases associated with deficiency in hepcidin. Kunitz-type serine protease inhibitors are natural inhibitors containing at least one Kunitz-domain binding with high affinity to TTSPs. As stand-alone domains with intrinsic stability, Kunitz-domains can be used as scaffolds for engineering protease inhibitors to be employed as biopharmaceuticals. In this study, a strategy based on the Kunitz-type inhibitors hepatocyte growth factor activator inhibitor (HAI)-1, HAI-2 and the amyloid

DPhG Annual Meeting 2016 Conference Book • 117 POSTERS

Method: A quantitative statistical analysis of the pharmaceutical care 3.5 Clinical Pharmacy documented in the case-report-forms from 39 DIDEMA patients over the study period of 6 months was conducted. The case report forms

were analysed according to the following parameters: Self-monitoring of POS.80 blood glucose (SMBG), daily insulin injections, average fasting blood Implementing preferred generic prescribing – community glucose levels, insulin- therapy-adherence, daily insulin dose and pharmacies’ view number of patients, which are following their nutrition plan or doing exercise or having hypoglycemic episodes. Inconsistent and imprecise

data were excluded from the statistical analysis. A Wilcoxon-signed- Breiholz, S.1; Eickhoff, C.1; Felberg, M.1; Klintworth, D.1; Müller, U.1; rank test or fisher-extact test with a significance level of alpha=0.05 Strunz, A. K.1; Schulz, M.1 1 Institute: Department of Medicine, ABDA – Federal Union of German Associations of were used to evaluate the differences between the study visits from Pharmacists, 10117 Berlin, Germany every month compared to the pre-study parameter characteristics. Results: The statistical analysis revealed that pharmaceutical care In 2011, the ABDA and the Federal Association of Statutory Health provided by community pharmacists result in more frequent SMBG, a Insurance Physicians (KBV) developed a concept that aims to improve greater amount of patients complying to their individual nutrition plan outcomes of drug therapy in patients with polymedication [1]. Against and injecting the correct insulin dose. Also, the insulin-therapy this background, a project named ARMIN (Arzneimittelinitiative adherence increased and fasting blood glucose levels were lowered. Sachsen-Thüringen, www.arzneimittelinitiative.de) was developed and However, all these positive changes did not lead to an increase in contracted for at first five years in two states, Saxony and Thuringia. hypoglycemic episodes. ARMIN consists of three components: (1) prescription of active Conclusion: The results show that pharmaceutical care can positively ingredients (INN) instead of brand products, (2) a medication catalogue alter disease specific parameters and improve patient behaviour to to select appropriate drugs based on guidelines, and (3) medication cope with the daily burden of type I diabetes mellitus in adolescents. management by pharmacists and physicians utilizing an electronic With this, the pharmacist as a health-communicator can substantially medication plan. support these individual adolescents by translating current treatment The preferred generic prescribing by physicians within this project was guideline into daily practice, giving answers to nutrition questions, implemented in July 2014. It aims to increase transparency for the reviewing the individual nutrition plan and thus set realistic goals for the patient and to reduce medication errors, considering the multitude of next visit. Consequently, better glycemic control should minimize the rebate contracts for generics between statutory health insurances and risk of short- and long-term diabetes related complications such as pharmaceutical companies. retinopathy or blindness for these young individuals [2]. Firstly, we developed a dataset including appr. 190 suitable active ingredients, defined in terms of active substances and formulations. References: Secondly, we specified how the description has to be filled in by the 1. Tamayo T, Rosenbauer J, Wild SH et al. Diabetes Res. Clin. Pract. 2014; 103:206-217. physician including a new six-figured code named WG14. This code 2. ISPAD Clinical Consensus Guidelines 2014; Pediatric Diabetes 2014:15(Suppl. 20):1-290. leads to a list of all fitting brand products when entered into the local 3. Obarcanin E, Krüger M, Müller P, Nemitz V et al. Int. J. Clin. Pharm. 2015: 37(5):790-8. software of any German pharmacy.

In February 2016, 1,552 community pharmacies were asked anonymously to evaluate the acceptance of this concept (Survey Monkey®). POS.82 Pharmacies were asked to assess the concept of generic prescribing in The impact of the implementation of an electronic general. Among others, participants were asked to indicate advantages prescribing software on the drug therapy safety in clinical and disadvantages of the concept for both pharmacies and patients. practice By the end of March 2016, a total of 628 pharmacies participated in the survey (response rate 40.5%). 69 % (n=433) assessed generic Ebbing, L.1; Kunze, T.1; Eisend, S.2 prescribing as useful or very useful. On average, the general concept of 1 Christian-Albrechts-University Kiel, Department of Clinical Pharmacy, Gutenbergstraße 76, preferred generic prescribing achieved a score of 2.4 on a scale of 1–6 24118 Kiel, Germany (1=very good to 6=very bad). Due to pharmacies major advantages for 2 University Medical Centre Schleswig-Holstein, Department Pharmacy, Arnold-Heller-Str. 3, 24105 Kiel, Germany patients are an enhancement of medication adherence and patient safety by avoiding unnecessary switching of the generic product A complex medication process can increase the amount of medication dispensed. errors, which might lead to a higher risk of adverse drug reactions. Prescribing an active ingredient instead of a brand name was highly Computerized physician order entry systems (CPOE-systems) may acknowledged by German pharmacies. Routine use of this concept in reduce medication errors - particularly prescribing errors - but may also combination with an electronic medication plan has the potential to increase them and thereof carry the risk of leading to patients harm [1]. enhance medication adherence and patient safety. The scientific observation of the implementation of electronic prescribing software is highly recommended [2]. References: In April 2014 the pharmaceutical employees of the University medical 1 ABDA-KBV-Modell/ARMIN: http://www.abda.de/themen/positionen-und-initiativen/armin/ centre Schleswig-Holstein started the rollout of the prescribing software MEONA with integrated clinical decision support system (CDSS). The objective of this study is to determine whether the implementation of MEONA has any impact upon the quantity and severity of medication POS.81 errors. Pharmaceutical care provided by community pharmacists Through visitation of various wards the operating cycles of the medical for adolescents with type 1 diabetes mellitus – follow up of and nursing personnel inside the medication process were documented DIADEMA and critical processes were identified. A medication error registration and classification system for prescription, transcription and dispensing Deters, M. A.1; Obarcanin, E.1; Läer, S.1 process was established. On four chosen wards over a period of each 1 Institue of Clinical Pharmacy and Pharmacotherapy Heinrich-Heine-University Duesseldorf four weeks total with handwritten prescriptions and four weeks after the implementation of the CPOE the occurrence of medication errors was Background: Diabetes mellitus is one of the most common diseases in recorded. childhood and the incidence increased over the past 20 years about 3- Drug-drug interactions were evaluated as one category of prescription 4% [1]. Micro- and macrovascular complications, due to poor metabolic errors by using pharmazie.com which is based on the ABDA-database. control, can lead to long-term complications such as high blood First results didn’t show any significant reduction - or increase - in pressure [2]. Especially in adolescence lower medication adherence is amount or severity of interactions, although the CDSS contains an a huge risk [2]. The DIADEMA study, a randomized controlled trial, has interaction-warning system. The evaluation of further medication error shown that community pharmacists can have a positive impact on categories is still in progress. therapy adjustment and glycemic control of adolescent diabetes patients [3].

118 • DPhG Annual Meeting 2016 Conference Book CLINICAL PHARMACY

References: 1. Bundesministerium für Gesundheit. Aktionsplan 2013/2015 zur Verbesserung der POS.84 Arzneimitteltherapiesicherheit in Deutschland. Bonn, 2013. Fostering interprofessional teamwork among pharmacy 2. AOK Bundesverband. HTA-Gutachten zur Beurteilung der Wirksamkeit von CPOE-Verfahren and medical students: A new patient-centred clinical zur Erhöhung der Arzneimitteltherapiesicherheit. Bremen, 2009. approach

Hopf, Y. M.1,2; Andraschko, M.1; Wahl-Schott, C.3; Fischer, M. R 2 1 University Hospital Munich, Department of Pharmacy, Marchioninistr. 15, 81377 Munich, Germany POS.83 2 University Hospital Munich, Institut für Didaktik und Ausbildungsforschung in der Medizin, Semi-mechanistic PK/PD modelling to characterise long- Ziemssenstr. 1, 80336 Munich, Germany 3 Ludwig-Maximilians-University, Department of Pharmacy, Pharmacology of Natural Sciences, term deterioration of neutropenia in cancer patients Butenandtstr. 5-13, 80336 Munich, Germany

Henrich, A.1,2; Joerger, M.3; Kraff, S.4; Jaehde, U.4; Huisinga, W.5; The WHO considers interprofessional education (IPE) as a prerequisite Parra-Guillen, Z. P.1; Kloft, C.1 for optimal patient care [1]. Studies have shown that IPE improves 1 Dept. Clinical Pharmacy & Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Kelchstr. 31, 12169 Berlin, Germany teamwork and encourages/promotes/facilitates understanding and 2 and Graduate Research Training program PharMetrX regard between different professions during working life [2-4]. The 3 Dept. of Oncology & Haematology, Cantonal Hospital St. Gallen, Rorschacher Strasse 95, standards published by the German Pharmaceutical Society (DPhG) 9007 St. Gallen, Switzerland 4 Dept. Clinical Pharmacy, Institute of Pharmacy, Universitaet Bonn, An der Immenburg 4, regarding education in the field of clinical pharmacy specifically request 53121 Bonn, Germany IPE sessions for pharmacy and medical students [5]. The Schools of 5 Institute of Mathematics, Universitaet Potsdam, Karl-Liebknecht-Str. 24/25, 14476 Potsdam Pharmacy and Medicine at the Ludwig-Maximilians-University (LMU) in Golm, Germany Munich partnered to foster a new IPE programme by joined and

simultaneous bedside teaching in clinical pharmacy. Background: Paclitaxel (PTX) exhibits complex pharmacokinetics The goals of this new IPE programme is to stimulate and encourage (PK), including non-linearity and high inter-individual variability. In interprofessional teamwork amongst undergraduates, to create an early addition, severe toxicity, especially neutropenia (pharmacodynamics opportunity to foster communication for medical and pharmacy (PD)) is observed, resulting in a narrow therapeutic window. The students, and to increase clinical experience for pharmacy students. combination of those two factors increase the need for dose- Preregistration medical students (post exam) were teamed up with individualisation and therapeutic drug monitoring. For the sparse PTX pharmacy students in their final year. The pharmacy students and neutrophil concentrations of the clinical trial “CEPAC-TDM”, a shadowed the medical students in their respective clinical settings. previously published PK/PD model [1] was evaluated to assess whether Students’ teams were based in all clinical settings, medical as well as it was able to describe the new data: Regarding PK, a slight surgical. Evaluation was done by pre/post testing of students’ underprediction of the PTX plasma concentrations was observed [2]; perception of IPE utilising SPICE 2D. Qualitative methods were used to regarding PD, worsening neutropenia over repeated chemotherapy evaluate additionally views and opinions regarding IPE after attending cycles was not captured by the PK/PD model. This behaviour was the course. Ethical approval was obtained from the LMU ethics hypothesised to be due to bone marrow exhaustion (BME) [2]. committee. Aim/Objectives: Based on the results of the external model evaluation, The pilot with 15 participants took place during summer term 2016, the first aim was to refine the PK model for the population in the since then further 110 students took part in the programme. SPICE 2D CEPAC-TDM trial. In a second step the PK/PD model was extended to evaluations in all groups showed an increase in appreciation of be able to describe long-term neutropenia by incorporating the interprofessional teamwork. Further statements from students after the hypothesised BME effect. IPE programme showed that participants in general enjoyed the Methods: In the CEPAC-TDM trial, non-small cell lung cancer patients interprofessional experience. Pharmacy students valued the “real life” (n=183) were treated with PTX (doses according to a published experience with discussion based upon real patients rather than case algorithm [1]) in combination with a carbo- or cisplatin every 3 weeks for studies. Medical students enjoyed in particular the exchange with a up to 6 cycles. PTX plasma concentrations (PK) were measured ~ 24 h different profession as well as the interest in patient care as displayed after drug administration, while neutrophil concentrations (PD) were by the pharmacy students. One particular point of criticism was that quantified on day 1 and 15±2 of each treatment cycle. For model pharmacy students were not able to apply their theoretical/university optimisation a stepwise analysis was performed: First, parameter knowledge within a new clinical context. Medical students were estimates from the published PK model were used as prior information disappointed as they hoped for more input from the pharmacy students utilised as prior information for re-estimation of the PK parameters with for example in terms of interactions, pharmacy students were often the new data assuming the same model structure. Second, the frustrated by seemingly too complex patient cases. Both student groups hypothesised BME was implemented in a semi-mechanistic way, by though wish for a continuation of the programme. extending the original Friberg et al. model [3]. For this purpose, one The IPE programme is set to be anchored in both curricula, pharmacy compartment accounting for slowly proliferating stem cells was added and medicine. Extended evaluation will take place another term in order as a first step in the maturation chain of neutropoiesis. For all modelling to implement and trial recommendations from previous term activities NONMEM 7.3 in combination with PsN 4.4 was used. evaluations. In addition to questionnaire evaluations two focus groups Results: An improved prediction of PTX concentrations was achieved will be conducted with the participants of summer term 2016. for the CEPAC-TDM study population. Further, the optimised PK/PD model, including the BME effect, was able to describe the observed Acknowledgements: We would like to thank Dr Patricia Raes, Dr Yvonne Kosanke and long-term worsening of neutropenia over the treatment cycles. For this Professor Angstwurm for their help in recruiting medical students. PK/PD model a high PD parameter precision (RSE < 10%) was References: achieved. The proliferation rate constant of the progenitor cells was 3.7- 1. World Health Organization. Learning together to work together for health-Report of a WHO fold higher than the one of the stem cells. Study Group on Multiprofessional Education of Health Personnel: the Team Approach. World Conclusions: An adequate description of PTX PK was achieved by Health Organization Technical Report Series 769, 1988. Geneva: WHO. combining previous knowledge from the former rich data with the 2. Olson R, Bialocerkowski A. Med. Educ. 2014;48:236-246. sparse CEPAC-TDM data. To account for long-term neutropenia, a 3. Stößel U KK, Kaba-Schönstein L. GMS Z. Med. Ausbild. 2006;23:Doc34. 4. Zwarenstein M, Goldman J, Reeves S. The Cochrane database Syst. Rev. mechanistically plausible PK/PD model was developed that adequately 2009;3:CD000072. described the hypothesised BME. Ultimately, the model shall be used to 5. Jaehde U (für die Fachgruppe Klinische Pharmazie der Deutschen Pharmazeutischen further improve individualised dosing of PTX to reduce toxicity. Further, Gesellschaft, DPhG). DAZ 2004, 144;15: 1743-1746. due to the mechanistic approach, the structure of the PK/PD model can also be beneficial for optimisation of long-term therapy of cancer patients with cytotoxic drugs exhibiting neutropenia as dose-limiting toxicity.

References: 1. Joerger, M. et al.: Clin. Pharmacokinet. 2012, 51(9): 607–17. 2. Henrich, A. et al.: PAGE 24 2015, Abstr. 3460. 3. Friberg, L. E. et al.: J. Clin. Oncol. 2002, 20(24): 4713–21

DPhG Annual Meeting 2016 Conference Book • 119 POSTERS

Phytomedicines as standard therapeutics – this has been the case long POS.85/SL.43 ago, once upon a time, when chemically defined medicines had not yet Evidence-based evaluation system for OTC drugs been discovered. So is the common view. But it is worthwhile to ask, whether this is also true today. Obviously, today, certain circumstances Achenbach, J.1; Culmsee, C.1 are needed to make this true. 1 Institut für Pharmakologie und Klinische Pharmazie, Philipps-Universität Marburg, Karl-von- A recent example is the therapy of functional gastrointestinal diseases. Frisch-Straße 1, 35032 Marburg Prokinetics had become standard therapeutics in the 1980ies and 1990ies, i.e. substances like cisapride, metoclopramide and For abstract see Short Lecture SL.43 domperidon, which act via serotonin receptors. Since 2000 cisapride, since 2014 metoclopramide and domperidon are no longer available in this indication due to rare but severe side effects. The phytomedicine Iberogast, which had been shown to be equivalent to metoclpramide POS.86 already in 1984, and to cisapride in 2002, and which had been included to the medical therapy guidelines in Germany already in 1999 resp. STW 5 in functional gastrointestinal diseases: 2001, has now in many cases taken the place of the former standard Metaanalysis of clinical trials therapeutics. Another example is St. John´s wort, which was included to the German 1 1 1 2 3 4 Müller, J. ; Rabini, S. ; Abdel-Aziz, H. ; Kelber, O. ; Storr, M. ; Kraft, K. national medical therapy guideline on unipolar depression 2009 after 1 Medical Affairs, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany equivalence to chemical standards had been shown in clinical trials 2 Innovation & Development, Phytomedicines Supply and Development Center, Bayer repeatedly. Also e.g. ginkgo has good reputation in its indication, the Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, therapy of dementia. Germany 3 Zentrum für Endoskopie Starnberg, Oßwaldstraße 1, 82319 Starnberg, Germany, und But as more than 80 % of all phytomedicines are used within the frame Medizinische Klinik und Poliklinik II, Klinikum der Universität München, Campus Großhadern, of self-medication, also the recommendation by the pharmacist has to Marchioninistraße 15, 81377 München, Germany be taken into account when looking for standard therapeutics. Here e.g 4 Zentrum für Innere Medizin der Universitätsmedizin Rostock, Lehrstuhl für Naturheilkunde, Ernst-Heydemann-Straße 8, 18057 Rostock, Germany cough and cold phytomedicines like primrose root/thyme combinations or marshmallow root extract are used widely. But here the way to Introduction: The gastro-prokinetics, such as metoclopramide, become standard therapeutics might not yet have been finalized. domperidone and cisapride, had been introduced to the therapy of Taken together, the uptake of phytomedicines to medicinal guidelines functional gastrointestinal diseases in the 70s and 80s, and for a long and their acceptance as standard therapeutics in daily practice rep. in time they seemed to be standard therapeutics in this indication. Since the recommendations by pharmacists has in some cases already taken they are no longer available for the use in this indication due to place in the last years. But there is still much more to be done in this restrictions of their marketing authorizations based on rare but severe field within the next few years. side effects, other well proven therapeutic options gain increased attention. One of these is STW 5 (Iberogast), for which more than 5 decades of therapeutic experience in more than 60 Mio patients are available. POS.88 Aims & Methods: Whether also the available clinical data comply to modern standards for a proof of efficacy was now tested by a meta- Does pre-coating of probes enable in vitro and in vivo analysis of the randomized placebo-controlled double blind trials microdialysis investigation of anidulafungin? available in the therapeutic indication functional dyspepsia. The individual datasets from all trials were entered and demografic data Weiser, C.1; Zeitlinger, M.2; Kloft, C.1 1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, and primary endpoints were evaluated (ANCOVA). Kelchstraße 31, 12169 Berlin, Germany Results: The primary outcome variable, the validated gastrointestinal 2 Dept. of Clinical Pharmacology, Medical University of Vienna, Währinger Gürtel 18-20, symptom score (GIS) [1], as well as the therapeutic dose (3 x 20 1090 Vienna, Austria drops/day) were identical in all trials, so allowing a uniform evaluation. The full analysis set (FAS) included 557 patients (272 resp. 285 for Objectives: Microdialysis (µD) is a minimally invasive technique, which placebo resp. verum). The mean age (48 resp. 49 years), the mean can serve in clinical settings to determine the drug concentrations body size (in both groups 168.7 cm), the mean body weight (72.0 resp. directly at the target-site e.g. in the interstitial fluid of various organs and 72.2 kg), the BMI (25.35 resp. 25.54), the gender disitribution (67.3 tissue. This technique enables the subsequent collection of unbound resp. 69.5 % females), the duration of the disease at the time of drug molecules, defined as the pharmacologically active fraction of the inclusion and the baseline of the GIS (11.6 resp. 11.5 points) were very drug, due to the diffusion through a semipermeable membrane. well comparable between both groups. For the primary variable GIS the Anidulafungin (AFG) is a lipophilic echinocandin antifungal drug and a difference between placebo and verum after 28 days of treatment treatment option for invasive candidiasis in non-neutropenic adult showed a highly significant (p< 0.0001) difference between placebo and patients and shows high adsorption on probe material as membrane verum (6.7 resp. 4.7 points). and tubing [1]. An in vitro µD investigation was performed to investigate Conclusions: This meta analysis therefore clearly shows the efficacy the influence of a (pre-)coating approach of µD probes to block the of STW 5 (Iberogast) according to present standards of evidence-based potential binding sites prior and during the actual in vitro µD medicine (EBM). In addition it emphasizes the high quality of the trials, investigation with AFG. Hence, the influence of (pre-)coating the probe which is indicated e.g. by the very good balance between both patient with AFG containing perfusate was investigated with regard to the groups. Additional insights can be expected from further analyses, as relative recovery (RR) of AFG in the µDialysate. e.g. the evaluation of different subgroups with specific predominant Methods: Two 8 h in vitro µD-investigations were performed symptoms or demographic properties. consecutively with the same CMA71 probes (100 kDa cut-off) and as perfusate and probe-surrounding medium a mixture of Ringer`s solution References: (RS) and human serum albumin solution (HSA) (0.5%, v/v). The AFG 1. Adam et al. Aliment Pharmacol Ther 2005; 22: 357-363 concentration in the probe-surrounding medium was 1 µg/mL for (i) uncoated probes and (ii) AFG coated probes. In-between (i) and (ii), probes were pre-coated with the AFG containing perfusate (1 µg/mL) in AFG-free probe-surrounding medium for 16 h. Coating was continued during the actual recovery investigation. Microdialysate (µDialysate) POS.87 was collected (nprobes=2) in 40 min intervals over each 8 h Phytomedicines as standard therapy - wishful thinking or investigation as well as 3 40 min samples at the end of the in-between reality? pre-coating period (flow rate of 1 µL/min). Quantification of AFG was performed with a developed and validated HPLC assay (XBridge BEH Kelber, O.1; Kraft, K.2 C18 column (50 x 3.0 mm, 2.5 µm), isocratic method with mobile 1 Innovation & Development, Phytomedicines Supply and Development Center, Bayer phase: methanol and ammonium dihydrogen phosphate 80:20 (v/v)). Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany The concentration of AFG in the µDialysate samples and the RR were 2 Zentrum für Innere Medizin der Universitätsmedizin Rostock, Lehrstuhl für Naturheilkunde, Rostock, Germany

120 • DPhG Annual Meeting 2016 Conference Book CLINICAL PHARMACY calculated and then results from (i) and (ii) were compared with each in urine of both dosing regimens for the first 24 hours after start of the other. treatment. Results: The mean (n=7 each) AFG concentration of the probe- Results: The results from the simulations performed with the adapted surrounding medium and of the perfusate of probe 1 was 1.139 µg/mL populationPK model showed a similar shape in the cumulated urine (CV 3.96%) and 1.149 µg/mL (CV 8.80%) and of probe 2 was 1.103 amounts published by Pea et al. (329.1, 388.6, 266.0 and 168 mg/L) for µg/mL (CV 4.17%) and 1.181 µg/mL (CV, %: 2.51), respectively. AFG the four different collection intervals: 0-2, 2-4, 4-8 and 8-12 hours [2]. was not detectable in the first µDialysate (0-40 min collection interval) For the mean Vu fixed to 1.71 L the concentrations for the four different sample of both uncoated probes for (i). RR of AFG in µDialysate during intervals were 460, 590, 480 and 320 mg/L. The maximum Vu of 2.35 L the 8 h of investigation for (i) increased over time from 1.57% to 32.5% resulted in smaller concentrations of 360, 450, 345 and 220 mg/L for (probe 1) and from 0.6% to 34.6% (probe 2) (n=11 µDialysate samples the different collection intervals and described the observed data more per probe). Compared to that, the AFG RR in µDialysate samples of (ii) adequately. For the simulations of the urine concentration-time profiles also increased over time and ranged from 62.2% to 107% (probe 1) and of LEV, we fixed the Vu to 2.35 L. The regimen 500 mg LEV bid resulted from 49.6% to 112% (probe 2) (n=12 µDialysate samples per probe). in a maximum LEV urine concentration around 425 mg/L after 14 hours After subtracting the mean AFG concentration from the µDialysate in and a minimal concentration of 125 mg/L after 12 hours before the next the 3 40 min in-between pre-coating period of 0.552 µg/mL (probe 1) LEV dose. The other regimen 750 mg qd showed a maximum LEV and 0.511 µg/mL (probe 2) from the AFG concentration from (ii), the concentration of 480 mg/L after 2 hours and a minimum concentration RR ranged from 13.7% to 58.9% (probe 1) and from 7.72% to 69.9% of 50 mg/L after 24 hours before the next dose. (probe 2), respectively. Conclusions: The urine concentrations from the simulations performed Conclusions: The comparison of RR in the two investigations showed with the adapted populationPK model were in agreement with the urine higher RR values in the pre-coated than in the uncoated probes. A concentrations by Pea et al.. Since we are interested in the patient plausible reason is that due to pre-coating, AFG molecules from population at risk for therapy failure, we performed simulations with the perfusate (partly) block the potential binding sites of diffused AFG higher Vu. The simulated concentration-time profiles of LEV were overall molecules from the probe-surrounding medium. Still, RR of AFG from plausible with regard to their maximal and minimal concentrations at the pre-coated probes increased during the 8 h investigated. With these respective time-points. The current investigation showed how in silico findings we conclude that pre-coating of probes with AFG in perfusate models streamline in vitro experiments exploring published clinical data helps blocking potential binding sites of AFG and increase the RR to investigate treatment efficacy at the target site, i.e. the antibiotic values but a constant RR value is not reached earlier than for uncoated effect on bacteria in catheterised ICU patients. probes, hence limiting its use in clinical µD studies in humans. References: References: 1. Schaeftlein, A. et al.: Abstract 2359, PAGE 21, Venice, Italy 2012. 1. Weiser et al.: 26th ECCMID, Amsterdam, Netherlands. 2016: EV0652 2. Pea, F. et al.: Journal of Chemotherapy 2003, 15(6): 563-567.

POS.89 POS.90 Treatment of catheterised ICU patients with levofloxacin- Identification of phase-II metabolites from serum samples How in silico models help to streamline in vitro of a human pharmacokinetic study with willow bark investigations of treatment efficacy at the target site. (Salicis cortex)

Goebgen, E. B.; Hartung, N.; Seeger, J.; Schaeftlein, A.; Kloft, C. Untergehrer, M.1; Knuth, S.1; Jürgenliemk, G.1; Heilmann, J.1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, 1 Faculty for Chemistry and Pharmacy; Pharmaceutical Biology; University of Regensburg; Kelchstr. 31, 12169 Berlin, Germany Universitätsstr. 31; D-93053 Regensburg; Germany

Objectives: Intensive care unit (ICU) patients are at risk of bacterial Willow bark (Salicis cortex, Salix ssp., Salicaceae) is used against low infections due to their impaired immune system. Catheterisation back pain and mild osteoarthritic and rheumatic complains as well as increases this risk with decreased susceptibility to antibiotics, as against minor articular pain, fever associated with common cold and catheterisation is highly linked to biofilm formation. Patients suffering headache. Whereas the efficacy of willow bark was shown in several from ventilator-associated pneumonia are often catheterised and are clinical studies, the principle of action is not fully understood. Although treated with antibiotics, e.g. the fluoroquinolone levofloxacin (LEV), with the anti-inflammatory active compound salicylic acid is known as a high renal elimination active against most probable pathogens. metabolite of salicylic alcohol derivatives, its plasma-concentration is Currently two dosing regimens of LEV are given in the ICU setting: 500 much too low to explain the efficacy of the whole willow bark extract [1]. mg bid i.v. and 750 mg qd i.v.. To investigate the pharmacodynamic To broaden the knowledge about the metabolisation of absorbed (PD) efficacy in urine of the different dosing regimens in vitro, the compounds from a willow bark extract, serum samples of a human concentration-time profile of LEV in urine is needed. However, data in pharmacokinetic study (10 volunteers, 12 h fasting time, controlled diet literature is only considering cumulated antibiotic amount from collection poor in phenolics, 13 blood withdrawals over a period of 24 h) [2] were intervals between 1.5 to 4 hours. Hence, this study aimed at using the analysed by LC-ESI-MS. A library of 116 possible phase-II metabolites concentration of the collection intervals to adapt an existing population concerning salicylic alcohol derivatives, flavonoids, and pharmacokinetic (populationPK) model to simulate the urinary proanthocyanidins was used to identify possible metabolisation concentration-time profile of LEV in silico in order to streamline in vitro products. In the serum samples, phase-II metabolites of naringenin (2x investigations. glucuronide, 2x sulfate, 2x glucuronide-sulfate), eriodictyol (3x Methods: LEV is known to rapidly penetrate into tissue and to be glucuronide, 1x sulfate), taxifolin (1x sulfate), catechin (1x sulfate, 1x linearly eliminated via the kidneys. These properties are accounted for glucuronide-sulfate), ferulic acid (1x sulfate), hydroxyphenyl-propionic in the 2 compartmental populationPK model [1] with a fast distribution acid (1x sulfate), saligenin (1x glucuronide, 1x sulfate), salicylic acid (1x rate constant (k12=2.78 h-1) from the central (V1=21.7 L) to the sulfate, 1x free, 1x salicyluric acid), and catechol (1x glucuronide, 1x peripheral (V2=64.4 L) compartment and a back-distribution rate sulfate) could be identified. As taxifolin, ferulic acid, and hydroxyphenyl- constant k21 (0.936 h-1). From V1, LEV is eliminated linearly propionic acid were not present in the extract, they could be (k13=0.512 h-1). The mean fraction of LEV excreted unchanged in urine metabolisation products of eriodictyol or naringenin and coumaric acid was fixed to 0.761 [2]. We extended the populationPK model by a or C-ring cleaved flavonoids, respectively, which could be found in the urinary tract compartment with the remaining urine volume in the willow bark preparation. No phase-II metabolites of procyanidins and no bladder (V3=0.15 L) and the LEV excretion rate constant (k34) to the genuine flavonoid glycosides could be detected in all serum samples. catheter bag as further parameters. The collected volume of urine (Vu) These results confirm the demand to use not only isolated compounds in 24 hours was fixed to 1.71 (±0.64) L or 2.35 L, respectively, from plant extracts for in vitro tests but also to consider their representing the mean (standard deviation) or the maximum value [2]. metabolisation products to increase the relevance of obtained Based on that, the rate constant k34 was derived from Vu eliminated per pharmacological data [3]. hour divided by V3. Simulations using these PK parameters were performed and compared with the data by Pea et al.. Finally the parameters were used to simulate the concentration-time profile of LEV

DPhG Annual Meeting 2016 Conference Book • 121 POSTERS

References: Acknowledgments: We gratefully acknowledge the contributions to sample and data collection 1. Nahrstedt, A. et al.; Wien Med. Wochenschr. 2007, 157: 348-351. made by volunteers and appreciate the excellent medical and technical assistance of A. Dehn, S. Griesbach, M. Kastner, J. Lockowandt, A. Steenpass, and J. Wiener. Dr Atzler acknowledges 2. Knuth, S. et al.: Planta Med. 2013, 79: 1489-1494. the support of the European Community under a Marie Curie Intra-European Fellowship for 3. Kroon, P. A. et al.: Am. J. Clin. Nutr. 2004, 80: 15-21. Career Development and Dr Choe was funded by an Else Kröner Memorial Stipendium from the Else Kröner-Fresenius-Stiftung. This work was funded by LMU Munich‘s Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative (DA).

References: POS.91 Atzler D et al.: Curr. Opin. Clin. Nutr. Metab. Care. 2015, 18(1):83-8. Evaluation of kinetic and dynamic parameters of oral L-homoarginine supplementation in young volunteers

Cordts, K.1,2; Schönhoff, M.1; Hoppe, J.3; Ortland, I.4; Hummel, F. C.3; Gerloff, C.3; Jaehde, U.4; Jagodzinsk, A. 2,5; Böger, R. H. 1,2; Atzler, D.1,2,6; Choe, C.2,3; Schwedhelm, E.1,2 1 Institute of Clinical Pharmacology and Toxicology, UKE, Martinistr. 52, 20246 Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Department of Neurology, UKE, Martinistr. 52, 20246 Hamburg, Germany 4 Institute of Pharmacy, Department of Clinical Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 5 Department of General and Interventional Cardiology, University Heart Center Hamburg- Eppendorf, Martinistr. 52, 20246 Hamburg, Germany 6 Vascular Biology, Institute for Stroke and Dementia Research, Klinikum der Universität München, Ludwig Maximilians-University of Munich

Background: L-Homoarginine (L-hArg) is a naturally occurring amino acid that differs from L-arginine by an additional methylene group. Formation of L-hArg from L-lysine and L-arginine is catalyzed by the L- arginine:glycine amidinotransferase (reviewed in Atzler et al. 2015). Median (25th; 75th percentile) plasma L-hArg concentration in healthy humans is 1.88 (1.47; 2.41) µM, and L-hArg is a natural component of pulses, e.g. Lathyrus species. L-hArg competes with L-arginine as a weak substrate for endothelial nitric oxide synthase and weakly inhibits arginase. Low L-hArg plasma concentrations have been associated with a higher risk for cardiovascular, cerebrovascular, and kidney diseases as well as for cardiovascular and all-cause mortality. The aim of this study was to investigate kinetic and dynamic properties of single and multiple oral doses of 125 mg L-hArg in twenty young healthy subjects. Subjects: Twenty apparently healthy Asian-Caucasian volunteers (11 female, 9 male) completed this study. The median (25th; 75th percentile) age was 28.5 (24.3; 48.8) years and the body mass index was 24.1 (22.9; 25.7) kg/cm2. Median hArg plasma concentration at baseline examination was 2.98 (2.32; 3.62) µmol/L. Study design: Participants received either 125 mg L-hArg (Wellnest International Ltd., West Sussex, UK) or placebo once daily in the morning for four weeks each in a randomized, double-blind, placebo- controlled cross-over design, interrupted by wash-out phases of four weeks. Primary endpoint was the evaluation of kinetic parameters, i.e. maximal concentration (Cmax), time-to-peak (Tmax), and area under the plasma concentration-time curve (AUC0-24hrs) after single and multiple doses of L-hArg. As secondary endpoints routine laboratory parameters, plasma L-arginine and asymmetric dimethylarginine (ADMA) concentrations, pulse wave velocity (PWV), augmentation index (AIx), flow-mediated vasodilatation (FMD), and transcranial magnetic stimulation (TMS) were evaluated at baseline and four weeks after each supplementation period. Results: Maximal L-hArg plasma concentrations were reached one hour after ingestion (Tmax). Oral single and multiple doses of 125 mg L- hArg increased baseline L-hArg plasma concentration by 8.74 4.46 [95% confidence intervals 6.65; 10.9] and 17.3 4.97 [14.9; 19.6] µmol/L (Cmax), respectively. AUC0-24hrs were 63.5 28.8 [50.0; 76.9] and 225 78.5 [188; 2624] µmol/L*h for single and multiple doses, respectively. Blood glucose was increased after L-hArg supplementation (p<0.05). Alkaline phosphatase activity was increased after placebo treatment and at four weeks of follow-up (p<0.05 for both). Other laboratory parameters, L-arginine, ADMA, PWV, AIx, FMD, TMS did not change significantly after four weeks of L-hArg ingestion. Adverse events were equally distributed in both study arms. Summary and Conclusion: Oral supplementation with 125 mg L-hArg once daily for four weeks is safe and suitable to elevate L-hArg plasma concentrations in humans. With this study we paved the way for larger, prospective clinical studies to investigate the benefit of L-hArg supplementation in patients with cardiovascular or metabolic disease.

122 • DPhG Annual Meeting 2016 Conference Book DRUG DESIGN/MEDICINAL CHEMISTRY

POS.93 3.6 Drug Design/Medicinal First 17β-Hydroxysteroid Dehydrogenase Type 14 Inhibitors and their 3D-Structures in Complex with the Chemistry Enzyme: Important Tools for the Characterization of a new Protein

POS.92 Marchais-Oberwinkler, S.1; Braun, F.1; Bertoletti, N.1; Möller, G.2; Adamski, J.2,3,4; Salah, M.5; Van Koppen, C. J. 5; Heine, A.1; Klebe, G.1 Discovery of orally available dual sEH/PPARγ modulators 1 Philipps University Marburg, Institute for Pharmaceutical Chemistry, Marbacher Weg 6, for simultaneous treatment of hyperglycemia and 35037 Marburg, Germany 2 Helmholtz Zentrum München, German Research Center for Environmental Health, hypertension in metabolic syndrome Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstraße 1, 85764 Neuherberg, Germany 3 1 1 1 1 1 Lehrstuhl für Experimentelle Genetik, Technische Universität München, 85350 Freising- Blöcher, R. ; Lamers, C. ; Wittmann, S. K. ; Merk, D. ; Hartmann, M. ; Weihenstephan, Germany Weizel, L.1; Diehl, O.1; Brüggerhoff, A.1; Boß, M.2; Kaiser, A.1; Schader, 4 German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany T.1; Göbel, T.1; Grundmann, M.3; Angioni, C.4; Heering, J.5; Abdul, H. 5 ElexoPharm GmbH, Campus A12, 66123 Saarbrücken, Germany K.6; Geisslinger, G.4; Wurglics, M.1; Kostenis, E.3; Brüne, B.2; Steinhilber, D.1; Imig, J. D.6; Schubert-Zsilavecz, M.1; Kahnt, A. S.1; 17β-HSD141 belongs to the SDR family and oxidizes the position 17 of Proschak, E.1 estradiol and 5-androstenediol using NAD+ as cofactor. As inhibitors are 1 Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, useful tools to characterize enzymes, the goal of this study was to D-60438 Frankfurt a. M., Germany. identify the first inhibitors of 17β-HSD14. In a preliminary study a library 2 Institute of Biochemistry I, Goethe-University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt a. M., Germany. of 17β-HSD1 and 17β-HSD2 inhibitors, selected with respect to scaffold 3 Institute of Pharmaceutical Biology, Rheinische Friedrich-Wilhelms-Universität Bonn, diversity, was tested for 17β-HSD14 inhibition. The most interesting hit Nussallee 6, D-53115 Bonn, Germany. was taken as starting point for chemical modification of the initial lead 4 Institute of Clinical Pharmacology, Goethe-University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt a. M., Germany. applying a ligand-based approach. The designed compounds were 5 Project Group Translational Medicine and Pharmacology TMP, Fraunhofer Institute for synthesized and tested for 17β-HSD14 inhibitory activity with a Molecular Biology and Applied Ecology IME, Theodor-Stern-Kai 7, D-60590 Frankfurt a. M., fluorescence-based assay using the recombinant purified protein, Germany. + 6 Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, estradiol as substrate and NAD as cofactor. The best inhibitors WI 53226, USA identified will be presented as well as their binding mode in the enzyme’s active site, which was elucidated after analysis of the The cardiometabolic syndrome (MetS) is a multifactorial disease cluster crystallographic structures2. The selectivity of the best compounds consisting of dyslipidemia, cardiovascular disease, type 2 diabetes towards 17β-HSD1 and 17β-HSD2 will also be addressed. mellitus and obesity. Pharmacological intervention in the MetS is dependent on numerous drugs, thus polypharmacy is an obvious References: problem in the treatment of MetS patients. This study focuses on the 1. Lukacik, P.; Keller, B.; Bunkoczi, G.; et al. Biochem. J. 2007, 402(3): 419–427 dual target approach to accomplish a more efficient therapy for MetS. 2. Bertoletti, N.; Braun, F.; Lepage, M.; et al. J. Med. Chem. 2016, DOI: The two targets addressed by dual ligand design are the soluble 10.1021/acs.jmedchem.6b00293. epoxide hydrolase (sEH) and the peroxisome proliferator-activated receptor type γ (PPARγ). In vivo studies could demonstrate that even though an inhibitor of sEH or PPARγ agonist have benefits when used individually, the combination is more beneficial for the multidisease POS.94 features in cardiometabolic syndrome [1]. Using a split-and-combine Design and synthesis of a second-generation clickable, strategy we designed a library of dual sEH/PPARγ modulators and proved that both targets can be simultaneously addressed by a merged photoreactive cholesterol analogue pharmacophore [2,3]. In a follow-up study, we designed lead-like 1 1 merged N-benzyl benzamides which were able to modulate sEH and Bartl, N. ; Bracher, F. 1 Department of Pharmacy – Center for Drug Research, Ludwigs-Maximilians-University, PPARγ. Structure activity relationship studies on both targets were Butenandtstr. 5-13, D-81377 Munich, Germany performed resulting in an equipotent submicromolar (IC50 (sEH) = 0.3 µM/ EC50 (PPARγ) = 0.3 µM) propionic acid N-benzyl benzamide Cholesterol is the most abundant sterol lipid in mammalian cells. It and derivative. Evaluation in vitro and in vivo displayed good ADME its metabolites play a major role in regulating diverse biological properties qualifying the novel dual modulator as pharmacological tool processes. Alterations in these processes are presumed to be compound for long term animal models of MetS [4]. 8-week evaluation associated with various diseases like arteriosclerosis or Alzheimer’s in spontaneously hypertensive obese rats (SHROB), a rat model of disease. Located in membranes, cholesterol is an essential structural MetS, demonstrated excellent efficacy including simultaneous reduction component, but also acts by direct interaction with membrane proteins of blood pressure, improvement of glucose tolerance, and organ [1]. Very limited knowledge exists about this regulating interaction, protection. These results could be confirmed in an 8-week curative mainly because it is not based on a classical ligand-receptor interaction study in ZSF1 rat model of MetS. [1,2]. In order to identify and examine more closely these sterol-binding proteins, we designed and synthesized a novel cholesterol-based molecule as a chemoproteomic tool. The synthesized molecule bears a photoreactive diazirine group for photoaffinity labeling (PAL) as well as a terminal alkyne moiety for the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) to conjugate any reporter azide (containing e.g. biotin or fluorescent dyes) via “click-chemistry”. To ensure that the sterol core is fully recognized by the target membrane proteins, we left the tetracyclic sterol structure untouched. Both the diazirine and the alkyne moieties were incorporated into the side chain, avoiding hydrolysis-labile functional groups and exceeding the size of the original

Acknowledgments: This work was supported by the Else-Kröner-Fresenius Foundation and cholesterol side chain too much. These two advantages, a native Deutsche Forschungsgemeinschaft (DFG; Sachbeihilfe PR1405/1-2; SFB 1039 Teilprojekt steroid core and hydrolytic stability, make this new molecule superior to A07). R.B., O.D. and M.B thanks to the graduate college Translational Research Inovation earlier described clickable, photoreactive sterol probes [3] and suitable Pharma (TRIP) for the PhD fellowship. The abstract figure is Courtesy of University of California Television. for bioorthogonal in vivo studies in any compartment of cells.

References: References: 1. Imig J.D. Exp Biol Med 2012, 237, 1402. 1. Song, Y. et al.: Protein Sci. 2014, 23(1): 1-22 2. Blöcher R. et al. J. Med. Chem. 2012, 55, 10771-10775 2. Peng, T. et al.: Curr. Opin. Chem. Biol. 2014, 21: 144-153 3. Blöcher R, et al. Med. Chem. Comm. 2016, 7, 1209-1216 3. Hulce, J. et al.: Nature Methods 2013, 10(3): 259-264 4. Blöcher R. et al. J. Med. Chem. 2016, 59, 61-81

DPhG Annual Meeting 2016 Conference Book • 123 POSTERS

POS.95 New approaches to β-carbolines via indole-2-Weinreb amides

Kamlah, A.; Lirk, F.; Bracher, F. Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians-University, Butenandtstr. 5-13, 81377 Munich, Germany

β-Carbolines are a large and structurally diverse class of alkaloids from All synthesized molecules were submitted to a homogenous plants, microorganisms, and marine organisms, showing a broad range fluorescence based assay for Sirt2 inhibition. of biological activities. Numerous approaches to 1-substituted β- carbolines have been worked out over the decades, and most of these Acknowledgments: This project was funded by DFG-Förderung GRK1976. approaches start from tryptophan or tryptamine derivatives. References: Unfortunately, tryptamines bearing additional substituents on the 1. K. Huber et al., J. Med. Chem. 2009, 53: 1383–1386. benzene ring are poorly available, and consequently, 1-substituted β- carbolines with additional residues on ring C are accessible only via multistep procedures. In recent projects, we identified highly substituted β-carbolines as lead POS.97 structures for the inhibition of the protein kinase DYRK1A [1] and as Development of benzothiazoles as dual 5-lipoxygenase and promising new antivirals based on the inhibition of the kinase CLK1 [2]. For being able to perform comprehensive analysis of structure-activity microsomal prostaglandin E2 synthase-1 inhibitors relationships in these chemotypes, we worked out new approaches to 1 2 2 1 polysubstituted β-carbolines. As starting materials we selected indole-2- Cheung, S.-Y. ; Werner, M. ; Werz, O. ; Schubert-Zsilavecz, M. , 1 carboxylates, which are, with a broad variety of substituents on the Hanke, T. 1 Goethe University of Frankfurt am Main, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, benzene ring, readily available via established methods. Conversion of Germany these compounds into Weinreb amides opened the opportunity to 2 Friedrich-Schiller University of Jena, Philosophenweg 14, 07743 Jena, Germany introduce variable residues R’ utilizing respective organometallic compounds, and completion of ring A (pyridine moiety) of the target Prostaglandins (PGs) and leukotrienes (LTs) are powerful bioactive lipid compounds was accomplished with different building blocks. mediators that have a large number of biological actions in the human body [1], [2]. The common precursor of PGs and LTs is arachidonic acid (AA). The 5-lipoxygenase (5-LO) and the microsomal prostaglandin E2 synthase-1 (mPGES-1) are both enzymes within the arachidonic acid cascade. 5-LO is the initial enzyme which catalyzes the conversion of AA to the corresponding LTs; whereas the mPGES-1 is responsible for the transformation of PGH2 into PGE2 which is one of the most prominent mediators of inflammation, pain and fever. A valuable pharmacological approach for anti-inflammatory therapy is the dual inhibition of 5-LO and mPGES-1. In contrast to the traditional NSAIDs References: the dual inhibition of PGs and LTs might be superior over single 1. Rüben K. et al.: PLOS ONE 2015, 10(7):e0132453. interference with PGs in terms of anti-inflammatory effectiveness as 2. Karlas A. et al.: Nature Communications 2016, 7:11320. well as regarding reduced side effects [3]. In the post area of selective

COX-2 inhibitors different approaches for dual inhibition of PGs and LTs

have been pursued, like dual COX/LO, dual COX-2/LTA4-Hydrolase or

dual 5-LO/mPGES-1 inhibitors [4, 5, 6, 7]. Within the dual 5- POS.96 LO/mPGES-1 inhibitors the pirinixic acid derivatives are the most Synthetic approach to novel 4-substituted advanced one. However pirinixic acid derivatives are well known + compounds with many various biological activities especially PPARα 3-arylideneindolin-2-ones as NAD dependent histone and PPARγ activation [8]. Therefore, in this series we replaced the deacetylase (sirtuin) inhibitors central scaffold of the pirinixic acid, the chlorinated pyrimidine core, by a benzothiazole, which was identified by a virtual screening approach [9]. Ong, N.1; Swyter, S.2; Jung, M.2; Bracher, F.1 Here we present the synthesis and in vitro pharmacological 1 Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-University, Butenandtstr. 5-13, 81337 Munich, Germany characterization of the benzothiazole derivatives and we were able to 2 Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, identify compounds, which are about equally potent to the most potent 79104 Freiburg i. Br., Germany pirinixic acid derivatives.

Previous work on 3-arylidene-indolin-2-ones in our group yielded two References: compounds showing promising biological activities on Sirt2 [1]. 1. Funk, C. D.: Science 2001, 294(5548): 1871−1875. 2. Samuelsson, B., Morgenstern, R., Jakobsson, P. J.: Pharmacol Rev 2007, 59(3): 207–224. 3. Koeberle, A., Werz, O.: Curr. Med. Chem. 2009, 16(32): 4274–4296. 4. Celotti, F., Laufer, S.: Pharmacol Res. 2001, 43(5): 429-436. 5. Chen, Z. et al.: J Med Chem 2011, 54(10): 3650-3660. 6. Koeberle, A. et al.: J Med Chem. 2008, 51(24): 8068-8076. 7. Hanke, T. et al.: J Med Chem. 2013, 56(22): 9031-9044. 8. Merk, D. et al.: Future Med Chem. 2015, 7(12): 1597-1616. 9. Waltenberger, B. et al.: J. Med. Chem. 2011, 54(9): 3163–3174. Docking studies indicated that Sirt2 contains additional space in binding pocket that could be covered by an additional substituent at C-4 of the indoline-2-one moiety. This prompted us to introduce diverse substituents at said position for SAR studies. As the established synthetic pathway was not suitable for synthetic POS.98 modifications at the said position we strived to develop a new synthetic Novel antifungal 2-arylindoles approach allowing the introduction of diverse substituents.

Luber, M.1; Stadler, M.1; Bracher, F.1 1 Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians-University, Butenandtstr. 5-13, 81377 Munich, Germany

In the course of a project aimed at the optimization of indole-derived inhibitors of the protein kinase CLK1, a series of 2-arylindoles was

124 • DPhG Annual Meeting 2016 Conference Book DRUG DESIGN/MEDICINAL CHEMISTRY prepared. Routine testing of all intermediates in our standard agar diffusion assay for antifungal and antibacterial activities gave surprising POS.100/SL.33 results for some of these indoles, as well as their synthetic precursors Rational design of thermodynamic and kinetic binding (hydrazones). High in vitro activity against some fungi (Yarrowia profiles by optimizing surface water networks coating lipolytica, Candida glabrata, Hyphopichia burtonii) was observed, but protein bound ligands the compounds did not show any antibacterial effects against gram- negative and gram-positive bacteria. Krimmer, S. G.1; Cramer, J.1; Betz, M.1; Fridh, V.2; Karlsson, R.2; Heine, The hydrazones were excluded from further investigations due to their A.1; Klebe, G.1 chemical instability and proposed toxicity. A systematic investigation of 1 Institute of Pharmaceutical Chemistry, University of Marburg, Marbacher Weg 6, 35032 Marburg, Germany structure-activity relationships was performed with the 2-arylindole 2 GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden chemotype. A systematic variation of the residues on the benzene moiety of the indole as well as the (hetero)aryl substituent at C-2 of the For abstract see Short Lecture SL.33 indole was performed, and selected chlorine-substituted compounds were shown to exhibit antifungal activity comparable to or better than the reference drug clotrimazole.

In addition to our experimental work we found out that the antifungal activity appears to be in relationship with theoretically calculated POS.101 Mulliken charges. The optimized structure parameters and the Mulliken Structure and Fragment Based Lead Discovery for New charges of each 2-(hetero)arylindole have been calculated at the Potential h17β-HSD14 Inhibitors. HF/STO-3G, B3LYP/STO-3G, B3LYP/6-31G(d), B3LYP/6-31G(d,p), B3LYP/6-311G(d) and B3LYP/6-311G(d,p) levels of theory, using the Bertoletti, N.1; Zara, L.1, Metz, A.1; Heine, A.1; Klebe, G.1, Marchais- Gaussian03 software package [1,2]. Oberwinkler, S.1 1 Philipps University Marburg, Institute for Pharmaceutical Chemistry, Marbacher Weg 6, 35032 Marburg, Germany

Human 17β-hydroxysteroid dehydrogenase type 14 (h17β-HSD14) is the latest identified 17β-HSD member of the Short-chain

Dehydrogenase-Reductase super family (SDR) [1,2]. In vitro, h17β- References: + 1. Chana, A. et al.: Chem. Res. Toxicol. 2002, 15(2): 1514-1526. HSD14 catalyses the NAD dependent oxidation at position 17 of 2. Buyukuslu, H. et al.: Spectrochimica Acta Part A 2010, 75(4): 1362-1369. estradiol (E2) and 5-androstene-3β,17β-diol (5-diol) to estrone and dehydroepiandrosterone, respectively. Northern blot experiments revealed that h17β-HSD14 is predominantly expressed in placenta, liver and brain [1]. As it is expressed in the brain, it may become a potential target for the treatment of neuronal diseases, POS.99 which are estradiol level dependent. Two variants of h17β-HSD14 are A novel approach to 2-substituted quinolin-4(1H)-ones via known. The first one was isolated from the retina and contains a serine nickel boride-mediated reductive ring transformation of (2- at position 205 (S205). An allelic variant differs only by a threonine at nitrophenyl)isoxazoles this position (T205), and was identified from melanotic melanoma cells. The in vitro turnover of both variants of h17β-HSD14 for E2 and 5-diol is Lohrer, B.1; Bracher, F.1 equal. 1 Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-University, Recently, we reported and characterized the apo (S205), holo (S205 Butenandtstr. 5-13, 81377 Munich, Germany and T205) and ternary complex crystal structures of h17β-HSD14 with

estrone as well as with a nonsteroidal inhibitor (T205) [3]. In addition, 2-Substituted quinolin-4(1H)-ones represent a promising class of we initiated a fragment-based lead discovery (FBLD) campaign by antimicrobial compounds. Their N-alkylated analogs for example show screening a 96 fragment library containing successful detected antimycobacterial activity [1,2]. Another interesting biological property is crystallographic screening hits and assembled considering the “Rule of their essential function in an intercellular communication system of 3” as a guideline. FBLD can explore the chemical space with respect to Pseudomonas aeruginosa called quorum sensing, e.g. by 2- scaffold diversity capable of binding to a protein binding pocket. The heptylquinolin-4(1H)-one (HHQ) [3]. small size of the fragments makes them suitable for optimizing activity

and selectivity for a target. We believe that crystallographic fragment O screening is a promising approach which may lead to the identification of more hits than other biophysical screening methods, especially for N those that have weak binding affinity [4], while providing essential H structural information about binding modes. We will present the binding 2-Heptylquinolin-4(1H)-one (HHQ) mode of the crystallographic hits from our fragment screen against h17β-HSD14 and will we discussed how this information can be used Here we report a novel method for the synthesis of 2-substituted for the improvement of our inhibitors. quinolin-4(1H)-ones starting from 5-(2-nitrophenyl)isoxazoles, which are readily available from 2-nitrophenylacetylenes via 1,3-dipolar cycloaddition. Key step is the simultaneous reduction of the nitro group and reductive ring cleavage of the isoxazole, followed by spontaneous cyclization to give the quinolin-4(1H)-one. Nickel boride was identified as the most suitable reductant for this transformation.

In contrast to standard methods (Conrad-Limpach and related syntheses), which build up the quinolin-4(1H)-one ring system via intramolecular ring acylation, this protocol avoids the formation of mixtures of isomers if additional substituents are present on the benzene ring.

References: 1. Wube, A. A. et al.: Eur. J. Med. Chem. 2011, 46(6): 2091-2101. 2. Wube, A. et al.: Molecules 2012, 17(7): 8217-8240.

3. Jerry Reen, F. et al.: Org. Biomol. Chem. 2012, 10(44): 8903-8910.

DPhG Annual Meeting 2016 Conference Book • 125 POSTERS

References: The target compounds were prepared from appropriately substituted 1. Lukacik, P. et al.: Biochem. J. 2007, 402(3): 419–427. benzaldehydes and isooctylamines by reductive amination and 2. Marchais-Oberwinkler, S. et al.: J. Steroid Biochem. Mol. Biol. 2011, 125(1-2): 66–82. subsequent precipitation with hydrogen chloride. 3. Bertoletti et al.: J. Med. Chem. 2016, Article ASAP; doi: 10.1021/acs.jmedchem.6b00293 The antifungal activity was evaluated in an agar diffusion assay and a 4. Schiebel et al.: ACS Chem Biol. 2016, 11(6):1693–1701. microdilution assay against Candida glabrata, Yarrowia lipolytica, and Aspergillus niger compared to clotrimazole and terbinafine.

POS.102 Pyridoisothiazolones as covalent modifiers of Lysine Acetyltransferase Activity

Simon, R. P.1; Furdas, S.1; Gajer, J.1; Sippl, W.2; Jung, M.1 1 Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany 2 Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, 06120 Halle, Germany

Lysine acetyltransferases (KATs) are epigenetic modifiers that catalyze References: the transfer of acetyl groups from their cofactor acetyl-CoA to the ε- 1. Nussbaumer, P. et. al.: J. Med. Chem. 1993, 36: 2115-2120. amino group of lysines in histones and other protein substrates. 2. Krauss, J. et. al. Arch. Pharm. Chem. Life Sci. 2014, 347: 1-8.

Aberrant activity of these enzymes has been correlated to the manifestation of several diseases, including cancer, inflammation, and neurodegenerative disorders.1, 2 Therefore, the development of small molecule modulators of KAT activity may provide useful tools for POS.104 probing the precise implication of these enzymes in pathogenesis and The Involvement of the Mitochondrial Amidoxime Reducing assess their potential as possible drug targets.3, 4 Component (mARC) in the Reductive Metabolism of Our group has developed N-substituted pyridoisothiazolones as Hydroxamic Acids covalent inhibitors of lysine acetyltransferases by combining virtual screening and molecular docking studies using the structural Ginsel, C.1; Plitzko, B.1; Jakobs, H. H.1; Froriep, D.1; Rothert, M.1; Stolfa, information of the hPCAF (KAT2B) catalytic domain. Initial structure- D. A.3; Jung, M.3; Mendel, R. R.2; Bittner, F.2; Havemeyer, A.1; Clement, activity-relationship studies focused on the substitution pattern of the B.1 isothiazolone nitrogen atom. We were able to show that N-phenyl 1 Christian-Albrechts-University Kiel, Department of Pharmaceutical Chemistry, derivatives inhibit KAT activity unselectively in low micromolar Gutenbergstraße 76, 24118 Kiel, Germany concentrations, whereas N-benzyl analogs were selective for hCBP 2 Department of Plant Biology, Braunschweig University of Technology, 38023 Braunschweig, Germany 5 (KAT3A). The inhibitory potency of these modulators was confirmed in 3 Albert-Ludwigs-Universität Freiburg, Institut für Pharmazeutische Wissenschaften, cellular assays as well as in a neuroblastoma xenograft mouse model.6 Albertstr. 25, D-79104 Freiburg, Germany To further improve the initial pyridoisothiazolone lead scaffold, a functionalized derivative was developed that allows derivatization of the The mitochondrial amidoxime reducing component mARC is a recently pyridine moiety. In this way, a small library of pyridine-substituted in our lab discovered molybdenum enzyme in mammals which in compounds was synthesized and tested for inhibition of recombinant concert with the electron transport proteins cytochrome b5 and NADH hPCAF using a heterogeneous antibody-based assay with time- cytochrome b5 reductase catalyzes the reduction of N-oxygenated resolved fluorescence readout in order to deduce initial structure- structures [1]. This three component enzyme system plays a major role activity-relationships. Further optimization of this promising class of in N-reductive drug metabolism and is thus the counterpart of compounds will possibly yield modulators with improved selectivity and cytochrome P450s and flavin-containing monooxygenases (FMO) physicochemical properties to serve as epigenetic tools. catalysed N-oxygenations [2]. Belonging to the group of N-hydroxylated structures hydroxamic acids are also potential substrates of the mARC- References: system (see figure 1). Hydroxamic acids show a variety of 1. Portela, A.; Esteller, M. Epigenetic modifications and human disease. Nat. Biotechnol. 2010, pharmacological activities and are therefore often found in drug 28, 1057-1068. candidates [3,4]. On the other hand they can also exhibit toxic 2. Dekker, F. J.; van den Bosch, T.; Martin, N. I. Small molecule inhibitors of histone properties as it is the case for many aryl hydroxamic acids formed acetyltransferases and deacetylases are potential drugs for inflammatory diseases. Drug during the metabolism of arylamides[5,6]. Thus, the metabolic stability Discovery Today 2014, 19, 654-660. 3. Simon, R. P.; Robaa, D.; Alhalabi, Z.; Sippl, W.; Jung, M. KATching-Up on Small Molecule of new hydroxamic acid containing drug candidates as well as the Modulators of Lysine Acetyltransferases. J. Med. Chem. 2016, 59, 1249-70. detoxification of toxic hydroxamic acid metabolites are of significant 4. Arrowsmith, C. H.; Bountra, C.; Fish, P. V.; Lee, K.; Schapira, M. Epigenetic protein families: interest. a new frontier for drug discovery. Nat. Rev. Drug Discovery 2012, 11, 384-400. Biotransformation assays with recombinant human proteins, subcellular 5. Furdas, S. D.; Hoffmann, I.; Robaa, D.; Herquel, B.; Malinka, W.; Świątek, P.; Akhtar, A.; porcine tissue fractions as well as in HEK293 cells were performed. The Sippl, W.; Jung, M. Pyrido- and benzisothiazolones as inhibitors of histone acetyltransferases mARC-dependent reduction of the model compound benzhydroxamic (HATs). MedChemComm 2014, 5, 1856-1862. 6. Gajer, J. M.; Furdas, S. D.; Grunder, A.; Gothwal, M.; Heinicke, U.; Keller, K.; Colland, F.; acid could be demonstrated. The N-reductive conversion of the Fulda, S.; Pahl, H. L.; Fichtner, I.; Sippl, W.; Jung, M. Histone acetyltransferase inhibitors block approved histone deacetylase inhibitor Vorinostat (SAHA) however, neuroblastoma cell growth in vivo. Oncogenesis 2015, 4, e137. was demonstrated to be rather low, thereby reflecting the relatively high metabolic stability and oral bioavailability of this compound. The toxic N- hydroxylated metabolite of the analgesic phenacetin, NOH- phenacetin[5], is not reduced by the mARC-system under the chosen conditions. This confirms the high toxicity of this component, as it needs

POS.103 to be detoxified by other pathways. As a consequence, for the Synthesis and biological evaluation of novel benzylamine- evaluation of the metabolic stability of new hydroxamic acid containing type antifungals drug candidates or for the evaluation of the potential risc for toxic metabolites it should be obligatory to monitor the N-reductive Krauss, J.; Stadler, M.; Bracher, F. metabolism by the mARC-system. 1 Department of Pharmacy LMU Munich This study underlines the hypothesis that mainly those N-oxygenated

compounds exhibit toxicity which are not easily reduced by the mARC A series of benzylamine derived antifungals with isooctyl side chain and enzyme system. alkyl or aryl ethers was synthesized and evaluated for antifungal activity So far our lab could demonstrate the reduction of several N-oxygenated as novel ergosterol biosynthesis inhibitors (SBI). An isooctyl side chain structures (N-hydroxyamidines, N-hydroxyguanidines, oximes, N- was introduced for imitating the alkyl side chain of ergosterol and its oxides, hydroxylamines, sulfhydroxamic acids). These functional groups precursors, alkyl or aryl ethers should imitate the rings A and B of are reduced significantly irrespective of the complete structure of the ergosterol. molecule. In contrast to this for hydroxamic acids the reduction rate might be low or undetectable depending on the structure. Further

126 • DPhG Annual Meeting 2016 Conference Book DRUG DESIGN/MEDICINAL CHEMISTRY

structure-activity studies will be necessary for the investigation of these References: influences. This might be helpful for the design of drug candidates 1. Bellentani, S. et al.: Dig Dis. 2010, 28: 155-161. avoiding deactivation by a mARC catalysed reduction. 2. Cave, M. C. et al.: Biochim. Biophys. Acta 2016, 1874: 30041-30044 3. Neuschwander-Tetri, B. A. et al.: Lancet. 2015, 385: 956-965. 4. Jahn, D. et al.: Dig Dis. 2016, 34(4): 356-363. 5. Tanaka, N. et al.: Biochim. Biophys. Acta 2015, 1852: 1242-1252. 6. Merk, D.& Lamers C. et al.: Bioorganic & Medicinal Chemistry 2015, 23: 499-514. 7. Lalloyer, F. et al.: Arteriosclerosis thrombosis and vascular biology. 2011, 31: 1573-1579.

POS.106 Pan retinoid X receptor agonist causes side-effects of lipid- lowering agent pirinixic acid Acknowledgments: We thank Melissa Zietz and Sven Wichmann (Christian-Albrechts-University Kiel, Germany) for technical assistance. Pollinger, J. C.1; Heitel, P.1; Kalinowsky, L.1; Proschak, E.1; Schubert- References: 1 1 1. Havemeyer, A. et al.: J Biol Chem 2006, 281(46), 34796-802. Zsilavecz, M. ; Merk, D. 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, (24), 8173-77. 2. Gruenewald, S. et al.: J Med Chem 2008, 51 60438 Frankfurt a. M., Germany; [email protected] 3. Ratner, M.: Nat Biotechnol. 2014, 32(9), 853-4 4. Barb, A., Zhou P.: Curr Pharm Biotechnol. 2008, 9(1), 9-15 5. Vaught, J. el al.: Cancer Res. 1981, 41(9), 3424-9 Retinoid X receptors (RXR) are part of the superfamily of nuclear 6. Hinson, J., Mitchell J.: Drug Metab Dispos. 1976, 4(5), 430-5 receptors and many of these nuclear receptors, including e.g. the farnesoid X receptor (FXR) and the peroxisome proliferator-activated receptors (PPAR), form heterodimers with RXR as counterpart. These heterodimers can be further divided into two categories: permissive and non-permissive heterodimers. The permissive heterodimers can be POS.105 activated by agonists of either partner and also the simultaneous Improvement of anthranilic acid derivates as a dual agonist presence of both RXR and partner receptor agonists resulting in a of FXR and PPARα synergistic activating effect [1]. Due to this, RXR plays a key role in the modulation of numerous physiological functions mediated by nuclear Gellrich, L. M.1; Schubert-Zsilavecz, M.1; Merk, D.1 receptors [2]. Although this seems to be a chance of influencing many 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, diseases, a pan-agonistic effect of RXR has only reached clinical 60438 Frankfurt a. M., Germany; [email protected] approval in the treatment of a few diseases such as cutaneous kaposi’s The prevalence of non-alcoholic fatty liver disease (NAFLD), one of the sarcoma or chronic hand eczema with alitretinoin as active ingredient most common liver disease, is rising constantly and currently amounts [3]. A selective agonist for one of the three subtypes has not been to 20-30% in the general population of Western countries [1,2]. The discovered so far but holds therapeutic potential for multiple indications. disease is particularly associated with obesity and insulin resistance Pirinixic acid is a dual PPAR α and γ agonist and has shown robust and constitutes the hepatic manifestation of the metabolic syndrome [2]. cholesterol lowering effects in rodent models [4]. But although its 5-20% of NAFLD patients develop a non-alcoholic steatohepatitis activity is similar to that of fibrates, pirinixic acid was never applied to (NASH) which may lead to fibrosis and later on to liver cirrhosis and humans, which might be explained by pleiotropic side effects [5]. hepatocellular carcinoma [1]. There are no approved drugs for the We have discovered that pirinixic acid is a partial agonist of all three treatment of NASH. The current therapy consists of Vitamin E and subtypes of retinoid X receptors and report a preliminary structure- thiazolidinediones. Regarding the drug pipeline Obeticholic acid, a activity relationship (SAR) for the agonistic interaction of pirinixic acid farnesoid X receptor (FXR) agonist, is the leading clinical candidate in and RXR. We have characterized our compound library of pirinixic acid phase III [3,4]. Additionally, GFT505, a dual peroxisome proliferator- derivatives in reporter gene assays for all RXR subtypes and observed activated receptor (PPAR) α/δ agonist, successfully completed a phase a significantly different SAR for RXR than for PPAR activation. IIb study [2,4]. Therefore, the nuclear receptors FXR and PPARα are Especially the 2,3-xylidino and the 4-chlorophenyl residues caused interesting targets for new active ingredients to treat NASH. activity on RXR. These findings might explain the side effects of pirinixic FXR is a bile acid receptor, whose activation decreases hepatic acid observed in preclinical development. Furthermore, our data gluconeogenesis and reduces circulating triglycerides [3]. The main indicates pirinixic acid as valuable lead compound to develop novel function of FXR is to control bile acid and cholesterol regulation. It was RXR modulators. observed that the hepatic expression of the receptor in NAFLD patients is decreased [2]. References: 1. Evans, R.M., Mangelsdorf, D.J.:Cell 2014, 157(1): 255-266 PPARα upregulates ß-oxidation and induces enzyme systems, that 2. Pérez, E. et al.: Biochimica et Biophysica Acta 2012, 1821(1): 57-69 protect cells against oxidative stress und suppress inflammation [5]. 3. Bubna, A.: Indian J Dermatol 2015, 60(5): 520 The receptor is activated by fatty acids and enhances their clearance in 4. Santilli, A., Scotese, A., Tomarelli, R.: Experientia 1974, 30(10): 1110-1111 the liver. PPARα knockout mice develop increased hepatic steatosis 5. Merk, D. et al.: Future Med. Chem. 2015, 7 (12): 1597-1616 and PPARα expression is low in patients with NAFLD. This is among other things a consequence of a low FXR level, because FXR is linked to the PPARα expression [2,7]. During the investigation of anthranilic acid derivates it was revealed that some agents of this substance group show a dual agonism of FXR and POS.107 Synthesis of folic acid derivatives for prostate cancer PPARα [6]. Since the bioavailability of these agents is not very drug- like, we substituted functional groups that are unimportant for the imaging receptor binding with more hydrophilic groups. The tert-butyl group of 1 1 the lead compound was replaced with some aliphatic amine Peric, N. ; Maison, W. 1 Pharmaceutical and Medicinal Chemistry, University of Hamburg, Bundesstraße 45, components, e.g. diethylamine or cyclic amine components, e.g. 20146 Hamburg, Germany azetidine. Furthermore, we aimed an approximation of the EC50 of FXR and Targeting tumor specific cell surface epitopes, so called tumor markers, PPARα, thus the carboxylic acid and the methyl group at the head with small molecules can lead to improved tools for cancer diagnosis group of the anthranilic acid were modified. Changes at the methyl and therapy. Elevated levels of prostate specific membrane antigen group at the head group assimilate the activation of FXR and PPARα (PSMA) are used as a tumor marker for prostate cancer [1]. PSMA is a and improve the maximum activation at FXR and PPARα in general. glycosylated type-II membrane protein that is present in high density on Elongation of the carboxylic acid at the head group and implementation the surface of malignant prostate cancer cells. Its expression increases of hetero atoms is another opportunity to improve the receptor with clinical stage, thus making it an extremely useful tumor marker [2]. activation. Both, small molecules and antibodies have been shown to be useful for PSMA targeting and have been used successfully for tumor imaging and immune therapy. Phosphinic acids like GPI, for example, can be

DPhG Annual Meeting 2016 Conference Book • 127 POSTERS used as modular ligands for the targeting of prostate cancer [3]. GPI focus on those functional groups that give access to carborane building binds with nanomolar affinity to PSMA and permits conjugation of blocks. Reacting these building blocks with organic compounds allows effector molecules like dyes without altering the binding properties [4]. us to create diverse organic-inorganic hybrid-libraries. The resulting However, GPI has suboptimal binding properties in vivo and needs to hybrid compounds are promising tools to study the applicability of be improved for imaging applications in animals or humans. GPI has carboranes as pharmacophores and identify unprecedented biological been developed as a transition state analogue of the native PSMA activities. substrate N-acetylaspartylglutamate (NAAG). In addition, PSMA has been found to act as a folate hydrolase and does thus recognize References: folylpolyglutamates in the same binding pocket as NAAG [5]. The poster 1. Scholz, M. et al.: Chem. Rev. 2011, 111: 7035-7062. highlights our efforts to design and synthesize improved modular PSMA 2. Scholz, M. et al.: ChemMedChem 2009, 4: 746-748. ligands. We have combined properties of the known ligand GPI with 3. Scholz, M. et al.: ChemMedChem 2011, 6: 89-93. 4. Scholz, M. et al.: Eur. J. Med. Chem. 2011, 46: 1131-1139. structural elements of folate and a conjugation site for effector 5. Scholz, M. et al.: Bioorg. Med. Chem. 2012, 20: 4830-4837. molecules according to docking studies. The synthesis involves the 6. Neumann, W. et al.: ChemMedChem 2016, 11: 175-178. arylation of benzylamines, which are notoriously difficult, yet highly important, starting materials for Pd-catalyzed arylations of the Buchwald Hartwig type.

POS.109 Investigation of structure-affinity relationships for novel GluN2A selective ligands based on the lead compound TCN-201

Müller, S. L.1; Schreiber, J.1; Wünsch, B.1; Seebohm, G.2; 1 PharmaCampus, Department of Pharmaceutical and Medicinal Chemistry, Corrensstraße 48, 48149 Münster, Germany 2 Institute for Genetics of Heart Diseases, Domagkstraße 3, 48149 Münster, Germany

NMDA receptors, ligand gated ion-channels, are expressed in the central nervous system as well as in the periphery. Due to the involvement of GluN2A subunit containing NMDA receptors in diseases like anxiety, depression, sinus arrhythmia and reduction of heart Acknowlegments: We kindly want to thank to Dr. Thomas Lemcke from Institute for Pharmaceutical and Medicinal Chemistry, University of Hamburg, for performing molecular contractility, the development of selective GluN2A antagonists is modeling studies. envisaged. [1,2] References: The sulfonamide 1 (TCN-201) is the lead compound for the synthesis of 1. Hilgenfeld R. et al.: EMBO J. 2006, 25(6), 1375-1384. GluN2A selective NMDA ligands. It shows a moderate GluN2A affinity 2. Heston W. D. et al.: Urology 2001, 57(6), 1179-1113. (IC50 = 158 nM) and additionally a high selectivity towards GluN2B 3. Coward J.K. et al.: J. Org. Chem. 2005, 70(17), 6757-6774. containing receptors (GluN2A : GluN2B > 300). [3] 4. Maison W. et al.: J. Med. Chem. 2009, 52(2), 544-550. To increase the activity and to investigate structure-affinity relationships 5. Carter, R. E., Feldman, A. R., Coyle, J. T.: PNAS 1996, 93(2), 749-753. for this compound class, 1 was modified in a systematic way. The

biological activity of the novel derivatives was investigated

electrophysiologically, i.e. the inhibition of the ion flux through GluN2A

containing receptors was recorded. POS.108 Medicinal Inorganic and Boron-Organic Chemistry: From Boronic Acids to Boron Clusters

Scholz, M. S. University of Bonn, Pharmaceutical Institute, An der Immenburg 4, 53121 Bonn, Germany

Pharmaceuticals based on inorganic elements are rare. However, these inorganic elements offer intriguing properties, which can be of high References: value for the application in drug molecules. Besides lithium and 1. Boyce-Rustay, J. M., Holmes, A.: Neuropsychopharmacology 2006, 31: 2405-2414. platinum complexes, boron compounds are becoming more and more 2. Bogdanova, A., Institute of Veterinary Physiology, University Zurich. popular. This is clearly underlined by the recent acquisition of Anacor 3. Bettini, E. et al.: J. Pharmacol. Exp. Ther. 2010, 335: 636–644. Pharmaceuticals by Pfizer for 5.2 billion dollars. Anacor Pharmaceuticals is specialized on boron compounds and is developing boronic acid drugs, which already reached the market or will be approved soon. The biological activity of boronic acids is attributed to POS.110 the electron-deficient nature of the element boron. This feature also Templated assembly of protein-binding fragments as a gives rise to versatile cluster compounds. These boron clusters begin to enter the field of drug development.[1] In addition to boronic acids, our method to reveal inhibitors of enteroviral 3C proteases research addresses the chemistry and biological activity of 12-vertex 1 1 1 dicarba-closo-dodecaborane (in short carborane) clusters (Fig. 1). Tauber, C. ; Becker, D. ; Rademann, J. 1 Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany

Using small-molecule fragments as starting points for the development of drugs is a tempting method that is able to provide ligands with improved potency, ligand efficiency, and selectivity. Unfortunately, it is usually restricted by the detection of these low affinity inhibitors. Our group was able to circumvent this by establishing dynamic ligation screening (DLS) [1]. Here a chemical reaction between fragments Fig. 1: Carborane Isomers. occurs whereas the equilibrium is shifted by addition of the protein as a template. Furthermore, DLS uses classical bioassays for the detection Earlier studies integrated carboranes in known bioactive lead of favourable fragment combinations. The approach works for various structures.[2-6] Now, we create unique carborane-based compound nucleophiles such as amines, thiols and alcohols reacting with libraries and screen for new applications. We develop chemical electrophilic fragments on a protein surface [2]. procedures to modify the cluster compounds at specific vertex Now, we extended the concept for the assembly of irreversible inhibitors positions, both at the cluster carbon and at the cluster boron atoms. We of Coxsackie virus B3 3C protease [3]. A protein-binding nucleophile 1

128 • DPhG Annual Meeting 2016 Conference Book DRUG DESIGN/MEDICINAL CHEMISTRY

reacts reversibly with a bis-electrophilic warhead 2, thereby positioning References: the second electrophile in close proximity to the active site. The attack 1. WHO, World Health Organization: World Malaria Report, 2015. of the nucleophilic thiolate of the viral cysteine protease (blue) results in 2. WHO, World Health Organization: Guideline, 2015. the covalent, irreversible deactivation of the enzyme. The assessment 3. Leven, M. et al.: J. Med. Chem., 2014, 57(19): 7971-7976. 4. Khankischpur, M., Kurz, T.: Synthesis, 2009, (23): 4068-4074. of the fragment combinations was based on measuring the enzyme 5. Korbad, B. L.; Lee, S.: Bull. Korean Chem. Soc., 2013, 34(4): 1266-1268. inactivation rate via FRET-assay and detection of covalent protein modification in mass spectrometry (Figure 1).

POS.112 Development of novel non-steroidal Farnesoid X Receptor (FXR) Antagonists

Schmidt, J.1; Schubert-Zsilavecz, M.1; Merk, D.1 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt a. M., Germany; [email protected]

The nuclear farnesoid X receptor (FXR) is a ligand-activated transcription factor, which acts as cellular sensor for bile acids and is Derivatives of the covalently bound fragments were designed, primarily expressed in liver, kidney and intestine [1-4]. It takes part in synthesized and tested for their inhibitory potential. Additionally, a the self-regulation of bile acids with the result that bile acid synthesis is crystal structure gave insight in the binding mode and proved blocked and their metabolism is enhanced when high levels of toxic bile assumptions of former docking studies. acids occur. Furthermore, FXR is involved in many other metabolic The protein-reactive electrophile was modified to various Michael systems such as glucose and lipid homeostasis and seems to have acceptors and optimization yielded a compound with submicromolar anti-inflammatory effects as well [4]. Since both, FXR activation with half-maximal inhibition as a potent broad-spectrum inhibitor against obeticholic acid (OCA) and whole-body FXR knockout mouse models enteroviral proteases. showed a metabolic improvement in obese mice, FXR holds promise in Further endeavours will concentrate on investigating the compatibility of the treatment of obesity and metabolic syndrome. However, the site of diverse biselectrophilic fragments with this assay with regard to their FXR activation appears to be essential for these effects [5]. Due to reactivity, chain length, and reversibility. recent studies, inhibition of intestinal FXR activity through glycine-β-muricholic acid (Gly-β-MCA) improved obesity, non-alcoholic References: fatty liver disease (NAFLD) and insulin resistance [5]. In obese mouse 1. Schmidt,M.F. et al.: Angew. Chem. Int. Ed. 2008, 47: 3275 –3278. 2. Burda, E.; Rademann, J.: Nat. Commun. 2014, 5:5170. models, oral treatment with Gly-β-MCA prevented weight gain and also 3. Becker,D. et al.: Nat. Commun. 2016, in revision. reduced absolute fat mass. In addition, blood glucose levels were reduced and insulin sensitivity improved. To prove the site of action, intestine specific FXR-knockout mice were equally treated and did not benefit from Gly-β-MCA [5-8]. Altogether, the study suggests significant therapeutic value of intestine specific FXR antagonism. However, the POS.111 intestinal stability and selectivity of Gly-β-MCA are questionable and Development of a new synthetic route for 3-hydroxy-N’- potent non-steroidal and selective FXR antagonists are required to arylidenepropanehydrazonamides as potential prove beneficial effects of FXR antagonism. antiplasmodial compounds In an in-house library screening, we discovered a benzamidophenylacetic acid derivative as lead compound for the Knaab, T. C.1; Held, J.2; Mordmüller, B.2; Kurz, T.1 development of non-steroidal FXR antagonists. It already offers 1 Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, respectable FXR antagonistic potency with a submicromolar IC50 value. Universitätsstraße 1, 40225 Düsseldorf, Germany 2 Institute for Tropical Medicine, Eberhard Karls University, Wilhelmstraße 27, 72074 Tübingen, Its structure can be divided into two building blocks, which allows a Germany systematic, antagonistic structure-activity relationship (SAR) compilation for each block. Our first investigations focused on the Malaria is still one of the most frequent causes of death in low income position and size of the acidic group while the benzamido moiety was tropical countries. Globally, there were approximately 214 million unaltered. By variation of the substitution pattern, we could improve malaria cases and 438 000 deaths in 2015 caused by the infections potency to a two-digit nanomolar IC50 value. with protozoan parasites of the genus Plasmodium.[1] The limited Since spatial extension is promising to enhance antagonistic potency, number of antimalarial drugs and increasing parasite resistance leads we systematically methylated all free positions of the scaffold to to an urgent need for new antimalarial drugs. The arylamino alcohol discover additional space for structural expansion. With this systematic quinine was the first antimalarial drug in clinical use and today its strategy, we draw a broad SAR of benzamidophenylacetic acids as analogue lumefantrine is part of the first-line antimalarial therapy novel class of highly potent FXR antagonists. Further structural (artemisinine-based combination therapy (ACT): lumefantrine & optimization will not only focus on antagonistic potency but also artemether).[2] Recently, we developed a new chemical class of consider solubility, metabolic stability and toxicity to develop a novel antiplasmodial compounds which mimics the general structure of FXR antagonistic tool compound for a more specific evaluation of FXR arylamino alcohols. The lead structure, a 3-hydroxy-N’- antagonism as therapeutic concept. arylidenpropanehydrazonamide derivative (see figure 1) demonstrated in vitro activity in single digit nanomolar range against P. falciparum References: strain 3D7 along with moderate in vivo activity in the P. berghei mouse 1. Seol, W., Choi, H. S., Moore, D. D.: Mol. Endocrinol. 1995, 9(1): 72–85. model.[3] Initially, a classical Pinner reaction was used as a key step to 2. Forman, B. M. et al.: Cell 1995, 81(5): 687–693. 3. Parks, D. J. et al.: Science 1999, 284(5418): 1365–1368. provide imidate hydrochlorides as precursor for subsequent amidrazone 4. Luciano Adorini, L., Pruzanski, M., Shapiro, D.: Drug Discov. Tod. 2012, 17(17): 988-997. synthesis.[4] Here we present an optimized synthetic protocol that 5. Jiang, C., et al.: Nat. Commun. 2015, 6: 1–18. enables a higher degree of chemical diversity for subsequent structure- 6. Lamers, C., Schubert-Zsilavecz, M., Merk, D.: Curr. Top. Med. Chem. 2014, 14(19): 2188– activity relationship (SAR) studies. Notably, trimethylaluminum [5] 2205. promoted amidation of β-hydroxynitriles provided β-hydroxamidines, 7. Merk, D., Steinhilber, D., Schubert-Zsilavecz, M.: Future Med. Chem. 2012, 4(8): 1015–1036. while amidrazones where accessible using hydrazine hydrate instead of 8. Huang, H., et al.: ChemMedChem. 2015, 10: 1184–1199. an amine.

Fig. 1: Intended structure modification of the lead structure

DPhG Annual Meeting 2016 Conference Book • 129 POSTERS

POS.113 POS.115/SL.41 Development of new versatile building blocks for the Analysing the framework of protein ligand interactions: pharmacokinetic optimization of PET-tracers Ligand-sensing cores and privileged scaffolds

1 1 2 3 Theiler, S. ; Wünsch, B. ; Faust, A. ; Höltke, C. Koch, O. 1 Institut für pharmazeutische und medizinische Chemie, Corrensstraße 48, 48149 Münster TU Dortmund University, Faculty of Chemistry and Chemical Biology, Otto-Hahn-Straße 6, 2 European Institute for Molecular Imaging – EIMI, Waldeyerstraße 15, 48149 Münster 44227 Dortmund, Germany 3 Institut für Klinische Radiologie, Universitätsklinikum Münster Albert-Schweitzer-Campus 1 / A16, 48149 Münster For abstract see Short Lecture SL.41 A fluorescent probe1 and a number of PET-tracers2,3 have been developed to image the endothelin A (ET-A) receptor which is a potential target for medical imaging as it is overexpressed in connection with many kinds of diseases, like cancer, inflammation and (cardio)vascular disorders. In comparison to the fluorescent probe, the POS.116 PET-tracers exhibit rather poor in vivo performance e.g. low Synthesis of 1- and 8-pyridinyl-substituted imidazo[1,5- accumulation in target tissue. This discrepancy is believed to result from a]quinoxalines as potential PDE10A-ligands for positron the molecular structure of the fluorophore. Small-molecular PET-tracers emission tomography (PET) usually consist of rather lipophilic structures whereas fluorescent probes consist of large heteroaromatic systems being e.g. negatively charged Franz, L.1; Scheunemann, M.2; Wagner, S.2; Lang, M.1; Brust, P.2; Briel, due to hydrophilic sulfonic acid groups. D.1 The aim of this project is to synthesize versatile building blocks 1 University of Leipzig, Institute of Pharmacy, Bruederstraße 34, 04103 Leipzig, Germany 2 Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, mimicking the molecular structure of fluorescent dyes in order to Permoserstraße 15, 04318 Leipzig, Germany enhance the hydrophilicity and hence the in vivo performance of the PET-tracers. First constructs were designed to contain an aromatic Phosphodiesterases (PDE´s) are second messenger hydrolysing system with one or more sulfonic acid groups (fig.1). This building block enzymes and important regulators of signal transduction mediated by can then be bound to a variety of ligands, including the developed these molecules. PDE10A, a cAMP and cGMP sensitive hydrolase, is endothelin receptor antagonist. It contains a carboxylic acid group (blue, primarily expressed in the striatum and was identified as drug target for fig.1) where a ligand can be bound via an amide bond and an alkyne the therapy of diverse disorders in the central nervous system (CNS) [1] group (red, fig.1) where 18F as the PET radionuclide can be introduced like schizophrenia or chorea huntington [2]. Recently, 1-arylimidazo[1,5- via a cycloaddition reaction with [18F]fluoroethyl azide. a]quinoxalines have been reported to be potent and selective inhibitors of PDE10A [3]. In terms of a potential use as 18F-labelled PET imaging agent new fluorine-containing substituted derivatives were synthesized. It has been shown that the methoxy substituted inhibitors are prone to metabolic oxidation, which leads to a loss of inhibitory potency or ability to cross the blood brain barrier [3]. To improve the metabolic stability of inhibitors the methoxy function in position 6 was exchanged by chlorine in the first synthesis step by electrophilic substitution. An electron deficient system was generated in Fig. 1: Naphthalene-based building block for PET-tracers, n=1-2. step 2 by oxidation of the amine to a nitro function to allow the nucleophilic aromatic substitution of fluorine by 4-methylimidazole in First radiosyntheses were performed successfully. Future studies will step 3. Afterwards, the amine was recovered by acidic reduction with examine the influence of different charges on the pharmacokinetic elementary iron in step 4 and acetylated in step 5. The Cyclisation in behavior in vivo. Therefore, building blocks with positive charges step 6 to create the quinoxaline was realized by a Bischler-Napieralski- (quarternary ammonium) or polar neutral compounds (polyalcohols) will reaction, the bromination of position 1 by electrophilic substitution in be developed. step 7.

References: NH2 NH2 NO2 NO2 NH2 1. Höltke, C. et al. Bioconjug. Chem. 2007, 18 (3): 685–94. F Cl F Cl F N N NCS NaBO3 · 4 H2O HN N Cl N Fe Cl N 2. Höltke, C. et al. Bioorg. Med. Chem. 2009, 17 (20): 7197–208. step 1 step 2 step 3 step 4 3. Michel, K. et al. J. Med. Chem. 2011, 54 (4): 939–48. X X X X X

N N NHAc X = -CF N NBS N POCl3 Ac2O 3 Cl N Cl N Cl N N -Br 6 1 6 1 Br 8 step 7 8 step 6 step 5 X X X POS.114/SL.42 A fluorescence polarization-based competition binding assay for detecting compounds interacting with inactive The derivatization of the quinoxalines was focused on position 1 and 8. mitogen-activated protein kinases and development of Finally, fluor-containing pyridinyl-groups were introduced by Suzuki- covalent inhibitors of c-Jun N-terminal kinase 3 Coupling with the corresponding boronic acid at the brominated positions to afford the monosubstituted (1, 2) and disubstituted (3) Koch, P.1 pyridinyl-derivatives. All compounds were characterized by high 1 Eberhard Karls Universität Tübingen, Institute of Pharmaceutical Sciences, Department of performance liquid chromatography, nuclear magnetic resonance Medicinal Chemistry, Auf der Morgenstelle 8, 72076 Tübingen, Germany. spectroscopy and mass spectrometry. It is expected that the new chlorinated derivatives have the same pharmaceutical effects as their For abstract see Short Lecture SL.42 methoxy analogues [4].

(1) (2) (3) N N N N N N Cl N Cl N Cl N 6 1 6 1 6 1 8 8 8 N N N CF3 Br F F F N F

Acknowledgments: Thanks are to J. Ortwein for HPLC analysis, Dr. L. Hennig for recording and analysis of NMR data and Dr. J. Preidl for LC-MS analysis.

130 • DPhG Annual Meeting 2016 Conference Book DRUG DESIGN/MEDICINAL CHEMISTRY

References: biosynthesis of lipid A, the lipophilic membrane anchor of LPS, 1. Liras, S.; Bell, A.S. Phosphodiesterases and Their Inhibitors (Wiley-VCH) 2014. represents therefore a promising strategy to combat Gram-negative 2. Schmidt, C.J. et al.: J. Pharm. Exp. Ther. 325 (2008) 681–690. bacteria. The enzyme LpxC, catalyzing the first committed step of lipid 3. Malamas, M. et al.: J. Med. Chem. 54 (2011) 7621–7638. A biosynthesis, has been explored as an optimal target for novel 4. Wagner, S. Eur. J. Med. Chem. 107 (2016) 97–108. antibiotics, due to its fundamental role for growth and viability of Gram- negative bacteria and its lack of homology towards mammalian enzymes [2]. A fragment-based drug design approach was used to develop novel POS.117 LpxC inhibitors. Using NMR spectroscopy, a library of fragments was Triazole-type ABCG2 modulators: approaches to improve screened in the presence of a known inhibitor and the LpxC enzyme. In drug-like properties ligand-observed NMR experiments interligand NOEs were detected [3]. The hits found were used to optimize the inhibitory activity of a known Antoni, F.1#; Stark, S.2#; Scholler, M.1; Bernhardt, G.1; König, B.2; LpxC inhibitor. Buschauer, A.1 1 Institute of Pharmacy, 2 Institute of Organic Chemistry, University of Regensburg, References: D-93040 Regensburg; #Authors contributed equally 1. Theuretzbacher, U.: Int. J. Antimicrob. Agents. 2012, 39: 295-299 2. Barb, A. W. et al.: PNAS. 2007, 104(47): 18433-18438 The efflux transporter breast cancer resistance protein (BCRP, ABCG2) 3. Harner, M. J. et al.: J. Biomol. NMR. 2013, 56(2): 65-75 is associated with the chemoresistance of malignant tumors. In addition, ABCG2 is highly expressed at the blood-brain barrier preventing the entry of numerous xenobiotics including drugs into the central nervous system. Previously, UR-MB108 [1, 2] was synthesized and characterized as a highly potent, selective and chemically stable POS.119 ABCG2 inhibitor. Synthetic phthalimide conjugation strategies for PROTAC development

Steinebach, C.1; Gütschow, M.1 1 University of Bonn, Pharmaceutical Institute, An der Immenburg 4, D-53121 Bonn, Germany

Proteolysis targeting chimeras (PROTACs) are used to link an E3 ubiquitin ligase to a specific target protein. These molecules have two recognition motifs separated by a linker: one terminal moiety recognizes specific target proteins and the other one binds to an E3 ubiquitin ligase. Ubiquitination of these proteins ultimately leads to their elimination via the ubiquitin/proteasome system (UPS). PROTAC technology in combination with an immunomodulatory drug (IMiD) as a low-molecular weight CRL4CRBN recruiter was first reported in 2015 for BET protein degradation in vitro and in vivo.1 A further heterodimer for the degradation of oncogenic BCR-ABL via the E3 ubiquitin ligase complex CRL4CRBN was described in 2016.2 Recently, PROTAC technology was proven to inhibit the growth of a solid tumor in vivo.3

From that perspective, it was considered to have the potential for a However, this compound shows high lipophilicity and low water platform technology in drug research.4 solubility. Aiming at improving drug-like properties, the tetrahydroisoquinoline moiety was replaced by a series of different saturated heterocycles. Furthermore, the quinoline moiety was replaced by smaller aromatic heterocycles. This approach resulted in modulators of lower molecular weights with activities in the three-digit nM range.

References: 1. Bauer, S. Quinoline carboxamides as modulators of breast cancer resistance protein (ABCG2): Investigations on potency, selectivity, mechanism of action, cytotoxicity, stability and drug-like properties. Ph.D. thesis, University of Regensburg, 2014; http://epub.uni- regensburg.de/29589/. 2. Bause, M. Synthesis of melanoma inhibitory activity protein inhibitors, ABCG2 transporter modulators, and neurotensin mimetics. Ph.D. thesis, University of Regensburg, 2015.

Herein we describe the development and synthesis of linker strategies

for phthalimide conjugation in view of PROTAC technology, which has POS.118 most recently received much attention in medicinal chemistry. We A fragment-based drug design approach for the evaluated literature known and new synthetic entries towards optimization of LpxC inhibitors bifunctional probes and examined protein data bank files with respect to phthalimide-cereblon (CRBN) complexes. To prepare various Agoglitta, O.1,2; Martin, B.1; Kalinin, D.1,3; Köhler, J.1; Holl, R.1,3 thalidomide derivatives containing functional groups e.g. nitro, amino, 1 Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstraße 48, hydroxy and carboxy, one-pot syntheses were applied starting from 48149 Münster, Germany commercially available phthalic anhydrides. These derivatives were 2 NRW Graduate School of Chemistry, University of Münster, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany investigated for the attachment of linkers by using different coupling 3 Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), University of Münster, Germany techniques. Furthermore, our study includes new protecting techniques for the glutarimide moiety, which were needed in case of electrophilic Multidrug resistant Gram-negative bacteria are becoming a global alkylation reactions at the non-glutarimide part of the molecule. threat. E.g., the treatment of healthcare-associated infections is steadily Our rational design of phthalimide-conjugated linker building blocks getting more and more challenging, due to the increasing number of includes different synthetic entries towards heterodimeric compounds. multidrug resistant bacteria. Additionally, the number of available and The synthetic implementation is presented herein. Our work is aimed at effective antibiotics is limited. Novel antimicrobial agents addressing so generating further examples which could hijack E3 ligases for the far unexploited bacterial targets are therefore urgently needed [1]. degradation of target proteins as a new therapeutic strategy. Being the main and fundamental component of the outer monolayer of the outer membrane of Gram-negative bacteria, lipopolysaccharides Acknowledgments: University of Bonn, Pharmaceutical Institute, Gilberg, E. (LPS) ensure an extremely effective permeability barrier towards chemical attacks. The inhibition of the enzymes being involved in the

DPhG Annual Meeting 2016 Conference Book • 131 POSTERS

References: 1. Winter, G. E. et al.: Science 2015, 348(6241): 1376-81. POS.121 2. Lai, A. C. et al.: Angew. Chem. Int. Ed. Engl. 2016, 55(2):807-10. New oxetane derivatives designed as p38 MAP kinase 3. Raina, K. et al.: Proc. Natl. Acad. Sci. USA. 2016, 113(26):7124-9. 4. Deshaies, R. J.: Nat. Chem. Biol. 2015, 11(9): 634-5. inhibitors

Rodrigues de Sá Alves, F.; Laufer, S. A. Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076, Tübingen POS.120 Aberrant kinase activity is implicated in a variety of human diseases, Synthesis and biological evaluation of neuroprotective 7-O- principally those involving inflammatory responses, such as rheumatoid esters of the flavonolignan silibinin arthritis. They could be treated through modulation of cytokines related to the protein kinase pathway such as tumor necrosis factor-a (TNF-α) Schramm, S.1; Huang, G.1; Kling, B.2; Heilmann, J.2; Decker, M.1 and interleukin-1β (IL-1β), whose biosynthesis and release are 1 Pharmazeutische und Medizinische Chemie, Institut für Pharmazie und Lebensmittelchemie, regulated by the activation of the p38α MAPK [1-2]. Laufer and co- Julius-Maximilians-Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany 2 Lehrstuhl für Pharmazeutische Biologie, Institut für Pharmazie, Universität Regensburg, workers have been reporting the design and pharmacological Universitätsstraße 31, D-93053 Regensburg, Germany evaluation of potent p38α MAPK inhibitors bearing the dibenzepinone, dibenzoxepine, dibenzosuberone and benzosuberone scaffolds [3-4]. In It is well established that oxidative stress, the formation of reactive order to discover new analogs of benzosuberones, we proposed the oxygen species (ROS), and subsequent neurotoxicity are key isosteric replacement of the carbonyl group by an oxetane moiety to processes in the pathophysiology of Alzheimer’s disease (AD). [1] fulfill the two selectivity requirements, the ‘linear binding’ mode and the There are reports that standardized extracts of Silybum marianum, glycine–flip at Gly110. Moreover, an oxetane group can elicit deep containing the silymarin complex and its main component silibinin changes in aqueous solubility, lipophilicity, metabolic stability, and (diastereomeric mixture of Silybin A 1a and Silybin B 1b, Scheme 1) conformational preference when replacing commonly employed and chemical derivatives of silibinin might exhibit neuroprotective functionalities like gem-dimethyl and carbonyl groups [5-6]. properties. [2,3] Ferulic acid represents a naturally occurring phenolic acid, that is a potent radical scavenger, measured in the physicochemical oxygen-radical absorbance capacity (ORAC) assay, [4] but it has been shown to lack neuroprotective activity in vitro after impairment of HT-22 cells with tert-butyl hydroperoxide. [5] To combine the potent antioxidant properties of silibinin 1 with ferulic acid, and structurally closely related compounds, and to produce putatively neuroprotective silibinin esters with antioxidant activity, 7-O-esters of silibinin 1 (2-5, Scheme 1) were synthesized. In order to establish SARs for the esters synthesized, their radical scavenging properties as well as Acknowledgments: University of Tübingen, DAAD-CNPq their neuroprotective effects have been determined. References: 1. Melnikova, I.; Golden, J:. Nat. Rev. Drug. Discov. 2004, 3(10): 993-994. O O 2. Laufer, S. A. et al.: J. Med. Chem. 2003, 46(15): 3230–3244. OH OH * * HO O * OMe O O * OMe 3. Laufer, S. A. et al.: J. Med. Chem. 2006, 49(26): 7912–7915. O R O 4. Karcher, S. C.; Laufer, S. A.: J. Med. Chem. 2009, 52(6): 1778–1782. OH OH OH OH OH O 1 OH O 2-5 5. Koeberle, S. C. et al.: Nat. Chem. Biol. 2012, 8(2): 141–143. 1a: silybin A (2R, 3R, 10R, 11R) 6. Wuitschik, G. et al.: J. Med. Chem. 2010, 53(8): 3227-3246. 1b: silybin B (2R, 3R, 10S, 11S)

R1 R1 R1 2 a-c) R1 = R2 = R3 = H 2 2 2 R R R 3 a-c) R1 = OMe, R2 = OH, R3 = H R = 1 2 3 R3 R3 R3 4 a-c) R = OMe, R = OH, R = OMe 1 2 3 a O b O c O 5 a-c) R = R = O2CH(CH3)2 , R = H Reagents and conditions: (I) acid, oxalyl chloride, CH2Cl2, rt; (II) silibinin 1, NEt3, THF, rt POS.122

Scheme 1: Reaction conditions and structures of synthesized 7-O-esters (2-5) Characterization of diazenylaryl sulfonic acids as neuraminidase inhibitors For the synthesis of the 7-O-esters several esterfication methods were investigated to get the desired regioselectivity without the use of a time Hoffmann, A.1; Richter, M.1; von Grafenstein, S.2; Walther, E.1; Xu, Z.1; consuming protecting group strategy. One of the first methods applied Schumann, L.1; Grienke, U.3; Mair, C. E.3; Kramer, C.2; Rollinger, J. M.3; previously was Mitsunobu esterification [3], which in fact yielded 7-O- Liedl, K. R.2; Schmidtke, M.1; Kirchmair, J.2,4 feruloylsilibinin (3a, Scheme 1) rather than the described 23-O-ester. 7- 1 Jena University Hospital, Department of Virology and Antiviral Therapy, Hans-Knoell-Straße 2, 07745 Jena, Germany Esterification can also be achieved using acyl chlorides in basic 2 University of Innsbruck, Centre for Chemistry and Biomedicine (CCB), Institute of General, conditions. [6] As this alternative method does not show as many Inorganic and Theoretical Chemistry, Innrain 82, 6020 Innsbruck, Austria unwanted side products [7] and is devoid of tedious chromatographic 3 University of Vienna, Department of Pharmacognosy, Althanstraße 14, 1090 Vienna, Austria 4 University of Hamburg, Center for Bioinformatics, Bundesstraße 43, 20146 Hamburg, purification that are necessary for Mitsunobu reactions, we decided to Germany use acyl chlorides for the syntheses of the 7-O-esters. Also compounds 3 and 4 could be obtained using the respective acid chlorides, which Neuraminidases (NAs) are valid targets to combat not only influenza but were prepared in situ without protection of the hydroxyl groups (Scheme also secondary Streptococcus pneumoniae infections causing 1). This procedure gave the 7-O-esters 2-5 consistently. pneumonia [1-3]. This study reports on the identification of diazenylaryl To investigate how stable the esters are under assay conditions, sulfonic acids as inhibitors of influenza A NAs and S. pneumoniae NA stability of 3a was determined via LCMS. (NanA) by virtual screening. Several reports link diazenylaryl sulfonic To determine the radical scavenging properties of esters 2-5, the ORAC acids to antiviral activity and the inhibition of influenza NA (e.g. [4]) but assay, which assesses their antioxidant physicochemical properties, their function and potency have never been thoroughly characterized. was employed. Neuroprotective effects of the compounds were We applied different biochemical and cell-based assays that confirmed investigated towards glutamate-induced oxidative stress in HT-22 the inhibitory activity of several of the 17 tested diazenylaryl sulfonic hippocampal neurons. Additionally, putative self-cytotoxic effects of the acids against influenza A virus NA and, in addition, revealed their compounds were studied. inhibitory activity against NanA. For the most active compound, NSC65847 from the National Cancer Institute’s (Bethesda, MD) References: compound library, the measured Ki values were <1 µM for both viral 1. Palmer, A.M.: Trends Pharmacol. Sci. 2011, 32: 141–147. and pneumococcal NAs. The compound also inhibited N1 virus variants 2. Yang, L.X. et al.: J. Med. Chem. 2009, 52: 7732–7752. containing NAI resistance-conferring substitutions. Via enzyme kinetics 3. Wang, F. et al.: Bioorg. Med. Chem. 2009, 17: 6380–6389. 4. Fang, L. et al.: Bioorg. Med. Chem. Lett. 2008, 18: 2905–2909. and nonlinear regression modeling, NSC65847 was suggested to impair 5. Kling, B. et al.: J. Nat. Prod. 2014, 77: 446–454. the viral NA as well as NanA with a mixed-type inhibition mode. 6. Gažák, R. et al.: J. Med. Chem. 2011, 54, 7397–7407. Diazenylaryl sulfonic acids appear to be binding to NAs in various 7. Huang, G. et al.: Beilstein J. Org. Chem. 2016, 12: 662–669. different orientations. Given its antiviral and antipneumococcal activity,

132 • DPhG Annual Meeting 2016 Conference Book DRUG DESIGN/MEDICINAL CHEMISTRY

NSC65847 was identified as a novel starting point for the development data were in good agreement with those obtained from the Hoechst of dual-acting NA inhibitors. 33342 assay.

References: 1. Kühnle, M. et al.: J. Med. Chem. 2009, 52, 1190-1197. 2. Bauer, S. et al.: ChemMedChem 2013, 8, 1773-1778. 3. Glavinas, H. et al.: Drug. Metab. Dispos. 2007, 35(9), 1533-1542. 4. Sakardi, B.; et al.: J. Biol. Chem. 1992, 267(7), 4854-4858.

POS.124 Applying machine-learning algorithms and high- throughput screening (HTS) on twelve targets for identifying potentially active compounds and reducing animal toxicity testing Acknowledgments: We thank the National Cancer Institute of the National Institute of Health (NIH) for the provision of free samples of compounds for testing and the Cambridge Sayed, A.1,2; Spahn-Langguth, H.3; Schramm, K. W. 1,4 Crystallographic Datacenter (CCDC) for providing us with a complimentary license for GOLD for 1Center for Life Sciences, Technical University Munich, Emil-Erlenmeyer-Forum 2, the purpose of this study. This work was funded by research grants P23051 and P24587 of the 85354 Freising, Germany Austrian Science Fund (FWF), the European Social Fund together with Thuringian Ministry of 2Rosettastein Consulting, Goethestrasse 2, 85354 Freising, Germany Economy, Labour and Technology (2011FGR0137). 3Department of Pharmaceutical Sciences/Pharmaceutical Chemistry, Karl-Franzens-University, Universitätsplatz 1/I, 8010 Graz, Austria References: 4Molecular EXposomics, German Research Center for Environmental Health, Helmholtz 1. Grienke, U. et al.: Sci. Rep. 2016, 27156. Zentrum München, Neuherberg, Germany 2. Walther, E. et al.: Int. J. Med. Microbiol. 2015, 305(3): 289-97. 3. Walther, E. et al.: Front. Microbiol. 2016, 7: 357. The regulatory necessity to fill information gaps about chemicals’ 4. Cheng, L. S. et al.: J. Med. Chem. 2008, 51(13):3878-3894. toxicity is increasing worldwide. The need for filling such gaps while reducing toxicity testing in animals is also becoming more predominant in risk assessment. Programs run by the multiple European and US agencies are encouraging the use of alternative approaches for toxicity POS.123 estimation. Recent legislations are accepting in silico approaches for Identifying triazole-type quinoline carboxamide derived predicting toxicological outcomes. As more data is being generated modulators of ABCG2 as inhibitors using an ATPase assay through High throughput screening (HTS) technologies, statistical in silico approaches are getting more popular as an alternative method for Scholler, M.1; Stark, S.2; Bause, M.2; Bernhardt, G.1; König, B.2; predictive toxicology. We present an automated workflow to construct Buschauer, A.1 and analyze Quantitative Structure Activity Relationship (QSAR) for 1 Institute of Pharmacy, University of Regensburg, 93040 Regensburg, Germany predicting the outcome of in vitro profiling of chemicals. We describe the 2 Institute of Organic Chemistry, University of Regensburg, 93040 Regensburg, Germany results of QSAR modeling efforts within Tox21 Data Challenge, which calculated the best balanced accuracy across all molecular pathway The breast cancer resistance protein (BCRP, ABCG2) is an efflux endpoints as well as the highest scores for ATAD5 and mitochondrial transporter highly expressed at the blood-brain barrier, preventing membrane potential disruption. xenobiotics including cytostatic drugs to enter the central nervous Twelve molecular pathway endpoints were investigated, which were system (CNS). In addition, ABCG2 overexpression by malignant cells is selected on the basis of toxicological relevance. The targets were associated with poor survival of cancer patients. Co-adminstration of experimentally screened as part of the Tox21 program and the resulting ABCG2 inhibitors with chemotherapeutics, which are substrates of this data library made accessible for competitors by the Tox21 Data transporter, harbours the potential to overcome multidrug-resistance Challenge organizers. Tox21 represents a multi- agency effort that uses and to reach effective concentration of cytostatics in the CNS. HTS assays for toxicity modeling and prediction in the US. The US Previously, our group reported on selective carboxamide-type ABCG2 Environmental Protection Agency (EPA), The National Institutes of modulators with activities in the two-digit nanomolar range [1, 2]. Health (NIH), The National Center for Advancing Translational Sciences Cellular uptake assays such the Hoechst 33342 assay do not (NCATS), The National Institutes of Environmental Health discriminate between ABCG2 substrates and inhibitors. To investigate Sciences/National Toxicology Program (NIEHS/NTP) and the Food and whether modulators of general structure 1 are substrates or act as Drug Administration (FDA) cooperate in screening chemical substances ABCG2 inhibitors, an ATPase assay was established. for some selected potential toxic effects. The data may then be used, The human ABCG2 was expressed in Sf9 cells by baculoviral infection with the assistance of in silico techniques, for providing an alternative [3] and membranes were prepared. ABCG2 expression was confirmed for expensive, time- consuming, and ethically-questioned animal by Western blot, and transporter activity was proven in a vesicular testing. transport assay, using the fluorescent ABCG2 substrate lucifer yellow. In this study we constructed models for 12 targets using a consensus of To quantify the inhibition of the efflux pump, the liberation of inorganic selected associative neural networks models from 10 descriptor phosphate upon transporter dependent ATP hydrolysis was measured packages. Automated QSPR workflow systems, OCHEM, the analytics photometrically [4]. When exploring different substrates, sulfasalazine platform, KNIME and the statistics software, CRAN R, were used to gave the highest signal-to-noise ratio. conduct the analysis and develop consensus models. All models were constructed using bootstrap aggregation. We also investigated multiple approaches for constructing consensus models and set criteria for including a model into consensus voting. The resulting consensus models yielded a balanced accuracy as high as 88.1% ± 0.6 for mitochondrial membrane disruptors. Such high balanced accuracy, in combination with applicability domain estimation, encourages the use of in silico modeling to guide future selection of chemical libraries for screening. The comprehensive statistics of all models are publicly available online at https://github.com/amaziz/Tox21-Challenge-

Publication while the developed consensus models can be accessed at Transport driven ATP hydrolysis decreased in the presence of the http://ochem.eu/article/98009. investigated carboxamide-type modulators in a concentration dependent manner, demonstrating that the investigated quinolone carboxamides are inhibitors, not substrates. The same holds for the reference compound fumitremorgin C (2). Compared to the latter (IC50 1.8 µM), the quinoline derivatives were considerably more potent as ABCG2 inhibitors (IC50 values < 100 nM) in the ATPase assay. The

DPhG Annual Meeting 2016 Conference Book • 133 POSTERS

[3] potential to be used as new anti-parasitic agents . Here, we report that POS.125 Schistosoma mansoni histone deacetylase 8 (SmHDAC8), the most 3D-QSAR analysis of Dual Leucine Zipper Kinase Inhibitors expressed class I HDAC isotype in this organism, is a functional acetyl- L-lysine deacetylase that plays an important role in parasite infectivity. Brinker, C.1; Lemcke, T.1 SmHDAC8 is a zinc depending enzyme. Linkerless aromatic 1 Institute of Pharmacy, University of Hamburg, Bundesstr. 45, 20146 Hamburg, Germany hydroxamates were identified by screening the ZINC-Database for small molecules containing zinc-chelating groups. Further investigation The dual leucine zipper kinase (DLK) is a MAP3-kinase which plays a of these compounds revealed the induction of apoptosis and mortality in crucial role in different MAPK signaling pathways, activating schistosomes. Crystal structures of smHDAC8 with several linkerless downstream kinases like p38 and JNK. DLK is especially expressed in hydroxamates could be resolved[4,5]. Based on structural data and the peripheral and central nervous system being required for modeling studies structure activity relationship analysis was established neurodegeneration and axon regeneration, and in the insulin-producing and used to guide the further synthesis[6]. This approach resulted in a beta cells, inhibiting the transcription factors CREB and MafA, important series of novel 3-amido-benzhydroxamates as nanomolar inhibitors of for insulin gene transcription and insulin secretion. Therefore, an SmHDAC8 with good selectivity regarding the major human HDACs overexpression of DLK contributes to a loss of beta-cell function and (HDAC1 and HDAC6) also inhibitory effects in phenotypic screening inferentially to diabetes mellitus. and promising pharmacokinetic properties. A well-established way to intervene in kinase signal transmission is the inhibition by ATP competitive small molecule inhibitors. A major drawback of known inhibitors is the lack of DLK selectivity and suitable ADME properties. [1] In connection with a project to identify new selective inhibitors of DLK by structure-based virtual screening, 3D-QSAR models were generated by a set of structurally various inhibitors known from literature. Docking of these structures to the ATP-binding site of different DLK X- ray structures[2] led to an alignment of likely biological active binding conformations. This alignment was used to generate CoMFA and CoMSIA models with different field combinations. Thorough model evaluation by crossvalidation, test set prediction and statistical analysis References: (R², R²cv, Q²,…) provided valid 3D-QSAR models which will be 1. Hotez PJ, Pecoul B: PLoS Negl Trop. 2010, 4: e718 [3] presented on this poster. 2. Doenhoff MJ, Cioli D, Utzinger J: Curr Opin Infect 2008, 21: 659-667 These models will be used in the postprocessing and compound 3. Andrews KT, Haque A, Jones MK: Immunol Cell Biol 2012, 90: 66-77 selection steps of the above mentioned virtual screening workflow. 4. Marek, M. et al.: PLOS Pathogens. 2013, 9: e1003645 5. S. Kannan, J. Melesina, A. Hauser et al.: J. Chem. Inf. and Mod., 2014, 54: 3005-3019 6. T. Heimburg, A. Chakrabarti et al.: J. Med. Chem., 2016, 59: 2423-2435

POS.127 Synthesis and in vitro characterization of hydroxamic acids as small molecule inhibitors for epigenetic parasitic targets

Bayer, T.1; Melesina, J.1; Chakrabarti, A. 2; Marek, M.3; Romier, C.3; Erdmann, F.1; Schmidt, M.1; Jung, M.2; Sippl, W.1 1 Institut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck – Straße 4, 06120 Halle (Saale), Germany 2 Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104 Freiburg, Germany Training set compound GNE-3511 in the ATP-binding site of DLK (5ceo) 3 IGBMC, Université de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch Cedex, France

References: Schistosomiasis, also known as bilharzia is caused by a blood-dwelling 1. E. Oetjen, T. Lemcke, Expert Opinion on Therapeutic Patents 2016, 26, 607–616. fluke of the genus Schistosoma. It uses a fresh water snail as an 2. S. Patel et al., J. Med. Chem. 2015, 58, 8182–8199. intermediate host and is transmitted through contaminated water [1]. 3. a) R. D. Cramer, D. E. Patterson, J. D. Bunce, J. Am. Chem. Soc. 1988, 110, 5959–5967; b) Taking into account that an estimate of 258 million people are infected G. Klebe, U. Abraham, T. Mietzner, J. Med. Chem. 1994, 37, 4130–4146. worldwide among who 280 000 die annually schistosomiasis is one of the most important parasitic diseases after malaria [2, 3]. Praziquantel is an anthelminthic which is effective against all human forms of schistosomiasis. Without any effective vaccine available and the POS.126 excessive use of praziquantel for the treatment of infected individuals Characterization of 3-amido-benzhydroxamates as as well as for preventive treatment the problem of resistant schistosome strains is arising [1]. modulators of epigenetic targets for the treatment of Histone deacetylases (HDACs) take an important part in epigenetics schistosomiasis since the state of acetylation of the histones correlates with transcriptional control [4]. The schistosome histone deacetylase 8 Heimburg, T.1; Chakrabarti, A.2; Melesina, J.1; Robaa, D.1; Hauser, A. (smHDAC8) was recently identified as a potential target for antiparasitic T.2; Schmidtkunz, K.2; Marek, M.3; Romier, C.3; Pierce, R.4; Erdmann, therapy and a large scale screening identified several hydroxamic acids F.1; Schmidt, M.1; Jung, M.2; Sippl, W.1 as active compounds on HDACs [5, 6]. Based on a screening hit J1075 1 Institut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck – Straße 4, 06120 Halle (Saale), Germany (3 - Chloro benzothiophene - 2 -hydroxamic acid) a series of 2 Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, hydroxamic acids with different scaffolds and different substitution Albertstr. 25, 79104 Freiburg, Germany patterns were synthesized and characterized. Here we present the 3 IGBMC, Universite de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch Cedex, France 4 Center for Infection and Immunity of Lille (CIIL), Université Lille Nord de France, Institut synthesis of these compounds and their phenotypical and in vitro Pasteur de Lille, 1 rue Professeur Calmette, 59019 Lille Cedex, France effects on schistomomes.

Schistosomiasis is one of the major human neglected parasitic diseases[1]. At the moment, there is no licensed vaccine available against Schistosomiasis and for Praziquantel, the one drug which is effective against all schistosome species, reduced efficiency and drug resistance are reported[2]. Currently inhibitors of human epigenetic enzymes are investigated as novel anti-cancer drugs and have the

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Several in vivo and in vitro studies have recently showed the ability of hSirt2 to catalyze long chain deacylation of lysine residues from histone and non histone proteins.[3,4] As consequence of the similarity with smSirt2 the investigation regarding the ability of the parasitic enzyme to catalyze deacylation of lysine residues and its results are reported. Furthermore since the treatment of schistosomiasis relies on the only available drug Praziquantel,[5] a screening of diverse compound libraries has been used as strategy for finding novel smSirt2 inhibitors and developing new drugs against schistosomiasis.

Acknowledgements: We thank our collaborators from the CO-factors RTG1976 (Deutsche Acknowledgements: SEtTReND, A-ParaDDisE Forschungsgemeinschaft) and A-ParaDDisE (EU FP-7 Health no. 602080) consortium for funding and support References: 1. Deribew, K. et al.: Int. J. Med. Med. Sci., 2013, 5 (3), 131-139 References: 2. Steinmann, P. et al.: LancetInfectDis, 2006, 6 (7): 411-425 1. Lancelot, J. et al. Schistosoma mansoni Sirtuins: characterization and potential as 3. WHO Fact sheet Schistosomiasis, updated February 2016 chemotherapeutic targets. PLoS Negl. Trop. Dis. 2013, 9: e2428. 4. KrennHrubec K. et al.: Bioorg Med Chem Lett. 2007, 17 (10): 2874 - 2878 2. Schiedel, M. et al. Fluorescence-based screening assays for the NAD+-dependent histone 5. Kannan, S. et al.: J. Chem. Inf. Model., 2014, 54 (10), 3005–3019 deacetylase smSirt2 from Schistosoma mansoni. J. of Biomol. Screen. 2015, 20: 112-121. 6. Marek, M. et al.: PLoS path. 2013, 9 (9), e1003645 3. Feldman, J.L. et al. Activation of the protein deacetylase SIRT6 by long-chain fatty acids and widespread deacylation by mammalian sirtuins. J.Bio.Chem. 2013, 288: 31350-31356. 4. Teng, Y. et al. BNature/ Sci. Rep. 2015, 5: 8529. 5. Wilson, M.S. et al. Immunophatology of schistosomiasis. Immunol. Cell. Biol. 2007, 85: 148- 154. POS.128

Selective histone deacetylase 6 (HDAC6) inhibitors using the phenothiazine system as cap group framework POS.130 1 2 2 1 Vögerl, K. ; Senger, J. ; Jung, M. ; Bracher, F. Structure-guided development of D2R/NTS1R heterodimer- 1 Department of Pharmacy, LMU Munich, 81377 Munich, Germany, 2 Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, 79104 Freiburg, selective ligands Germany Möller, D. Histone deacetylases catalyze the removal of acetyl groups from lysine Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander Universität residues in histone and additionally in several non-histone proteins [1]. Erlangen-Nürnberg, Schuhstraße 19, 91052 Erlangen, Germany Tubastatin A has previously been described as potent and highly selective inhibitor of histone deacetylase 6 (HDAC6), a HDAC Dopamine D2 receptors (D2Rs), which belong to family A of G protein- isoenzyme mainly involved in the acetylation status of non-histone coupled receptors (GPCRs), regulate numerous physiological functions proteins. Structure-based drug design combined with homology and are involved in the pathophysiology of severe neuropsychiatric modeling techniques were used to develop this selective HDAC6 disorders including schizophrenia and Parkinson’s disease. Along with inhibitor consisting of a tetrahydro-γ-carboline as a surface recognition an increasing number of other GPCRs, D2Rs have been proven to not cap group, a p-tolyl group linker, forcing the cap to lie against the only exist as isolated entities but to form both homo- and heterodimers catalytic channel rim and bearing the zinc-binding group (ZBG) [2]. within the plasma membrane. Among receptors interacting with D2Rs in Inspired by this work we investigated various alternative polycyclic cap the central nervous system (CNS), the neurotensin receptor subtype 1 group frameworks. Based on the phenothiazine system in conjunction (NTS1R) together with its endogenous ligand neurotensin has gained with an aromatic hydroxamic acid we developed a new chemotype of substantial interest over the past decades. Both GPCRs are known to selective HDAC6 inhibitors with enhanced potency. be closely associated and highly co-localized in vivo. The specific targeting of heterodimers with bivalent ligands represents a powerful approach for the development of novel tool compounds and drugs for GPCRs. Driven by the binding energy of two recognition elements, a carefully designed bivalent ligand bridging the orthosteric binding sites of two adjacent protomers should exhibit extremely high binding affinity, and therefore high tissue selectivity for heterodimer- expressing cells over areas that express only one individual receptor. The growing number of recent high-resolution GPCR x-ray crystal structures and the increase in computational power available for homology modeling and molecular dynamics enabled us to design a References: collection of bi-orthosteric ligands addressing D2R/NTS1R heterodimers. 1. Dallavalle, S. et al.: Biochemical Pharmacology 2012, 84, 756-765 By means of solid state-supported synthesis NT(8-13), the active 2. Butler, K. V. et al.: Journal of the American Chemical Society 2010, 132, 10842-10846 fragment of neurotensin, was linked to three different D2R-specific pharmacophores employing -amino acid-functionalized polyethylene

glycol (PEG) spacers (Fig. 1). Within this series of D2R/NTS1R- recognizing heterobivalent ligands, only compounds with a sufficient POS.129 spacer size exhibited affinities in the low picomolar range and up to 10,000-fold selectivity for D2R/NTS1R-coexpressing cells compared to Investigation about the Schistosoma mansoni sirtuin 2 cells expressing D2R only. The preferential recognition of the (smSirt2) deacylase activity and its inhibitors D2R/NTS1R heterodimer over monomeric binding modes was confirmed in binding studies with membranes from striatal tissue, underpinning the Monaldi, D.1; Schiedel, M.1; Marek, M.2; Romier, C.2; Jung, M.1 biological relevance of the approach. 1 Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104, Freiburg, Germany. 2 Département de Biologie Structurale Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UDS), CNRS, INSERM, Illkirch, France.

Schistosoma mansoni sirtuin 2 (smSirt2) is a NAD+-dependent lysine deacetylase which has been recently identified as therapeutic target for the treatment of schistosomiasis, a parasitic disease caused by the flatworm Schistosoma mansoni.[1] Phylogenetic analysis have defined smSirt2 as orthologous of the human Sirtuin 2 (hSirt2)[1] and even if a crystal structure of SmSirt2 has been not solved yet, homology models have reported high similarity with the human isoform.[2]

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TRPML3 and increased selectivity either for human or for mouse TRPML3.

References: 1. Grimm, C. et al, Chemistry and Biology, 2010, 17(2): 135-148.

POS.132 Fig. 1: Overview of the D2R/NTS1R heterodimer geometry modelled based on recent x-ray crystal structures of the closely related D3R, the NT(8-13)-bound NTS1R and the synthesized Development of a G-protein biased mu opioid agonist with series of heterobivalent ligands targeting D2R/NTS1R. reduced side effects

Using bivalent ligands containing a D2R-agonist substructure, we could Dengler, D.1; Manglik, A.2; Lin, H.3; Aryal, D. K.4; McCorvy, J.4; Corder, demonstrate that Gi/Go-promoted D2R signaling was attenuated in the G.5; Levit, A.3; Kling, R. C.1; Bernat, V.1; Hübner, H.1; Huang, X. P.4; D2R/NTS1R-coexpressing cells, while the compounds behaved as full Sassano, M. F. 4; Giguere, P. M.4; Löber, S.1; Duan, D.2; Scherrer, G.5; D2R agonists in absence of NTS1R. In contrast, our bivalent ligands Kobilka, B. K.2; Roth, B. L.4; Shoichet, B. K.3; Gmeiner, P.1 showed atypical dose-response curves in a -arrestin recruitment 1 Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich- assay and were able to significantly enhance -arrestin recruitment to Alexander University, Schuhstraße 19, 91052 Erlangen, Germany 2 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, the D2R/NTS1R heterodimer. In summary our results suggest a highly Stanford, CA 94305, USA selective binding profile and distinct signalling behavior for our bi- 3 Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158, orthosteric heterobivalent ligands compared to monovalent congeners USA 4 Department of Pharmacology, UNC Chapel Hill Medical School, Chapel Hill, NC 27514, USA addressing D2R or NTS1R, thereby opening a promising perspective 5 Department of Anesthesiology, Stanford University School of Medicine, Stanford, CA 94305 towards novel tools for GPCRs. USA.

References: Morphine and related opioids are well-known traditional analgesics and 1. Hübner, H., Schellhorn, T., Gienger, M., Schaab, C., Kaindl, J., Leeb, L., Clark, T., Möller, play until today an essential role in pain therapy. Their powerful 2. D.*, Gmeiner, P.* Nat. Commun., 2016, doi: 10.1038/ncomms12298, in press. analgesic effect is mainly referred to their ability to activate the μ opioid receptor (μOR). While the wide range of μOR agonists are alleviating pain extremely efficacious, they all suffer from similar adverse effects like respiratory suppression, constipation, sedation, physical POS.131 dependence and tolerance. The side effects often limit the dose for opioid analgesics resulting in an inadequate pain management. Small molecule activators of TRPML channels Recent studies with morphine in β-arrestin-2 knock out mice suggest that the analgesic effect results from stimulation of the G-protein Plesch, E.1; Keller, M.1; Chao, Y. K.2; Chen, C. C.2; Grimm, C.2#; pathway, while many side effects may be an outcome of activated β- Bracher, F.1# arrestins. Thus, functionally selective μOR agonists with bias towards 1 Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität, Butenandtstraße 5-13, 81377 München, Germany G-protein promoted signaling are required. [1] 2 Department of Pharmacy - Center for Drug Research and Center for Integrated Protein The determination of the μOR crystal structure [2] provides an Science Munich (CIPSM), Ludwig-Maximilians-Universität, Butenandtstraße 5-13, opportunity to seek μOR ligands with new chemotypes via structure- 81377 München, Germany # shared last based approaches. In silico screening of over three million commercially

available compounds against the orthosteric binding pocket and further TRPML channels, also called mucolipins, are endolysosmal cation structure-based optimization of initial docking hits led us to the channels. Mutations of TRPML1 in humans can cause the identification of a novel μOR agonist scaffold. Starting from the neurodegenerative lysosomal storage disorder mucolipidosis type IV originally determined hit as an isomeric mixture, we were able to while TRPML3, when mutated, causes deafness and pigmentation develop an optically pure, subtype specific and G-protein biased μOR defects. The discovery of small molecule activators in a recent high- agonist (PZM21) by an appropriate synthetic strategy. Based on the throughput screen [1] has significantly fostered and facilitated TRPML crystal structure of the μOR in the active state [3], the binding mode of channel and thus endolysosomal ion channel research. Currently PZM21 could be established using SAR and molecular dynamics available TRPML channel agonists are non-selective and activate all studies involving diffusible and covalently binding analogs. PZM21 three TRPML channel isoforms [1]. Through systematic chemical shows an unique efficacy profile in mice models. The compound modifications of a selected lead structure, we have now designed generates substantial analgesia with only little suppression of potent and selective TRPML channel agonists. respiration compared to morphine. Results of mouse open field Our lead structure SN2 [1] potently activates human and mouse locomotion and conditioned place preference experiments indicate that TRPML3 but lacks selectivity for mouse TRPML1 and human TRPML1 PZM21 may have less reinforcing activity than classic opioids. Uniquely, and TRPML2 (Figure 1). With the aim to further improve the potency PZM21 seems also to be able to differentiate between the affective and selectivity profile of SN2-type compounds, we set out to synthesize component of pain and reflexive behaviors. Hence, this novel scaffold and test >50 chemically modified versions of SN2. may serve both as a probe to investigate μOR signaling and as a

therapeutic lead for a new class of opioid analgesics devoid of many of the dose-limiting side effects. [4]

References: 1. L.M. Bohn et al., Science 1999, 286: 2495-2498 2. A. Manglik et al., Nature 2012, 485: 321-326 3. W. Huang et al., Nature 2015, 524: 315-321 4. A. Manglik et al., Nature 2016, accepted for publication

These modifications comprise variations of the substitution pattern of the aryl-ring, variations of the aliphatic norbornane ring system, aromatisation of isoxazoline to an isoxazole fragment, introduction of polar substituents as well as replacement of the isoxazol(in)e ring by other heterocycles. Following synthesis, we tested the compounds in overexpressing HEK293 cells using calcium imaging. We thus identified novel compounds with an increased efficacy for both mouse and human

136 • DPhG Annual Meeting 2016 Conference Book DRUG DESIGN/MEDICINAL CHEMISTRY

In an attempt to address this need, we performed an antagonist POS.133 screening campaign investigating compounds from our in-house Identification and in vitro characterization of GPCR ligands compound library in both cAMP accumulation and β-arrestin by a covalent docking approach recruitment assays at the human GPR84 recombinantly expressed in Chinese hamster ovary (CHO) cells. As a result, we identified several Stößel, A.1; Fish, I.2; Hübner, H.1; Shoichet, B. K.2; Gmeiner, P.1 hit compounds which blocked the receptor, the most potent compound 1 Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich- showing an IC50 value of 1.40 µM in cAMP assays (versus 20 µM Alexander University, Schuhstraße 19, 91052 Erlangen, Germany 2 Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158, decanoic acid corresponding to its EC80). The new antagonists USA represent useful pharmacological tools to further investigate the (patho)physiological roles of GPR84. Furthermore, these structures will G protein-coupled receptors (GPCRs) are the largest family of cell- serve as starting points for optimization and development of more surface receptors and remain a highly relevant drug target. Covalent potent GPR84 antagonists. molecular probes have proven to be useful tools to gain further insights Moreover, in order to gain a better understanding of the signalling in structural and functional properties of GPCRs. They have shown pathways involved in the immune response triggered by MCFAs, we enhanced potency or selectivity towards the targeted protein and have evaluated a series of MCFAs as well as hydroxylated MCFAs in cAMP demonstrated the ability to stabilize ligand-receptor complexes for and β-arrestin assays. The results revealed that the structure- crystallization. [1] activity relationships of the MCFAs obtained in cAMP assays differ from Recently, a new approach (DOCKovalent) for the screening of large those observed in β-arrestin assays suggesting biased signalling of virtual libraries of electrophilic small molecules was developed and the natural agonists. In contrast to the non-hydroxylated MCFAs, the applied to discover covalent ligands for different enzymatic systems. [2] hydroxylated fatty acids showed higher activity in cAMP assays than in We successfully adopted this structure-based virtual screening method -arrestin assays, leading to the assumption that the hydroxylated to identify covalent molecular probes for three different class A GPCRs. MCFAs are Gi-biased. This may have implications with regard to the Therefore, a library of approximately 800,000 commercially available or modulation of inflammatory responses by MCFAs. synthetically accessible small molecules bearing a reactive group (Michael system or alkyl halide) were virtually screened against the µ- References: opioid receptor, the ß2-adrenergic receptor (H2.64C mutant) and the 1. Wang, J et al.: J. Biol. Chem. 2006, 281: 34457-34464. 5HT2A receptor (S2.61G, T2.64C double mutant). 2. Bouchard, C et al. Glia 2007 55: 790-800. The high-ranking candidates were selected for experimental validation 3. Suzuki, M et al.: J. Biol. Chem. 2013, 288: 10684-10691. in a radioligand depletion assay indicating an irreversible blocking of the 4. Dietrich, P.A. et al.: Blood 2014, 124(22): 3284-3294. orthosteric binding site. The identified hits were further characterized by 5. Nicol, L.S.C. et al.: J. Neurosci. 2015, 35(23): 8959-8969. 6. Audoy-Rémus, J. et al.: Brain Behav. Immun. 2015, 46: 112-120. kinetic and functional studies, and used as templates for an additional screening round of close analogs. Taking advantage of this concept, we have identified specific covalent hits indicating that the covalent docking approach can be applied prospectively on several targets and may have broad utility for the POS.135 discovery of covalent ligands or fragments for GPCRs. Identification of sphingosine-1-phosphate receptor ligands using a combined approach of molecular dynamics and pharmacophore-based virtual screening

Bermudez, M.1; Leutz, S.1; Nguyen, T. N.1; Wolber, G.1 1 Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2, 14195 Berlin, Germany

Belonging to the class of G protein-coupled receptors (GPCRs)1, sphingosine-1-phosphate receptors (S1PRs) represent promising thus

References: challenging targets for the treatment of various inflammatory diseases 2 1. Weichert, D.; Gmeiner, P.: Covalent Molecular Probes for Class A G Protein-Coupled and autoimmune disorders . However, only few orally bioavailable Receptors: Advances and Applications. ACS Chem Biol 2015, 10(6), 1376-1386. ligands are known to date and only fingolimod is therapeutically used 2. London, N. et al.: Covalent docking of large libraries for the discovery of chemical probes. Nat for the treatment of multiple sclerosis. The aim of this study is to Chem Biol 2014, 10(12), 1066-72. discover novel S1PR ligands by computer-aided drug design as both pharmacological tools and potential drug candidates. To date only one out of 5 S1PR subtypes has been crystallized3. Starting from the crystal structure of the S1PR 1 subtype we developed POS.134 a structure-based virtual screening workflow that combines chemical feature-based 3D-pharmacophores and conformational sampling by Pharmacological tools for the immune-stimulatory orphan molecular dynamics (MD) simulations4,5. Due to the availability of a G protein-coupled receptor 84 crystal structure we initially focused on the S1PR 1 subtype and identified 10 potential ligands with high chemical diversity. In the second Köse, M.1; Schiedel, A. C.1; Brandt, S.1; Hoffmann, K.2; von Kügelgen, step we investigate structural differences between the 5 subtypes by I.2; Müller, C. E.1 means of homology modeling, docking and MD simulations. This 1 PharmaCenter Bonn, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, information will be essential for the future development of ligands with 53121 Bonn, Germany 2 PharmaCenter Bonn, Department of Pharmacology, University of Bonn, Sigmund-Freud- subtype specific effects. Straße 25, 53127 Bonn, Germany Taken together we present and discuss the virtual screening work-flow, model validation, experimental challenges and the MD-based The G protein-coupled receptor (GPCR) 84 (GPR84) is a Gi-coupled enhancement of the static pharmacophore models that were used to class A GPCR which is highly expressed in the bone marrow and in study S1PRs. microglial cells; and upregulated under inflammatory conditions. GPR84 can be activated by medium-chain fatty acids (MCFAs, 9-14 carbon References: chain length) leading to an enhanced cytokine (IL-12 p40, IL-8 and 1. M. Bermudez, G.Wolber, Bioorg. Med. Chem., 2015, 14, 3907-3912. TNF-α) production in myeloid cells.1-3 Recent in vivo experiments 2. R. Proia and T. Hla, J. Clin. Invest., 2015, 125, 1379-1387 have indicated that GPR84 may play an important role in myeloid 3. M.Hanson et al., Science, 2012, 335, 851-855 4, 5 6 4. A. Bock and M.Bermudez et al.: J. Biol. Chem., 2016, in press, doi: leukemia neuropathic pain and Alzheimer´s disease. Thus, GPR84 10.1074/jbc.M116.735431 might be a promising drug target, e.g., for the treatment of inflammatory 5. M. Bermudez et al.: Drug Discov. Today, 2016, in press, doi: 10.1016/j.drudis.2016.07.001 diseases. Besides MCFAs, which activate GPR84 in the micromolar concentration range, only very few ligands are available. However, selective pharmacological tools are required for further evaluation of GPR84 as a potential new drug target.

DPhG Annual Meeting 2016 Conference Book • 137 POSTERS

ACh is regulated by cholinesterases (ChEs), namely POS.136 acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). A Design and synthesis of OH-substituted benzo[7]annulen- therapeutic approach is to inhibit these two hydrolytic enzymes. Well 7-amines as GluN2B selective NMDA receptor ligands known AChE inhibitors are tacrine, donepezil, galantamine and rivastigmine, with tacrine being the most potent derivative in the Temme, L.1; Wünsch, B.1 series [5]. Unfortunately, the use of tacrine is currently limited by its 1 PharmaCampus, Department of Pharmaceutical and Medicinal Chemistry, WWU Münster, serious hepatotoxicity [6] and the clinical effectiveness of AChEIs is still Corrensstr. 48, 48149 Münster, Germany under debate [7]. It is therefore desirable to develop new and highly effective drugs for AD. The function of the ionotropic NMDA receptor is important for synaptic Promising candidates are bipharmacophoric hybrid compounds in which plasticity. Whilst its hyperactivity leads to diseases like Alzheimer´s and the inhibitory potency of tacrine can be combined with other Parkinson´s disease, its hypoactivity is for example involved in the pharmacological benefits, such as reduced hepatotoxicity [8]. In this development of schizophrenia. Therefore it is necessary to reconstitute context, compound 1, consisting of tacrine and the muscarinic the function of the NMDA receptor. Several strategies have been superagonist iperoxo [9] (imitating the endogenous ligand ACh) linked employed to reduce the hyperactivity and therefore prevent the resulting by a C10 polymethylene spacer, was found to have excellent damaging of neurons (excitotoxicity). Whereas open channel blockers anticholinesterase activities for both AChE (IC50 = 0.155 nM) from lead to a total block of the NMDA receptor and therefore cause electric eel and isolated rat brain as well as for BChE (IC50 = 1.797 nM) unwanted side effects like hallucinations, GluN2B selective NMDA from equine serum, respectively. Additionally, this hybrid shows less receptor antagonists reduce the excitotoxicity by stabilizing the agonist- cytotoxicity than the corresponding tacrine-related dimers. Docking bound receptor in a desensitized state and preserving the physiological experiments provide a structural model to rationalize the inhibitory function. [1] power towards AChE. Compound 1 is a promising candidate for further drug development investigations.

Based on the lead structure ifenprodil a conformational restriction approach was persued with the aim to overcome its low selectivity. [2] Encouraged by the promising results this approach was applied on a Acknowledgments: Thanks are due to the Institute for Molecular Infection Biology of the University of Würzburg (Germany) for evaluation of the cytotoxicity of the tacrine-related distinct and but also highly potent GluN2B antagonist, Ro 25-6981. The compounds. aminoalcohol 1 shows high GluN2B affinity (Ki=16 nM), inhibitory References: activity (IC50=12 nM) and selectivity towards σ1 and σ2 receptors. The 1. Deutsche Alzheimer Gesellschaft, Factsheet 2014. phenol 2 is even 10 times more affine (Ki=1.6 nM). [3, 4] Additionally, 2. Hardy, J.: J. Neurochem. 2009, 110(4): 1129-1134. molecular docking studies showed promising interactions between the 3. Bulic, B. et al.: Angew. Chem. Int. Ed. Engl. 2009, 48(10): 1740-1752. OH moieties of 1 and 2 and crucial amino acids in the binding pocket of 4. Gella A., Durany, N.: Cell Adh. Migr. 2009, 3(1): 88-93. the receptor. [5] Therefore further compounds with structure 3 were 5. Grimmer T., Kurz A.: Drugs Aging 2006, 23(12): 957-967. designed to combine the promising properties of 1 and 2 with the 6. Watkins, P.B. et al.: JAMA: J. Am. Med. Assoc. 1994, 271(13): 992-998. 7. Munoz-Torrero D.: Curr. Med. Chem. 2008, 15(24), 2433-2455. objective to increase affinity, selectivity and inhibitory activity. The 8. Nepovimova E. et al.: J. Med. Chem. 2015, 58(22): 8985-9003. poster will show the synthesis of ligands 3, their GluN2B affinity and 9. Schrage R. et al.: Br. J. Pharmacol. 2013, 169(2): 357-370. further relationships between the structure and the GluN2B affinity.

Acknowledgements: Cells-in-Motion Cluster of Excellence in Münster, Prof. Wolfgang Sippl and his working group in Halle References: POS.138 1. Zhu, A. et al.: Cell 2016, 165: 704–714. Synthesis, receptor-subtype-selectivity and species- 2. Fischer, J. et al.: Pharmacol. Exp. Ther. 1997, 283: 1285–1292. 3. Benner, A. et al.: ChemMedChem 2014, 9, 741–751. selectivity of dimeric hetarylpropylguanidine-type- 4. Gawaskar, S. et al.: Bioorg. Med. Chem 2014, 22, 6638–6646. histamine H2 receptor agonists (hH1,2,3,4R, gpH1,2R, rH2R) 5. The docking studies were performed in cooperation with Prof. Dr. Wolfgang Sippl, University of Halle. Pockes, S.1; Buschauer, A.1; Elz, S.1 1 Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany

Since the 1970s 3-(1H-imidazol-4-yl)propylguanidine (SK&F-91,486 POS.137 (1)[1]) is known as the prototypic pharmacophore of highly potent Novel Hybrid Inhibitors of Cholinesterases as Potential histamine H2-receptor (H2R) agonists of the guanidine class of Anti-Alzheimer Agents compounds including, e.g., impromidine and arpromidine.[2] In order to understand more of the structure-activity relationships of alkylated Messerer, R.1; Dallanoce, C.2; Matera, C.2; Wehle, S.1; Flammini, L.3; analogues of SK&F-91,486, we characterized 78 newly synthesized Barocelli, E.3; Decker, M.1; Sotriffer, C.1; De Amici, M.2; Holzgrabe, U.1 derivatives including several bivalent compounds (e.g., 2-5) by using 1 Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, different pharmacological in-vitro methods.[3] University of Würzburg, Am Hubland, 97074 Würzburg, Germany 2 Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica “Pietro Pratesi”, The already mentioned dimers were equipped with different spacer Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy lenghts (C3-C12) and various heteroaromatic 5-ring systems. We 3 Dipartimento di Farmacia, Università degli Studi di Parma, Parco Area delle Scienze, 27/A, replaced the initial imidazol-4-yl group of SK&F-91,486 (1) by the 43124 Parma, Italy bioisosteric 2-amino-4-methylthiazol-5-yl and 2-aminothiazol-5-yl moieties, respectively, to study their influence on histamine receptor Alzheimer`s disease (AD) is the most prominent form of dementia subtype selectivity. affecting worldwide about 9% of the population aged over 65 and the The potential H2R agonists were subjected to a broad screening number of patients will rise to estimated 81.1 million by 2040 [1]. AD is procedure utilizing radioligand binding assays with membranes of Sf9 a multifactorial disease and several theories about its pathogenesis are cells[4] (hH1,2,3,4R). Compounds were also functionally characterized in discussed, mostly including the ß-amyloid cascade [2], the -protein the [35S]GTPS assay (hH2R, Sf9 cell membranes).[5] Receptor subtype hyperphosphorylation hypothesis [3], oxidative stress, free radical selectivity vs. hH1,3,4R was determined for selected derivatives also formation and neuroinflammation [4]. Today’s medication is based on using this technique. The most promising dimers (2-5) were also tested the cholinergic hypothesis, asserting that a decline of acetylcholine with gpH2R- and rH2R-Sf9 cell membranes in the [35S]GTPS assay to (ACh) in the brain leads to cognitive and memory deficits. The level of characterize the species profile of the ligands.

138 • DPhG Annual Meeting 2016 Conference Book DRUG DESIGN/MEDICINAL CHEMISTRY

Organ bath studies (gpH1R (ileum), gpH2R (right atrium)) yielded functional data in a more physiological environment. The major part of POS.140 the new SK&F-91,486 analogues displayed partial or full agonism via A divergent synthesis of oxoaporphine and hH2R, rH2R, and gpH2R, respectively. The most potent analogue, oxoisoaporphine alkaloids via direct metalation of bivalent thiazole-type bisguanidine 5, was a partial agonist (Emax = alkoxyisoquinolines 88%) and 250-times (pEC50 = 8,56) as potent as histamine vis-à-vis the gpH2R. Attempts to antagonize the positive chronotropic effect of Melzer, B.1; Bracher, F.1 (partial) agonists by preincubation with cimetidine, or by adding a 1 Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians University, cimetidine bolus at the end of the concentration-response curve, Butenandtstr. 5-13, D-81377 Munich, Germany respectively, were successful and furnished pA2 values for the antagonist (5.87 – 6.38) which are in accordance with literature data. The tetracyclic oxoaporphine and oxoisoaporphine alkaloids are a small class in the wide field of benzylisoquinoline alkaloids. Due to their antibacterial, antifungal, anticancer, antiangiogenic and other biological Nr. R1 R2 n NH NH NH n activities they are of high pharmaceutical relevance [1,2]. N 2 A A 8 N NH2 R1 N N N N R2 H H H H H 3 A A Herein we describe a novel and divergent synthetic approach to HN 12 (1) (2-5) 4 B B 8 oxoaporphine and oxoisoaporphine alkaloids starting from 5 B C 8 S S alkoxyisoquinolines. Direct metalation as key-step using the Knochel- N H N = A2 = B H2N = C HN N N Hauser base (TMPMgCl·LiCl) led to 1-magnesiated isoquinolines [3,4], CH3 which in turn afforded 1-iodoisoquinolines by quenching with iodine or References: aryl(isoquinolin-1-yl)carbinols upon trapping with aromatic aldehydes 1. Parsons, M.E. et al.; Agents Actions 1975, 5, 464. [4], respectively. Photochemical cyclization of ortho-bromo 2. Buschauer, A.; J. Med. Chem. 1989, 32, 1963-1970. aryl(isoquinolin-1-yl)carbinols under reductive conditions gave the 3. Pockes, S.; Dissertation, Univ. Regensburg 2015. oxoaporphine alkaloids lysicamine and oxoglaucine. For the synthesis 4. Seifert, R. ; J. Pharmacol. Exp. Ther. 2003, 305, 1104-1115. 5. Schneider, E.H.; Seifert, R.; Pharmacol. Ther. 2010, 128, 387-418. of oxoisoaporphine alkaloids, the required intermediate 1- arylisoquinolines were obtained by palladium-catalyzed Suzuki cross- coupling of the 1-iodoisoquinolines with the appropriate arylboronic acid pinacol ester building blocks. Subsequent hydrolysis of the ester to a carboxylic acid, directly followed by Friedel-Crafts type cyclization with POS.139 Eaton’s reagent afforded the 6-O-demethylated oxoisoaporphines 6-O- Computational simulations guided by experiments - a demethylmenisporphine and dauriporphinoline. Re-methylation of these strong alliance for GPCR research alkaloids using Kunitomo’s protocol [5] with methyl iodide and silver oxide gave the alkaloids menisporphine and dauriporphine. Bermudez, M.1; Wolber, G.1 1 Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2, 14195 Berlin, Germany

G protein coupled receptors (GPCRs) are integrative and highly dynamic signaling machines, transferring information across membranes via multiple signaling pathways [1]. Driven by the hypothesis form follows function the complexity of GPCR signaling roles requires a large conformational ensemble of distinct receptor states. Over the last decade, specific receptor-ligand complexes were determined by crystallography [2] providing an indispensable structural view on this protein class. However, these crystal structures represent single static conformations of highly flexible proteins. For a better understanding of receptor functionality we need functional models that consider GPCRs as dynamic entities. By means of computational simulations and 3D-pharmacophore analysis we show a dynamic view on GPCR signaling [3,4]. Taking muscarinic acetylcholine receptors as model systems we link conformational characteristics to distinct receptor functions [5]. Guided by pharmacological experiments, we developed mechanistic models to show that partial agonists for the muscarinic M2 receptor stabilize distinct fractions of inactive agonist-bound receptors [6]. Using bitopic ligands as pharmacological tools, we clearly identified a dualsteric and a purely allosteric binding mode stabilizing active and inactive receptor states, respectively. Modulation of the resulting ligand References: 1. Stévigny, C., Bailly, C., Quetin-Leclercq, J.: Curr. Med. Chem.: Anti-Cancer Agents 2005, binding ensembles by various means reveals that agonist’s preference 5(2): 173-182. for inactive receptor complexes decreases its overall efficacy. Our 2. Liu, Y. et al.: Curr. Top. Med. Chem. 2013, 13(17), 2116-2126. findings suggest a more general role of ligand binding ensembles in 3. Metzger, A., Schade, M. A., Knochel, P.: Org. Lett. 2008, 10(6), 1107-1110. determining agonist efficacy and may pave the way towards the rational 4. Melzer, B., Bracher, F.: Org. Biomol. Chem. 2015, 13, 7664-7672. design of partial agonists. 5. Kunitomo, J., Satoh, M.: Tetrahedron 1983, 39(20), 3261-3265. Independently from this effect, we demonstrate on a molecular level how specific signaling pathways can be addressed in a ligand- dependent and predictable manner. Our data indicate a restriction of the conformational flexibility based on the chemical structure of a ligand POS.141 resulting in a shift of the physiologically imprinted signaling preference. This chemically encoded conformational selection potentially represents Total synthesis of Rubrolide R and S a rational explanation for biased signaling, not only for different G proteins, but also for other signaling pathways including β-arrestins or Schacht, M.; Boehlich, G. J.; De Vries, J.; Schützenmeister, N. Universität Hamburg, Pharmaceutical and Medicinal Chemistry, Bundesstraße 45, GPCR-interacting proteins. D-20146 Hamburg

References: The increasing emerge and distribution of resistant pathogens primary 1. A. Bock et al.: Trends Pharmacol. Sci., 2014, 35, p630–638. demands the discovery and development of novel antimicrobial agents 2. C. Piscitelli et al.: Mol. Pharmacol., 2015, 88, 536-551. 3. M. Bermudez, G.Wolber, Bioorg. Med. Chem., 2015, 14, 3907-3912. and therapeutic approaches [1]. A promising source in this subject are 4. M. Bermudez et al.: Drug Discov. Today, 2016, in press, doi: 10.1016/j.drudis.2016.07.001 represented by rubrolides, which are biological active metabolite from 5. M. Bermudez et al.: Mol. Inform., 2015, 8, 526-530. marine derived organisms [2]. In 2014 rubrolide R 1 and S 2 have been 6. A. Bock and M.Bermudez et al.: J. Biol. Chem., 2016, in press, doi: isolated from the marine-derived fungus Aspergillus terreus (OUCMDZ- 10.1074/jbc.M116.735431

DPhG Annual Meeting 2016 Conference Book • 139 POSTERS

1925).[3] Both rubrolide structures reveal biological activity against influenza A and rubrolide S 2 has also shown activity against the POS.143 tobacco mosaic virus.[4] The total synthesis of these two novel natural Investigation of the hemagglutinin cleaving type-II products is essential for further biological investigations, especially in transmembrane proteases TMPRSS2 and matriptase as the field of antiviral as well as antibiotic testings. The rubrolide scaffold potential targets for influenza treatment can be dissected into three building blocks. A short synthesis avoiding toxic tin compounds and unnecessary protecting groups has been Keils, A.1; Böttcher-Friebertshäuser, E.2; Magdolen, V.3; Steinmetzer, established. The key steps of the synthesis are a Suzuki-Miyaura cross T.1 coupling [5] and a vinylogous Knoevenagel condensation [6]. 1 Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35037 Marburg, Germany 2 Institute of Virology, Philipps University Marburg, Hans-Meerwein-Str. 3, 35043 Marburg, Germany 3 Department of Gynecology, Technical University of Munich, Ismaninger Str.22, 81675 München, Germany

The activation of the surface glycoprotein hemagglutinin (HA) by host proteases is indispensable for influenza virus infection. HA is synthesized as a precursor protein (HA0) and cleaved into the subunits HA1 and HA2 either in the trans-Golgi network (TGN) or on the cell surface. Whereas the HA of highly pathogenic avian influenza viruses (HPAIV) is cleaved by furin-like proprotein convertases, the HA of the seasonal and pandemic human viruses as well as low pathogenic avian influenza viruses (LPAIV) is activated by trypsin-like serine proteases, such as TMPRSS2 and matriptase. Therefore, these proteases References: 1. European Center for Disease Prevention and Control Antimicrobial surveillance in Europe emerged as potential targets for influenza treatment [1]. 2013, Annual Reprort oft he European TMPRSS2 and matriptase belong to the type II transmembrane serine Resistance Surveillance Network (EARS-Net), 2014. proteases (TTSPs), which contain an N-terminal cytosolic segment, 2. S. Miao et al.: J. Org Chem. 1991, 56, 6275-6260. followed by a transmembrane domain, a variable “stem region” and a C- 3. W. Zhu et al.: J. Antibiot. 2014, 67, 315-318. terminal extracellular protease domain of the chymotrypsin S1-fold. The 4. Q.-F. Hu et al.:, Heterocycles 2014, 89, 2177-2183. TTSPs are synthesized as single chain zymogens and are activated by 5. J. Boukouvalas, L.C. McCann, Tetrahedron Lett 2010, 51, 4636-4639. autocatalytic cleavage after an arginine or lysine residue, with the 6. J. Boukouvalas, F. Maltais, N. Lachance, Tetrahedron Lett. 1994, 43, 7897-7900. P4/P’4 cleavage site RQAR↓VVGG for matriptase and RQSR↓IVGG in case of TMPRSS2. Through a conserved disulfide bond, linking the protease domain with the “stem region”, these TTSPs are likely to remain membrane-bound following activation. Active matriptase can be POS.142 detected on the cell surface and after shedding also in the extracellular GluN2B selective antagonists: Enantiomerically pure space. In contrast, TMPRSS2 activity is principally limited to the inside tetrahydro-3-benzazepines derived from (S)-DOPA of the cell where it accumulates in the TGN. Using an expression plasmid encoding the protease domain with an N- terminally located histidine-tag, followed by an enterokinase cleavage Rath, S.1; Wünsch, B.1 1 PharmaCampus, Institut für Pharmazeutische und Medizinische Chemie, Westfälische site, matriptase was conveniently expressed in form of inclusion bodies Wilhelms-Univ. Münster, D 48149 Münster, Germany in E.coli. After refolding, purification, and activation by enterokinase sufficient material for kinetic measurements and structure Due to overaging global population neurodegenerative diseases like determinations could be obtained. In contrast, the preparation of active Alzheimer’s disease and Parkinson’s disease have attracted major TMPRSS2 using E. coli expression systems is problematic, detailed attention. Thus the development of selective therapeutic drugs for information about TMPRSS2 remains rare and is mainly based on treatment is of high interest. A promising approach is to address the subcellular full-length expression in mammalian cell systems. GluN2B subunit of the NMDA receptor. Overactivation of the NMDA Therefore, we are working on an appropriate mammalian expression receptor leads to excitotoxicity which is associated with system for TMPRSS2 using different constructs to obtain active neurodegenerative diseases. Ifenprodil served as promising lead protease for further biochemical characterization and the development compound for the design and development of novel GluN2B of new inhibitors. antagonists. Although ifenprodil shows high affinity (Ki = 10 nM, IC50 = 13.3 nM) its selectivity is rather low.[1,2,3,4] References: Based on the ifenprodil structure our group developed antagonists 1 1. Garten W. et al.: EurJ Cell Biol. 2015 94(7-9): 375-83 with a 3-benzazepine scaffold. Several compounds of type 1 possess high GluN2B affinity and antagonistic activity. The phenyl butyl derivative 1a (R1 = CH3, R2 = H, R3 = (CH2)4Ph) with a Ki value of 5.4 nM and an IC50 value of 360 nM belongs to the most active and POS.144 selective GluN2B antagonists.[5] Based on these encouraging results, a chiral pool synthesis of CRISPR/Cas9 Bid knockout reveals a key role for Bid- diastereo- and enantiomerically pure 3-benzazepines 2 starting from mediated mitochondrial damage in ferroptosis (S)-DOPA was envisaged. The additional substituents in position 1 and 4 should allow the fine tuning of the GluN2B affinity. Moreover, the Jelinek, A.1; Neitemeier, S.1; Hoffmann, L.1; Ganjam, G. K.1; Culmsee, introduction of [18F] to generate a tracer for positron emission C.1 tomography (PET) is possible. Evaluation of binding affinity is currently 1 Institute of Pharmacology and Clinical Pharmacy, Biochemical-Pharmacological Center, Philipps-University Marburg, Karl-von-Frisch-Straße 1, 35043 Marburg, Germany in progress. Targeted genome engineering by CRISPR/Cas9 is an evolving tool for generating specific knockout cell lines by DNA cleavage and introduction of insertion or deletion mutations. In this study, we exploited this tool to generate a Bid (BH3-interacting domain death agonist) knockout cell line in neuronal HT-22 cells. Bid determines regulated cell death in paradigms of oxidative stress by glutamate toxicity (oxytosis) in neurons, where its activation and mitochondrial References: translocation mediates mitochondrial damage, subsequent release of 1. Kemp, J. A., McKernan, R. M.; Nat. Neurosci. 2002, 5, 1039–1042. Apoptosis inducing factor (AIF) and cell death. In the present study we 2. Stark, H., Graßmann, S., Reichert, U., Pharm. UZ 2000, 29, 159–166. generated a Bid CRISPR/Cas9-knockout cell line to elucidate the 3. Schepmann, D. et al., J. Pharm. Biomed. Anal. 2010, 53, 603–608. potential role of BID in a model of ferroptosis in HT-22 cells. Ferroptosis 4. Borza, I., Domány, G., Curr. Top. Med. Chem. 2006, 6, 687–695. has recently been characterized as an iron-dependent form of oxidative 5. Tewes, B. et al., ChemMedChem 2010, 5, 687–695.

140 • DPhG Annual Meeting 2016 Conference Book DRUG DESIGN/MEDICINAL CHEMISTRY stress induced cell death [1] and involves cystine/glutamate antiporter (Xc-) inhibition, impairment of GpX4 (glutathione peroxidase 4) activity and subsequently increased lipid peroxidation. So far, however, the key mechanisms mediating cell death as a consequence of ROS accumulation and potential involvement of mitochondrial death pathways in paradigms of ferroptosis have not been reported. In order to investigate mechanisms of ferroptosis in neuronal HT-22 cells, we applied erastin as a pharmacological inhibitor of Xc- and analyzed glutathione depletion (GSH assay), lipid peroxidation (BODIPY - FACS analysis), Bid translocation to the mitochondria (confocal fluorescent microscopy), mitochondrial ROS formation (MitoSOX - FACS analysis), mitochondrial morphology (fluorescence microscopy), and cell death (Annexin V/PI - FACS analysis). In this paradigm of ferroptosis in neuronal cells we detected time-dependent GSH depletion, followed by increases in lipid peroxidation and mitochondrial impairments which all preceded cell death. Using the CRISPR/Cas9-Bid-knockout HT-22 cell line we found that Bid knockout prevented erastin-induced cell death as well as upstream events, e.g. lipid peroxidation, loss of mitochondrial membrane potential (TMRE - FACS analysis), mitochondrial ROS production as well as mitochondrial fission. In contrast, erastin-mediated early glutathione depletion was not affected by Bid knockout. In addition, we analyzed the effects of the established pharmacological inhibitors ferrostatin-1, liproxstatin-1 and the BID inhibitor BI-6c9 on paradigms of mitochondrial parameters in wild-type cells compared to the effects in Bid-knockout cells after induction of ferroptosis with erastin. BI-6c9 inhibited erastin-induced morphological changes and also prevented cell death. Similar protective effects were achieved in a concentration-dependent manner with ferrostatin-1 and liproxstatin-1. FACS analysis demonstrated that the applied inhibitors abolished lipid peroxide formation and reduced mitochondrial ROS production in both conditions of oxidative cell death, i.e. erastin-induced ferroptosis and glutamate-induced oxytosis. In conclusion, the present study exposes the mitochondrial transactivation of BID as a key molecular link between oxidative stress and mitochondrial pathology in the model of ferroptosis considerably resembling established characteristics of cell death in paradigms of oxytosis in neuronal cells.

References: 1. Dixon, S. J. et al; Cell 2012, 149: 1060–1072.

DPhG Annual Meeting 2016 Conference Book • 141 POSTERS

References: 1. Magalhaes, A., Dunn, H., Ferguson, S.: Br. J. Pharmacol. 2011, 165(6): 1717-36. 3.7 GPCR/Ion Channels 2. Bock, A. et al.: Nat. Commun. 2012, 3:1044 doi: 10.1038/ncomms2028. 3. Antony, J. et al.: FASEB J. 2009, 23:442-450. 4. Thomas, R. et al.: J. Pharmacol. Exp. Ther. 2008, 327(2):365-74. POS.146 5. Schrage, R. et al.: Br. J. Pharmacol. 2013, 169(2):357-70. Dualsteric compounds to induce signaling pathway selectivity in CHO-M1 cells

1 2 2 3 Bödefeld, T. ; Matera, C. ; Dallanoce, C. ; Messerer, R. ; Holzgrabe, U.3; De Amici, M.2; Mohr, K.1; Schrage, R.1 POS.147 1 Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, Differential modulation by allosteric receptor epitopes of Gerhard-Domagk- Straße 3, 53121 Bonn, Germany; dualsteric ligand binding and signaling at human 2 Department of Pharmaceutical Sciences, University of Milan, Via Mangiagalli 25, 20133 Milan, Italy; muscarinic M1 acetylcholine receptors 3 Institute of Pharmaceutical and Medicinal Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany Holze, J.1; Klöckner, J.2; Holzgrabe, U.2; Decker, M.2; Mohr, K.1; Tränkle, C.1 Muscarinic acetylcholine receptors (mAChRs) belong to the large 1 Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, superfamily of G protein-coupled receptors (GPCRs). Stimulation of Gerhard-Domagk-Str. 3, 53121 Bonn, Germany GPCRs leads to a conformational change in the receptor protein 2 Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany allowing the receptor to transduce extracellular stimuli onto intracellular adaptor proteins which then further propagate the signal inside the cell The five muscarinic receptor subtypes are metabotropic GPCRs, with [1]. GPCR-induced signaling can be the result of the interplay between the M1 being most abundantly expressed in the central nervous system rather complex molecular events as several GPCRs can activate [1]. Recently, we investigated the bitopic/dualsteric hybrid ligands JK multiple different adaptor proteins, for instance different classes of 550 and JK 537 (cf. Table 1) containing the orthosteric binding block heterotrimeric G proteins. Additionally, GPCRs can be targeted by iperoxo and BQCA-derivated compounds as allosteric moieties [2]. It is compounds not only via the binding site for the endogenous messenger known that dualsteric ligands are able to bind in two different (orthosteric binding site) but also via distinct druggable “allosteric” sites orientations to the receptor which are characterizable by different [2]. binding constants: the dualsteric binding pose which stabilizes the In the present work, we investigated the influence of spatial restriction active state with the affinity measure Kactive and the allosteric binding of the allosteric vestibule located in the extracellular loops of the pose preferring the inactive state with Kinactive [3]. Based on the receptor protein on muscarinic M1 acetylcholine receptor (M1 mAChR) - operational model of agonism for dynamic ligands [4] we have shown mediated signaling pathways. To this end, we employed several earlier that increasing the spacer length (n = 3 to n = 5, cf. Table 1) dualsteric compounds which are able to simultaneously occupy the between the orthosteric and the allosteric moiety favored the active orthosteric and an allosteric binding site of mAChRs, thereby dualsteric orthosteric/allosteric receptor binding pose at the human hM1 constraining flexibility of the extracellular vestibule which constitutes the receptor [2]. To elucidate these findings we studied the binding and common allosteric binding site [3]. At the muscarinic M2 receptor, such signaling properties of JK 550 and JK 537 in intact live Flp-InTM-Chinese spatial restriction of the allosteric area of the receptor protein has been hamster ovary cells (Flp-InTM-CHO) stably expressing either hM1 wild- demonstrated to allow selective activation of particular signaling type or hM1 receptors single point mutated at two allosteric epitopes. In pathways [2]. Here, we want to transfer this principle to the muscarinic detail, receptor binding experiments were carried out applying [3H]N- M1 subtype, which preferentially signals into Gq/11-mediated pathways, methylscopolamine ([3H]NMS, 0.2 nM) and, functionally, receptor- but can also promiscuously stimulate Gs and Gi proteins [4]. We mediated signaling was quantified as compound induced IP1- investigated M1 receptor-mediated signaling induced by dualsteric accumulation via FRET measurement in wild-type hM1, hM1-Y179A and compounds consisting of highly affinitive and efficacious agonist iperoxo hM1 E401A cells. Data analysis based on the operational model of [5] as an orthosteric building block linked to an allosteric phthalimide agonism for dynamic ligands (e.g. Kactive, Kinactive) [4] or a four parameter (phth) moiety or a bulkier naphthalimide (naph) residue through alkyl logistic equation (Emax). chains of different lengths (6, 7, 8 and 10 methylene units). Gq/11- and Gs-dependent signaling pathways were analyzed in HTRF-based IP1 and cAMP accumulation assays, respectively. We found that, in general, Gs protein activation in CHO-M1 cells was highly sensitive to the restriction of spatial flexibility of the extracellular receptor area, because application of the bulky allosteric naph residue compromised M1 receptor-mediated cAMP production to a greater extent than IP1 accumulation. Moreover, compounds with a rather short linker length (i.e. C6) displayed weaker potency and efficacy for both Gq/11- and Gs- mediated signaling than dualsteric ligands with elongated linker chains (i.e. C8). In particular, iper-6-phth was a partial agonist for the Gq/11 For both hybrids the binding experiments revealed that in the allosteric pathway, but totally lost affinity for Gs-mediated signaling, whereas iper- M1-E401A mutation, Kinactive was significantly increased compared to the 7-phth was a good partial agonist for both pathways under investigation. M1-wt resulting in a significant increase in the orientation ratio Rpose,binding For compounds carrying the rather voluminous naph moiety as (= -log(Kactive/Kinactive) (cf. Table). Vice versa, for JK 550 but not JK 537 in allosteric residue an even stronger impact of a short linker length was the allosteric M1-Y179A mutation, Kinactive was significantly decreased observed. Indeed, iper-6-naph acted as a neutral antagonist on both compared to the M1-wt resulting in a significant decrease of the pathways investigated up to a concentration of 10 µM, although it was orientation ratio Rpose,binding. When the global regression data analysis able to bind to the M1 mAChRs with an affinity in the low micromolar was extended to also include the functional signaling data, in no case range. Interestingly, previous studies demonstrated that this ligand is a the Rpose,function values were different from the corresponding Rpose,binding partial agonist at M2 and M3 subtypes of mAChRs [2 and unpublished values. The lower value of Kinactive in the M1-Y179A mutation for JK 550 data]. Dualsteric compounds with 7, 8 and 10 polymethylene spacer resulted in a shortfall of its maximal receptor occupancy (max. occ.) in chains were all good partial agonists for Gq and moderate partial the dualsteric pose to 45% compared to 93% in M1-wt. Functionally, this agonists for Gs protein activation. We conclude that a C6 linker translated into a significant decrease of the maximum effect Emax from connecting the allosteric and the orthosteric moiety compromises 53% in M1-wt to 16% in M1-Y179A, and it strongly reduced max*, i.e. the receptor flexibility to the greatest extent and thus has the strongest efficacy of JK 550 at 100 % receptor occupancy in the active pose. impact on M1-mediated signaling. Interestingly, the longer hybrid JK 537 did not show decreased Rpose Taken together, we present iper-6-naph as the first dualsteric values in the M1-Y179A mutant. However, the - compared to the M1-wt - compound with functional subtype selectivity at mAChRs, as it is a identical maximum receptor occupancy of the dualsteric pose of JK 537, rather good partial agonist at M2 [2] and M3 receptors, but clearly acts i.e. 97 vs. 94%, did not translate into any IP1 signaling (cf. Table). as a neutral antagonist at the M1 subtype. Furthermore, our findings In conclusion, our findings reveal that Y179 in the extracellular loop 2 show that it is possible to control M1-receptor mediated signaling by (ECL2) of the hM1 receptor plays a key role for the binding orientation restriction of the conformational flexibility in the allosteric area of the and the extent of signaling induced by a given dualsteric ligand. receptor protein.

142 • DPhG Annual Meeting 2016 Conference Book GPCR/ION CHANNELS

References: excitability of many types of neurons. Recent findings have shown that 1. Bymaster, F.P. et al.: Neurochem. Res. 2003, 28(3-4), 437-442. the Fragile X mental retardation protein (FMRP) directly activates Slack 2. Chen, X. et al.: J. Med. Chem. 2015, 58(2), 560-76. channels. Deletion or mutation of FRMP results in Fragile X syndrome 3. Bock, A. and Mohr, K.: Drug Discov. Today Technol. 2013, 10(2), 245-252. (FXS), the most common form of inherited intellectual disability and 4. Bock, A. et al.: Nat. Chem. Biol. 2014, 10(1), 18-20. autism in humans. A mouse model of FXS, the Fmr1 knockout mouse, displays some phenotypical features of the human FXS. Interestingly, + + Na -activated K currents are decreased in Fmr1-deficient mice suggesting that Slack may contribute to the neuronal abnormalities in POS.148 FXS. Knockdown of the psychiatric susceptibility gene Cacna1c Experimental Procedures: To identify phenotypes of the Fmr1 mediates protection of mitochondria against glutamate- knockout mouse that may rely on decreased Slack activity, we induced stress in neuronal cells compared autistic and anxiety-related phenotypes of Slack- and Fmr1- deficient mice, using well-established behavioral experimental tests Michels, S.1; Ganjam, G. K.1; Martins, H.2; Braun, M. D.3; Kisko, T. M.3; (e.g. elevated-plus maze test, open field test, nest building test and Schwarting, R. K. W.3; Wöhr, M.3; Schratt, G.2; Culmsee, C.1 social interaction tests). 1 Institute for Pharmacology and Clinical Pharmacy, Philipps-University, Karl-von-Frisch-Str. 1, Results: We did not find any altered anxiety levels in Fmr1-deficient 35043 Marburg, Germany mice, but our findings reveal a trend to decreased anxiety-related 2 Institute of Physiological Chemistry, Philipps-University, Karl-von-Frisch-Str. 1, 35043 Marburg, Germany behaviors of Slack-deficient mice in all anxiety tests. Fmr1 knockouts 3 Experimental and Physiological Psychology, Philipps-University, Gutenbergstr. 18, build nests of poor quality and show lack of preference for social novelty 35037 Marburg, Germany in a three-chamber social interaction test while performance of Slack knockouts is normal in these experimental setups. Conversely, Slack- Several genome wide association studies have identified CACNA1C as deficient mice show social dominance in a tube test version whereas one of the strongest genetic risk factors for affective disorders [1,2]. It there is no difference between WT and Fmr1 knockout mice. has recently been shown that the main SNP rs1006737 is associated Conclusion: We could not detect any similarities between autism- and with increased mRNA expression of CACNA1C [3]. However, its role in anxiety-related phenotypes of Slack- and Fmr1-deficient mice. Our disease pathogenesis is still largely unknown [4]. CACNA1C codes for findings do not support the hypothesis of a contribution of Slack to the α1C subunit of CaV1.2, which is the major L-type voltage-gated these behavioral deficits seen in FXS. calcium channel in the brain, and underlies key neuronal functions such as dendritic development, cell survival, and synaptic plasticity [5]. Furthermore, mitochondrial dysfunction is also linked to psychiatric disorders and associated with the deregulation of intracellular calcium levels [6]. POS.150 The aim of the present study is to investigate the effects of siRNA- Approach to Map the P2X7 Receptor Interactome by mediated knockdown of Cacna1c gene expression on mitochondrial Protein Cross-Linking Mass Spectrometry function combined with glutamate-induced oxidative stress in immortalized mouse hippocampal HT-22 cells. We analyzed cell Stocklauser, R.1; Schmidt, A.2; Zhang, J.1; Imhof, A.2; Nicke, A.1 viability and mitochondrial parameters using real-time impedance 1 Walter Straub Institute of Pharmacology and Toxicology, LMU Munich, Germany 2 measurements, colorimetric and luminescence-based assays, and flow Munich Center of Integrated Protein Science, Biomedical Center, LMU Munich, Germany cytometry with different fluorescent dyes. The P2X7 receptor (P2X7Rs) is a trimeric ATP-gated cation channel. It We found that the downregulation of Cacna1c mRNA levels significantly is involved in inflammatory processes by triggering the release of pro- protected the neuronal HT-22 cells from glutamate-induced cell death. inflammatory cytokines and has been shown to contribute to a variety of In detail, glutamate challenged HT-22 cells transfected with Cacna1c pathologies, such as neurodegenerative processes and inflammatory siRNA showed reduced mitochondrial ROS formation and diminished bowel disease. Therefore, it represents a promising target for the rise in mitochondrial Ca2+ levels compared to control. Moreover, loss of development of novel drugs. In contrast to all other P2XR family mitochondrial membrane potential upon glutamate treatment was members, it has a low affinity for extracellular ATP and a long attenuated in Cacna1c siRNA transfected cells. Confirming these cytoplasmic C-terminal domain, which is supposed to be crucial for results, inhibition of L-type calcium channels with isradipine also targeting of the receptor to the plasma membrane, interactions with prevented glutamate-mediated excitotoxicity in primary rat cortical other proteins as part of a larger signalling complex and its ability to neurons. Accordingly, several current publications suggest L-type induce effects such as the formation of large pores and membrane calcium channel antagonists as an approach to innovative blebbing. Despite its importance as a drug target, the proteins involved pharmacotherapy of mood disorders [7,8]. The molecular mechanisms in its localization, P2X7R signalling and functional modulation under underlying the effects of Cacna1c on mitochondrial performance remain physiological conditions are still unclear. to be elucidated. To address this issue we generated a P2X7R BAC transgenic mouse

Acknowledgments: Supported by DFG FOR2107 model, in which the receptor is fused via a streptag-heptahistidyl-linker to an EGFP reporter protein. This BAC transgenic approach permits References: 1. Green, E. et al.: Mol. Psychiatry. 2010, 15(10): 1016-22. P2X7R-EGFP expression at near physiological or moderate over- 2. Ferreira, M. et al.: Nat. Genet. 2008, 40(9): 1056-8. expression levels under its endogenous promotor. Biochemical and 3. Yoshimizu, T. et al.: Mol. Psychiatry. 2015, 20(2): 162-9. immunohistochemical experiments show that expression patterns and 4. Harrison, P.: Curr. Opin. Neurobiol. 2016, 36: 1-6. levels of P2X7R-EGFP correlate with endogenous P2X7Rs in different 5. Bhat, S. et al.: Prog. Neurobiol. 2012, 99(1): 1-14. tissues. The complex glycosylation of the protein as well as specific 6. Manji, H. et al.: Nat. Rev. Neurosci. 2012, 13(5): 293-307. membrane localization indicate its correct folding and integration into 7. Cipriani, A. et al.: Mol. Psychiatry advance online publication. 2016 the plasma membrane. Using the P2X7R-EGFP fusion protein as a bait, 8. Zamponi, G.: Nat. Rev. Drug. Discov. 2016, 15(1): 19-34. endogenous P2X7R subunits are efficiently and specifically co-purified by immunoprecipitation against EGFP, confirming their coassembly. Although immunoprecipitation-coupled mass spectrometry (IP-MS) is nowadays the standard for the identification of protein complexes, POS.149 protein-protein interactions with high dissociation rates are typically lost Autistic and anxiety-related behaviors of Slack and Fmr1 due to extensive washing. Especially for membrane proteins, knockout mice detergents are essential to efficiently extract the protein complex from the plasma membrane and therefore impairing its native environment. Bausch, A.1; Zerfass, P.1; Nann, Y.1; Dieter, R.1; Ruth, P.1; Lukowski, Chemical crosslinking of proteins prior to the actual purification process R.1 reduces the loss of transient interactions. To further analyse the P2X7R 1 Pharmakologie, Toxikologie & Klinische Pharmazie, Institut für Pharmazie, Universität interactome, we adapted this approach to our P2X7R BAC transgenic Tübingen mouse model. Initial biochemical experiments show that the P2X7R can be crosslinked using the membrane permeable homo-bi-functional Background: Sodium-activated Slack channels are highly expressed NHS-ester disuccinimidyl suberate (DSS), leading to the formation of throughout the brain and modulate firing patterns and general stable di- and trimers of endogenous P2X7R and the P2X7R-EGFP

DPhG Annual Meeting 2016 Conference Book • 143 POSTERS fusion protein. The optimization of crosslinking conditions is ongoing and will be presented. Data obtained from protein crosslinking coupled with mass spectrometry (XL-MS) experiments will not only be used to identify novel interaction partners, but also to define intra- and intermolecular interaction domains and will therefore provide a vital insight in the organization and function of the P2X7R signalling complex.

144 • DPhG Annual Meeting 2016 Conference Book NATURAL COMPOUNDS/CHEMICAL BIOLOGY

the endothelial migration by 60 %. Oleic acid as a representative of 3.8 Natural Compounds/ MUFAs had no influence. In summary, we could show for the first time that the enzyme ACC plays a crucial role in the migration of endothelial cells. Inhibition or Chemical Biology knockdown of ACC1 strongly inhibited the migratory process. This inhibition is clearly based on the shift of the fatty acid composition of the endothelial cell membrane.

POS.152 Acknowledgement: This work was supported by the German Research Foundation (DFG, FOR The acetyl-CoA carboxylase (ACC) inhibitor soraphen A 1406, FU 691/9-2). blocks the migration of primary endothelial cells by shifting the cell membranes’ composition of fatty acids

Glatzel, D.1; Koeberle, A.2; Werz, O.2; Ashtikar, M.3, Müller, R.4; Fürst, POS.153 R.1 1 Institute of Pharmaceutical Biology, Biocenter, Goethe-University, Max-von-Laue-Str. 9, 60438 Crystallization and structure elucidation of plant type III Frankfurt am Main, Germany polyketide synthase enzymes 2 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University Jena, Philosophenweg 14, 07743 Jena, Germany 1 2 1 1 3 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group Khalil, E. ; Lukat, P. ; Liu, B. ; Beerhues, L. Translational Medicine and Pharmacology (TMP), Goethe-University, Max-von-Laue-Str. 9, 1 Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, 60438 Frankfurt am Main, Germany Mendelssohnstrasse 1, 38106 Braunschweig, Germany 4 Helmholtz Institute for Pharmaceutical Research Saarland, Department of Microbial Natural 2 Struktur und Funktion der Proteine, Helmholtz-Zentrum für Infektionsforschung, Products and Department of Pharmaceutical Biotechnology, Saarland University, Campus E8.1, Inhoffenstrasse 7, 38124 Braunschweig, Germany 66123 Saarbrücken, Germany Among the huge number of natural products distributed all over the Acetyl-CoA carboxylase catalyzes the first step in the biosynthesis of world, i.e. in bacteria, fungi and plants, polyketides provide one of the fatty acids in bacterial and eukaryotic cells, i.e. the conversion most important families. They include many powerful drugs such as (carboxylation) of acetyl-CoA into malonyl-CoA. ACC-generated lovastatin, tetracycline and daunorubicin and their broad biological malonyl-CoA functions as a substrate for de novo lipogenesis and acts activities make them good candidates for new drug discovery. They are as an inhibitor of mitochondrial β-oxidation of fatty acids. Because of its biosynthesized through a serious of decarboxylative condensation role in lipid metabolism this enzyme has become an interesting target in reactions by a group of enzymes called polyketide synthase (PKS) drug discovery in the field of metabolic diseases and cancer. Despite enzymes, which are further divided into three main groups: type I, type this interest in ACC, no attention has as yet been given to the role of II and type III PKSs [1].Type III PKSs have, compared to the other ACC in endothelial cells. types, drawn much attention, as they are smaller and simpler but still We aimed to investigate the role of ACC in endothelial cell migration, a maintain the catalytic function of polyketide chain elongation and functional key aspect of angiogenesis. We used the ACC inhibitor cyclization. In 1999, chalcone synthase (CHS) from Medicago sativa soraphen A, a polyketidic natural compound isolated from the (alfalfa) was the first enzyme of this class that was crystallized and still myxobacterium Sorangium cellulosum, as well as an RNAi-based is the most well studied [2]. Recently, many CHS-like enzymes have approach to inhibit the function of ACC. Primary human umbilical vein been crystallized, which revealed the structural basis for the functional endothelial cells (HUVECs) were used as in vitro model. First, we diversity of this enzyme family. In this study, we have successfully analyzed the action of soraphen A on cell viability. The compound did solved the structures of two important enzymes of the same family, neither lower the metabolic activity of HUVECs up to a concentration of benzophenone synthase (BPS) and biphenyl synthase (BIS). BPS and 100 μM after 24 and 48 h (CTB assay) nor increase in the apoptosis BIS were cloned and expressed in our workgroup from cell cultures of rate after 24, 48, or 72 h up to 100 μM. Measuring adenosine Hypericum androsaemum and Malus domestica, respectively: Both triphosphate (ATP) levels revealed that 30 μM soraphen A does not enzymes utilize benzoyl-CoA as starter substrate, which is a rare starter alter the ATP levels in HUVECs after 24 h treatment. In contrast, a 48 h unit for type III PKSs. After reaction with three molecules of malonyl treatment significantly lowered the ATP levels by 12 %. Also gene CoA, BPS forms 2,4,6-trihydroxybenzophenone via a Claisen silencing of ACC1 in HUVECs attenuated the ATP levels by 11 %. condensation mechanism, but BIS forms 3,5-dihydroxybiphenyl using Mitochondrial membrane potential (MMP) assays showed decreased an aldol condensation mechanism. Biphenyls are compounds with MMP levels (10 %) in soraphen A-treated cells after 24 h. antimicrobial activity and represent the phytoalexins of the Rosaceae Interestingly, the compound inhibited the migration of endothelial cells plant family, which includes important fruit trees like apple and pear. in a wound healing/scratch assay by 65 %. Gene silencing of ACC1 in The production of these compounds helps the plants to resist microbial HUVECs strongly decreased endothelial migration, whereas a diseases. Furthermore, benzophenones and polyprenylated knockdown of ACC2 had no influence. Furthermore, Boyden chamber benzophenones are important biological compounds, exemplified by assays revealed that soraphen A can also lower chemotactic migration guttiferone F which exhibits antimicrobial and anti-HIV effects [3, 4]. In by 34 %. Since actin rearrangement is necessary for migratory this project, we were also able to solve the structure of a BPS single processes, we analyzed the F-actin cytoskeleton (microscopy) and site mutant which converted the wild type enzyme into a new type III found that soraphen A decreases the number of filopodia by 60 %, but PKS, called phenylpyrone synthase (PPS), which produces 6-phenyl-4- did not influence stress fiber formation. Soraphen A inhibited the hydroxy-2-pyrone as a major product after only two condensation activation of the migration-regulating kinase Erk (Western blot analysis), reactions with malonyl-CoA [5]. Our work will help understand the whereas the activation of Akt was not influenced. variations in the sequential condensation and cyclization mechanisms Liquid chromatography-mass spectrometry (LC-MS) showed that of type III PKSs, leading to product variability although using the same soraphen A leads to a shift of the membrane lipid composition of starter substrates. HUVECs. Most notably, the levels of phosphatidylglycerol (PG) were lowered by 54 %. To establish a causal link between the reduction of PG levels and the observed migration-inhibiting action of soraphen A, a rescue experiments was performed: Substitution of dioleoylphosphatidylglycerol (10 μM and 100 μM, multilamellar vesicle) completely reversed the action of soraphen A. Moreover, soraphen A- treated cells also showed a shift in the degree of membrane lipid desaturation. Among the saturated fatty acids, the levels of the species phosphatidylcholine (PC), phosphatidylethanolamine (PE), and Acknowledgments: This work was supported by a scholarship from the Libyan government to phosphatidylinositol (PI) were decreased by 10 %, 6.5 %, and 15 %, Ebtisam Khalil and a grant from the Deutsche Forschungsgemeinschaft (DFG). respectively. In contrast, soraphen A increased PI levels within the References: class of monounsaturated fatty acids (MUFA) by 37 % as well as PC 1. Shen B: J. Curr Opin Chem Biol. 2003, 7: 285-295. and PE within the group of polyunsaturated fatty acids (PUFA) by 55 % 2. Ferrer, JL. et al.: J. Nat Struct Biol. 1999, 6: 775-784 and 21 %. Interestingly, the treatment of HUVECs with the PUFA 3. Chizzali, C. et al.: J. Plant physiol. 2012, 158: 864-875 4. Liu, B. et al.: J. Plant J. 2003, 34: 847-855 linolenic acid (100 μM) mimicked the effect of soraphen A and lowered 5. Klundt, T. et al.: J. Biol. Chem. 2009, 284 (45): 30957-30964

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POS.154 POS.157 Permeation characteristics of hypericin is influenced by The heritage of 19th and early 20th century explorers: Hypericum perforatum extract historical diaries, letters and manuscripts as tools to identify pharmaceutically active natural compounds Butterweck, V.1; Verjee, S.1; Kelber, O.3; Abdel-Aziz, H.3 1 Institute for Pharma Technology, School of Life Sciences, University of Applied Sciences Helmstädter, A. Northwestern Switzerland, Gründenstrasse 40, 4132 Muttenz, Swizzerland Institute of Pharmaceutical Chemistry, Goethe University, Frankfurt am Main, Germany 2 Innovation & Development, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany Diaries, letters and publications of pioneering explorers sent form 3 Medical Affairs, Phytomedicines Supply and Development Center, Bayer Consumer Health, Europe throughout the world in the 19th and early 20th century, are Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany containing information about the medicinal uses of plants now and then.

This has been already shown for the botanist Berthold Seemann (1825- Hypericin is a polycyclic quinone found in Hypericum perforatum L. 1871) [1], the anthropologist Mellville William Hilton-Simpson (1881- Although hypericin reportedly has numerous pharmacological activities, 1938) [2], the physician Arthur William Francis Kerr (1877-1942), and only a limited number of studies have been performed on the absorption the reverend Joseph John Freeman (1794-1851). It is well known that and transport characteristics of this compound [1], presumably, historical sources may help to unveil traditional uses of medicinal plants because hypericin is a highly lipophilic compound which is poorly which may then serve as promising guidelines for phytopharmaceutical soluble in aqueous solutions. Recently we have shown [2] that screening and the search for new lead structures [3]. These sources quercitrin and isoquercitrin, but not hyperoside, quercetin or rutin include historical herbals [4], the heritage of Christian missionaries [5] increased the uptake of hypericin in Caco-2 cells. The major aim of this and many others. It has been shown that the heritage of pioneering study was to get a detailed understanding of the exposure and fate of explorers is serving that purpose as well. Reports have been screened hypericin in the Caco-2 cell system when combined with extract matrix. and the information given has been compared with state-of-the-art The permeation characteristics of hypericin (5 μM) in absence or ethnopharmacological, phytochemical, and pharmaceutical knowledge presence of H. perforatum extract STW 3-VI (7.5, 29 and 58 μg/ml) in order to (i) estimate the degree of reliability of the historical records were studied in the absorptive direction. and (ii) to identify medicinal plants traditionally used but scarcely Following application of hypericin to the apical side of the monolayer investigated with modern methods. Obviously, the sources investigated only negligible amounts of the compound were found in the basolateral exert a significant degree of reliability, as many uses and indications compartment. The amount of hypericin in the basolateral compartment could be scientifically confirmed. On the other hand, many plants increased concentration-dependent in the presence of extract matrix described as medicinally useful have never been investigated so far. (from 0 to 7.5 %). The majority of hypericin was found after cell Thus it seems reasonable to prefer these plants in phytochemical and extraction (44% in absence and 76% in presence of the extract). The pharmacological screening programs as investigations should lead to precise mechanism through which hypericin might overcome the useful results with higher probability than screening at random. Several hydrophobic barrier of cell membranes remains to be elucidated. The suggestions can already be made. experiments with the extract STW 3-VI as a matrix showed that besides flavonoids there seem to be further compounds in the extract (e.g. Acknowledgments: This study was partially funded by the Wellcome Trust (Grant No. phenolic acids or proanthocyanidins) which substantially improve the WT106016AIA). Studies were done at the Archives of Kew Gardens,The Royal Anthropological permeation characteristics of hypericin. Institute of Great Britain, London, and the Pitt Rivers Museum, Oxford. References: References: 1. Helmstädter, A.: Ethnopharmacological information from the botanical correspondence of 1. Butterweck V et al., Planta Med 2003 69 (3):189-192 Berthold Seemann (1825-1871). Pharmazie 2015, 70(9): 616-626. 2. Verjee S et al. Planta Med 2015; 81: 1111-1120 2. Helmstädter, A.: Ethnopharmacology in the work of Melville William Hilton-Simpson (1881- 1938) – historical analysis and current research opportunities. Pharmazie 2016, 71(6): 352-360. 3. Buenz, E.J.; Schnepple, D.J.; Bauer, B.A. et al.: Techniques: Bioprospecting historical texts by hunting for new leads in old tomes. Trends Pharmacol. Sci. 2004, 25(9): 494-498. 4. Adams, M.; Berset, C.; Kessler, M.; Hamburger, M.: Medicinal herbs for the treatment of POS.155/SL.29 rheumatic disorders – a survey of European herbals form the 16th and 17th century. J. Ethnopharmacol. 2009, 121(3): 343-359. Genome mining-guided drug discovery: from proteasome 5. Anagnostou, S.: Forming, transfer and globalization of medical-pharmaceutical knowledge in to protease inhibitors south east Asian mission (17th to 18th c.) – historical dimensions and modern perspectives. J. Ethnopharmacol. 2015, 167: 78-85. Kaysser, L.1,2; Zettler, J. 1,2; Zubeil, F.3; Leipoldt, F. 1,2; Wolf, F.1,2; Schorn, M.4; Bauer, J.1,2; Bendel, T.1,2; Kulik, A.5; Dorrestein, P. C.6; Moore, B. S.4,6; Gross, H.1,2; Grond, S.3 1 Dept. Pharmaceutical Biology, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany 2 German Centre for Infection Research (DZIF), partner site Tübingen 3 Inst.. Organic Chemistry, University of Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany 4 Scripps Institution of Oceanography, UC San Diego, 9500 Gilman Drive, La Jolla CA, 92093-0204, USA 5 Interfaculty Institute of Microbiology and Infection Medicine Tübingen (IMIT), University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany 6 Skaggs School of Pharmacy, UC San Diego, 9500 Gilman Drive, La Jolla CA, 92093-0751, USA

For abstract see Short Lecture SL.29

POS.156/SL.14 The virulence factor LecB varies in clinical isolates: consequences for ligand binding and drug discovery

Titz, A.1 1 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus E8.1, 66123 Saarbrücken, Germany

For abstract see Short Lecture SL.14

146 • DPhG Annual Meeting 2016 Conference Book BIOPHARMACEUTICS

3.9 Biopharmaceutics Baclofen (BAC), a specific GABAB-receptor agonist, has successfully been used for decades for the treatment of spastic disorders. However, the fraction of an oral or i.v. dose that reaches the CNS is negligible, POS.158 since BAC does not sufficiently permeate through the blood-brain barrier (BBB). As a consequence intrathecal administration is needed to Physicochemical performance and skin penetration obtain appropriate biophase levels. kinetics of different acyclovir formulations analysed by More lipophilic analogues (e.g. ester derivatives) usually exhibit confocal Raman microscopy considerably higher membrane permeability than parent BAC. Hence, as potential CNS-targeting prodrugs, BAC methyl- and propyl-ester Jung, N.1; Namjoshi, S. N.2; Mohammed, Y. H.2; Grice, J. E.1; Raney, S. carbamates were studied in vivo in intravenously dosed rats, where - as G.3; Roberts, M. S.2,4; Windbergs, M.1 opposed to results with the respective esters - a significant amount of 1 Helmholtz-Institute for Pharmaceutical Research Saarland and Saarland University, Campus E BAC was detected in brain tissues following prodrug dosage, 8.1, 66123 Saarbrücken, Germany 2 Therapeutics Research Centre, University of Queensland, BrisbaneQld 4072, Australia particularly in the case of the methyl carbamate. 3 U.S. Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, Maryland The purpose of the current studies, mainly comparing the methyl- vs. 20993, USA the propyl carbamate, was the characterization of physicochemical 4 University of South Australia, 101 Curie St, Adelaide SA 5001, Australia properties in-silico and in-vitro as well as membrane permeabilities in For development and quality control of topically administered cell monolayer-based models for intestinal and CNS transport. Test formulations, in vitro analysis of penetration of drugs and excipients into systems used were Caco-2 cell monolayers for studies on intestinal and through the skin is indispensable. Generally, this involves in vitro permeabilities as well as hCMEC/3D cell monolayers as model for the permeation testing (IVPT) using Franz diffusion cells with subsequent BBB. drug quantification by HPLC. As this procedure is destructive, drug Solubility studies yielded a significant solubility decrease with increasing quantification is limited to the endpoint of the study or requires high lipophilicities (BAC, 25 mg/ml, BAC methyl carbamate, 0.41 mg/ml, 2- numbers of individual Franz cells which are dismantled at predefined propyl carbamate, 0.09 mg/ml) with the butyl carbamate being very time points. Further, such tests do not allow to investigate interactions poorly soluble, experimental logP values ranged from -0.56 for BAC, 1.5 of the formulation with the skin sample. Therefore, novel non- for BAC methyl carbamate to 3.4 for BAC 2-propyl carbamate. destructive analytical approaches are highly valuable alternatives. Membrane permeability studies confirmed that BAC and BAC In this study, confocal Raman microscopy was used as a chemically carbamates have no relevant affinity to P-glycoprotein, while BAC, yet selective and non-destructive technology to assess the penetration of not the ester carbamates, appears to be a substrate for the taurine six different commercially available acyclovir creams into excised transporter. Apparent apical-to-basolateral permeabilities for BAC human skin. The creams were rubbed into full thickness skin samples methyl carbamate in Caco-2 cell monolayers were appr. 20-fold higher with a circular motion simulating the clinical “in use” situation. After than for BAC, apparent permeabilities in the BBB membrane model for predefined time intervals, the cream was gently removed from the skin the methyl carbamate were double as high as for parent BAC with a surface and confocal Raman microscopy was used to track the tendency to increase with higher lipophilicities. compounds of the formulations within the skin tissue. The depth of In summary, for BAC in Caco-2 cells the impact of the taurine penetration of the cream components was determined by acquiring transporter was lower than expected and that of the P-glycoprotein virtual cross sections in xz-direction throughout the skin sample, as well transporter basically nonexistent. Moreover, no relevant impact of as line scans in the z-direction downward. transport systems was detected so far in the permeability studies with While no significant difference in penetration depth among the creams ester carbamates up to logP = 3.4. Hence, neither lipophilicities nor was found after 4 hours, the reference products showed a relatively apparent permeabilities or transporter affinities would oppose against higher accumulation in the stratum corneum after 24 hours, In addition, CNS targeting, yet support the concept that ester carbamates may deeper penetration into the upper epidermis could be detected for the represent suitable BAC prodrugs. reference products as compared to the test drug products. The data acquired with confocal Raman microscopy correlated well with findings from IVPT studies performed with the same formulations in static Franz diffusion cells using heat separated human epidermis samples. POS.160 As physicochemical characteristics of the formulations are prone to influence the skin penetration kinetics, the different formulations were A novel disintegration testing setup for solid oral dosage investigated by confocal Raman microscopy with respect to their forms including adjustable hydrodynamics and pressure compound distribution and phase behavior. Interestingly, the dispensing forces process was found to influence the phase behaviour and consequently the skin penetration kinetics. Rach, R.1; Nawroth, T.1; Langguth, P.1 Based on the data, confocal Raman microscopy shows a high potential 1 Institute of Pharmacy and Biochemistry, Department of Pharmaceutical Technology and Biopharmacy, Johannes Gutenberg-University, 55128 Mainz, Germany to serve as an innovative approach for label-free and non-destructive analysis of physicochemical formulation characteristics as well as the Disintegration testing is a widely used technique to characterize and skin penetration behaviour of drugs as well as of excipients. analyze solid dosage forms, not only during formulation development but also as a tool for quality control. The disintegration apparatus Acknowledgments: Funding for this project was made possible, in part, by the Food and Drug Administration through grant U01FD005226-01. The views expressed in this abstract do not described in PhEur and USP is commonly utilized. However, the device reflect the official policies of the U.S. Food and Drug Administration or the U.S. Department of was introduced 50 years ago and it is not surprisingly, that the Health and Human Services; nor does any mention of trade names, commercial practices, or knowledge about the human gastrointestinal tract conditions, e.g., organization imply endorsement by the United States Government. gastric motility and media composition, has vastly increased over the last decades and the significant impact of those parameters on the disintegration of orally administered dosage forms and the dissolution of POS.159 drugs was acknowledged [1]. It is hence obvious that the compendial device cannot reflect the current status of biopharmaceutical science, Baclofen carbamate prodrugs: Physicochemical properties as there were no significant changes implemented since. Therefore, it is and membrane permeabilities in cell monolayer-based highly necessary to develop a biopredictive disintegration tester that studies on intestinal and CNS transport accounts for hydrodynamics, movement pattern and pressure forces. As our group presented previously, hydrodynamics and forces in the Pendl, E.1,2; Hänggi, S.2; Mosad, S.3; Niess, R.3; Imanidis, G.2; compendial disintegration tester are highly variable and non-controllable Abdelaziz, A.4; Spahn-Langguth, H.1,2,3 [2]. The need for disintegration testing with modifiable movement 1 Department of Pharmaceutical Sciences/Pharmaceutical Chemistry, Karl-Franzens-University, patterns and pressure forces that accounts for hydrodynamics and Universitätsplatz 1/I, 8010 Graz, Austria 2 Institute of Pharma Technology, University of Applied Life Sciences, Gründenstrasse 40, mechanical forces under controllable conditions motivated the 4132 Muttenz/Basel, Switzerland development and introduction of an in house build device with a 3 Faculty of Pharmacy & Biotechnology, German University in Cairo - GUC, Main Entrance El- modified probe chamber and programmable moving unit. Tagamoa El-Khames, New Cairo City, Egypt 4 Life Sciences Center Weihenstephan, Technical University Munich, Emil-Erlenmeyer-Forum 2, This work shows the results of disintegration studies in the modified 85354 Freising, Germany device with various immediate release (IR) solid dosage forms. The

DPhG Annual Meeting 2016 Conference Book • 147 POSTERS

influence of pressure forces on disintegration time was investigated in References: combination with different hydrodynamics. Distinct IR formulations were 1. Debieux. C. M. et al.: Proceedings of the National Academy of Sciences of the United States used with diverse disintegration mechanisms, in particular mechanical of America, 2011, 108: 13480-13485 force dependent, as well as independent formulations. The aim was to 2. Baesman. S.M. et al.: Extremophiles, 2009, 13: 695-705. 3. Borghese. R. et al.: J Hazard Mater, 2014, 269: 24-30. demonstrate the effect of applying pressure on the dosage form during 4. Aruguete. D.M. et al.: Environ Sci-Proc Imp, 2013, 15: 93-102. the disintegration process. The studies were performed and analyzed 5. Shamim. A.N. et al.: Abstr Pap Am Chem S, 2009, 237: 66-66. with the help of a DoE software (MODDE). 6. Estevam. E.C. et al.: Molecules, 2015, 20: 13894-13912. These studies are part of a project that aims to develop orally 7. Biswas. K.C. et al.: J Microbiol Meth, 2011, 86: 140-144. administered formulations with maximum bioavailabilities with the help 8. Hunter. W.J. et al.: Curr Microbiol, 2008, 57: 83-88. of a biopredictive in vitro disintegration tool. However, to validate the 9. Ramya. S. et al.: J Trace Elem Med Biol, 2015, 32: 30-39. modified disintegration setup and define biopredictive operating conditions an increased knowledge about in vivo disintegration times is needed. POS.162 References: 1. Koziolek, M. et al.: Mol. Pharmaceutics 2013, 10(5): 1610–1622. Friction-related protein particle formation in compounding 2. Kindgen, S. et al.: Journal of Pharmaceutical Sciences 2015, 104(9): 2956–2968. and filling steps

Brückl, L.1,2; Hahn, R.2; Schröder, T.1; Sergi, M.1; Sonderegger, C.1; Scheler, S.1 1 Department of Pharmaceutical Development, Sandoz GmbH, 6336 Langkampfen, Austria POS.161 2 Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, 1190 Vienna, Austria Nature’s red remedy towards microorganisms Protein aggregation and particle formation by physical degradation is a 1,2,3 1 1 1 Griffin, S. ; Nasim, M. J. ; Estevam, E. C. ; Denezhkin, P. ; Lilischkis, common concern during the compounding and filling steps of 4 2 3 1 R. ; Keck, C. M. ; Schäfer, K. H. ; Jacob, C. biopharmaceuticals. Many factors are currently under discussion to 1 Division of Bioorganic Chemistry, School of Pharmacy, Saarland University, D-66123 Saarbuecken, Germany affect the physical stability of proteins during compounding and filling, 2 Institute of Pharmaceutics and Biopharmaceutics, Philipps-Universität Marburg, Marburg however, the contribution of each factor and their interactions are still 35032, Germany unclear. A highly debated topic is protein denaturation and subsequent 3 Department of Biotechnology, University of Applied Sciences, Kaiserslautern, 66482 Zweibruecken, Germany aggregation or particle formation by the effect of shear, which is an 4 Department of Information Technology and Microsystem Technology, University of Applied omnipresent factor as it occurs in form of velocity gradients in moving Sciences, Kaiserslautern, 66482 Zweibruecken, Germany liquids. However, recent studies using in situ and real-time circular dichroism spectroscopy could show that the structural conformation of Introduction: Staphylococcus carnosus has the potential of producing representative proteins in solution is unaltered by shear rates up to at elemental chalcocogen nanoparticles [1-3]. S. carnosus excels its trade, least 104 s-1 [1]. Thus, it becomes clear that the stability of proteins in in particular, producing elemental selenium nanoparticles. These free solution is most likely unaffected during processing and that deposits of selenium are, however, toxic for the host microorganisms investigations should focus on interfaces, which impart enough energy and ultimately result in cell mortality. Besides the unwanted toxicity to unfold proteins upon adsorption. As the mechanism of protein against the host organisms, these nanoparticles might be beneficial for aggregation at air/liquid interfaces has already been characterized, we the treatment of other diseases caused by microorganisms [4-6]. are focusing here on friction-related protein degradation at solid Aim: Therefore, the aim of this study was to investigate if bacterial surfaces. Friction at solid surfaces occurs at various spots in the based selenium nanoparticles possess anti-microbial activity against manufacturing of biopharmaceuticals, e.g. inside sliding bearings of various different microorganisms and to investigate if mechanically bottom mounted magnetic type stirrers which are nowadays widely generated selenium particles could show a similar efficacy. used for compounding. Methods: Bacterially cultivated nanoparticles were obtained by For the first time custom designed small scale bearings, made of incubation of S. carnosus in media containing various concentrations of different materials, in combination with an IgG1 antibody as model Na2SeO3. Series of centrifugation and sonication steps were performed protein were used as a realistic model for lab scale investigation of the for the isolation of elemental selenium. Mechanically generated degradation mechanism inside the bearing. In the course of various particles were obtained by bead milling. The size and morphology was experiments we could exclude protein denaturation caused by frictional analysed using dynamic light scattering, laser diffraction and light heat generation, by the possible presence of extraordinary high shear microscopy. The anti-microbial activity was performed against S. rates in free solution as well as heterogeneous nucleation of IgG1 carnosus, Escherichia coli, Saccharomyces cerevisiae and particles on SiC micro/nano particles shed from the bearing. Abrasion of Steinernema feltiae. adsorbed proteins [2] by contact sliding was identified as prevailing Results: In the recent studies, different strains of S. carnosus have protein degradation mechanism and was quantified by an increase in shown the capability of reducing inorganic selenites into elemental turbidity and by monomer loss. The time course of protein degradation selenium particles [7-8]. Keeping this in view bacterial selenium in the bearing was found to be saturable and to follow Michaelis-Menten nanoparticles were produced and cultivated with considerable efficacy. kinetics. Interestingly, the time a molecule requires for conformational The particles possessed a size of about 440 nm. The results of the change after surface adsorption in relation to the touching frequency of antimicrobial experiments showed that bacterial particles were more the bearing was found to be most probably the crucial parameter effective against the gram positive host. The bacterial and yeast assays determining the dependence of protein degradation from the stirring showed considerable activity while the least activity was seen with the rate. Furthermore, protein degradation was highly dependent on nematodes. The mechanically modified particles were, in comparison, combinations of the material of the bearing and excipients. Thus, a test less effective over all, which may be related to the quality (i.e. larger system was introduced which allowed to systematically study these size) of these particles. effects with a wider variety of materials and to investigate the Conclusions: In short, the study shows effective production and effectiveness of stabilizers for different surface materials. The test cultivation of selenium nanoparticles from S. carnosus. Apart from this, system consists of spheres of a certain size and material, which are the toxicities against the host as well as other common microorganisms placed inside a sample-containing, rotating container under the have demonstrated a possible application in the field of medicine and exclusion of air. The rotational movement of the container causes agriculture [9]. The natural particles were shown to be more toxic friction between the spheres, which is finally leading to protein against the test microorganism, while the mechanically manufactured degradation and particle formation. Results from the test system using particles were less efficient, which might be due to the larger size. IgG1 and recombinant human growth hormone confirmed a protective Therefore, future studies will aim at producing smaller sized particles to effect of polysorbate 80 against friction-mediated particle formation. improve the efficacy. Ultimately, the bacterially cultivated selenium Polysorbate 80 most likely acts by a reduction of protein adsorption, nanoparticles are an effective natural source in the fight against showing higher protection in combination with a highly hydrophobic diseases caused by microorganisms. sliding material (PTFE) in contrast to silicon carbide and glass. Interestingly, monomer loss in presence of Fe3+-ions, which were Acknowledgments: The authors acknowledge the financial support from the University of Saarland and the CAPES Foundation, Ministry of Education of Brazil. introduced upon contact sliding of stainless steel, was even facilitated in

148 • DPhG Annual Meeting 2016 Conference Book BIOPHARMACEUTICS the presence of polysorbate 80, cancelling out its protective effect against abrasion. Finally, a comparison of degradation products from POS.164 various stresses by infrared spectroscopy (ATR-FTIR) revealed a high Is the unintended targeting of polymeric and lipid Nano- similarity between friction-related degradation products in general. DDS to ovarian and adrenal tissues an artefact or a Therefore, abrasion of adsorbed proteins is very likely the prevailing common phenomenon? physical degradation mechanism in processing steps where contact sliding occurs. Summarizing, our findings can directly be applied in the Mäder, K.1; Lucas, H.1; Weiss, V.1; Chytil, P.2; Etrych, T.2; Kressler, J.3, optimization of production steps, the future design of compounding Müller, T.4 vessels and the test system can be used as tool for formulation 1 Institute of Pharmacy, Martin Luther University Halle-Wittenberg, W. Langenbeckstr. 4, development 06120 Halle (Saale), Germany 2 Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovský Sq. 2, 162 06 Prague 6, Czech Republic, Acknowledgments: Biophysical Characterization group in Oberhaching (Sandoz) 3 Institute of Chemistry, Martin Luther University Halle-Wittenberg, Von-Danckelmann-Platz 4, References: 06120 Halle (Saale), Germany 4 Dept. of Internal Medicine IV, Martin Luther University Halle-Wittenberg, Ernst Grube Str. 40, 1. Brückl, L. et al.: Journal of Pharmaceutical Sciences 2016, 105 (6): 1810-1818. 06120 Halle (Saale), Germany 2. Sediq, A., Duijvenvoorde, R., Jiskoot, W. : Journal of Pharmaceutical Sciences 2016, 105 (2) : 519-529 Nanoscaled drug delivery systems (Nano-DDS) are made from different materials (e.g. lipids and polymers). They offer unique opportunities to optimize the efficiency of drug delivery. One of the main goals includes the accumulation of the drug loaded carrier in the target tissue (e.g. in POS.163 tumor tissue or in inflamed or infected tissue). It is well known, that Engineering of Bioinspired Nanoparticles for Drug Delivery many Nano-DDS show unintended accumulation in the liver, spleen and lung. Unfortunately, small organs are often neglected because of the Haryadi, B. M.1; Winter, G.1; Engert, J.1 efforts to separate them and the problem of poor drug content due to 1 Pharmazeutische Technologie und Biopharmazie, Ludwig-Maximilians-Universität (LMU) the small size. However, despite their small size these organs possess München, Butenandtstraße 5, 81377 München Germany important functions. Using the dye DIR and optical imaging, we described previously, that several Nano-DDS might accumulate in the Nanoparticles have gained a lot of interest in drug delivery. Besides ovaries [1]. Our findings were confirmed with other Nano-DDS (lipid size and charge, nowadays other parameters, such as geometry, nanodispersions) by the group of Benoit, who used two different probes surface properties, deformability, and degradability, are considered for and independent detection techniques (NIR and γ-counting) [2]. nanoparticle advancement [1]. Recent reports in the literature show that Furthermore, Merian et al. showed that triple labeled (3H, 14C and ellipsoidal, cell membrane-attired, and soft nanoparticles were least fluorescence) lipid nanodispersions accumulate on the adrenals and phagocytized by macrophages and last longer in the circulation system ovaries [3]. In order to understand this surprising accumulation better [2-4]. By implementing all parameters in one system, we developed and to increase the evidence of our findings on polymeric Nano-DDS, bioinspired nanoparticles. A mechanical stretching technique for we extended our efforts to other polymeric nanoparticles and also to fabricating elongated polylactic acid (PLA) or poly(lactic-co-glycolic polymer micelles. We designed also double labelled nanoparticles, acid) (PLGA) nanoparticles with diameters and lengths around 200 and where one dye was covalently linked and one highly lipophilic dye was 700 nm, respectively, was applied. For functionalization of non covalently dissolved in the polymer matrix (Fig. 1 left). Ex vivo nanoparticles, a top-down biomimetic method was applied by optical images show that both dyes accumulate in the adrenals and smothering biodegradable polymeric nanoparticles with natural ovaries (Fig. 1 right). erythrocyte membranes, inclusive of membrane lipids and associated membrane proteins. The structure, size and surface zeta potential and protein analysis of the particles were examined by transmission electron microscopy, dynamic light scattering, as well as gel electrophoresis, consecutively. The bioinspired nanoparticles may offer a distinctive and elegant way in drug delivery.

Fig.1: Left: Principle structure of double labelled nano-particles. DY-676 is covalently bound to the amphiphilic HPMA derivative. The highly lipophilic dye DiR is dissolved in the FA-PGA core of the nanoparticles. Right: Ex- vivo images of ovaries with fallopian tubes (top, a-c) and kidneys with adrenals (bottom, d-f). From left to right are shown respectively: (a,d) Intensity weighted single spectrum images of DiR. (b,e) Intensity weighted single spectrum images of DY-676. (c,f) Unmixed composite images with overlaid signals (red color: DY-676; green color: DIR).

Our on different nanoscaled drug carriers indicate that polymeric micelles, polymer nanoparticles, nanocapsules and even nanoemulsions might accumulate in the ovaries and the adrenals. This observation has been made on different mice strains and on rats. Other Fig. 1: Transmission electron micrograph of bare ellipsoidal (above) and erythrocyte groups made similar observations with lipid nano-carriers [2, 3]. Our membrane-coated (below) elongated nanoparticles. current results indicate no clear impact of the particle size and the zeta potential. Further studies are necessary to evaluate the key parameters Acknowledgements: DAAD (Deutscher Akademischer Austauschdienst) is highly thanked for B.M.H.’s doctoral scholarship. The authors are grateful to Barbara Kneidl and PD Dr. med. Lars for this accumulation in order to design Nano-DDS which either avoid Lindner (Arbeitsgruppe Liposomen, Medizinische Klinik und Poliklinik III, Klinikum der LMU this uptake or to design specific carriers for ovarian or adrenal drug München) for use of Liposofast-Basic. Dr. Wei Zhang (Department of Pharmaceutical delivery. Biotechnology, LMU München) is kindly acknowledged for providing negative staining material for TEM analysis. The authors would like to thank Dr. Markus Döblinger (Department of Chemistry and Center for NanoScience (CeNS), LMU München) for assistance with TEM Acknowledgements: The support from the German Research Foundation (DFG grants MA investigations. 1648/7 and MA1648/8) and the Grant Agency of the Czech Republic (grant No. P207/12/J030) is gratefully acknowledged. References: 1. Blanco, E. et al.: Nature Biotechnol. 2015, 33 (9), 941-951. References: 2. Mathaes, R. et al.: Int. J. Pharm. 2014, 465, 159-164. 1. Schädlich, A. et al. J. Contr. Rel. 2012, 160, 105–112. 3. Hu, C. et al.: Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 10980-10985. 2. Hirsjärvi, S. et al., Int. J. Pharm. 2013, 453, 594–600. 4. Anselmo, A. et al.: ACS Nano. 2015, 9, 3169-3177. 3. Mérian, J.; et al. J. Nucl. Med.2013, 54,1996-2003.

DPhG Annual Meeting 2016 Conference Book • 149 POSTERS

POS.165 POS.167 Layer-by-Layer Stabilization of Cylindrical Microparticles Stabilization of Oligonucleotide-loaded Gelatin Using DNA Nanoparticles by Lyophilization

Möhwald, M.; Pourasghar, M.; Schneider, M. Geh, K. J.1; Hubert, M.2; Winter, G.1 Department of Pharmacy, Biopharmaceutics and Pharmaceutical Technology, Saarland 1 Ludwig-Maximilians-Universität München, Munich, 81377, Germany University, Campus A4 1, 66123 Saarbrücken, Germany 2 Umeå University, Umeå, 901 87, Sweden

In recent years many novel shapes have been introduced for particulate Introduction: In several studies, gelatin nanoparticles (GNPs) loaded drug delivery systems. Changing the geometry of particles allows the with immunomodulatory cytosine-phosphate-guanosine oligonucleo- creation of new drug release profiles, improved targeting and altered tides (CpG-ODNs) showed excellent clinical effects in the treatment of cell interactions [1 – 3]. Therefore innovative engineering techniques recurrent airway obstruction in horses1-3. We could establish a large have to be established, allowing full control of shape and size as well as scale production of GNPs4 in order to supply extensive clinical studies the incorporation of active compounds. We previously introduced a in equine patients. A topical application is ensured by the administration bottom-up, template-assisted engineering technique for the production of CpG-GNPs via inhalation after nebulization. However, one main of cylindrical, nano-composed microparticles [4, 5]. Within a shape- disadvantage is the maximal colloidal stability of 48 h of CpG-loaded defining template, nanoparticles of different materials can be GNPs in dispersion. Therefore, the purpose of this study was to develop interconnected by alternating polymer deposition (Layer-by-Layer), of a freeze-dried CpG-GNP formulation in order to allow long-term leading to cylindrical micron-sized particulates. For the attachment of storage. the polymers, electrostatic interactions can be the driving forces. In this Methods: GNPs were prepared according to one-step desolvation4 as a recent study, we applied amorphous silica nanoparticles (SNPs) modification of the common two-step desolvation method. After providing negative surface charges. For the polymer coating we chose cationization, GNPs were loaded with CpG-ODNs. Sucrose and chitosan (CHT) as a biocompatible and biodegradable polyelectrolyte trehalose were used as drying adjuvants and lyophilisation of the providing positive charges due to tertiary amines. As the counterpart we formulations was performed according to Zillies et al.5. Samples were used a herring testes DNA (HT-DNA) with negative charges due to the stored at room temperature as well as at 2-8°C. Formulations were phosphate-sugar backbone. The resulting polymer coating after the characterized in terms of particle size and size distribution (PDI) using deposition of six double-layers (CHT/HT-DNA counted as one double- dynamic light scattering, loading efficiency (UV spectroscopy) and CpG- layer) can be observed in Figure 1. ODN stability using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). Results: GNPs showed particle sizes between 225 nm and 245 nm and PDI values below 0.37 after loading with CpG-ODNs. Interestingly, after reconstitution of the lyophilized samples, particle sizes (208 – 236 nm) and PDI values (< 0.28) decreased (Figure 1). This phenomenon may be caused by the formation of a porous structure in the lyophilisates and subsequent shrinking of the particles, which is not completely reversible following rehydration. Particle sizes and PDI values remained stable upon storage for 6 months at room temperature or at 2-8°C. No difference between the drying agents sucrose and trehalose could be seen. MALDI MS data revealed that the integrity CpG-ODNs was not Fig. 1: Electron microscopy (SEM) visualization of nano-composed microparticles stabilized by affected by the freeze drying process no the storage conditions (Figure chitosan and HT-DNA building up six double-layers; A] After the mechanical manipulation of the 2). Furthermore, loading efficiencies of the samples were not impaired particles, the polymer matrix interconnecting the nanoparticles can be observed; B] A close-up by lyophilization or storage and remained above 90%. of the particles shows polymer deposition in fine layers on the nanomaterial

The coating approach including HT-DNA lead to stable, cylindrical microparticles representing the geometry of the applied template. The electron microscopy visualization clearly shows the polymers building up a matrix immobilizing the SNPs to a corn-like architecture. Due to the biodegradability of chitosan, a release of the DNA can be achieved potentially qualifying the system as a novel delivery platform for nucleic- acid based drugs.

References: Conclusions: Lyophilization has been demonstrated as a suitable 1. Sarah L. Clark et al.: Ionic Effects of Sodium Chloride on the Templated Deposition of method to stabilize CpG-ODN-loaded GNPs. The drying procedure had Polyelectrolytes Using Layer-by-Layer Ionic Assembly, Macromolecules 1997, 30: 7237–7244 no negative impact on particle characteristics or CpG integrity. MALDI 2. S. S. Shiratori, M. F. Rubner, pH-Dependent Thickness Behavior of Sequentially Adsorbed MS could successfully be used to investigate the stability of CpG-ODNs Layers of Weak Polyelectrolytes, Macromolecules 2000, vol. 33: 4213–4219 bound to the particle surface. Furthermore, stability of the lyophilized 3. S. E.A. Gratton et al.: Nanofabricated particles for engineered drug therapies: A preliminary biodistribution study of PRINT™ nanoparticles, J. Control. Release 2007, vol. 121: 10–18 CpG-GNPs at different storage conditions (room temperature and 2- 4. C. Tscheka et al.: Macrophage uptake of cylindrical microparticles investigated with 8°C) could be shown for 6 months. Bioactivity of freeze-dried correlative microscopy, Eur. J. Pharm. Biopharm. 2015, vol. 95: 151-155 formulations is currently being assessed in an in vitro assay using 5. D. Kohler et al.: Template-assisted polyelectrolyte encapsulation of nanoparticles into equine cells from bronchoalveolar lavage. dispersible, hierarchically nanostructured microfibers, Adv. Mater. 2011; vol. 23: 1376–1379 References: 1. Klier, J., et al., Journal of Veterinary Internal Medicine, 2015. 29(1): p. 286-293. 2. Klier, J., et al., Pharm Res, 2012. 29(6): p. 1650-1657. 3. Klier, J., et al., Equine Veterinary Journal, 2015. 47(S48): p. 26-26. POS.166/SL.57 4. Geh, K.J. in 10th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology. 2016. Glasgow, GB. Oligoaminoamide-based siRNA carriers for in vivo tumor 5. Zillies, J.C., et al., European Journal of Pharmaceutics and Biopharmaceutics, 2008. 70(2). targeting and gene silencing

Lee, D. J.1,2; Kessel, E.1,2; He, D.1,2; Klein, P. M.1; Lächelt, U.1,2; Wagner, E.1,2 1 Department of Pharmacy & Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 Munich, Germany 2 Nanosystems Initiative Munich, Schellingstraße 4, 80799 Munich, Germany

For abstract see Short Lecture SL.57

150 • DPhG Annual Meeting 2016 Conference Book BIOPHARMACEUTICS

Acknowledgments: Financial support by the German Research Foundation (HE 7440/2-2 and POS.168/SL.50 HE 3119/9-1), Bundesministerium für Bildung und Forschung (NanoMatFutur 13N12561) and the Austrian Science Fund (2259-B26) is gratefully acknowledged. Toward Biopredictive Dissolution for Enteric Coated References: Dosage Forms 1. Eckl, K.M.: et al.: J. Invest. Dermatol., 2011, 131(9): 1938–1942. 2. Witting, M.: et al.: Nanomed. Nanotech., Biol. Med., 2015, 11(5): 1179–1187 Al-Gousous, J.1; Amidon, G. L.2; Langguth, P.1 1 Insititute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudinger Weg 5, 55099 Mainz, Germany. 2 Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan 48109, USA. POS.170 For abstract see Short Lecture SL.50 alpha-Synuclein at the Blood-Brain Barrier in Parkinson`s Disease

Mahringer, A.; Fricker, G. Department of Pharmaceutical Technology and Biopharmaceutics, Ruprecht-Karls University of POS.169 Heidelberg, Heidelberg, Germany

Thermoresponsive nanogel mediated protein replacement Localised at the brain microvessel endothelium, the blood-brain barrier therapy restores skin-barrier function to full-thickness skin (BBB) provides a precise homeostatic neuronal environment and models derived from Transglutaminase 1 deficient patients means a key determinant in drug transport to the brain. Solute carriers, efflux transporters (P-glycoprotein (P-gp), Breast Cancer Resistance Yealland, G.1; Plank, R.2,3; Obst, K.1; Miceli, M.4; Molina, M.4; Eckl, Protein (Bcrp), Multidrug Resistance Protein 4 (Mrp4)), tight junctions K.5,2,3; Calderón, M.4; Hennies, H. C.2,5; Hedtrich, S.1 (TJs, Occludin, Claudin-5, Zonula occludens (ZO-1)) as well as 1 Institute of Pharmaceutical Sciences Freie Universität Berlin, Germany; endocytotic processes selectively deliver essential nutrients to the brain 2 Dept. of Biological Sciences University of Huddersfield, UK; 3 Medical University of Innsbruck, Austria; or remove neurotoxic agents. These BBB elements respond to a variety 4 Institute of Chemistry & Biochemistry Freie Universität Berlin, Germany; of regulatory signals making them susceptible to profound changes that 5 Dept of Biology, Edge Hill University, UK; occur during CNS diseases or pharmacotherapy [1]. 6 Cologne Centre for Genomics, University of Cologne, Germany In Parkinson`s Disease, alpha-Synuclein (αS) is a 14.4 kDa Mutations in the Transglutaminase 1 (TGase1) gene, TGM1, cause neuropeptide that forms neurotoxic deposits in dopaminergic neurons autosomal recessive congenital ichthyosis (ARCI), a rare skin and is prone to aggregation into fibrils or Lewy bodies [2]. Particularly, cornification disorder characterised by severe epidermal scaling and the A53T mutant conformation leads to an early-onset of motor significant impairment to skin-barrier function. Current therapies offer dysfunction and cognitive impairment symptoms. It has been shown only symptomatic relief. Delivery of functional TGase1 to the viable recently that αS is present in cerebral blood vessels of cerebral amyloid epidermis might rectify those dysfunctions caused by TGM1 mutations. angiopathy patients as well as in the cerebrospinal fluid (CSF) and Whilst topical application would be the most direct route of blood plasma [3,4]. administration, proteins are unable to penetrate into the skin due to their The following project provides insights into changes of BBB elements physicochemical properties, calling for the use of novel delivery evoked by αS in Parkinson`s disease and allows conclusions on the strategies. involvement of pathophysiologically altered BBB clearance mechanisms Full-thickness skin models were generated from fibroblasts and in αS brain accumulation. Their restoration to healthy control levels keratinocytes derived from ARCI patients possessing TGM1 mutations could imply new targets in the therapy of Parkinson`s disease and [1]. Relative to healthy controls these demonstrated increased extend established neurologic treatments to a vascular approach. permeability to testosterone and clear histopathological aberrations A biphasic effect of both human native and A53T mutant αS monomers analogous to those seen in ARCI. As an experimental protein on the expression and function of the efflux transporters P-gp, Bcrp and replacement therapy, thermoresponsive-hyperbranched polyglycerol- Mrp4 as well as of TJs in an in vitro and ex vivo model of porcine brain poly(N-isopropylacrylamide) nanogels (NG) were loaded with TGase1 capillary endothelial cells, isolated rat brain capillaries as well as in and applied topically to skin models [2]. These were subsequently A53T αS transfected rats (SD-Tg(SNCA*A53T)268Cjli) was determined exposed to a temperature gradient emulating that found across the in the present study: Both native and mutant αS isoforms significantly epidermal layers of human skin. increased P-gp, Bcrp and Mrp4 RNA, protein-expression and -function Following four applications over nine days (1, 5 or 10µg/cm2 TGase1 at lower concentrations after short-term incubation which turned into a applied in 500µg/cm2 NG), skin-barrier function was improved in a decline at higher concentrations and after exposure for 48 to 72h (1- manner proportional to TGase1 concentration (Fig 1). Improvements 1000ng/ml, 1-72h); this process was accompanied by an initial were markedly greater than those seen following application of TGase1 tightening of the BBB at low αS concentrations but followed by a in PBS alone (Fig 2), in keeping with previous work demonstrating gradual opening at higher concentrations after long-term incubation enhanced protein delivery to the viable epidermis by NG [2]. Application (Occludin). In parallel to the up-regulation of the efflux transporters an of unloaded NG saw no effect to skin-barrier function. An increased RAGE (receptor of advanced glycation end-products) immunohistochemical activity assay confirmed the delivery of functional expression was observed, which emerged together with the secretion of TGase1 to the SC and outer viable epidermis in NG, but not PBS inflammatory TNFα and the induction of NFκB. Additionally, low αS treated skin models. concentrations caused a transient reduction (1-24h) of LRP1 (low Assessment of NG interactions with cell monolayers demonstrated density lipoprotein receptor-related protein 1) expression in capillary uptake of unloaded NG in healthy keratinocytes in a concentration endothelial cells. dependant manner, which could be attenuated by endocytic inhibition. Transport experiments across the BBB in vitro indicated an increased No apparent cytotoxic induction was seen in healthy keratinocytes or efflux rate and extent of both native and mutant αS monomers from the fibroblasts incubated with TGase1 loaded NG, as assessed by MTT, brain to the blood compartment relative to their uptake. This was also LDH release and BrDU incorporation assays. confirmed by the calculation of Kp,uu in vitro. Inhibitors of clathrin- Mutant TGM1 skin models demonstrate deficient skin-barrier properties mediated endocytosis decreased the passage from brain to blood and that can be restored by topical application of functional TGase1 vice versa implicating a receptor-mediated mechanism (e.g. RAGE, encapsulated within thermo-responsive NG. Given their apparent LRP1). Last, αS uptake was higher in Parkinson rats after tail vein biocompatibility, the use of thermo-responsive NG for topical protein injection which can be linked to the pathological modifications at the replacement therapies presents a promising avenue in the treatment of BBB. severe congenital skin disorders such as ARCI. References: 1. Danemann R., Prat A.: The Blood-Brain Barrier (Cold Spring Harb. Perspect. Biol.) 2015. 2. Peelaerts W. et al.: Nature 2015, 522(7556): 340-344. 3. Tamo W. et al.: Neurosci. Lett. 2005, 326: 5-8. 4. Simonsen A.H. et al.: Biomark. Med. 2016, 10(1): 19-34.

Fig. 1 and 2: Skin model permeability to Testosterone following treatment with (1) TGase1 dissolved in PBS or (2) TGase1 loaded in thermo-responsive NG

DPhG Annual Meeting 2016 Conference Book • 151 POSTERS

phenols are major components in volatile oils which are used as 3.10 Pharmaceutical fragrances, insect repellents, pheromones or aroma in many different products [2]. The pharmacological action of monoterpenes is very broad, including antimicrobial [3], anti-inflammatory and anticancer Technology and activity [4]. In the late 1960s glucoconjugated forms of monoterpenes were firstly detected in rose petals. These conjugates represent Biomaterials nonvolatile precursors of the monoterpenes and open the possibility to retard the release of the actives in all fields of their application [5]. Despite this, the use of these compounds has been hindered until POS.171 know, because only recently an innovative method to produce these Molecular interactions in pharmaceutical compounds compounds by biotechnological means was developed [5]. Potential applications of these compounds were recently reviewed by Schwab et Edkins, K.1 al., also suggesting a potential use as surfactant, due to its related 1 School of Medicine, Pharmacy and Health, Durham University, University Boulevard, chemical structure to decyl glucoside, a surfactant frequently used in Stockton-on-Tees, TS17 6BH, United Kingdom cosmetic industry [5]. Aim: Therefore, the aim of this study was to investigate whether Small organic molecules, especially in the pharmaceutical sciences, monoterpenyl glucosides can be used as surfactants, which would tend to crystallise in a plethora of different crystal forms, either as pure make these compounds to actives with added value in pharma, food compounds or with the inclusion of solvent molecules. Due to their and cosmetics. changed physico-chemical characteristics, such as melting point, Materials and methods: Two monoterpenyl glucosides, geraniol compressibility, solubility and thus bioavailability, and physical and glucoside and thymol glucoside, as well as 2-phenylethanol glucoside chemical stability, different crystal forms can pose a problem to the were investigated. The possibility to form an emulsion was investigated manufacture of medicines.[1] It is thus crucial to understand the by producing emulsions consisting of 10% (w/w) oil (Miglyol 812) and crystallisation behaviour and manufacturability of these compounds in water to up to 100% (w/w). The amount of surfactant was varied from order to avoid problems in the life-time of the medicine and costly 1% to 5%, respectively. The emulsions obtained were analysed recalls comparable to ritonavir[2] or rotigotine.[3] Bioactive molecules regarding droplet size and physical stability by using light microscopy and pharmaceuticals typically have multiple functional groups, enabling and macroscopic observations. The emulsion quality was compared to them to interact with receptors and thus show pharmacological action. emulsions produced with Tween 80 as stabilizer. The CMC was In the solid-state, the interactions through these functional groups are investigated by determining the surface tension at different the driving forces of molecular recognition. By applying X-ray and concentrations using a tensiometer equipped with a wilhelmy plate. As neutron diffraction methods as well as thermoanalysis, vapour sorption references the stabilizers Sulfopon 1216g (MW 330) and Tween 80 and spectroscopic analysis in combination with computational (MW 1310) were used. techniques, we are probing the strong and weak interactions within the Results: The emulsions produced with Tween 80 resulted in droplet crystal forms and during the crystallisation in order to understand and sizes of about 10 µm, independent on the concentration of stabilizer predict their characteristics. Using the pharmaceutical compounds used. The emulsions showed a slight physical instability, i.e. a slight Piroxicam, Theophylline and Diatrizoic acid, the influence of the creaming was observed after one day of storage. In addition a strong molecular interactions within the crystal structure of their respective foam formation during production of the emulsions was observed. The hydrated crystal forms on the observed macroscopic characteristics will emulsions produced with geraniol glucoside as stabilizer showed similar be discussed. A series of tetrahydrocarbazolone derivatives are used results, indicating a good emulsification efficacy. Emulsions stabilized as model compounds to investigate the destabilising effect of steric bulk with 2-phenylethanol glucoside resulted in emulsions with similar sizes on the predominant hydrogen bonding interaction in the solid-state and to the control, however, a phase separation shortly after production was solution. observed. Emulsification with thymol glucoside stabilizer did not lead to Recent interest in medicinal chemistry in using bio-isosteric homogeneously sized emulsions. Interestingly, the amount of foam replacement as tool for lead structure optimisation has led to the created during production was directly correlated to the ability to form replacement of functional groups with more or less related substituents emulsions, i.e. most of the foam was created by Tween 80 and geraniol without change of overall pharmacological effect. However, the glucoside, some foam was created by 2-phenylethanol glucoside and implications that a changed substituent have on the solid-state no foam was created with thymol glucoside. Measurements of the CMC characteristics of the new compound are rarely investigated. We are values for Sulfopon and Tween 80 confirmed the results obtained from systematically elucidating the substituent influence on the crystal form the literature, with values of about 50µM/l and 1mM/l (i.e. 0.03%), landscape in order to predict changes in processability and respectively. The CMC of geraniol glucoside was found to be about manufacturability of novel drug compounds. A series of 10mM/l (i.e. 0.3%). The CMC of 2-phenylethanol glucoside is even triphenylimidazole derivatives are used as model compounds to reveal higher and no CMC was detected for thymol glucoside, because the the influence of halogen substitution on solvent inclusion as well as the solubility of thymol glucoside was < 1%. However, for all compounds a relationship of the respective desolvated crystal forms. strong reduction in the surface tension, even at low concentrations, was observed. The highest reduction in surface tension was obtained for References: Sulfopon and geraniol glucoside (about 30mN/m), followed by thymol 1. Hilfiker R, Polymorphism: In the Pharmaceutical Industry (Wiley-VCH Verlag GmbH & Co. glucoside, Tween and 2-phenylethanol glucoside, respectively. KGaA, Weinheim, Germany), 2006. 2. Chemburkar SR et al., Org. Proc. R. & D. 2000, 4, 413-417. Conclusion: Results confirm that monoterpene glucosides possess 3. Rietveld IB and Ceolin R, J. Pharm. Sci. 2015, 104, 4117-4122. amphiphilic properties. Depending on their chemical structure they might be used as emulsifiers, stabilizers or wetting agents. Therefore, this class of excipients might be used in the future for the production of innovative healthcare products, combining a medical or cosmeceutical and processing action in only one compound. In addition with other POS.172 actives they might lead to synergistic effects, i.e. an improved medical Monoterpenyl and related glucosides – surfactants with or cosmeceutical activity and/or less required excipients. The possibility added value in pharma and cosmetics? of tailor-made release profiles from these compounds further increases the scientific interest in this kind of compounds, suggesting more Mohr, C1; Dauer, K2.; Schwab, W.3; Huang, F.-C.3; Hoffmann, T.3; research in this field. Fischer, T.3; Keck, C. M.1 1 Institute of Pharmaceutics and Biopharmaceutics, Philipps-Universität Marburg, References: 35032 Marburg, Germany 1. Raskin, I. et al.: Trends Biotechnol. 2002, 20(12): 522–531. 2 Applied Pharmacy, University of Applied Sciences Kaiserslautern-Campus Pirmasens, 2. Isman, M.: Crop Prot. 2000, 19(8-10): 603-608. 66953 Pirmasens, Germany 3. Park, S. et al.: Anaerobe 2012, 18(3): 369–372. 3 Biotechnology of Natural Products, Technische Universität München, 85354 Freising, 4. Salminen, A. et al.: Cell. Mol. Life Sci. 2008, 65(19): 2979–2999. Germany 5. Schwab, W. et al.: Appl. Microbiol. Biotechnol. 2015, 99:165–174.

Introduction: Terpenes and especially monoterpenes as well as aryl alcohols are secondary plant metabolites with manifold applications in the fields of food, healthcare and cosmetics [1]. Monoterpenes and

152 • DPhG Annual Meeting 2016 Conference Book PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS

i.e. bi- oder trimodal distribution and larger agglomerates, were POS.173 obtained, indicating that the particle counter is able to provide useful Is a particle counter an alternative to laser diffractometry information of the size and size distribution of microsized particles. for size analysis? Conclusion: Results confirm that the Mastersizer 3000 is a highly accurate instrument for size analysis. However, as shown in previous Stumpf, F.1; Ibraimi, M.1,2; Paulke, B.3; Keck, C. M.1 studies, results proved again that the size results can be highly 1 Institute of Pharmaceutics and Biopharmaceutics, Philipps-Universität Marburg, misleading if parameters for analysis are incorrect. Another hazard of 35032 Marburg, Germany 2 Institute of Pharmaceutical Technology, Anadolu University, 26470 Eskisehir, Turkey this method is the possibility that larger particles might not be detected 3 Fraunhofer-Institute for Applied Polymer Research, 14476 Potsdam-Golm, Germany by this method. The Mastersizer X and the particle counter showed accurate results and led to reliable information about the presence of Introduction: Nanosized drug carriers, i.e. liposomes, nanoemulsions, larger particles. Therefore, especially if the presence of larger particles lipid nanoparticles, nanocrystals or polymeric nanoparticles, possess should be investigated, these techniques are recommended. special properties when compared to larger particles. Besides the Advantages of the particle counter are significantly lower costs of the development, formulation and application of these carriers, size equipment, a simple setup of the instrument and fast measurement characterization is one of the major fields of interest. Size procedures. characterization aims not only on the analysis of the main size, but also on the size distribution and on the detection of possible larger particles. Acknowledgments: Mirsad Ibraimi thanks ERASMUS for providing the scholarship. We thank Matthias Wojcik for the REM images. Especially the detection of possible larger particles is highly important, because larger particles might influence the physical stability of the References: nanosized formulation (e.g. Ostwald-ripening). Therefore, methods 1. Keck, C. M., Müller, R. H. Int. J. Pharm. 2008, 355 (1-2): 150-63. 2. Keck, C. M. Int. J. Pharm. 2010, 390 (1):3-12. which detect larger particles highly reliable are highly important. In most 3. Acar Kübart, S., Keck, C. M., J Pharm Tech Drug Res 2013, 2(17) dx.doi.org/10.7243/2050. cases microscopic techniques and laser diffraction are used for such purposes. The advantages and disadvantages of these techniques were investigated in previous studies [1-3]. The aim of this study was to investigate if a particle counter, normally used for counting particles in infusion systems, can also be used for this purpose. POS.174 Materials and methods: Latex samples with different sizes (2-3µm Sortase-A mediates VHH – conjugation to differently and 3-6µm) were analysed separately and as mixture (1:1) using PEGylated liposomes different sizing technologies. Laser diffraction measurements were performed using a Mastersizer X and a Mastersizer 3000 (Malvern Wöll, S.1,2; Schiller, S.1; Scherließ, R.2 Instruments, UK). Particle counting was performed using a PAMAS 1Merck KGaA, Frankfurter Straße 250, 64293 Darmstadt, Germany SVSS (Pamas Partikelmess- und Analysesysteme, Germany). Particles 2Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, were further characterized using light microscopy (Zeiss, Germany) and Germany raster electron microscopy (Hitachi High-Technologies Europe GmbH, Introduction: Liposomes are widely used as drug delivery system Germany). The results obtained were compared to each other. (DDS) in research for cancer therapy. Enclosing cytotoxic compounds Results: Microscopic analysis confirmed the size of the samples (Fig. into physiologically indifferent carriers releasing their cargo at the site of 1). In addition, a few agglomerates of the particles in the range from action increases safety and efficacy of anti-cancer drugs [1]. Equipping about 10 µm – 100 µm were detected. Results obtained from the the DDS with ligands binding to specific proteins on tumour cells Mastersizer 3000 revealed monodisperse distributions when analysed enables a targeted delivery. Common chemical coupling approaches with the universal mode using Mie theory and correct optical properties make use of any of the reactive side chains of amino-acids (e.g. for latex particles. Modification of the results, e.g. analysis in the special maleimide or EDC-NHS chemistry), leading to an undefined binding and “spherical particles” mode and/or without the additional backscattering decreased activity of the targeting moiety [2]. A sterically defined

conjugation in non-paratop regions of an antibody would ensure preservation of activity as well as steric accessibility of the antigen binding regions. Therefore, we make use of a Sortase-A mediated directional transpeptidation of a LPETG (leucine-proline-glutamate- threonine-glycine) modified anti-eGFP single-domain antibody (VHH) to pentaglycine-modified liposomes, where the antibody is conjugated to the outer leaflet with high steric selectivity [3]. Four liposome concepts are tested for enzyme reaction with regard to develop a DDS platform with various in-vivo behaviour controlled by PEGylation status and surface charge (Fig 1). Conjugation and activity of the antibody is confirmed by an eGFP-binding assay.

Fig. 1: REM images of mixed latex sample, containing particles from 2µm – 6µm. technique (blue light) led to more accurate results, which were in agreement with the results obtained from microscopic observations. Fig. 2: Liposome formulation concepts However, the larger agglomerates were not detected by this technique. The reason for this might be the stirrer of the instrument which might Methods: Liposomes were prepared by a continuous solvent injection have provided enough forces to destroy the agglomerates during the technique. Formulations consisted of DPPC, Cholesterol, DSPEmpeg- measurement. Results obtained from the Mastersizer X were similar to 2000 or DPPG and DMA-G5 or DMA-PEG-G5 in a molar ratio of the results from the Mastersizer 3000 with optimized size analysis, i.e. 59.4 : 34.65 : 4.95 : 1. Physical properties were controlled by dynamic the results showed bi- or trimodal distributions, which corresponded to light scattering and Laser Doppler electrophoresis, and pentaglycine the results obtained from microscopy. In contrast to the results obtained content was determined using RP-HPLC ELSD. After incubation with from the Mastersizer 3000, results obtained from the mastersizer X Sortase-A and anti-eGFP VHH, liposomes were purified using dialysis. revealed a small fraction of larger sized particles, indicating that the Reaction success was confirmed by mass spectroscopy. Binding ability Mastersizer X was more appropriate to detect a few larger particles was shown using HP-SEC by determination of unbound eGFP in a within a small sized main population. The reason might the smaller liposome – target protein mixture. sample cell of the Mastersizer X which uses only a small magnetic Results and Conclusion: Liposomes were prepared in a size range stirrer. Thus, the agglomerates were not destroyed during the suitable for in-vivo administration (<200 nm, PDI < 0.25) and measurement. The results obtained from the Pamas instrument were functionalized with the targeting ligand while retaining physicochemical highly depended on the concentration used for the measurement. Most properties (Fig. 2). Mass spectroscopy confirmed conjugations of anti- appropriate dilutions were found to be in the range from 1:10,000 to eGFP VHH with a plane and a PEG-spaced pentaglycine modified lipid 1:20,000. By using these dilutions, similar results to the Mastersizer X, (Table 1). HP-SEC showed activity of the targeting ligand on the surface of the liposomes and revealed superior binding of eGFP to

DPhG Annual Meeting 2016 Conference Book • 153 POSTERS targeted formulations compared to unspecific incubated DDS. The shaker 3031 (GFL, D- Burgwedel) at 39 °C and 60 rpm, using a visking present work therefore shows a possible liposomal platform for the dialysis tubing MWCO 12-14 kD in PBS pH=7.4. Samples were investigation of therapeutic VHHs with respect to the different in-vivo analyzed by RP-HPLC with UV detection at 220 nm. properties (circulation time, cellular uptake) of the drug delivery system Results and Conclusion: The peptide release requires the penetration due to the adaptable surface properties. of aqueous body fluids and dissolution at the muscle tissue/oil vehicle interface. The incorporation of HPMC, CMC and HPC at the higher concentration counteracts the encapsulation caused by the AlSt gelled MCT, thus increasing the diffusion of water into the oil depot. In addition to this, the HPC: peptide complex (10:1) showed a nearly complete and sustained release of the peptide, reaching approximately 70% after 14 days. In contrast, the reference formulation without polymer showed an incomplete release of the peptide which levelled off at 45% after 14 days. Upon contact with water nanostructures are formed. HPC is able Fig. 3: Physicochemical properties of VHH modified liposomes to adsorb at the oil/water interface to the area of the nanostructures that are not fully covered by PL90H, thus affecting the release [2]. This creates a diffuse layer around the structures promoting a steric Conjugation repulsive effect between the formed nanodroplets and therefore Expected Mw Determined Mw Product contributes to their stability and the complete release of the peptide DMA-VHH 14179 14179 from the oil depot [3]. Furthermore, the oil vehicle containing the 2% to 0.2% HPC to peptide particles exhibited a zero-shear viscosity of 443 DMA-PEG-VHH 15836 15842 mPas at 39°C after a linear ramp shearing, 1 to 500 s -1 for 3 min, which ultimately reduces the spreading of the oil depot after application. Further in vitro studies are required in order to elucidate whether the Table 1: Mass spectroscopy of targeting ligands observed effect on the release can be correlated to the polymer chain

length or to a specific polymer class. Acknowledgements: The authors thank Lee Kim Swee, Christopher Bachran, Matthias Schröder and Lena Conrad of BioMedX for providing proteins and helpful discussion about the experiments. References: 1. Beckjunker, J. and Kauffold, J.: Anim Reprod Sci 2007, 97:84-93. References: 2. Mezdour, S.:Colloids Surfaces A Physicochem Eng Asp 2008, 331:76–83. 1. Barenholz, Y.: J. Control. Release, 2012, 160 (2), 117-134. 3. Karlberg, M., Thuresson, K., Lindman, B.: Colloids Surfaces A Physicochem Eng Asp 2005, 2. Saha, B., T.H. Evers; M.W. Prins: Analytical chemistry 2014, 86 (16), 8158-8166. 262:158–167. 3. Ta, H.T. et al: Circ. Res. 2011, 109 (4), 365-373.

POS.175 POS.176 ® rd Development of a New Depot Formulation for Peptide SmartLipids - 3 generation lipid nanoparticles with high Delivery loading capacity for lipophilic actives

1 1 2 1,2 1 2 3 1 Bacher, L. ; Arntjen, A. ; Hespeler, D. ; Nguyen, T. M. H. , Keck, C. Yordanova, Y. ; Kauffold, J. ; Zaremba, W. ; Frieß, W. 3 1 LMU Munich, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, M. 1 Butenandtstr.5-13, 81377 Munich, Germany, 2 University of Leipzig, Faculty of Veterinary Applied Pharmacy, Hochschule Kaiserslautern-Campus Pirmasens, 66953 Pirmasens, Medicine, An den Tierkliniken 29, 04103 Leipzig, Germany Germany 2 3 Veyx Pharma GmbH, Söhreweg 6, 34639 Schwarzenborn, Germany Institute of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Freie Universität Berlin, 12169 Berlin, Germany 3 Institute of Pharmaceutics and Biopharmaceutics, Philipps-Universität Marburg, Introduction: A new oil based parenteral sustained release formulation 35032 Marburg, Germany of Gonadorelin [6-D-Phe] in a multi-dose container for the manipulation of the reproduction cycle in the swine was developed [1]. In a previous Introduction: Geranylgeranyl-2-propanol (GGP) is a terpenic alcohol study the correlation between viscosity and release rate from pure oil and acts as a metabolism facilitator by regulating the activity of various mixtures composed of castor oil and medium chain triglycerides (MCT) cellular proteins and their location in different cellular compartments [1]. was evaluated. In vitro and in vivo data showed that a sustained release The mechanism of action is not fully understood. However, present data of the peptide can be achieved. However, a relatively high initial burst prove the high potency of this compound to reduce oxidative stress at release of the peptide from the pure oil mixtures was observed. the cellular level [1]. Oxidative stress is a main cause of aging and Furthermore, the rather high viscosity at 25 °C and Newtonian flow known to promote many diseases. Therefore, GGP might be a potent behavior posed difficulties upon multiple dose application. Therefore, a active compound to prevent and treat various oxidative stress related second generation of oil suspensions was composed of aluminium diseases in the future. The compound is lipophilic and possesses poor distearate (AlSt) gelled MCT with the addition of wetting agents aqueous solubility, leading to poor bioavailability. Thus, the aim of this phospholipon® 90H (PL 90H), Tween® 80 and Span® 80 as well as the study was to formulate GGP as smartLipids to improve its bioactivity. resuspendibility enhancer Kolliphor® ELP (KP ELP). These components Materials and Methods: SmartLipids are referred to be the third improved considerably the applicability, resulting from the shear-thinning generation of lipid nanoparticles. Similar to the first and second behavior of the formulation compared to pure oil mixtures.The current generation of the lipid nanoparticles, they improve the bioavailability of study aims to evaluate the use of suspended particles made of polymer poorly soluble compounds. However, due to their chaotic smartLipid and peptide incorporated in the developed vehicle containing MCT, 3% matrix they enable a higher loading capacity of the active compound (m/m) AlSt, 1% (m/m) PL 90H, 5% (m/m) KP ELP in order to obtain a [2,3]. Until now only limited data are available for such smartLipid self-emulsifying sustained/controlled release formulation. matrices, which should be composed of many different solid and liquid Materials and methods: Tested polymers: Hydroxy- lipids. In this study eight solid waxes with different chemical natures propylmethylcellulose (HPMC) K4M, sodium carboxymethylcellulose (microcrystalline waxes, paraffin waxes, plant waxes) were blended and (CMC) 7LP EP, sodium carboxymethylcellulose (CMC) 7M8SF, used as a solid lipid matrix. Different amounts of various semisolid and hydroxypropylcellulose (HPC), 2-hydroxypropyl-β-cyclodextrin HP7 liquid waxes were systematically added to the solid matrix and the (Ashland, D-Düsseldorf) and hydroxyethyl starch (HES) 200/0.5 for mixtures were evaluated in regard to homogeneity, hardness and parenteral use (Fresenius Kabi, A-Linz). Solutions of 0.2% peptide and appearance. Suitable mixtures were transferred into smartLipids by hot 0.2% or 2% polymer mixtures were freeze dried using a Christ Epsilon high pressure homogenization using Plantacare 2000 as stabilizer. The 2-6 D freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH, D- most suitable formulation, leading to small sized particles with a narrow Osterode) (100 mTorr; primary drying: 60 h, -25°C; secondary drying: size distribution and a sufficient physical stability, was used for the 12 h, 35°C). The lyophilisates were cryo ground with a Retsch Cryo Mill development of the GGP-loaded smartLipids. (Retsch Technology, D-Haan) and suspended in the oil vehicle using an Results: The mix of eight different solid waxes led to very hard and Ultra-Turrax T-10 basic (IKA-Labortechnik, D-Staufen) for 5 min at 2000 bridle mixtures. Addition of semisolid and/or liquid waxes up to 50% rpm. The in vitro release experiments were performed in an incubated (w/w) improved the processability of the blends, decreased the

154 • DPhG Annual Meeting 2016 Conference Book PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS hardness and increased the stickiness. Most appropriate blends investigate skin penetration from these formulations, ex vivo penetration consisted of 50% solid lipid phase and 50% semisolid and/or liquid studies were carried out using postauricular porcine skin. The drug waxes. For some mixtures blends consisting of even 70% liquid and penetration was compared to that from a conventional semisolid semisolid wax were found to be appropriate for further investigations. formulation. After incubation, the stratum corneum was removed by skin Homogenisation of these blends revealed small sized smartLipids with surface biopsy. Deeper skin layers were segmented via cryosectioning. sizes below 300 nm. Matrices containing semisolid paraffin waxes The drug in the different skin layers was extracted and quantified by formed agglomerates, indicating that these types of waxes might not be HPLC-UV/VIS. suitable to be used in smartLipid matrices. The best formulations were The penetration experiments showed that film-forming formulations are obtained from a blend containing equal parts of eight solid waxes able to ensure a continuous penetration of the drug into the skin while (3.75% each) and a blend of lanolin alcohol, mineral oil, diisopropyl reaching therapeutically suitable drug levels in the viable epidermis. dilinoleate and isopropyl lanolate, contributing to 70% (w/w) of the This implies that film-forming formulations may allow more convenient smartLipid matrix. The loading capacity of GGP in the matrix was dosage regimens and hence may improve the patients’ compliance. determined to be 28% (w/w). The production of smartLipids with 28% GGP loaded into the matrix resulted in particles with a size of about 160 Acknowledgements: PD Dr. Martin Schenk is acknowledged for the donation of pig ears. This project was supported by the European Social Fund and by the Ministry of Science, Research nm and a polydispersity index < 0.2. However, the zeta potential was and the Arts Baden-Wuerttemberg slightly different, when compared to the non-loaded formulation, indicating an overloading of the matrix, i.e. excess GGP might be located at the outside of the matrix. These particles remained stable for only one week, further indicating the overload of the matrix with GGP. In contrast, formulations containing 25% GGP possessed a similar small POS.179 size, possessed a zeta potential similar to the non-loaded particles and Advanced ex vivo test models elucidate the impact of lack remained physically stable for at least 6 months. Conclusion: GGP-loaded smartLipids were successfully developed in of substantivity on cutaneous absorption of drugs this study and are now available for further in vitro and in vivo studies. 1 1 1 1 The GGP-smartLipid matrix contains twelve different lipids and enables Hermann, S. ; Schmidberger, M. ; Daniels, R. ; Lunter, D. 1 Department of Pharmaceutical Technology, University of Tuebingen, Auf der Morgenstelle 8, a loading capacity of 28% for GGP. This data supports the theory of the 72076 Tuebingen, Germany smartLipid concept, i.e. a chaotic lipid matrix increases the loading capacity of lipophilic actives. Results also showed that smartLipids Skin diseases are usually treated using topical formulations that contain should be produced with a drug load that is slightly below the maximum one or more active substances. Frequently, multiple applications per loading capacity of the lipid matrix. Therefore a complete encapsulation day are necessary, as up to 90 % of the formulation (and thus of the of the active compound into the matrix can be improved, which active) are withdrawn from the skin by contact with the environment. decreases expulsion of the active compound during storage and They are then no longer available for penetration into the skin and the increases the physical stability of the formulation. effectiveness of the treatment may thereby be impaired. It would thus be advantageous to develop formulations with enhanced substantivity. Acknowledgments: The project was partly funded by ZIM KF2161909SK4. Such formulations would stay on the treated site for a longer period of References: time and would thus reduce the application frequency and enhance 1. Fournial A et al., EP2555743 A2, 2010. patient compliance. However, methods that effectively characterize the 2. Ruick R, PhD-Thesis, Freie Universität Berlin, 2015. substantivity are still lacking. 3. Junmahasathien, T. PhD-Thesis, Freie Universität Berlin, 2015. We thus developed ex vivo methods that probe the substantivity of topical formulations by simulating skin-to-skin or clothing-to-skin contact and enable the determination of the amount of formulation that is withdrawn from the skin due to the contact. Furthermore, we simulated POS.177/SL.38 the impact of skin-to-clothing contact on cutaneous absorption ex vivo. Three different types of formulations were used to validate the Skin penetration analysis by confocal Raman experimental setups: A conventional semisolid cream [1] and two microspectroscopy – potentials and pitfalls sustained release formulations. The first sustained release formulation was an oil-in-silicone oil-emulsion [2] and the second one a film forming Lunter, D.1 formulation [3]. Nonivamide was used as a model drug. 1 Department of Pharmaceutical Technology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany It was shown that the methods are capable of detecting differences in substantivity between the three model formulations. Furthermore, the For abstract see Short Lecture SL.38 lack of substantivity was found to lead to reduced permeation of the drug through the skin. Additionally, the extent to which the formulation was removed from the skin due to lack of substantivity was found to correlate very well with the reduced amount that permeated the skin [4]. POS.178 Acknowledgements: PD Dr. Martin Schenk is acknowledged for the donation of pig ears. This Film-Forming Formulations with Sustained Skin project was supported by the European Social Fund and by the Ministry of Science, Research Penetration of an Antipruritic Drug and the Arts Baden-Wuerttemberg References: Heck, R.1; Lunter, D.1; Daniels, R.1 1. Bundesvereinigung Deutscher Apotheker, Hydrophile Capsaicinoid Creme 0,025 % / 0,05 % / 1 Department of Pharmaceutical Technology, University of Tuebingen, Auf der Morgenstelle 8, 0,1 % (NRF 11.125), Neues Rezeptur Formularium, D-Eschborn, 2010. 72076 Tuebingen, Germany 2. Rottke M, Lunter D, Daniels R, Eur J Pharm Biopharm, 2014 86: 260-266. 3. Heck R, D Lunter and R Daniels, Skin Forum 14th annual meeting, 2014. Chronic pruritus is a common symptom accompanying various chronic 4. Hermann S, Daniels R, Lunter D, Pharm Dev Technol 2016, DOI: skin diseases. Conventionally, it is treated with antihistamines and local 10.3109/10837450.2015.1135346. anesthetics. However, these drugs often cannot provide sufficient relief. As an alternative, capsaicinoids can be used. Their long-lasting antipruritic effect is caused by continuous stimulation of transient receptor potential vanilloid 1 (TRPV1) at the epidermal pain conducting POS.180 fibers. To achieve this, currently available formulations need to be applied 4-6 times a day. This is inconvenient and results in poor patient Premix membrane emulsification for the preparation of compliance. colloidal lipid emulsions as drug delivery systems The aim of our study was thus to develop a film-forming formulation with sustained penetration for dermal use making it easy to treat large areas Gehrmann, S.; Bunjes, H. of affected skin over a long period. Technische Universität Braunschweig, Institut für Pharmazeutische Technologie & Zentrum für Pharmaverfahrenstechnik, Mendelssohnstraße 1, 38106 Braunschweig, Germany Film-forming formulations were prepared by incorporating an oily drug solution into mesoporous silica and subsequently incorporating these An increasing number of new drug substances are poorly water soluble carriers into a plasticized film-forming polymer dispersion. To and thus difficult to effectively administer to patients. A promising option

DPhG Annual Meeting 2016 Conference Book • 155 POSTERS for the administration of such substances is loading them into colloidal operated impinging jet mixers [1], multi-inlet vortex devices [2] or even a lipid emulsions. Premix membrane emulsification is a possibility to simple Y-junction mixing setup [3] and by using microfluidic devices [4]. prepare these colloidal carriers with the required size and a narrow The aim of this study was to evaluate the solvent shifting method for the particle size distribution. In this process, a coarse pre-emulsion is preparation of triolein (TO) nanoemulsions without and with lecithin extruded through the pores of a membrane, yielding smaller emulsion (E80S) as stabilizer by using a simple Y-junction mixing setup. droplets [1]. 1 ml of ethanol precursors containing 1-20 mg/ml TO without and with In order to gain deeper understanding of the parameters influencing the lecithin (0-10 mg/ml Lipoid E80S at fixed TO concentration of 5 mg/ml) routine applicability of premix membrane emulsification the effect of were rapidly mixed with 19 ml water preserved with 0.02% w/v sodium different emulsifiers and membrane filter materials on the quality of the azide via a Y-junction (r = 0.25 mm). Under these conditions (mixing resulting emulsions was studied. An instrumented small scale time was about 8 s), the Re number was about 5000. All samples were membrane extruder [2] was used in the experimental setup which prepared in triplicate, stored at 4-8 °C and analyzed by dynamic light allowed precise process control. The emulsions consisted of 20% scattering (DLS, average size and zeta potential), asymmetrical flow Miglyol 812 (MCT) (or peanut oil in some cases) stabilized with 5% field-flow fractionation coupled to multi-angle laser light scattering surfactant in double distilled water containing 0.01% thiomersal as (AF4/MALLS, size distribution), optical microscopy and single particle preservative. Sodium dodecyl sulfate (SDS), poloxamer 188, Tween 80, optical sizing (detection and quantification of µm-sized particles). tyloxapol, and sucrose laurate were used as surfactants. After The size of surfactant-free TO NPs increased with increasing TO dispersion with an Ultra-Turrax the pre-emulsions were processed 21 concentration in the ethanol precursor with diameters (DLS) between 40 times through different disposable 200 nm membrane filters and 300 nm. Polydispersity indices (PDI) were below or close to 0.1 (polycarbonate (PC), polyester (PE), polyethersulfone (PES), indicating rather narrow size distributions, which was confirmed by polysulfone (PS), polyvinylidene fluoride (PVDF), nylon and cellulose AF4/MALLS. Moreover, no particles in the µm-size range were acetate (CA)) at a flow rate of 0.25 ml/s. For some additional detected by optical microscopy. The nanoemulsions were stable for up investigations a 200 nm alumina membrane was employed. to 30 days. The stability of such surfactant-free oil droplets has been The process led to different particle sizes and size distributions suggested to be by adsorption of OH- ions onto the droplet surface [5]. indicating that the polymeric membrane materials could be classified Addition of lecithin resulted in smaller NPs with decreasing size with into groups. The first group led to nanoparticles with all emulsifiers increasing lecithin concentration (Figure 1). Interestingly, addition of under investigation (nylon, CA). The second group (PC, PS and PVDF) lecithin in low concentration caused a destabilization of the emulsion yielded submicron particles only with SDS. Membranes out of PES and droplets (Figure 1). The most stable formulation was obtained at an PE were between the two groups, whereby the PES membrane tended E80S/TO mass ratio of 0.20 (droplet size about 60 nm, PDI around to be part of the second group and PE of the first group. As confirmed 0.18). Calculations predict that a TO droplet with a diameter of about by scanning electron microscopy the track-etched PC and PE 60 nm would be covered completely with a lecithin monolayer at a mass membranes have a uniform pore size of 200 nm, whereas the other ratio of 0.24, assuming a spherical particle shape, a lecithin monolayer membranes have a more sponge-like membrane structure containing thickness of 2 nm and a TO density of 0.91 mg/ml. Taken into account pores of 1-2 µm diameter in some cases. The differences in emulsion that lecithin is not a pure substance, the calculated ratio is overall in quality can thus not solely be explained by the membrane structure but good agreement with the experimental results. A sudden increase of the the effect may include an interaction between emulsifier and membrane PDI was observed at E80S/TO mass ratios ≥ 1 where the formation of surface. To identify potential correlations, contact angles between mixed structures or vesicles may be expected. emulsifier solution and membrane material were measured. The In conclusion, TO nanoemulsions with droplet sizes in the lower nm- combinations of emulsifier solution and membrane material with a size range and rather narrow size distributions were obtained by a contact angle less than 49° led to MCT emulsions with a median simple, low-energy preparation process. However, as the solvent particle size below 500 nm in all cases, whereas the particle size was shifting method results in highly diluted dispersions, on-going work always above 1 µm for contact angles higher than 55°. Basically focusses on the evaluation of up-concentration of the nanoemulsions in analogous results were obtained with peanut oil emulsions albeit with a addition to investigations on the NP morphology and drug incorporation. slightly shifted transition area between successful and unsuccessful formulation/ membrane combinations. The wetting of the membrane with the continuous phase of the emulsion seems, therefore, to be of special importance. Additional experiments with an alumina membrane confirmed this assumption as this setup did not only lead to colloidal emulsions in all cases but also to the best results with all emulsifiers except for poloxamer. Taking the correlation between wetting properties of the formulation with the membrane into consideration colloidal lipid emulsions could thus be obtained with all emulsifiers under investigation. Membranes from CA, nylon and, in particular, alumina, appear particularly promising for routine use as they can be successfully be applied with many different formulations.

Fig. 1: Z-average diameters and PDI of the nanoemulsions of independently prepared Acknowledgments: This study was performed in the context of the DFG research group 856 mikroPART. We thank Simone Schulze (TU Braunschweig, Institute for Chemical and Thermal samples (n = 3). The TO concentration in the ethanol precursor was 5 mg/ml. Process Engineering) for taking the SEM images. References: 1. Joseph, S., Bunjes, H.: J. Pharm. Sci., 2012, 101(7): 2479-2489. Acknowledgements: The authors thank the Danish National Research Foundation, Denmark, for 2. Gehrmann, S., Bunjes, H.: Chem. Eng. J., 2016, 284: 716-723. financial support of the project. References: 1. Han, J. et al.: J Pharm Sci-Us. 2012, 101(10):4018-23. 2. Liu, Y. et al.: Chem Eng Sci. 2008, 63(11):2829-42. 3. Gillian, J.M. et al: Chem Eng Commun. 2008, 195(12):1553-74. POS.181 4. Zhigaltsev I.V. et al.: Langmuir. 2012, 28(7):3633-40. 5. Marinova, K.G. et al.: Langmuir. 1996, 12(8): 2045-51. Evaluation of solvent-shift generated surfactant-free and lecithin-stabilized triolein nanoparticles

Glud, K.1; Kuntsche, J.1 1 University of Southern Denmark, Campusvej 55, Odense M 5230, Denmark

Solvent shifting is a method to precipitate compounds by introducing a solution containing the compound into a non-solvent. Under conditions resulting in high Reynolds numbers (Re), the solvent shifting method usually yields nanoparticles (NPs) in the lower nanometer size range. Such conditions can be obtained both by rapid mixing using hand-

156 • DPhG Annual Meeting 2016 Conference Book PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS

acids as further nonviral vectors. This concept is based on the POS.182 hypothesis that a minimal positive charge of the carrier is associated Delivery system for the targeting of ocular with less toxic effects on cells. The formation of the complex with neovascularizations monocationic peptides is envisioned to be induced via electrostatic interactions between the negatively charged phosphate groups of Haunberger, A.1; Göpferich, A.1 nucleic acid and the positive charge at the side chain of the peptide. 1 Institute of Pharmacy, Department of Pharmaceutical Technology, University of Regensburg, The complex should be further stabilized via hydrophobic interactions Universitätsstr. 31, 93053 Regensburg, Germany between the aromatic side chains of neighbouring peptide molecules. Material and Methods: DNA/peptide complex formation was induced by Background: The proliferative forms of age-related macular mixing DNA (eurofins) and peptides (synPeptide, Peptide 1: Ac-Lys- degeneration (AMD) and diabetic retinopathy (DR) are both among the Gly-Phe-Leu-Trp-Leu-Phe-Ser, Peptide 2: Ac-Lys-Gly-Phe-Leu-Trp- leading causes of blindness in the industrialized world. The newly Leu-Phe-Cys) in different NP ratios (NP ratio is the number of formed vessels, which are sprouting from the choroid in AMD and from negatively charged phosphate groups of DNA to the number of the retinal capillaries in DR respectively, are often leaky, which causes positively charged amino groups of the peptide) and incubation at room edema and haemorrhage. Also tensile stress and the breakdown of the temperature. The complex formation was determined via gel retardation blood-retina barrier affect the surrounding tissue and can lead to fast assay with a 1% agarose gel stained with ethidium bromide. For the visual impairment. Today´s therapies like laser coagulation or anti- visualization of the complex transmission electron microscopy was used VEGF-therapeutics often suffer from unsatisfying outcomes and are not (Libra 120, ZEISS). free from side effects [1, 2]. Results and Discussion: Two peptides consisting of eight amino acids In our approach, we focus on a combination of Cyclosporin A (CsA) and which only differ in the carboxy terminus were used for the complex Itraconazole (Itra), as these active substances selectively and formation with a short double stranded DNA sequence (22bp). The synergistically inhibit endothelial cell proliferation and different steps of peptides were used in different concentrations yielding different NP angiogenesis [3]. The two therapeutics were incorporated in lipid ratios. In a gel retardation assay complex formation was detected at nanocapsules (LNC); to enable an accumulation of these nanoparticles every NP ratio, but only at higher NP ratios (10, 20, 30) the whole DNA in proliferating vessels, the surface of the nanocapsules was decorated was bound in a complex (data not shown). Also the measurements with with the cyclic peptide c(RGDfC) (cyclo(Arg-Gly-Asp-D-Phe-Cys)), the transmission electron microscope showed a difference between a which is a ligand of the αvβ3-integrin. This receptor is up-regulated in pure peptide (rodlike filaments) solution and a DNA-peptide-mixture proliferating endothelial cells [4] and the presentation of c(RGDfC) on (helical strands), which is a proof for complex formation (Figure 1). the surface of nanoparticles even leads to their accumulation in normal retinal and choroidal capillaries [5]. Methods: CsA and Itra were encapsulated into LNC via two different methods during the nanoparticle preparation in a phase-inversion process [6]. The particle size was determined by dynamic light scattering (DLS). For the quantification of the encapsulation efficiencies for CsA and Itra, respectively, the LNC were purified via ultrafiltration and size exclusion chromatography to remove non-encapsulated drug. The CsA and Itra content of purified and non-purified LNC was determined via HPLC. For the c(RGDfC)-decoration of nanocapsules, the peptide was coupled to a PEGylated phospholipid which was then incorporated into the LNC shell via post insertion [7]. To confirm the enhanced binding to endothelial cells, human dermal microvascular Fig. 1: Images of transmission electron microscopy with negative staining. A: complex between endothelial cells were incubated with c(RGDfC)-carrying and blank DNA and peptide 2 (scale bar 100 nm); B: pure peptide 2 solution (scale bar 100 nm). fluorescently labeled LNC and the binding was quantified via flow cytometry. Conclusion: Both peptides are suitable for the formation of a complex Results: We were able to incorporate CsA and Itra in lipid nanocapsules with DNA. Therefore they are potentially convenient for the use as a with high encapsulation efficiencies (about 70 % for CsA and 100 % for new drug delivery system for nucleic acids. Itra, respectively). The size of the prepared LNC was about 50 nm and did not change due to drug encapsulation. The c(RGDfC)-modified Acknowledgments: Margit Schimmel nanocapsules showed 5-6 times increased binding to human dermal microvascular endothelial cells.

Acknowledgments: This work was supported by the German Research Foundation (DFG; GO 565/18-1). POS.184 References: 1. Hagedorn, C.; Adelmann, R.: Age-Relate Macular Degeneration in Diabetic Retinopathy in Influence of surfactants and formulations on stratum Tombrain-Tink,, Barnstable (Ed.): Ocular Angiogenesis (Humana Press) 2006. corneum lipids 2. Ma, J.; Zhang, S.: Endogenous Angiogenic Inhibitors in Diabetic Retinopathy in Tombrain- Tink,, Barnstable (Ed.): Ocular Angiogenesis (Humana Press) 2006. Zhang, Z.1; Lunter, D.1 3. Nacev, B.; Liu, J.: PLoSONE 2011, 6(9): e24793. 1 Department of Pharmaceutical Technology, Auf der Morgenstelle 8, 72076 Tübingen, 4. Chavakis, E. et al.: Diabetologia 2002, 45(2):262-267. Germany 5. Pollinger, K. et al.: pnas 2013, 110(15):6115-6120. 6. Heurtault, B. et al.: Pharm. Res. 2002, 19(6):875-880. Stratum corneum (SC), which consists of corneocytes and intercellular 7. Hirsjärvi, S. et al.: Eur. J. Pharm. Biopharm. 2014, 87(1):152-9. lipids, plays an essential role as a barrier to the external environment. However, treatment with surfactants on SC can induce lipid extraction and other property changes, leading to barrier defect. To investigate these influences, basic cream DAC was chosen as model formulation. It POS.183 contains two kinds of non-ionic surfactants: glycerol mono stearate 60 (GMS) and macrogol-20-glycerol mono stearate (PEG20GMS). Water Complex formation between short peptide sequences and and sodium lauryl sulfate (SLS) were used as negative and positive DNA for nucleic acid delivery control. In this study, GMS, PEG20GMS, GMS and PEG20GMS mixture, SLS Haas, V.1; Goepferich, A.1 solution and basic cream DAC as well as its oil phase (OP) and 1 Department of Pharmaceutical Technology University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany aqueous phase (AP) were applied onto excised porcine skin during incubation in Franz diffusion cells. Subsequently, SC was isolated Introduction: The development of nonviral vectors for the application of according to ref [1]. Dehydrated SC sheets were subjected to confocal gene and RNAi therapies is of great interest. Lipidic or polymeric Raman microscopy (CRM) to detect SC lipid content and differential carriers are widely used for such drug delivery systems and led to scanning calorimetry (DSC) measurements to evaluate SC lipid significant research for improvements [1,2]. In this study small ordering. A pyranine fluorescence staining technique [2] was also monocationic peptides were used for the complex formation with nucleic conducted to visualize the barrier impairment.

DPhG Annual Meeting 2016 Conference Book • 157 POSTERS

Various kinds of surfactants show different capabilities of barrier The process conditions such as die table speed, die diameter as well as damage. SLS exhibits the strongest ability among all the studied material characteristics e.g. particle size and size distribution, cohesion, surfactants. It withdraws 38% (w/w) lipids from SC and reduces the friction and restitution coefficients were systematically changed to second lipid thermal transition temperature by 13 °C (average mean evaluate their influence on the die filling process. The results give both value). Fluorescence staining experiment reveals the brightest image qualitative (powder flow pattern from the feeder to the dies) and for SLS, also indicating the least SC barrier integrity after treatment. quantitative (tablet mass and mass variation, mass flow rate etc.) GMS and PEG20GMS mixture exhibits stronger ability compared to insights into the die filling process. The numerical results indicate that either component by extracting 35% (w/w) lipids and shifting thermal faster die table speeds and smaller die diameters give rise to lower transition to lower temperature by 7 °C. Interestingly, OP shows tablet mass with significantly increased mass variation, which could stronger barrier destroying capability than AP from both CRM and DSC eventually increase the challenges in meeting the specifications. On the results, although this difference is not obvious in fluorescence staining. other hand, cohesion reduces the tablet mass and increases its However, basic cream DAC does not show significant difference in variation. The friction between the particles and geometry influences the contrast with water treated samples. This might imply that, barrier particle rearrangement in the feeder thus the dead zone is less impairing ability of surfactants are weakened while they interact with prevalent. other formulation components, e.g. due to emulsification. In summary, this study gives references to the CQA of die filling from an open feeder of a rotary tablet press and emphasizes the importance of Acknowledgments: PD Dr. Martin Schenk is acknowledged for the donation of pig ears. This blend characterization in early stage of formulation development to project was supported by the European Social Fund and by the Ministry of Science, Research and the Arts Baden-Wuerttemberg and the China Scholarship Council. ensure a high tableting process performance. In addition, such numerical studies will support product quality control concurrently with References: 1. Kligrnan, A. M. and Christophers, E.: Arch. Dermatol. 1963. 88(6): 702-705. established means of pharmaceutical development. 2. Pagnoni, A., Kligman, A., Stoudemayer, T.: J. Cosmet. Sci. 1998. 49: 33-38.

POS.186 POS.185 Investigation of powder flowability in a model die filling Numerical analysis of powder flow in a lab-scale tableting process machine Fritsch, A. K.1,2; Hildebrandt, C.3; Gopireddy, S.R.1; Rackl, M.2; Hildebrandt, C.1; Gopireddy, S. R.2; Scherließ, R.1; Urbanetz, N. A.2 Urbanetz, N. A.1 1 Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, 1 Pharmaceutical Development, Daiichi-Sankyo Europe GmbH, Luitpoldstr. 1, Germany 85276 Pfaffenhofen a. d. Ilm, Germany 2 Daiichi-Sankyo Europe GmbH, Pharmaceutical Development, Luitpoldstrasse 1, 2 Department of Materials Handling, Material Flow and Logistics, Technical University of Munich, 85276 Pfaffenhofen, Germany Boltzmannstr. 15, 85748 Garching, Germany 3 Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany Related to the fact that tablets constitute the top sold drug delivery system, the demand of high production rates while guaranteeing quality Tablets constitute the most common pharmaceutical dosage form inter is increasing over the last decades. One major challenge in tablet alia because of high patient compliance and most commonly produced production and from a patient safety point of view is the accurate using a rotary tablet press, which consists of three distinct stages. One content and content uniformity of the active pharmaceutical ingredient of them is the die filling in which the powder blend is deposited into the (API) in the final dosage form. Out of the different stages in the tableting die or cavity of the turret formed by lowering the lower punch. The unit operation, namely die filling, compaction and ejection, die filling amount of powder collected in the die depends on the material represents the most crucial step since it is the last stage to control and properties and process conditions, and it is the main factor which specify the mean and range of tablet mass and API content. Numerical controls the tablet production rate. Due to its relevance, interest in simulations offer an alternative solution to process analytical tools in understanding the die filling process has drawn more and more pharmaceutical formulation development to investigate critical quality attention over the last years. attributes (CQA) in the tableting unit operation. In this study, the die The current study investigates the die filling process experimentally filling process from an open feeder of a lab-scale rotary tablet press using a model die filling system. In this system, the die translates was investigated using the Discrete Element Method (DEM) to elucidate the CQA with respect to process parameters as well as material properties by which the process understanding is being enhanced. The DEM considers particles as individual elements, thus collisional forces between the particles as well as with the geometry are computed to find all the particles trajectories. The particle motion is captured by the DEM as particles travel from the feeder, represented by an inclined chute connected to a rectangular box, to the dies of the turret.

Fig. A. The die filling ratio over the die speed. The dashed lines indicate where the fitted curve starts to descend, and thus mark the critical die speed.

Fig. B. Mean particle diameter (x50) plotted over vc. The equation shows the linear relationship between vc and x50, and dashed lines indicate the 95% confidence interval band.

underneath the fixed shoe at a constant speed which can be varied Fig. 1: Visualization of the particle discharge from the feeding box in the die (view from right to from 50 to 500 mm/s simulating a simplified tablet press. left). The die table moves at a constant rotational speed of 20 rpm in the anti-clockwise direction Several commonly used excipients in direct compression formulations (indicated by the arrow). Particles are coloured according to their velocity (red: high, blue: low). were investigated, comprising fillers/binders of different grades of lactose, mannitol and microcrystalline cellulose. The powder mass In Fig. 1 the process of die filling is visualized by colouring the particles which was deposited at increasing die speeds was weighed, normalized according to their velocities. As soon as the die enters the bottom of the to the maximum possible filling level, determined by the bulk density, feeder (Fig. 1, t = 0.00 s), the particles slowly start to fall into the die and plotted against the die speed, and it is shown in Fig. A. A general mainly due to gravity. During the filling process, the top right corner of trend of exponentially decreasing die filling at increasing die speeds the feeder (portion of the box in which particles move with highest was observed. The highest die speed, at which the filling ratio first starts velocity, Fig. 1, t = 0.05 s) is getting emptied, which implies that the to decrease, is called the critical die speed (vc). The vc value for a given powder filled into the die is mostly composed of particles originating material can be extracted from Fig. A as indicated by the dashed lines. from this region. This difference in particle motion causes a confinement For all tested materials, the vc value was determined and plotted of particles on one side of the feeder and empty space on the other side against the mean particle diameter (x50), see Fig. B. The results hence may bear the risk of segregation. illustrate that there is an almost perfect linear correlation between the

158 • DPhG Annual Meeting 2016 Conference Book PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS mean particle diameter and the critical die speed. Furthermore, the Acknowledgments: The work was supported by the Federal Ministry of Education and Research results reveal that a hundred percent increase in particle size would (BMBF 03X0104D and E - NanoMed and BMBF 03XP0003 - NanoBEL). facilitate at least double the critical die speed. In this given setup, a minimum particle size threshold (34.2 µm) exists at which no complete die filling will be possible. Such a simple model die filling system helps in characterizing the POS.188 powder flowability specific to the tableting process. Hence it supports the product and process development by e.g. formulation optimization Suitability of Microemulsions as Vehicle System for Dermal through providing an estimation about a particle size distribution that Protein Application yields a higher production rate.

Albold, D. 1; Scherließ, R.1 1 Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany

POS.187 Introduction: Formulating a protein for dermal application (e.g. The hen’s egg test as a model for optical detection of vaccination or intra-dermal effects) is challenging due to the protein`s particle behavior under dynamic flow conditions molecular weight, hydrophilicity and sensitivity to external conditions, which mostly results in a short product shelf-life [1]. Here pre- Warncke, P.1; Müller, R.2; Stranik, O.2; Schlenk, F.1; Werner, S.1; microemulsion concentrates are promising topical formulations. Due to Malsch, D.2; Bergemann, C.3; Fritzsche, W.2; Fischer, D.1 their spontaneous formation upon addition of water they facilitate a 1 Department of Pharmaceutical Technology, Institute of Pharmacy, Friedrich-Schiller-University reduced contact time between the API and aqueous phase. Lipoid® S Jena, Otto-Schott-Str. 41, 07745 Jena, Germany LPC 80 was chosen as surfactant with high self-emulsifying power, 2 Leibniz Institute of Photonic Technology, Department of Nanobiophotonics, Albert-Einstein-Str. 9, 07745 Jena, Germany besides its enhanced skin penetration effects and good tolerability [2, 3 Chemicell GmbH, Eresburgstraße 22-23, 12103 Berlin, Germany 3]. Successful penetration of the protein-loaded microemulsion (ME) will be supported by a microneedle system, stimulating a local irritation. Iron oxide particles are attracting high interest for applications such as Therefore, an ideal formulation should balance between good skin magnetic drug targeting, hyperthermia or MRI imaging. Differences tolerability and high drug stability. between in vitro tests under static conditions and the dynamic Aim of the Study: The present study focused on stability of a model conditions in the blood stream were suggested to be responsible for a protein in a ME-system analysing changes in structure and activity. limited transfer of 2D-cell culture data to the in vivo situation. Blood flow Based on tolerability studies with 3D-human skin models the “natural”- may compromise the interaction between nanoparticles and target cells, based phospholipid-ME (PL) will then be compared with a formulation especially for particles with low binding affinities. Furthermore, cell including well-known PEGylated surfactants (PEG). culture systems do not sufficiently represent the complexity of Methods: The ME were prepared in a fixed ratio (surfactants:lipophilic interactions between nanomaterials and the different blood components phase:aqueous phase) containing catalase as model protein. After relevant for systemic administration. To mimic the behaviour of particles preparing the final formulations, changes in protein structure were with and without the presence of a magnetic field in vivo, the chick analysed at predefined time points via nanoDSF (NanoTemper embryo was used as an easy accessible, vascular network system technologies). This technique is an advanced differential scanning using optical microscopy. fluorimetry technology which offers the opportunity to detect smallest Different fluorescent iron oxide particles and magnetic silica beads conformation changes in biomolecules, measured as shifts in thermal (both chemicell GmbH, Berlin) with varying surface characteristics were unfolding transition temperature (Tm). Under same conditions the investigated regarding their flow characteristics and optical properties. catalase activity was examined, utilising the ability of active enzyme to Particle stability in blood, the tendency to agglomerate and degrade hydrogen peroxide (assay modified after Beers and Sizer [4]). hemocompatibility were tested in red blood cell assays. The in vivo Results & Conclusion: Fig. 1 demonstrates that both formulations situation was investigated with the shell less HET-CAV (hen’s egg test- have an obvious influence on protein structure and their activity. chick area vasulosa) model. After systemic injection of particles, the Changes of catalase conformation were detectable as shift in the Tm blood vessel systems were screened under an optical microscope compared to Tm of a stable solution (Fig. 1 B, C). Directly after (AxioImager Z1.m, Carl Zeiss, Jena) operated in the dark field or the preparation and 24 hr later the protein showed a reduced Tm and loss of fluorescence mode. A PCO-Sensicam connected to the microscope activity in both systems (Fig. 1 A). At 3 hr later the Tm of PL-ME turned was used to record particle behavior in blood vessels. A magnetic field back close to the active maximum, corresponding to an increasing was generated by a cylindrical NdFeB magnet next to the objective. activity (Fig. 1 B, blue curve). In comparison, the PEG formulation Data analysis was realized with TrackMate, a versatile SPT plugin for revealed higher structural changes, resulting in lower melting curves the image processing package Fiji. (Fig.1 C) but in a similar activity profile. According to first findings the The HET-CAV model with its planar area vasculosa allowed the optical observed rise in activity within 3 hrs could be explained with an inspection of the whole blood vessel system. Particles with emission equilibration time of protein in its new environment. wavelengths of the fluorescent dye >500 nm were optimal for optical Further studies with other proteins will examine this phenomenon and microscopy due to the background fluorescence of the embryonic characterise in more detail the time dependency of protein activity in a tissue. Moreover, particle sizes of 1 µm in combination with a 10x ME system. NanoDSF technology offers quick and reproducible objective were optimal for single particle tracking. The measurements in presence of lipophilic ME ingredients. Does a hemocompatibility testing of the particles indicated their suitability for chromatographic method, like size exclusion chromatography, produce administration directly into the systemic circulation. None of the additional and comparable results? This and more questions will be particles did show any detectable disturbance of the erythrocyte discussed in the present study with a final focus on assessment of ME membranes nor erythrocyte aggregation up to 10-fold higher as vehicle system for proteins along with tolerability studies on 3D- concentration than in the ex ovo model. Tracking analysis allowed the human skin models. detection of particle flow profiles depending on the particle coating, agglomeration behavior, and the dimensions and type of the blood vessels. In the presence of a magnetic field a hydrodynamic resistance was caused that led to a decreasing velocity of the particles near the vascular wall and strong agglomeration caused by magnetic forces. After removal of the field the agglomerates started to move again with a partial disintegration of the agglomerates. As in vitro in microfluidic channels these effects were not visible, they were suggested as surface Fig.1: Comparison of catalase activity (A) and nanoDSF of PL (B) and PEG (C). All interactions of particles with the complex blood environment depending measurements were performed in comparison to 100 % catalase activity (red lines). B and C on the coating of the particles. show on y-axis the first derivative of F350/F330 data, next to temperature changes from 20 °C In conclusion, the shell-less HET-CAV model is a suitable dynamic up to 95 °C on x-axis. model by offering a planar surface making the entire vascular network accessible. Transport of magnetic particles under the influence of a Acknowledgements: The authors would like to thank NanoTemper Technologies GmbH, Lipoid magnetic field can be observed with fluorescence microscopy. GmbH and Gattefossé for their support. This project is financed by the Phospholipid Research Center.

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References: As alternatives to polymeric systems, we also developed lipid based 1. Frokjaer, S.: Nature Reviews Drug Discovery 2005, 4(4): 298-306. oleogels as alternative systems [3]. The main component of the oleogel 2. Kogan, A., Garti, N.: Adv. Coll. Interface Sci. 2006, 123: 369-385. is a pharma-grade oil (e.g. sesame or peanut oil, MCT). The oil is 3. Paolino, D. et al.: Int. J. Pharm. 2002, 244(1): 21-31. solidified by an oleogelator (e.g. hydroxy-stearic acid). Direct injectable 4. Beers, R.F.Jr., Sizer, I.W.: J. Biol. Chem. 1952, 195: 133-140. oleogels (DIOGs) contain no organic solvents. It is possible to incorporate proteins via an emulsification process and to obtain a prolonged release in vivo (Fig. 2), [3].

POS.189/SL.13 Application of a dried H1N1 vaccine by epidermal powder immunization in piglets using a novel pyrotechnically driven applicator elicits antigen-specific antibodies

1 1 1 2 2 3 Engert, J. ; Anamur, C. ; Engelke, L ; Fellner, C. ; Lell, P. ; Henke, S. ; Stadler, J.4; Zöls, S.4; Ritzmann, M.4; Winter, G.1 1 Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Fig.2: Mutispectral Optical Imaging of in vivo albumin release (ALEXA Fluor R 680-BSA) from Ludwig-Maximilians-University Munich, Butenandtstr. 5, D-81377 Munich, Germany DIOGs. The DIOGs contained no organic solvent and were s.c. injected into mice. The release 2 PyroGlobe GmbH, Hauptstr. 15, D-85276 Hettenshausen, Germany rate is determined by the selected oil and the oleogelator concentration. 3 IIS Innovative Injektionssysteme GmbH, Lohmannstr. 2, D-56626 Andernach, Germany 4 Klinik für Schweine, Ludwig-Maximilians-University Munich, Sonnenstr. 16, D-85764 Oberschleißheim, Germany In situ forming Oleogels (ISFOGs) contain a lower percentage of a biocompatible organic solvent (e.g. NMP). The oleogel is formed in situ For abstract see Short Lecture SL.13 by diffusion of the NMP into the outer environment which causes a precipitation of the oleogelator.Compared to DIOGs, ISFOGs permit higher concentrations of the oleogelator and therefore, stronger gels. The results of our preclinical in vivo studies show, that ISFOGs biodegrade with high reproducibility over several weeks. In conclusion, POS.190 DIPOs, DIOGs and ISFOGs are promising carriers for parenteral DIPOs, DIOGs and ISFOGs as novel direct injectable and controlled drug release. biodegradable drug delivery systems for parenteral controlled release References: 1. Weiss, V.M. et al.; J.Contr.Rel. 2012, 158, 156–164 2. Mäder, K. et al., DE102014005782 (A1) 1 1 1 1 1 Mäder, K. ; Windorf, M. ; Kutza, J. , Weiss, V. ; Rodrigues, A. G. ; 3. Mäder, K.; Kutza,, J.; Windorf, M; DE102013018193 (A1) Wersig, T.1; Kressler, J.2 1 Institute of Pharmacy, Martin Luther University Halle-Wittenberg, W. Langenbeckstr. 4, 06120 Halle (Saale), Germany 2 Institute of Chemistry, Martin Luther University Halle-Wittenberg, Von-Danckelmann-Platz 4, 06120 Halle (Saale), Germany POS.191 The demand for optimized parenteral controlled release is increasing Needle-free injection of vesicular phospholipid gels both for local and systemic acting drugs. Currently, the selection of the material is very limited. Polyesters made from lactic and glycolic acid Breitsamer, M.1; Winter, G.1 are mainly used (PLA and PLGA). However, due to the high acidity of 1 Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, the monomers, autocatalytic degradation processes occur which often Ludwig-Maximilians-University, Butenandtstr. 5, 81377 Munich, Germany leads to complex release kinetics and acidic microenvironments (e.g. pH values 2). The acidic microenvironment might lead to the drug Vesicular phospholipid gels (VPGs) are highly concentrated semisolid degradation and the formation of covalent linkages between drugs and aqueous dispersion of phospholipids in buffer [1]. They have been used polymer degradation products. To decrease production complexity and in the past as depot formulations for the sustained release of proteins costs, it would be desirable to inject a drug loaded solution which forms and peptides [2-4]. Their composition and manufacture is extremely a depot after injection. Clinically used in situ implants involve the use of elegant and cheap and with release rates over several weeks they organic solvents (e.g. NMP) which is undesirable. Therefore, the represent a most desirable functionality. The only drawback of such development of new biodegradable and direct injectable drug carriers formulations is the fact that VPGs have a high viscosity, which is with reduced monomeric microacidity and the avoidance or decrease of influenced by the amount of phospholipids incorporated into the organic solvents is highly desirable. We developed direct injectable formulation and further by the encapsulated drug [5]. The viscosity polymers (DIPOs), direct injectable oleogels (DIOGs) and in situ influences the release behaviour of drugs from the VPG and forming oleogels (DIOGs) as alternative systems. compromises the easy administration of the formulations through a thin Our DIPOs systems are based on linear biodegradable polyester s.c. needle. A practical and elegant solution to overcome administration backbones with free OH-groups, which are obtained via enzymatically problems for such highly viscous drug formulations is the use of needle- synthesis by the reaction of polyols with dicarboxylic acids. free injection devices, which, like the Biojector 2000, have been Furthermore, the modification of the free OH-groups with fatty acids approved by the FDA and other regulatory agencies. provides a versatile platform for biodegradable drug carriers with In the present study several VPG formulations composed of egg lecithin tuneable properties, including the lipophilicity and the physical state [1]. and PBS buffer pH 7.4 with a total phospholipid amount between 25% In addition to their use as nano- and microparticles, we synthesized and 55% were prepared with a Dual Asymmetric Centrifuge (DAC). hydrophobic liquid polymers, which can be directly injected [2]. The Methylene blue (MB) and Sulforhodamine B (SRB) were used as dyes. polymers were well tolerated and provided in preclinical studies a Viscosity of the formulations was measured with a rotational rheometer controlled release over several weeks (Fig. 1). and injectability with a conventional needle and syringe into pig skin was measured with a Texture Analyzer. A Biojector 2000 with disposable needle-free syringes was used for the administration of VPGs into different in vitro models. Gelatin blocks were used as models with high transparency for first investigations of the injectability of the formulations with the needle free-injector. Further, the skin of a piglet (post mortem injections) was used for injections of VPGs. The injection depth was measured directly after administration of the VPGs with the Biojector 2000 by cutting through the injection site and measurement Fig.1: Multispectral optical in vivo Images of the DIR loaded liquid DIPO formulations after s.c. injection in mice. The lipophilic fluorescent dye DIR was released over several weeks from fatty with a scale. acid modified Poly-(glycerol-adipate). The modification degree of the polymeric backbone with Injection forces for the injection of VPGs into pig skin showed, the fatty acid was different between (a) and (b). Therefore, the release kinetics can be expectedly, increasing forces with increasing phospholipid controlled by the DIPO composition. concentration, reaching maximum forces of about 50N for the formulation with 50% phospholipids. The formulation with 55%

160 • DPhG Annual Meeting 2016 Conference Book PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS phospholipids was not injectable. MB-VPGs with a phospholipid content depletion of surface material assuming that degradation is linked to a of 25% to 55% were successfully injected into gelatin blocks with the loss or degradation of the surface coating. In addition, the fate of the Biojector 2000. We observed decreasing injection depth with increasing nanoparticles was followed by Raman spectroscopy. amount of phospholipids. Further, we were able to inject SRB-VPGs In conclusion, the combination of the different techniques constitute an with phospholipid amounts of 25% to 50% into pig skin with the needle- in vitro prediction model that simulates the biodegradation of iron oxide free injector. The VPGs with 25% to 45% phospholipids penetrated into based magnetic nanoparticles. the subcutaneous and muscle tissue and formed a longish shaped depot, while injections of formulations with 50% phospholipids were Acknowledgements: The authors would like to thank A. Mohn and J. Grabow for their excellent technical assistance. We acknowledge the Federal Ministry of Education and Research (project intradermal and partially subcutaneous and formed a spherical shaped 03XP0003) for financial support. depot. Again we could observe that injection depth decreased with References: increasing phospholipid amount. 1. Marques, M. R. C. et al.: Dissolution Technol. 2011, 18(3): 15-28. Needle-free injection significantly simplifies the injection of highly viscous VPG formulations or makes it even possible. With that, previous restrictions in the use of VPGs can be overcome and the full potential of this controlled release concept can be used in the future. POS.193 Acknowledgments: Lipoid GmbH, Frigenstraße 4, 67065 Ludwigshafen, Germany; Veterinary ® Clinic for Swine, Ludwig-Maximilians-University Munich, Sonnenstr. 16, 85764 smartPearls for dermal delivery – control of loading Oberschleißheim, Germany. procedure with amorphous actives References: 1. Brandl, M. et al: Chemistry and Physics of Lipids, 1997, 87(1): 65-72. Pyo, S. M.1; Keck, C. M.2; Müller, R. H.1 2. Tian, W. et al: Journal of Controlled Release, 2010, 142(3): 319-325. 1 Freie Universität Berlin, Institut für Pharmazie, Kelchstr. 31, 12169 Berlin, Germany 3. Buchmann, S. et al: BMC Musculoskeletal Disordes, 2015, 16: 82. 2 PharmaSol GmbH, Stubenrauchstr. 66, 12161 Berlin, Germany 4. Zhang,Y. et al: Drug Development and Industrial Pharmacy, 2015, 42(7): 1042-1049. 5. Neuhofer, C., Dissertation, LMU München, 2015. Loading of mesoporous materials (silica, e.g. Neusilin) has been used to keep poorly soluble drugs in the amorphous state, to increase their oral bioavailability (e.g. CapsMorph® technology [1]). The amorphous state proved to be physically stable for more than 7 years of storage. Re-crystallization is hindered by the space restriction within the pores POS.192 (2-50 nm). Bioavailability is increased due to the increased saturation Simulation of the biodegradation of iron oxide and iron solubility Cs, and fast dissolution velocity dc/dt of the amorphous oxide-silica nanoparticles in artificial body fluids actives (no dissolution limitation). Recently this approach has been transferred to dermal delivery, the smartPearls® [2]. Surprisingly the Rabel, M.1; Cialla-May, D.2; Müller, R.2; Kurland, H.-D.3; Thamm, J.1; amorphous state remained after incorporating into dermal formulations, Bergemann, C.4; Grüttner, C.5; Müller, F. A.3; Fischer, D.1 despite the presence of the solute water (low solubility solute, but still 1 Friedrich Schiller Universtity, Department of Pharmaceutical Technology, potentially sufficient to generate crystallization). The penetration into Otto-Schott-Strasse 41, 07745 Jena, Germany human skin was similar or even superior to nanocrystals, e.g. 2 Leibniz Institute of Photonic Technology, Albert-Einstein-Strasse 9, 07745 Jena, Germany 3 Friedrich Schiller University, Otto Schott Institute of Materials Research, Löbdergraben 32, cyclosporin A. The technology can be applied to cosmetics, consumer 07743 Jena, Germany care and pharma products. 4 chemicell GmbH, Eresburgstrasse 22-23. 12103 Berlin, Germany There are several procedures for loading the actives into the pores, e.g. 5 micromod Partikeltechnologie GmbH, Friedrich-Barnewitz-Strasse 4, 18119 Rostock, Germany co-grinding, solvent methods (immersion, wetness impregnation) or loading with supercritical carbon dioxide. The wetness impregnation Iron oxide based magnetic nanoparticles have attracted interest in method is a simple process; drug solution is added to the porous many medical and biological applications such as contrast agents for material, blended and then subsequently evaporated (typically in MRI, stem-cell tracking or magnetic drug targeting. Since the multiple steps). This leads to precipitation of the active inside the pores, biodegradation of nanoparticles is a critical parameter that effects and partially on the surface. Preferred is the localization in the pores to potential risks and limitations for biological purposes it is necessary to avoid potential re-crystallization on the surface in case too much active develop models to simulate the degradation behaviour of magnetic is surface located. The pore volumes are relatively high, e.g. up to nanoparticles in biological systems. about 1.7ml per g carrier, being equivalent to a maximum theoretical In this study, nanoparticles with varying core materials (iron oxide or loading of about 60-70% w/w by an assumed drug density of 1.5g/cm3. iron oxide in a closed silica matrix), biodegradable and biopersistent However, before obtaining this theoretical maximum, crystal formation coatings (starch, polyethylene glycol, glucuronic acid, polyethylene occurs in practice above a “critical threshold”. Thus the loading process imine, dextran, silica) as well as different surface charges were used. needs to be controlled to obtain a pure amorphous product, verifiable by These nanoparticles were extensively characterized physicochemically x-ray diffraction and differential scanning calorimetry (DSC). regarding their hydrodynamic size, surface charge, morphology and Parameters of the loading procedure itself can also affect the critical magnetic properties using photon correlation spectroscopy, laser threshold, e.g. amount of solvent added per step, active concentration Doppler anemometry, vibrating sample magnetometry and electron in the solvent. Industrially friendly is loading with high solvent amounts microscopy. The nanoparticles were aged and degraded in different and high active concentrations, however bearing the increased risk of simulation fluids mimicking the lysosomal compartment at different surface localization of active during evaporation and resulting in crystal stages (artificial lysosomal fluid, ALF, pH = 4.5 and 5.5) and the plasma formation. compartment (simulated body fluid, SBF, pH = 7.4) [1] over a period of In this study Syloid® SP53D 12008 was loaded with coenzyme Q10 up to 28 days. Degradation and changes of the core and the shell were (Q10) as model active, using the impregnation method. Ethyl acetate evaluated by iron quantification, electron microscopy, Raman and containing 15% Q10 was used for loading, the amount of solvent used infrared spectroscopy as well as magnetic measurements. in each step was equal to 100%, and alternatively only to 50% All tested nanoparticles showed comparable hydrodynamic diameters in theoretical pore filling. The 50% filling should lead to faster uptake by aqueous dispersion with different neutral, cationic and anionic surface the excess pore volume and less surface localization. However, charges depending on the coating material, and with controlled stability industrially it requires the double number of addition steps and thus is in artificial fluids. The degradation of the core-shell nanoparticles was more costly. With Q10 in ethyl acetate it was found that both loading found to be dependent on the surface type, core materials and the pH methods lead to almost similar loading of about 40% in the amorphous and composition of the degradation medium. At pH 7.4 in SBF all state, as shown by x-ray and DSC. In a second study, to assess nanoparticles remained unchanged over 28 days regarding potential localization of active on the carrier surface, light microscopy aggregation, zeta potential, magnetism and core-shell structure. In was applied. As model active curcumin was used, because of its strong lysosomal fluids at pH 4.5 and 5.5 the degradation was found to yellowish color easy to detect. The microscopic appearance after each increase with decreasing pH. The core degradation in acidic media solvent addition and evaporation step was investigated. First, minor seemed to be dependent on the biodegradability, water permeability surface localization was detected, and the microscopic pictures can be and hydration, as well as the acid/base character of the shell. finally assessed by comparing with x-ray and DSC data. Furthermore, the degradation of the core correlated with a decrease of In conclusion, a control of the loading procedure is essential not only to the magnetization. Infrared spectroscopy was utilized to evaluate the ensure loading in the amorphous state, but also to identify loading parameters which are most economical. In this case loading with

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maximum solvent amount still leads to an amorphous product. References: Combination of x-ray, DSC and light microscopy proved sensible, 1. Wei, Q., Keck, C. M., Müller, R. H.: Int. J. Pharm. 2015, 482(1-2): 11–20. because first detectable surface localization does not lead already to 2. Nolte, M. et al.: PCT/EP2009/057688, 2009. crystallization (i.e. too thin surface film of active on carrier). Using the 3 3. Monsuur, F., Höfer, H. H., Keck, C. M.: US provisional patent application no. 62050587, 2014. methods, the surface coverage can be identified still not leading to 4. Staufenbiel, S. et al.: 1st European Conference on Pharmaceutics 2015, #42: 26. detectable crystallization.

References: 1. Wei, Q., Keck, C. M., Müller, R. H.: Int. J. Pharm. 2015, 482(1-2): 11–20. 2. Monsuur, F., Höfer, H. H., Keck, C. M.: US provisional patent application no. 62050587, POS.195 2014. Overcoming the mucus barrier – Antibiotic nanocapsules as drug delivery system for the treatment of pulmonary infections in Cystic Fibrosis

POS.194 Torge, A.1,2; Wagner, S.3; Chaves, P. S.2; Oliveira, E. G.2; Titz, A.3; smartPearls® for dermal delivery of amorphous actives – Beck, R. C. R.2; Schneider, M.1 effect of loading concentration on stability & release 1 Department of Pharmacy, Biopharmaceutics and Pharmaceutical Technology, Saarland University, Campus A4 1, 66123 Saarbrücken, Germany 2 Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul (UFRGS), Jin, N.1; Pyo, S. M.1; Keck, C. M.2; Müller, R. H.1 Av. Ipiranga, 2752, 90610-000 Porto Alegre, RS, Brazil 3 1 Freie Universität Berlin, Institut für Pharmazie, Kelchstr. 31, 12169 Berlin, Germany Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Chemical Biology of 2 PharmaSol GmbH, Stubenrauchstr. 66, 12161 Berlin, Germany Carbohydrates, Campus E8 1, 66123 Saarbrücken, Germany

Amorphous drugs have well known advantages for drug delivery The treatment of pulmonary infections is an essential part of Cystic (bioavailability increase etc.), but often face the problem of physical Fibrosis therapy, as recurrent lung infections caused by biofilm forming stability during shelf life of the formulations. The amorphous state could bacteria are associated with a decreased lung function and lower be nicely stabilized by encapsulating the drugs in mesoporous materials survival rates [1]. However, effectiveness of inhaled antibiotics is limited (silica, e.g. Neusilin [1]), or macropores of small size, due to the space due to low diffusion rates and a possible deactivation of the drug in the restriction in the pores. Physical stabilities of up to 7 years are reported. highly viscous mucus [2, 3]. To overcome this biological barrier we Materials used are e.g. various silicas (e.g. Syloid, company Grace), loaded ciprofloxacin into lipid-core nanocapsules with a core consisting having the advantage of regulatory acceptance. The activities since of oleic acid and sorbitan monostearate and a poly-ε-caprolactone shell 2008 focused solely on the oral administration route, to increase stabilized by polysorbate. bioavailability (e.g. CapsMorph technology [2]); in 2014 the technology Ciprofloxacin-loaded nanocapsules were prepared by interfacial was transferred to dermal delivery [3]. An identical or superior dermal deposition of the polymer, yielding 180 nm particles with a drug penetration was shown compared to both nanocrystals and even encapsulation efficiency of 86.9%. Permeability was found to be amorphous nanoparticles, e.g. cyclosporine A [4]. increased inside respiratory horse mucus compared to a suspension of The more intensive use of the porous particle technology for drug the drug. The nanocapsules showed a sustained drug release. The delivery is just emerging since a few years, thus there is still a lack of minimum inhibitory concentrations against Pseudomonas aeruginosa basic knowledge how properties of the materials (e.g. pore size, and Staphylococcus aureus were similar for the drug-loaded functionalization of pore surface, etc.) or parameters of the loading nanocapsules and the free ciprofloxacin. Thus, antibacterial activity is procedure affect important pharmaceutical properties (e.g. drug loading, not affected by encapsulation. Interestingly, formation of biofilm-like physical stability, solubility properties, release, etc.). In this study, the aggregates was observed upon treatment of S. aureus with the free wetness impregnation method was used to load azithromycin (AZ) as drug, while aggregate formation was avoided when bacteria were model active onto Syloid SP53D 11920 particles (Grace, US). The incubated with ciprofloxacin-loaded nanocapsules. active was dissolved in ethanol 96%, using two concentrations of AZ for In comparison to the free ciprofloxacin, our drug-loaded nanocapsules the loading (50% saturated solution Cs (Cs50%), and 25% saturated show several advantages: (1) higher permeability through respiratory solution (Cs25%)). The AZ solutions were admixed in multiple steps, mucus, (2) sustained drug release and (3) a biofilm-preventing effect. followed by subsequent drying in an oven. The formulations were stored The combination of these benefits might enable a highly effective at room temperature, and the physical stability of the amorphous state antibiotic therapy at a reduced dosing frequency. monitored also by x-ray. The achieved drug loading was determined by HPLC. The saturation solubilities of the formulations prepared with Acknowledgments: The DAAD and BMBF (FiDel project, FKZ 13N12530) are thanked for Cs50% and Cs25% solutions were determined in a shaking test and the financial support. dissolution velocities in a release test. References: With both drug concentrations Cs50% and Cs25% in the loading 1. Ahlgren H.G. et al.: Bmc. Pulm. Med., 2015, 15:67, 1-6. 2. Levy J.: J. Pediatr., 1986, 108(5), 841-846. solution, a similar drug loading of about 30% was achieved, determined 3. Bhat P.G., Flanagan D.R., Donovan M.D.: J. Pharm. Sci.,1996, 85(6), 624-630. by HPLC. Also the saturation solubility Cs was identical for both formulations (1310 µg/ml and 1302 µg/ml, resp.). Based on this, one could decide to use the higher concentrated loading solution, because this reduces the number of loading cycles (3 vs. 7 loading steps). However, the properties such as stability and release were clearly POS.196 different. Re-crystallization was observed already after 1 week with the Microfluidic analysis for the testing of penetration and Cs50% formulation, whereas the Cs25% formulation remained stable efficacy of antibiotic loaded PLGA micro- and for more than 1 year stored at room temperature. Also the release nanoparticles in biofilms velocities were different, being faster for the Cs50% formulation, especially in the first 40 minutes. Ernst, J.1; Klinger-Strobel, M.2; Arnold, K.1, Markarewicz, O.2; Pletz, M. Based on this, the use of lower concentrated loading solutions seems to W.2; Fischer, D.1 be sensible for the production of formulations with higher physical long- 1 Department of Pharmaceutical Technology, Friedrich-Schiller-University Jena, term stability. This might be explainable by preferential localization of Otto-Schott Str. 41, 07745 Jena, Germany the active in the pores, whereas in higher concentrated solutions the 2 Center for Infectious Diseases and Infection Control, Jena University Hospital, beginning evaporation after solvent addition to the silica leads to more Erlanger Allee 101, 07747 Jena, Germany surface localization of the drug. Surface-localized drug is more prone to Biofilm embedded bacteria benefit from the protective environment and form crystals, compared to the one in the space-restricted pores. This thus reveal in strongly reduced susceptibility to antibiotics. Herein, higher surface localization can also explain the faster release observed biodegradable micro- and nanoparticular carriers loaded with for the Cs50% formulation. These results provide a direction for optimal antimicrobials are a promising approach to overcome the biofilm barrier loading of porous materials. They show also the opportunity to [1]. For analyzing antibiotic activity, static biofilm models are widely modulate release by different loading parameters, and highlight the used but suffer from some disadvantages e.g. no continuous nutrients need for more intensive systematic investigations for better supply or missing physiological shear flow. This can be avoided by the understanding of basic mechanisms of this novel delivery system. use of dynamic models like microfluidic flow-chamber systems

162 • DPhG Annual Meeting 2016 Conference Book PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS simulating in vivo conditions more closely. This study was aiming to potential of BNC as drug delivery system for such applications could be develop poly(lactic-co glycolic acid) (PLGA) - PEG-PLGA nanoparticles demonstrated [2]. For the same reason, flat BNC fleeces should be (NP) and microparticles (MP) for the hydrophilic drug tobramycin (Tb) to loaded with nucleic acids and the applicability as GAM (“gene activated test penetration and antimicrobial activity in a novel microfluidic matrix”) should be examined. The main focus was the evaluation of the technique for biofilms of Burkholderia cepacia. DNA loading efficiency and the release behaviour. Additionally, the Particles loaded with Tb were prepared by a double-emulsion ability of the BNC fleeces to protect DNA against nucleases as well as evaporation method using the Ultra-Turrax T25 (IKA-Werke, Staufen, the biological compatibility and activity should be examined. Germany) for homogenization [2]. Additionally, drug-free particles were BNC fleeces were biosynthesized by static cultivation of prepared as controls. Obtained MP and NP were characterized by Komagataeibacter xylinus in 24-well plates [3]. After alkaline purification scanning electron microscopy, dynamic light scattering and and washing until pH neutrality, BNC fleeces were loaded with plasmid zetapotential measurement (Zetasizer Nano ZS, Malvern Instruments, DNA (pDNA, pGL3 or pSV-β-Gal) using an injection or reswelling Herrenberg, Germany). The drug content was analyzed by HPLC. For method. In some cases, the pDNA was complexed with linear biofilm experiments a microfluidic system (BioFlux 48-well microfluidic poly(ethylene imine) (2.5 kDa) to polyplexes in a N/P ratio of 20 [4]. plates, Fluxion Biosciences, South San Francisco, USA) was used to BNC fleeces were characterized regarding morphology and evaluate penetration and efficiency of antibiotic loaded particle physicochemical properties using scanning electron microscopy or by suspensions in B. cepacia biofilms in a non-static environment. measuring the compression stability. Release behaviour was Moreover, 24-Well Tissue Culture Chambers (Sarstedt AG, Nümbrecht, investigated under agitated conditions in PBS pH 7.4 at 70 rpm and Germany) were used as static model for comparison with the room temperature. Protection against nucleases was tested by microfluidic system. To track the particles PLGA was covalently labelled incubation of loaded BNC samples in a DNase I solution (1.5 U/µg with 7-amino-4-methyl-3-coumarinylacetic acid (AMCA) [3]. These blue DNA) for different time periods, inactivating DNase I by heating this fluorescent particles could be clearly distinguished from LIVE/DEAD mixture to 70 °C and extraction of the pDNA. Integrity of pDNA was (green/red) stained bacteria by confocal laser scanning microscope analysed afterwards using agarose gel electrophoresis. Additionally the (cLSM; LSM510, Carl Zeiss Jena GmbH, Jena, Germany). In vitro cell biocompatibility was investigated by local application of loaded BNC viability was analyzed by CellTiterGlo® assay with the human lung fleeces onto the chick area vasculosa (CAV) of fertilized hens eggs ex endothelial cell line A-549 (ACC107, DSMZ). ovo [5]. The transfection efficiency was evaluated in vitro in The particles exhibited mean hydrodynamic diameters between 700 nm dependence of the incubation time using CHO-K1 cells. and 1000 nm for MP resp. ca. 250 nm for NP with narrow size By optimizing the applied loading methods, different parameters could distributions. Tb incorporation increased the zetapotentials to about -10 be identified to control the efficiency and speed of loading. Thus, the mV compared to drug-free particles of about -30 mV. The cLSM BNC fleeces could be loaded with up to 50 µg pDNA using injection or experiments showed that MP and NP were distributed in deep layers of reswelling techniques. Neither the three-dimensional network structure the biofilms indicating their penetration ability. In contrast to static nor the mechanical properties of BNC were affected. The release chamber slide experiments, the microfluidic system contained an array behaviour could be controlled in dependence of the loading strategy. of microfluidic flow channels arranged on a well plate format with each Either a fast release with an initial burst within the first 24 h or a long- flow channel connected to an input well and an output well on the plate. term release up to 336 h with an almost linear release profile could be To begin an experiment, bacterial cells were loaded into the input wells. obtained. Loaded BNC samples were able to protect pDNA against The BioFlux Pressure Interface coupled to the top of the well plate nucleases effectively over 8 h (injection loaded BNC) or 72 h (reswelling applied a controlled pneumatic pressure which drives the fluid through loaded BNC), respectively. After local application of loaded BNC on the the channels. The flow profile in BioFlux plates was found to be uniform CAV of shell-less hen’s eggs no harmful influences could be observed. and laminar. Surprisingly, despite a fast drug release Tb loaded NP and In transfection experiments with CHO-K1 cells, different transfection MP showed superior penetration and efficacy in biofilm-embedded efficiencies were obtained, in accordance to the release behaviours. microbes compared to the free Tb or a blend of Tb and particles. The By means of this study it could be shown for the first time that BNC is blends had no antimicrobial activity on B. cepacia indicating that only Tb applicable as GAM for local gene delivery purposes. By loading BNC loaded particles can reach the biofilm embedded bacteria. No with pDNA or polyplexes using different loading techniques, the release cytotoxicity of NP or MP in A-549 cells could be observed up to a kinetics could be specifically modified and the pDNA could be protected concentration of 1 mg/mL and 24 h incubation time. against degradation by nucleases. The excellent biocompatibility of In conclusion, the microfluidic system BioFlux could be successfully BNC was not affected by loading with pDNA or polyplexes and the established for biofilm experiments utilizing LIVE/DEAD staining of the transfection efficiency could be demonstrated in vitro as a first proof-of- bacteria and blue fluorescent labelling of the polymeric carrier under concept. more physiological conditions compared to static models. For both models, non-static and static, penetration in biofilm embedded bacteria Acknowledgments: We would like to thank JeNaCell GmbH for providing the K. xylinus culture. and antimicrobial activity could be effectively tested. Moreover, it could References: be shown that biodegradable polymers such as PLGA and PEG-PLGA 1. Jorfi, M., Foster, E.J.: J. Appl. Polym. Sci. 2015, 132(14). exhibit a great potential as micro- and nano-carrier systems for the 2. Moritz, S., et al.: Int. J. Pharm. 2014, 471(1–2): 45-55. encapsulation of antibiotics to improve their deposition and bacterial 3. Kralisch, D., et al.: Biotechnol. Bioeng. 2010, 105(4): 740-7. killing in deeper biofilm layers to provide therapeutic benefit in biofilm- 4. Fischer, D., et al.: Pharm. Res. 1999, 16(8): 1273-1279. 5. Müller, R., et al.: J. Magn. Magn. Mater. 2015, 380: 61-65. associated chronic pulmonary B. cepacia infections, like cystic fibrosis.

Acknowledgments: This work was supported by the Deutsche Forschungsgemeinschaft (DFG), grants PL 320/3-1 and FI 899/4-1. References: POS.198 1. Klinger-Strobel, M. et al., Expert Opin. Drug Deliv. 2015, 12:1351-74. 2. Ungaro, F. et al., J. Control. Release 2012, 157:149-59. Spray drying microencapsulation of polyethylenimine 3. Klinger-Strobel, M.; Int. J. Nanomed. 2016, 11:575-83. (PEI)-based nanoparticles into a poly(vinyl alcohol) matrix

Schulze, J.1; Aigner, A.1 1 Rudolf Boehm Institute for Pharmacology and Toxicology, Clinical Pharmacology, Leipzig University, Härtelstrasse 16-18, D-04107 Leipzig, Germany POS.197 Bacterial nanocellulose as gene activated matrix The nucleic acid-based treatment of diseases offers great opportunities, even in the case of targets considered as otherwise undruggable. Due Pötzinger, Y.1; Rahnfeld, L.1; Kralisch, D.1; Fischer, D.1 to its potential applications, the interest in gene therapy, and 1 Friedrich-Schiller-University, Department of Pharmaceutical Technology, consequently in gene delivery, has grown continuously over the past Otto-Schott-Straße 41, 07745 Jena, Germany years. Among non-viral delivery systems, polyethylenimine (PEI) is a promising candidate due to its high biological activity. The combination The biomedical application of bacterial nanocellulose (BNC) is currently of this cationic polymer with a lipid delivery system further improves the under intensive investigation due to the high purity, biocompatibility and properties of the formed nanoparticles (NPs), by combining the special mechanical properties of this material. Its applicability as tissue beneficial features of a lipid system and the advantages of PEI. or cartilage replacement, as implant or wound dressing is particularly studied [1]. By loading different active ingredients, e.g. antiseptics, the

DPhG Annual Meeting 2016 Conference Book • 163 POSTERS

An important problem related to polyplexes and lipopolyplexes is their tendency to aggregate, resulting in a significant loss of biological activity. As a consequence of their short shelf life, nanoparticles must be prepared freshly prior use and administered within a short time frame. Addressing this issue, we developed a spray drying method to embed PEI-based polyplexes or lipopolyplexes in a microparticulate poly(vinyl alcohol) formulation. With spray drying, we use an efficient, well-established and facile tool to produce large amounts of our NiMDS Fig. 1: A hydrocyanine-spacer-PNA is hybridized with a ROS-insensitive dye connected to the complementary PNA strand. (Nanoparticles-in-Microparticle Delivery System). In the present study, we show the feasibility of a spray drying process for the generation of PVA microparticles containing PEI-based nanoparticles, and analyze References: their physicochemical properties and biological efficacies. 1. Kundu K, et al. Angew. Chem.-Int. Edit. 2009, 48: 299-303. A crucial point of embedding NPs in a polymeric matrix was to investigate whether this is accompanied by changes in nanoparticle size or surface charge upon their release from the polymeric matrix. The characterization of the size of released nanoparticles shows no effect of this manufacturing procedure on lipopolyplexes (LPP). In contrast, the size of encapsulated polyplexes (PP) slightly increases, indicating a corona effect of the PVA during the spray drying process. For both, LPP and PP, a decrease of the zeta potential was observed. Regarding the biological activity, we demonstrate that the encapsulated nanoparticles are not only just as efficient as their unformulated counterparts, but show even better transfection results. Another desirable feature achieved with this easy-to-handle nanoparticle-in-microparticle delivery system (NiMDS) is its long-term storability. Even upon storage of the dry powder up to three months at room temperature, the biological activity remains high. Additionally, the cytotoxicity of PEI nanoparticles was profoundly reduced after their embedding into the PVA matrix and subsequent release. The microparticles, forming the PVA matrix, show a mean diameter of 3 – 5 µm, a preferable particle size for pulmonary administration. Notably, we successfully performed an in vivo study in which BALB/c ByJ mice were exposed to the microparticulate powder in a self-constructed inhalation chamber, showing promising transfection results. Taken together, spray drying proves to be a suitable method for producing high amounts of this easy-to-handle formulation and also turns out to improve the potency and biocompatibility of PEI-based DNA nanoparticles. We further demonstrate that these NiMDS allow long-term storability of the embedded polyplexes / lipopolyplexes.

POS.199 A modular ratiometric fluorescent probe for detection of extracellular reactive oxygen species

Andina, D.1; Brambilla, D.1; Leroux, J. C. 1; Luciani, P.1,2 1 Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093, Zurich, Switzerland 2 Institute of Pharmacy, Friedrich Schiller University Jena, Lessingstraße 8, 07743, Jena, Germany

A well-established first line defense mechanism during acute and chronic inflammation is the oxidative burst, during which reactive oxygen species (ROS) are released. The specific detection of the extracellular radicals has, however, remained a challenge. A diagnostic tool to monitor ROS-rich areas quantitatively and selectively might help to understand the progress of inflammation and evaluate the efficacy of antioxidant therapies. Thus, a new modular ratiometric fluorescent probe to detect extracellular ROS was designed. The contrast agent consists of a ROS-sensitive dye connected to a negatively charged PEG-polyamino acid-peptide nucleic acid (PNA) and a ROS-insensitive dye coupled to the complementary PNA strand (Figure 1). Hydrocyanines were shown to selectively detect superoxide and hydroxyl radicals [1] and were, therefore, used in this project as ROS-sensitive probe. The ROS-insensitive dye (Chromis dye series, Cyanagen) allows to calibrate the system and to trace the probe even when in a ROS-free environment. Hybridization of the two labelled complementary strand yielded a ratiometric fluorescent probe responsive to ROS in cell-free assays as well as in a stimulated colon carcinoma cell line.

164 • DPhG Annual Meeting 2016 Conference Book PHARMACOLOGY

3.11 Pharmacology POS.201 Crtc1-deficient mice show reduced cardiac function which POS.200 is ameliorated by isoprenaline treatment

Cardiomyocyte cGMP and mitochondrial BK channels exert Morhenn, K.1,2; Geertz, B.3; Eschenhagen, T.2,3; Oetjen, E.1,2,4 cardioprotection against ischemia/reperfusion injury 1 Department of Clinical Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany 2 DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck 1 1 1 1 2 Frankenreiter, S. , Mohr, E. ; Kniess, A. ; Ruth, P. ; Krieg, T. ; Friebe, 3 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, A.3; Lukowski, R.1 University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany 1 Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, 4 Institute of Pharmacy, University of Hamburg, Bundesstraße 45, 20146 Hamburg, Germany University of Tuebingen, Tuebingen, Germany 2 Department of Medicine, University of Cambridge, Cambridge, United Kingdom Cardiac hypertrophy leads to heart failure, one of the common causes 3 Department of Physiology, Julius Maximilian University, Wuerzburg, Germany for hospitalization in the western world. Chronic -adrenergic signaling Background: The Ca2+- and voltage-activated potassium channel of contributes to the pathogenesis of cardiac hypertrophy, as evidenced by big-conductance (BK), encoded by the KCNMA1 gene, is usually the therapeutic success of β-adrenoceptor antagonists. The cAMP present at the plasma membrane of cells. However, in cardiomyocytes Regulated Transcriptional Coactivator 1 (CRTC1) is regulated by (CMs) BK channels are localized exclusively at the inner mitochondrial increases in cAMP and calcium/calcineurin, as elicited by -adrenergic membrane (mitoBK) [1]. By studying hearts obtained from BK-null mice signaling, both known to participate in the development of cardiac (BK-KO) in an ex vivo Langendorff perfusion setup we and others hypertrophy [1]. Our previous data show an increase in CRTC1 protein previously found evidence for mitoBKs as infarct-limiting factors [1;2]. It content in hearts of mice and humans under conditions of maladaptive is well established that canonical BK channels are directly stimulated by hypertrophy. Mice globally deficient in Crtc1 show signs of hypertrophy the cyclic guanosine-3',5'-monophosphate (cGMP)/cGMP-dependent indicated by a higher ratio of heart weight to tibia length and increased protein kinase type I pathway; however, it is unclear whether myocyte size while they show no signs of fibrosis. cardioprotection afforded by cardiomyocyte cGMP in vivo requires In the present study, cardiac morphology and function of Crtc1-deficient mitoBK. mice were echocardiographically assessed. Methods: Using the Cre/loxP recombination system we generated Our findings show a decreased cardiac function in Crtc1-deficient mice animals with a CM-restricted deletion of BK channels (CMBK-KO) [3] compared to their wild type littermates measured by a decrease in and of the nitric oxide (NO)-sensitive guanylyl cyclase (CMsGC-KO) [4]. ejection fraction, fractional shortening and cardiac output by 38±9%, The susceptibility of the conditional BK and sGC mutants to 40±9% and 36±10%, respectively. Left ventricular volume and left ischemia/reperfusion (I/R) injury was compared to age- and litter- ventricular inner diameter were increased in Crtc1-deficient mice during matched controls (CMBK-CTR and CMsGC-CTR) as well as to global systole and diastole. These parameters indicate a systolic dysfunction BK-KO and BK wild-type (BK-WT) mice. An open chest in situ model of in Crtc1-deficient mice. Wall thickness increase between diastole and myocardial infarction was applied to determine differences in infarct size systole was reduced in Crtc1-deficient mice indicating a reduced at baseline and upon ischemic pre-/postconditioning (iPre/iPost) or contractile function. pharmacological interventions using the BK blocker paxilline and the BK As a model for β-adrenergic induced cardiac hypertrophy, isoprenaline opener NS11021 as well as cGMP modulating drugs like the PDE5- was administered to Crtc1-deficient mice and their wild type littermates inhibitor sildenafil or the sGC activator cinaciguat. As additional for seven days (30 g/day/g bodyweight); control mice received NaCl. cardiovascular parameters we assessed blood pressure and heart Long term isoprenaline treatment ameliorated the decreased cardiac function of the conditional mutants. function in Crtc1-deficient mice: In Crtc1-deficient mice, the increase in Results: After 30 min of ischemia followed by 120 min of reperfusion ejection fraction, fractional shortening and cardiac output between NaCl the infarct size between CMsGC-KO and their control hearts did not or isoprenaline treated mice was 1.28 fold, 1.25 fold and 2 fold higher, differ. I/R, however, provoked significantly more cardiac damage in respectively, compared to wild type littermates. global BK-KO and CMBK-KO mice than in age- and litter-matched BK- Meanwhile, mRNA expression of Crtc1 showed a tendency to increase WTs and CMBK-CTRs, respectively. This could also be shown by an in wild type mice after isoprenaline treatment, suggesting a further increase in apoptosis in the CMBK-KO hearts. The BK blocker paxilline increase after longer isoprenaline treatment. mRNA expression of increased cardiac damage, whereas the NS compound provoked a collagen 11, collagen 3 1 or the profibrotic Ctgf was not increased reduction in infarct size in CMBK-CTR but not in CMBK-KO hearts. after isoprenaline treatment in wild type nor in Crtc1-deficient mice but Short repetitive episodes of ischemia applied directly after infarction showed a tendency to an increase as well. (iPost) significantly reduced the myocardial damage in WT and all CTR In conclusion, our data indicate a systolic dysfunction with a reduced mice, whereas the protective effect of this intervention was much less contractile function in Crtc1-deficient mice which is ameliorated by long pronounced in hearts that lacked functional BK channels in CMs and term isoprenaline treatment. This might suggest, that the loss of CRTC1 was completely absent in mice lacking CM NO-sGC or global BK contributes to cardiac dysfunction. Considering that propranolol inhibits channel expression. Interestingly, cardioprotection elicited either by CRTC1 activation [own unpublished data], reduced cardiac function sildenafil or by cinaciguat seem to require mitoBK as well as NO-sGC in after treatment with -adrenoceptor antagonists might in part be due to CM. Finally, CMBK-KO mice presented with a reduced blood pressure impaired CRTC1 signaling. and a minor impairment of the left ventricular contractility seen under physiological conditions. Acknowledgments: The Crtc1-deficient mice were a kind gift by Jean-René Cardinaux, Center Conclusion: The presented findings suggest lack of BK channels in for Psychiatric Neuroscience, Prilly-Lausanne, Switzerland. CMs as a cause for mild cardiac dysfunctions. Additionally, absence of References: CM mitoBK renders the heart more susceptible to I/R injury. 1. Screaton, RA. et al.: Cell 2004, 119(1): 61-74

Cardioprotection elicited by the BK opener NS11021 suggests that BK channels may be promising drug targets that interfere with the causes and/or consequences of myocardial ischemia. Interestingly, both CM NO-sGC and BK are important to allow the protective signaling events POS.202 triggered either by short, repetitive episodes of ischemia or by drugs Distinct cardiac metabolic shifts in two mouse models of modulating the cGMP signaling pathway. Further studies are needed to elucidate whether cardioprotection via NO-GC and BK are linked by a cardiac hypertrophy common cGMP pathway in the CM itself. 1,2 1,2 1,2,3 Gundler, A. L. ; Morhenn, K. ; Oetjen, E. 1 Department of Clinical Pharmacology and Toxicology, Cardiovascular Research Center, References: University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany 1. Singh, H. et al.: Proc Natl Acad Sci USA 2013, 110(26): 10836-10841. 2 DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck 2. Soltysinska, E.: PLoS One 2014, 9(7). 3 Institute of Pharmacy, University of Hamburg, Bundesstraße 45, 20146 Hamburg, Germany 3. Agah, R.: J Clin Invest, 1997, 100(1): 169-179. 4. Takefuji, M. et al.: Circulation 2012, 126(16), 1972-1982. Heart failure is one of the major reasons for hospitalization, and maladaptive hypertrophy plays a pivotal role in the pathogenesis of heart failure. The condition of a higher energy demand in cardiac

DPhG Annual Meeting 2016 Conference Book • 165 POSTERS hypertrophy provokes changes in the metabolism of cardiomyocytes. cells and cleaved caspase-3 positive cells as a marker for initiated The heart returns to a fetal like pattern of using energy supplies. apoptosis. DLK V364A mutant and wild-type transfected HIT cells were Oxidation of fatty acids decreases while the usage of glucose to provide treated with the calcineurin inhibitors cyclosporin A, tacrolimus and ATP increases. This metabolic shift provides enough energy in the short hydrogen peroxide as a reactive oxygen species. Treatment of DLK term, however, in the long term it seems to aggravate the development wild-type with the proinflammatory cytokine TNF increases -cell of heart failure [1,2]. apoptosis and nuclear translocation [5] and was used as a positive Our previous data show that mice deficient in CRTC1 (cAMP Regulated control. In cells transfected with the DLK V364A mutant an increase in Transcriptional Coactivator 1) develop cardiac hypertrophy and -cell apoptosis and nuclear translocation compared to cells transfected dysfunction. Moreover, humans and mice under conditions of with DLK wild-type was observed. TNF and the calcineurin inhibitors maladaptive hypertrophy exhibit elevated CRTC1 protein content in the increased apoptosis and nuclear translocation in DLK wild-type heart, e. g. the Mybpc3-targeted knock-in (KI) mice, a common model transfected cells, but they were not able to increase apoptosis and for HCM (hypertrophic cardiomyopathy). To investigate the role of nuclear translocation in DLK V364A mutant transfected cells. CRTC1 in cardiac metabolism, Crtc1-deficient (KO) mice were studied To investigate whether DLK and calcineurin interact, a protein protein and compared to their wild-type (WT) littermates as well as to the KI interaction assay was performed. Recombinant produced MBP (maltose mice. binding protein) fused DLK proteins and 6x-His tagged calcineurin To investigate the state of cardiac metabolism, mRNA levels of protein were purified by affinity chromatography. In the protein protein transcription factors and enzymes for different metabolic pathways were interaction assay purified MBP fused DLK wild-type protein interacted analyzed via reverse transcription-qPCR. The mRNA content of two with 6x-His tagged calcineurin protein compared to MBP (negative transcription factors contributing to the expression of enzymes for the β- control), whereas the DLK V364A mutant did not. oxidation, PGC-1α (peroxisome proliferator activated receptor γ These findings show that DLK interacts with the phosphatase coactivator 1-α) and PPARα (peroxisome proliferator activated receptor calcineurin via the V364 within the LxVP-type calcineurin interaction α), was reduced by 57.3±5.7% and 48.7±7.3%, respectively, in KI mice, domain. This is the same region which calcineurin interacts with but remained unchanged in KO mice compared to wild-type mice. There cyclosporin A and tacrolimus leading to inhibition of phosphatase was no change in the mRNA levels of GLUT1 (glucose transporter 1) activity [4]. These results show that the mutation of V364 to A within and CPT1B (carnitine palmitoyltransferase 1B). Both proteins are DLK interferes with calcineurin-immunosuppressant binding on involved in the uptake of either glucose or fatty acids. However, the calcineurin. Disruption of the DLK-calcineurin interaction increased DLK insulin dependent glucose transporter GLUT4 (glucose transporter 4) kinase activity, and induced its nuclear translocation and -cell was reduced in KI mice by 43.8±3.8%. As a general marker for energy apoptosis. Taken together with our previous data, these results suggest demand, the mRNA content of AMPK (AMP-activated protein kinase) that DLK dephosphorylation by calcineurin contributes to the was examined. The mRNA expression of the γ2-subunit of AMPK was phosphatase’s -cell protective effect. In addition, a novel mechanism increased by 31.9±7.1% in KO mice compared to WT mice, but did not for regulating DLK activity was detected. differ in the KI mice. These findings suggest a shift away from β-oxidation in the KI mice. References: However, the mRNA expression of GLUT4 was reduced as well. It 1. Oetjen, E. et al.: Mol. Pha. 2003, 63(6): 1289-95 might be conceivable that the higher requirement for energy, complied 2. Plaumann, S. et al.: Mol. Pha. 2008, 73(3): 652-9 with higher use of glucose in the KI mice over a long time period could 3. Oetjen, E. et al.: Diabetologia 2006, 49(2): 227-236 have led to alterations in glucose metabolism. Although the KO mice 4. Rodríguez, A. et al.: Mol. Cell. 2009, 33(5): 616-26 seem to have an augmented need for energy as well, changes in the 5. Walbach, M. et al.: Cell. Signal. 2016, 28(4): 272-83 mRNA levels of the tested markers were not observed. Further investigations might show how CRTC1 regulates cardiac metabolism.

Acknowledgments: We thank Jean René Cardinaux (Center for Psychiatric Neuroscience, Site Cery, 1008 Prilly-Lausanne, Switzerland) for the kind gift of the KO mice and Lucie Carrier POS.204 (Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, High throughput screening (HTS) of “Nature Bank” University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany) for the kind gift of the KI mice. fractions at ecto-nucleotide pyrophosphatase1 and 3 References: (NPP1 and 3) to identify novel drug-like inhibitors 1. Neubauer, S.: N Engl J Med. 2007, 356(11): 1140-51. 2. Taegtmeyer, H.: Ann N Y Acad Sci. 2010, 1188:191-8. Hinz, S.1; Lee, S.1; Serive, B.2; Mak, T.2; Vial, M.2; Böttcher, S.2; Di Capua, A.2; Scott, S.2; Pham, N. B.2; Quinn, R.J.2; Müller, C. E.1 1 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 2 Eskitis Institute for Drug Discovery, Griffith University, 46 Don Young Rd, Nathan QLD 4111, Australia POS.203 Disrupting the interaction between calcineurin and the dual The ecto-nucleotide pyrophosphatase 1 and 3 (NPP1 and NPP3) are leucine zipper kinase (DLK) increases kinase activity able to modulate purinergic signaling through the hydrolysis of nucleotides yielding AMP, which is further degraded to adenosine by Duque Escobar, J.1,3; Lemcke, T.2; Oetjen, E.1,2,3 ecto-5’-nucleotidase (CD73).[1] NPP1 is expressed in osteoclasts, 1 Department of Clinical Pharmacology and Toxicology, Cardiovascular Research Center, chondrocytes, and adipose tissue, and has been reported to be University Medical Center Hamburg Eppendorf, Martinistr. 52, 20246 Hamburg, Germany 2 Institute of Pharmacy, University of Hamburg, Bundesstraße 45, 20146 Hamburg, Germany upregulated in human gliomas. NPP1 inhibitors have therefore been 3 DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck proposed as new therapeutic targets for the treatment of cancer. Most of the known NPP1 inhibitors are nucleotide derivatives and analogs Diabetes mellitus type 2 incidence and prevalence are still increasing which are difficult to develop as orally bioavailable drugs. Others, e.g., worldwide. Decrease in -cell mass and function are the most important oxadiazole or biscoumarine derivatives, display low affinity and lack factors for the clinical manifestation of type 2 diabetes. The structurally selectivity.[2] NPP3 is abundantly expressed in basophils and mast distinct immunosuppressive drugs tacrolimus and cyclosporin A inhibit cells,[3] and has been reported to be overexpressed in bile duct and the calcium-calmodulin dependent phosphatase calcineurin thereby colorectal cancers.[4,5] Therefore, NPP3 inhibitors have been aimed for decreasing insulin gene transcription and inducing -cell apoptosis [1, the treatment of allergy and cancer. For NPP3 no potent and selective 2]. In addition, both drugs enhance the activity of the mitogen-activated inhibitor has been published so far. Our goal has been to identify and protein kinase kinase kinase 12 (DLK, dual leucine zipper kinase) [2, 3]. develop small-molecule heterocyclic compounds with high potency and Our Previous data show that mutation of V364 to A within the putative selectivity for either NPP1 or NPP3.[2,6] calcineurin interaction domain 362-LPVP-365 (LxVP) resulted in an At the Eskitis Institute for Drug Discovery in Brisbane over 200,000 increase of DLK kinase activity. In the present study, the direct lead-like enhanced fractions obtained from extracts of endemic plants regulation of DLK by calcineurin was investigated. and marine organisms with drug-like properties potentially suitable for To study the effect of DLK V364A DLK mutant on -cell apoptosis, peroral application are available. Each fraction contains only 4-5 expression vectors for flag epitope tagged DLK wild-type and DLK individual compounds, which display the desired lipophilicity and V364A mutant were transiently transfected into the -cell line HIT. solubility profile. These fractions are part of a library, the so-called Double immunocytochemistry was performed to detect DLK transfected Nature Bank, which can be utilized for high throughput screening at pharmacological targets.[7,8] Because natural products have remained

166 • DPhG Annual Meeting 2016 Conference Book PHARMACOLOGY to constitute an important source of drugs, especially in the left ventricular outflow tract obstruction. Moreover, interstitial fibrosis development of anticancer and antimicrobial agents, we set up a joint was significantly stimulated by Ang II in CRP4-KO and HET hearts, project between University of Bonn and Griffith University (Brisbane, whereas the production of anti-fibrotic factors such as BNP was Australia) to discover NPP1 and NPP3 inhibitors. diminished. Besides a direct interaction of CRP4 and cGKI, Co-IPs To this end, a spectrophotometric HTS assay was developed, identified CRP4 to interact with numerous sarcomeric and cytoskeletal established and validated, by which the “Nature Bank” fractions were proteins such as cardiac myosin-binding protein C3 (Mybpc3) troponin I screened for NPP-1 and -3 inhibition. The developed HTS assay led to and troponin T, α-actinin 2, myosin 7 and vinculin, which are well known the identification of several fractions with high inhibitory potency at genetic hot spots to cause HCM and/or DCM in humans as well. NPP1 or NPP3. The next step will be to isolate single active Together, the increased susceptibility of CRP4-deficient hearts to compounds, elucidate their structures, synthesize and optimize them. chronic Ang II exposure identifies CRP4 as a novel anti-hypertrophic/- fibrotic factor regulated by cGMP/cGKI. The loss of CRP4 might be at Acknowledgments: We thank the Heinrich-Hertz foundation for support least partly compensated by a cardiac-restricted amplification of the References: highly related LIM-protein CRIP1 (cysteine-rich intestinal protein 1). 1: Zimmermann, H., Zebisch, M., Sträter, M.: Purinergic Signal. 2012, 8(3): 437-502. CRIP1 upregulation seems to protect CRP4-deficient hearts from an 2: Chang, L. et al.: J. Med. Chem. 2014, 57(23): 10080-10100. even more severe outcome, since the knockdown of CRIP1 (and CRP4, 3: Stefan, C., Jansen, S., Bollen, M.: Trends Biochem. Sci. 2005, 30(10): 542-550. respectively) in zebrafish larvae results in massive cardiac 4: Yano, Y. et al.: Int. J. Mol. Med. 2003, 3(5): 763-767. 5: Yano, Y. et al.: Cancer Lett. 2004, 207(2): 139-147. abnormalities and premature death. So far, our studies discovered two 6: Lee, S. et al.: Bioorg. Med. Chem. 2016, 24(14): 3157-3165. novel LIM proteins acting as modifiers of the phenotypic characteristics 7: Camp, D. et al.: Comb. Chem. High Throughput Screen. 2014, 17(3): 201-209. of cardiac remodeling. 8: Harvey, L.A., Edrada-Ebel, R., Quinn, R. J.: Nat. Rev. Drug Discov. 2015, 14(2): 111-129. References: 1. Lukowski, R. et al.: Proc. Natl. Acad. Sci. USA 2010, 107 (12): 5646-5651. 2. Patrucco, E. et al.: Proc. Natl. Acad. Sci. USA 2014, 111(35): 12925-12929. 3. Huber, A. et al.: J. Biol. Chem. 2000, 275 (8):5504-5511. POS.205 4. Zhang, T. et al.: J. Biol. Chem. 2007 282 (46): 33367-33380. 5. Geier, C. et al.: Circ. Res. 2003 107 (10):1390-1395. Cysteine-rich LIM-only protein 4 (CRP4), a novel cardiac cGMP/cGKI effector protein in Angiotensin II induced myocardial remodeling

Straubinger, J.1; Deng, L.1; Boldt, K.2; Krattenmacher, D.3; Ruth, P.1; POS.206 Just, S.3; Lukowski, R.1 Acid sensing Ion channels as targets in gastroesophageal 1 Institute of Pharmacy, Department of Pharmacology, Toxicology and Clinical Pharmacy, University of Tübingen, Germany reflux disease 2 Institute for Ophthalmic Research, Molecular Biology of Retinal Degenerations, and Medical Proteome Center, University of Tuebingen, Germany Ulrich-Merzenich, G.1; Sherbakova, A.2; Kelber, O.3; Abdel-Aziz, H.4 3 Molecular Cardiology, University of Ulm, Germany 1 Medizinische Klinik III, UKB, Friedrich Wilhelms-Universität Bonn, Germany 2 Department of Forestry, Volga State Technical University, Yoshkar-Ola, Russia Background: Cardiac hypertrophy is an adaptive response of the heart 3 Innovation & Development, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, to many cardiovascular disorders including hypertension, myocardial Germany infarction, defects of the valves and mutations in proteins of the 4 Medical Affairs, Phytomedicines Supply and Development Center, Bayer Consumer Health, sarcomere. Elevated levels of cardiac cyclic guanosine-3',5'- Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany monophosphate (cGMP) activate cGMP-dependent protein kinase I (cGKI), which reportedly opposes detrimental cardiac remodeling Cytokines are relevantly involved in the signal transmission of the induced by growth-promoting neurohormonal signals and stresses like intestinal immune system. In earlier studies in a rat model it was shown that modulators of cytokines, as especially IL-8, play a decisive role in Angiotensin II (Ang II) via Gαq-coupled receptors [1,2]. However, the molecular details underlying beneficial effects of cGMP/cGKI remain the pathogenesis of reflux esophagitis. IL-8 is a small heparine binding largely elusive. Hence we investigated the function of cardiac cysteine- protein, which triggers activation and migration of neutrophiles fom the rich LIM-only protein 4 (CRP4), a known phosphorylation target of cGKI blood to the tissue, so having a pro-inflammatory effect. By measuring in smooth muscle cells [3,4]. Additionally, CPR4 is a highly related the release of IL-8 from HET-1A cells the anti-inflammatory action of the homologue of the muscle LIM protein CRP3/MLP, which has been herbal combination medicine STW 5 was compared to its components. linked to dilated (DCM) and hypertrophic (HCM) cardiomyopathies in STW 5 is used in functional dyspepsia and irritable bowel syndrome mice and humans [5]. and is a combination of extracts from Iberis amara (L.), Mentha piperita Methods: We investigated the role of CRP4 in neurohormonal induced (L.), Matricaria camomilla (L.), Glycyrrhiza glabra (L.), Angelica heart hypertrophy upon Ang II infusions (2 mg/kg/d) using CRP4 archangelica (L.), Carum carvi (L.), Silybum marianum (L.) Gaertn., knockout (KO), wild type (WT) and heterozygous (HET) littermates. The Melissa officinalis (L.) und Chelidonium majus (L.). extent of cardiac remodeling processes was defined by examining Synchronized HET1A cells (20.000) were incubated with 4 changes in the heart weight to the body weight (HW/BW), as well as the concentrations each of STW 5 or the component extracts in equivalent growth response of the myocytes and the determination of myocardial concentrations for 18 h, alone or concomitantly with the pro fibrosation. Alterations in cardiovascular performance were assessed by inflammatory stimulator capsaicin (80µM). The IL-8 release was echocardiography and telemetric blood pressure measurements. enhanced by the incubation with capsaicin from 46.3±5 to 85.8 Hypertrophic marker genes, putative effects of Ang II on components of ±14pg/ml (p<0.005). Treatment of the cells with the component extracts the cGMP/cGKI pathway and the expression pattern of other members led to an IL-8 release between 20.4 ± 8 and 441 ± 177 pg/ml. The of highly related LIM proteins were analyzed in total mRNA and protein viability of the cells was not influenced by the experimental conditions. preparations isolated from healthy and hypertrophic ventricles. Since The highest IL-8 release was observed for the Iberis amara extract. LIM domains are generally considered as protein binding interfaces, we When incubated together with capsaicin it caused an IL-8 relese of up used mass spectrometry supported Co-Immunoprecipitation (Co-IP) to 625±121 pg/ml. In contrast, the combination STW 5 (59µg/ml), which studies to identify interaction partners of CRP4 at baseline and upon contains Iberis amara in an equivalent concentration led to an IL-8 hypertrophic Ang II-stimulation. Moreover, the importance of CRP4 in release of 55.3±2 pg/ml. STW 5 accordingly suspended the release of cardiac development was determined by morpholino-induced Il-8 induced by capsaicin as well as by specific single extracts. The knockdown experiments in zebrafish larvae as an appropriate model for mechanisms leading to a reduction of IL-8 release, which is an studying inherited cardiovascular defects. undesired pharmacological effect in this model, by STW 5, but not by Results & Conclusion: CRP4 is abundantly expressed in the cardiac some of its component extracts, is reduced, and is intended to be muscle of WT mice, whereas mRNA and protein levels were reduced further on. significantly reduced in HET hearts and absent from KO muscles. Acknowledgments: A. Shcherbakova was supported by DAAD. The study was supported by a HW/BW ratios of all three genotypes did not differ at baseline, however, research grant of Steigerwald Arzneimittelwerk GmbH, a part of Bayer group. cardiomyocyte size and heart ratios were elevated in CRP4 HET and

KO animals in response to the Ang II infusions. Irrespective of the blood pressure, CRP4-deficient mice developed a HCM-like phenotype with a

DPhG Annual Meeting 2016 Conference Book • 167 POSTERS

References: 1. Abdel-Aziz H, Schneider M, Neuhuber W, Kassem AM, Khailah S, Müller J, Gamaleldeen H, Khairy A, Khayyal MT, Efferth T, Ulrich-Merzenich G. Mol Med. 2015. doi:10.2119/molmed.2015.00098

POS.207 Plasma Trough Concentrations of Azole Antimycotics in Lung Transplant Recipients

Stelzer, D.1,2; Weber, A.2; Ihle, F.1; Ceelen, F.1; Zimmermann, G.1; Kneidinger, N.1; Schramm, R.3; Winter, H.4; Zoller, M.5; Vogeser, M.6; Andraschko, M.2, Behr, J.1, Neurohr, C.1 1 Department of Internal Medicine V, LMU-Munich, Marchioninistrasse 15, 81377 Munich, 2 Germany, Comprehensive Pneumology Center, Member of the German Center for Lung Research DZL 3 Hospital Pharmacy, LMU-Munich, Marchioninistrasse 15, 81377 Munich, Germany 4 Department of Cardiac Surgery, LMU-Munich, Marchioninistrasse 15, 81377 Munich, Germany 5 Department of Thoracic Surgery, LMU-Munich, Marchioninistrasse 15, 81377 Munich, Germany 6 Department of Anesthesiology, LMU-Munich, Marchioninistrasse 15, 81377 Munich, Germany Institute of Laboratory Medicine, LMU-Munich, Marchioninistrasse 15, 81377 Munich, Germany

Background: The purpose of this retrospective study was to analyze azole plasma trough levels (APL) in patients following lung transplantation. Methods: From July 2012 to July 2015 all measured APL of itraconazole, voriconazole, posaconazole liquid and posaconazole tablets in lung transplant recipients of the Munich Lung Transplant Program were evaluated. Target APL were defined as follows: itraconazole: ≥700ng/ml, voriconazole: 1000-5500ng/ml and posaconazole: ≥700ng/ml (prophylaxis) and ≥1000ng/ml (therapy). Results: 806 APL of 173 lung transplant recipients were evaluated (95 (55%) male, 119 (69%) double-LTx, age 51.4±13.4 years, underlying disease: lung fibrosis (n=70; 40%), chronic obstructive pulmonary disease (n=47; 27%), cystic fibrosis (n=31; 18%), pulmonary hypertension (n=6; 3%) and other (n=19; 11%)). Median APL for itraconazole, voriconazole, posaconazole liquid and posaconazole tablets are shown in Table 1. The highest median APL were achieved with posaconazole tablets and voriconazole. The lowest median APL were observed for posaconazole liquid, which were below the target APL for therapy and prophylaxis. Voriconazole and posaconazole tablets showed the greatest percentage of APL above the target threshold, whereas the lowest number of APL above the target APL was noted for posaconazole liquid.

Table 1: Mean and median APL

daily n mean (±SD) median min max APL > indication azole dose (pat) (ng/ml) (ng/ml) (ng/ml) (ng/ml) TPL (mg) prophylaxis ITR 400 79 1155 (±852) 1055 20 4203 62%

VOR 400 4 1826 (±846) 2107 600 2800 85%

POS- 600 13 808 (±596) 592 50 1933 49% Liq POS- 300 7 2709 (±2906) 2123 50 8698 76% Tab

therapy ITR 400 10 779 (±506) 801 30 1669 50%

VOR 400 60 2173 (±2061) 1628 20 11878 70%

POS- 800 31 930 (±682) 765 30 2424 38% Liq POS- 300 25 2509 (±1495) 2107 405 4843 82% Tab

ITR=itraconazole; VOR=voriconazole; POS-Liq=posaconazole liquid; POS-Tab=posaconazole tablets; n=number; pat=patient; SD=standard deviation; APL=azole plasma trough levels; min=minimum APL; max=maximum APL; TPL=target plasma trough level

Conclusions: Our study showed that achieved APL and APL above the target level in lung transplant recipients vary considerably between the different azoles analyzed. Even if most patients treated with voriconazole or posaconazole tablets reached APL above the target threshold, up to 30% of these APL were still below the required target levels for therapy and prophylaxis. Therefore, therapeutic drug monitoring should be integrated in the post lung transplantation management, to identify patients at risk for low APL.

168 • DPhG Annual Meeting 2016 Conference Book REGULATORY SCIENCES/INDUSTRIAL DRUG DEVELOPMENT

3.12 Regulatory Sciences/Industrial Drug Development

POS.208

Commissioned testings of ACE inhibitors in the former Fig. 1: Liquid forms of phytomedicines may contain ethanol, which is, due to its solubility in GDR during the 1980s water and its advantages as solvent of for the extraction of herbal drugs and as excipient, recommended by all leading pharmacopoeias. Kreiss, C. Aims & Methods: The aim was therefore to assess to which extent According to present guidelines, ACE Inhibitors (Ramipril, Enalapril etc.) phytomedicines contribute to the ethanol exposition in this age group, in still play an important role for the therapy of essential hypertension and comparison to the normal uptake with usual food items. congestive / chronic heart failure up to this day. That fact is reflected by By evaluation of data from the use of phytomedicines in liquid form and the impressive number of more than 5 billion DDD (defined daily doses) by generation of a scenario for the exposure by food items based on alone in Germany. new analytical data, exposition values for a 6 years old child were The first federal registration of Captopril in 1980 (Europe), respectively estimated. in 1981 (USA), by the US based company Squibb was soon followed by Results: When using phytomedicines, a 6 years old child is exposed to further developments of various international pharmaceutical amounts of ethanol between 70 and 180 mg with a single dose. Whith 3 companies. Many of these substances were tested in numerous clinical times daily dosing this is 210 - 540 mg. Related to a body weight [b.w.] studies that were conducted worldwide with a large number of patients, of 20 kg, this is 10.1 – 27.0 mg/kg b.w. [1]. regarding the safety, efficacy and the therapeutic value of this An evaluation of side effects of these medicinal products, collected in medication in comparison to placebo and various established standard non-interventional studies in more than 50.000 children, and of the treatments. spontaneous reports from their use in about 3 Mio. children, dit not Some of these clinical studies (mostly phase III) with ACE inhibitors reveal ethanol related side effects. were conducted in various medical facilities / hospitals of the GDR For evaluation of the uptake of ethanol with food items commonly used during the 1980s. These commissioned testings in children, the ethanol content of these items was determined by gas ("Auftragsuntersuchungen") were mirrored by massive media coverage chromatography. E.g. in fruit juices, up to 770 mg/L were found, in during the last years, especially in Germany. bakery products up to 1200 mg/100 g [3]. Based on these data a The following exemplary analysis of these clinical studies was scenario for the mean ethanol exposure was developed, using data on conducted at the Institute for History of Pharmacy (Marburg) and tries to nutritional habits from USA and Germany. investigate the question if the selection of the involved patients, the The resulting mean ethanol exposure was 10.3 mg/kg b.w.. Assuming applied inclusion / exclusion criteria, the form of 'informed consent', the an exposure in the upper range of this scenario, it was 12.5 – 23.3 mg individual study design and practical aspects met the German / ethanol per kg b.w.. international standards of the 1980s. Another focus lies on the federal Conclusion: According to these data, the ethanol uptake with regulations of the GDR, concerning medicinal products and the phytomedicines in children is in the same order of magnitude like approval of clinical studies on human subjects by the governmental everydays exposure with usual food items. agency ZGA ("Zentraler Gutachterausschus für Arzneimittelverkehr"). From this point of view, it is conclusive that ethanol related side effects, The source materials that were used for the analysis of these which are not observed by the average daily ethanol intake via food, commissioned testings of ACE inhibitors consist mainly of documents can likewise not be expected by the use of phytomedicines authorized originating from the federal archive of Germany (Bundesarchiv) and for the use in children. The exposure to ethanol resulting from the use represent files of the ministry of health of the former GDR as well as of of these products therefore is no cause for toxicological concerns. exemplary CRF (case report forms) that were provided through the archive of the former pharmaceutical company Hoechst for a research Acknowledgments: This contribution is dedicated to Prof. Dr. Dr. h. c. mult. Heinz Schilcher, Immenstadt/Bayern, Germany, who to our great regret deceased on 17 June 2015. His textbook project that was conducted at the institute of history of medicine at the Leitfaden Phytotherapie [4] gives a useful summary of the subject of ethanol in herbal medicinal Charité in Berlin. products for children.

References: 1. Kelber O et al. PharmInd. 2008, 70: 1124-7 2. Kelber O et al. Wien Med Wochenschr 2016, DOI: 10.1007/s10354-016-0474-x POS.209 3. Gorgus E et al. J Analyt Toxicol. 2016, DOI: 10.1093/jat/bkw046 Ethanol exposure in children: Food as by far more relevant 4. Schilcher H et al. Leitfaden Phytotherapie (Elsevier Urban & Fischer) 2016 source than phytomedicines

Kelber, O.1; Hittinger, M.2; Gorgus, E.2; Schrenk, D.2 1 Innovation & Development, Phytomedicines Supply and Development Center, Bayer POS.210 Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany Similarity question of nanomedicines: Physicochemical 2 Food Chemistry and Toxicology, University of Kaiserslautern, Erwin-Schrödinger-Straße 52, 67663 Kaiserslautern, Germany comparison of parenteral iron generic products with their innovators Introduction: Liquid dosage forms of medicinal products are well suitable for children, as they allow adapting the dose to the age group. Schnorr, J.1; Fütterer, S.1; Langguth, P.1 But in many cases, especially also in herbal products, they contain 1 Pharmaceutical Technology and Biopharmaceutics, J. Gutenberg-University Mainz ethanol (Fig. 1), which has repeatedly been triggering critical questions. Parenteral iron complexes have a pivotal role in the treatment of anaemia in patients with chronical kidney diseases [1]. Although all marketed parenteral iron complexes are based on a nano-sized ferric iron oxide core surrounded by a stabilising, varying carbohydrate as gluconate, dextran or saccharate, they show clear differences in clinical [2, 3] and non-clinical studies [4-6]. Even for generic iron products with identical carbohydrate fractions, clinical differences have been reported [7]. The interchangeability of iron products is therefore questionable.The purpose of this investigation is to compare generic parenteral iron

DPhG Annual Meeting 2016 Conference Book • 169 POSTERS products with their originators. Ferrlecit® (HFeD) was tested against Sodium Ferric Gluconate Complex in Sucrose Injection (HFeD_G), Dexferrum® (FeG) against Ironate® (FeG_G). Products were compared with respect to particle size (DLS, TEM), particle and core morphology (XRPD, Mößbauer Spectroscopy,TEM), charge (ζ –potential) and in vitro liberation of labile iron in plasma (Ferrozine assay). For HFeD and HFeD differences were obtained in size (DLS), charge, particle morphology (TEM) and in vitro liberation of iron in plasma. In case of ferrozine assay HFeD_G liberated 41,8% more labile iron in the simulated 500mg dose than HFeD. FeG and FeG_G differentiate in size (DLS), particle shape (TEM) and labile iron content. Taken together, these results suggest that there may be clinical relevant differences between the tested generics and originators.

References: 1. Macdougall, I. C.: J. Ren. Care 2009, 35, Suppl. 2: 8–13. 2. Hayat A.: Clin. Med. Res. 2008, 6 (3-4), p. 93–102. 3. Okam, M. M. et al.: Am. J. Hematol. 2012, 87 (11), p. 123-124. 4. Fütterer, S. et al.: J. Pharm. Biomed. Anal. 2013, 86, p. 151–160. 5. Neiser, S. et al. : Biometals 2015, 28(4), p. 615‐635 6. Spicher, K. et al.: Regul. Toxicol. Pharmacol. 2015,3 (1), p. 65–72. 7. Stein, J.; Dignass, A.; Chow, K. U.: Curr. Med. Res. Opin. 2012, 28 (2), p. 241–243.

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170 • DPhG Annual Meeting 2016 Conference Book ANTIINFECTIVES

References: 1. Bartels, K. Diploma thesis (FU Berlin) 2015. 3.13 Antiinfectives 2. Abbanat, D. et al.: Antimicrobial Pharmacodynamics in Theory and Clinical Practice, 2nd ed. (Informa Healthcare USA, Inc.) 2007. 3. CLSI: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that grow aerobically POS.211 (ISBN 1-56238-784-7 [Electronic]). Preventing new resistance development in antibiotic 4. Schwalbe, R.; Steele-Moore, L.; Goodwin, A. C.: Antimicrobial susceptibility testing protocols (CRC Press) 2007. therapy: Which concentration of levofloxacin should Escherichia coli be exposed to?

Goebgen, E. B.1; Kloft, C.1 POS.212 1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Influence of the peptide chain release factor Kelchstr. 31, 12169 Berlin, Germany methyltransferase PrmC on virulence associated metabolic

Objectives: Increasing resistance to antibiotics is one of the challenges pathways of Pseudomonas aeruginosa PA14 we are currently facing. In 2013, 10% to <25% of the tested isolates of 1 2,3 1 Escherichia coli (E. coli), one of the pathogens known for increasing Depke, T. ; Häußler, S. ; Brönstrup, M. 1 Department of Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, resistances, expressed resistance to fluorquinolones in Germany [1]. Germany Thus, there is a clear need of dosing strategies which are (i) capable of 2 Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, eradicating the bacteria and furthermore (ii) prevent new resistance Braunschweig, Germany 3 Institute of Molecular Bacteriology, Twincore, Centre for Clinical and Experimental Infection development. As predictors, determining the pharmacodynamic (PD) Research, Hannover, Germany potency of antibiotics, critical thresholds are used. The two major thresholds are: the minimum inhibitory concentration (MIC) and Metabolomics is an interdisciplinary field of study that applies analytical minimum bactericidal concentration (MBC), also known as 4x MIC [2]. chemistry and multivariate data analysis to examine the entirety, certain Therefore, this in vitro study aimed for testing different thresholds for subsets or characteristic profiles of small molecules (<1500Da) in the fluorquinolone levofloxacin (LEV) for their ability to ensure biological systems under specific conditions [1]. These metabolite eradication of the bacterial load without any regrowth. profiles represent a “snapshot” of cellular biochemistry that is very Methods: As preliminary investigation, the MIC of the clinical E. coli useful for molecular phenotyping and understanding of biochemical isolate used was determined using the standard CLSI microdilution reactions to various stimuli. method [3]. The main investigations were performed in an in vitro We apply a metabolomics approach using gas chromatography and infection model (IVIM) with absence (growth control (GC)) or presence ultra-high performance liquid chromatography coupled to (tandem) (time-kill curve (TKC)) of constant antibiotic concentrations of 1, 2, 2.5, mass spectrometry to examine virulence and quorum sensing related 3, 3.5 and 4x the MIC of the clinical E. coli isolate used. The static IVIM metabolic processes in the opportunistic Gram-negative pathogen consisted of a cell-culture flask filled with cation-adjusted Mueller-Hinton Pseudomonas aeruginosa. We aim to achieve a broad coverage of the Broth (CA-MHB). At the start of the experiment, 100 µL of bacteria P. aeruginosa metabolome that enables us to comprehensively suspended in 0.9% NaCl were added to 8.9 mL CA-MHB and pre- characterise changes in metabolite levels upon knock-out of virulence incubated at 37 °C for 2 hours to yield an inoculum of 106 colony associated genes such as pqsE and prmC which are potential targets forming units (CFU)/mL at time point zero. Subsequently, 1 mL for anti-virulence drugs. antibiotic solution or 1 mL CA-MHB was added to TKC or GC, The peptide chain release factor methyltransferase PrmC is essential respectively, which was followed by incubation for 24 hours. Samples for P. aeruginosa virulence without being directly involved in virulence (n≥6) were taken according to a predefined sampling schedule. factor or quorum sensing signal biosynthesis. Deficiency in PrmC leads Bacterial concentrations were determined using the droplet plate to increased stop codon readthrough and a phenotype with strongly counting method, explained in [4]. The LEV concentrations were reduced pathogenicity. It has been reported that prmC knock-out strains measured using a fluorescence assay at the start and at the end of the produce significantly lower amounts of the redox-active small-molecule experiments [1]. All experiments were performed in triplicate and virulence factors from the phenazine class while maintaining an intact graphically evaluated with bacterial or LEV concentration plotted versus quorum sensing system [2]. Our studies reveal that PrmC deficiency time in RStudioTM. leads to decreased levels of alkylquinolone quorum sensing signal Results: Preliminary investigations found a LEV MIC for the tested molecules in the cell but not in the growth medium suggesting that isolate of 4 µg/mL which translated into antibiotic concentrations of 4, 8, PrmC affects non-quorum sensing functions of alkylquinolones making 10, 12, 14 and 16 µg/mL in the TKC. All performed GCs showed a it an even more interesting protein to study in the context of virulence maximum bacterial load around 1010 CFU/mL at the end of the regulation and anti-virulence strategies. experiments. LEV concentrations remained stable over the whole Besides these new insights into the role of PrmC in P. aeruginosa experiment, without relevant degradation. Initially, all TKC resulted in an virulence, we discovered several previously undescribed members of approximately 3 log10-fold reduction of bacterial load after 2 hours. For the alkylquinolone family of quorum sensing signal molecules and TKC with up to 2x MIC, the initial bacterial killing was followed by developed an untargeted LC-MS/MS method that allows for the regrowth which resulted in a final bacterial load of approximately simultaneous analytical characterisation of various small molecule 109 CFU/mL after 24 hours. After an initial reduction, the bacterial virulence factors and quorum sensing signal molecules. concentration of the intermediate antibiotic concentration of 2.5x MIC persisted at a concentration of 10³ CFU/mL. Antibiotic concentrations Acknowledgments: This work is supported by a scholarship of the Studienstiftung des higher than 2.5x MIC were capable of fully eradicating the bacteria with deutschen Volkes (German National Academic Foundation) providing financial and non-material support to T.D. bacterial concentrations of ≤10² CFU/mL after 24 hours. Conclusions: Overall the GCs were in agreement with previous results References: and confirmed that optimal growth conditions for the bacteria were 1. Patti, G.J., O. Yanes, and G. Siuzdak: Nat. Rev. Mol. Cell Biol. 2012, 13(4): 263-269 2. Pustelny, C., et al: Environ. Microbiol. 2013, 15(2): 597-609. chosen. The regrowth present in TKC with 1 and 2x MIC seemed to indicate a development of secondary resistance, which was prevented in the TKC with higher concentrations (2.5-4x MIC). Since the intermediate LEV concentration (2.5x MIC) did not fully eradicate the bacteria, a concentration of at least 3x MIC should be used to ensure POS.213/SL.46 both eradication and prevent further resistance development. These The value of pharmacometrics in development, investigations showed that the threshold MBC is capable of preventing an increase in resistance and eradicate the bacterial load in 24 hours in optimisation and clinical use of anti-infective therapies. an in vitro setting. Further investigations are needed to confirm this 1 finding for the in vivo setting. Wicha, S. G. 1 Dept. of Pharmaceutical Biosciences, Uppsala Universitet, Husargatan 3, 75124 Uppsala, Sweden

For abstract see Short Lecture SL.46

DPhG Annual Meeting 2016 Conference Book • 171 POSTERS

5. EMEA, Committee for Medicinal Products for Human Use: Guideline on bioanalytical method POS.214 validation. 2012, 44 (July 2011). The effect of levofloxacin 500 mg bid and 750 mg qd against resistant Escherichia coli in the in vitro dynamic infection model POS.215/SL.12 Seeger, J.1; Goebgen, E. B.1; Kloft, C.1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Inhibitors of the bacterial translocase MraY as potential Kelchstr. 31, 12169 Berlin, Germany novel antibiotics

Background and objectives: Optimising the dosing regimens of anti- Ducho, C.1 infectives is one strategy to face the challenge of increasing 1 Saarland University, Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, antimicrobial resistances. Escherichia coli (E. coli) is one of the most Campus C2 3, 66123 Saarbrücken, Germany prevalent causative pathogens for nosocomial infections of the urinary tract and the emergence of fluoroquinolone resistant strains is a For abstract see Short Lecture SL.12 widespread problem all over Europe. Still, levofloxacin (LEV) is a commonly used agent for the treatment of complicated urinary tract infections (UTIs). For the treatment of severe UTIs, known to be caused by less susceptible strains, a 500 mg twice a day (bid) dosing regimen is recommended [1]. However, a dosing regimen of 750 mg once a day POS.216 (qd) is used in the US [2]. This in vitro investigation aimed to explore Antimicrobial β2,2-amino acid derivatives eliminate whether a 750 mg qd LEV regimen is superior to a 500 mg bid LEV Staphylococcus aureus biofilms by membrane disruption regimen in eradicating resistant E. coli and preventing the emergence of and biomass removal new resistances. For this, the in vitro dynamic infection model (IVIM) was used to imitate human pharmacokinetic (PK) profiles and to Ausbacher, D.1; Lorenz, L.2; Goeres, D. M.2; Stewart, P. S.2; Strøm, M. observe their pharmacodynamic (PD) effects, such as time-kill B.1; Vuorela, P. M.3; Fallarero, A.3 behaviour and emergence of resistances. 1 Natural Products and Medicinal Chemistry Research Group, Department of Pharmacy, Material and methods: The used IVIM was developed and validated UiT - The Arcitc University of Norway, Tromsø, 9037, Norway 2 Center for Biofilm Engineering, Montana State University, Bozeman, Mt, 59717, USA for LEV against E. coli at our department [3, 4]. To mimic the 3 Pharmaceutical Design and Discovery Group, Division of Pharmaceutical Biosciences, concentration-time (C-t) profiles of LEV in urine resulting from a 500 mg University of Helsinki, Helsinki, 00014, Finland bid and a 750 mg qd regimen, an adapted previously published LEV PK-model was used [5]. The experimental settings, such us pump rates, Bacterial biofilms account for up to 80% of all human microbial have been calculated in RStudio™. After 2 hours of pre-incubation, infections (1). In patients, medical devices like catheters, artificial heart E. coli was exposed to the two different LEV C-t profiles for 24 hours. valves or prosthetic joints are prone to bacterial attachment and the Samples were taken at predefined time points in order to determine development of biofilms. Antibiotic treatment of biofilm infections is LEV concentrations by a fluorescence assay [4]. For simultaneous challenging and high intrinsic tolerance to antibiotics leads to insufficient bacteria quantification, an automatic plate counting method using the reduction of the biofilm, recurrence of infection and increased risk for schuett colonyQuant® was developed and validated according to the the development of resistance (2). Promising compounds for fighting EMA guideline on bioanalytical method validation [6]. In preliminary biofilm bacteria are antimicrobial peptides due to their rapid and investigations and after each experimental run, the minimal inhibitory membrane disruptive mode of action. However, antimicrobial peptides concentration (MIC) of the isolate or the surviving bacteria, respectively, have the disadvantage of being easily degraded by proteolytic enzymes was assessed using the microdilution method. of microbes or the human body. Results: The automatic counting method was successfully validated in Based on the pharmacophore of short antimicrobial peptides, we have agreement with the EMA guideline for up to 100 colony forming units of synthesized small amphipathic β2,2-amino acid derivatives (3). These E. coli, but failed for higher colony counts. Therefore manual counting derivatives have a molecular weight below 500 Da, are proteolytically was used for bacteria quantification. The initial MIC of the investigated stable and potent against gram-positive bacteria including antibiotic E. coli strain was 8 mg/L, while the MIC increased for bacteria which resistant strains like MRSA and MRSE. The activity spectrum also survived the exposition to the 500 mg bid regimen. comprises Staphylococcus aureus (S. aureus) biofilms and both in vitro The used experimental settings were able to mimic the C-t profiles of and microscopy studies showed that derivatives A1 - A3 are promising the two dosing regimens. In both cases, C was achieved after 2 – lead derivatives (4). 4 hours and LEV was able to initially reduce the bacteria load about ≥ 3log10-fold. However, in the 500 mg bid regimen, bacterial eradication was followed by regrowth. On the contrary, the C-t profile resulting from the 750 mg qd regimen inhibited the bacterial growth for the entire 24 hours. Conclusion: Discrimination between the efficacy of the investigated C-t profiles in eradicating resistant bacteria and preventing the development of secondary resistance was possible by mimicking them in the IVIM. A 500 mg bid LEV regimen was not fully capable of eradicating a In the present study, we investigated how our small, antimicrobial resistant E. coli. The observed regrowth and the increase in MIC value peptide mimicking derivatives act on S. aureus biofilms. We used 96- after the performed experiment indicates the emergence of secondary well plate based assays and fluorescence microscopy to investigate kill resistance mechanisms, such as the expression of efflux pumps. The and reduction of biofilm bacteria treated with A1 - A3. Furthermore, we investigation points towards a superiority of the 750 mg qd LEV regimen applied a CDC biofilm reactor system to prepare biofilms of a green in preventing the development of secondary resistances. Therefore, the fluorescent protein (GFP) expressing S. aureus strain on glass coupons once a day dosing of the high LEV concentration seems to be a for flow cell studies. We constructed a dye leakage indicator system by promising dosing regimen for further investigations in the in vivo setting. utilizing expression of the 27 kDa GFP and the small dye filmtracerTM calcein-red (Mw 790 Da). Real-time confocal laser scanning microscopy References: was used to observe the impact of different treatments including our 1. Pea, F. et al.: Journal of Chemotherapy 2003, 15 (1120-009X): 563-567. lead derivative A3. 2. Anderson, V. R., Perry, C. M.: Drugs 2008, 68 (4): 535-565. Our results indicated that the β2,2-amino acid derivatives both kill and Gloede-Michael, J.: Pharmacodynamic in vitro studies contributing to the rational use of remove S. aureus biofilms. Observations of membrane disintegration in linezolid in infections by vancomycin resistant Enterococcus faecium. Diss. 2011. biofilm bacteria and the dye leakage experiments suggested that 3. Bartels, K.: Validierung des dynamischen In-vitro -Infektionsmodells zur Bestimmung des treatment results in membrane collapse without preceding formation of pharmakodynamischen Effekts von Levofloxacin auf Escherichia coli. Diss. 2015. 4. Schaeftlein, André: Neue Wege in der Modellierung von Mikrodialysatdaten im Menschen : pores. Charakterisierung der klinischen ADMER-Prozesse von Moxifloxacin , Levofloxacin und The high potency based on kill and removal of biofilm bacteria make Linezolid in Gesunden und Hochrisikopopulationen. Diss. 2013. β2,2-amino acid promising lead molecules to fight recalcitrant biofilm infections.

172 • DPhG Annual Meeting 2016 Conference Book ANTIINFECTIVES

Acknowledgements: The authors thank Betsey Pitts for excellent technical assistance. The project was supported by the Research Council of Norway “fellesløftet” grant 214493/F20 and the personal overseas research grant for D.A. References: 1. Römling, U., Balsalobre, C., J. Intern. Med. 2012, 272(6): 541-561. 2. Stewart, P.S. Pharmaceuticals, 2015, 8(3): 504-511. 3. Hansen, T., et al. J. Med. Chem., 2011, 54(3): 858-868. 4. Ausbacher, D. et al. Biofouling, 2014, 30(1)

DPhG Annual Meeting 2016 Conference Book • 173 POSTERS

oxidative stress triggers molecular mechanisms of regulated necrosis 3.14 Neurological Disorders that involve the activation of receptor interacting protein 1 (RIP1) independently of death receptor activation. Here, we show in neuronal HT-22 cells that erastin-mediated formation POS.217 of reactive oxygen species (ROS) [1] triggers mechanisms of regulated Knockdown of glucose-regulated protein 75 (Grp75) necrosis independent of TNF-signaling. In this model system of ferroptosis, erastin promotes glutathione depletion and lipid protects against glutamate toxicity in neuronal HT22 cells peroxidation followed by activation of RIP1 and subsequent RIP1-RIP3 necrosome formation which is regarded as a hallmark of regulated Honrath, B.1; Culmsee, C.1; Dolga, A.2 necrosis [2]. Silencing of RIP1 by siRNA or by the RIP1 inhibitor 1 Institute of Pharmacology and Clinical Pharmacy, University of Marburg, Karl-von-Frisch necrostatin-1 prevented erastin-induced cell death. In contrast, the Strasse 1, 35043 Marburg, Germany 2 Department of Molecular Pharmacology, University of Groningen, Antonius Deusinglaan 1, ferroptosis inhibitor ferrostatin-1 failed to protect cells against TNF- 9713 AV Groningen, The Netherlands induced classical necroptosis, a form of programmed cell death that is mediated RIP-kinases downstream of death receptor activation (e.g. Glucose-regulated protein 75 (Grp75) is a protein with multiple cellular tumor necrosis factor receptor TNFR) [2, 3]. Recently, a genome-wide functions that has been linked to neurodegenerative disorders such as siRNA screen linked cylindromatosis (CYLD) to RIP1/RIP3-dependent Parkinsons’ disease [1], and in tumor pathology as a prognostic marker necroptosis [4]. In the present paradigm of ferroptosis, CYLD depletion for the differentiation of neuroblastoma [2]. Grp75 is ubiquitously promoted neuronal survival and decreased RIP1-RIP3 complex expressed and is localized in the cytosol, at the endoplasmic reticulum formation. Furthermore, increasing evidence suggests a role of and in the mitochondria [3]. It has been associated with the mitochondria in erastin-induced cell death pathways. Here, we revealed mitochondrial protein import machinery and mitochondrial integrity, cell the involvement of mitochondrial fission regulating dynamin-related death induction, activation of MAPK signaling pathways, and the protein 1 (DRP-1) since genetic depletion or pharmacological inhibition formation of ER-mitochondrial contact sites. In tumor and neuronal cell of DRP-1 protected HT-22 cells against erastin toxicity. Additionally, lines, Grp75 depletion was shown to induce oxidative stress and knockout of CYLD via the CRISPR/Cas9 system prevented erastin- mitochondrial fragmentation, and to increase the sensitivity to various induced DRP-1 translocation to mitochondria, suggesting that CYLD cell death inducers [1, 4, 5, 6]. On the contrary, knockdown of Grp75 and DRP-1 can link mechanisms of regulated necrosis to mitochondrial was also shown to be protective against α-synuclein toxicity in neuronal pathways of cell death in paradigms of oxidative stress. cells [7]. Thus, the exact function of this protein in different types of cells remains elusive. In this study, we investigated effects of genetic Grp75 References: ablation in neuronal HT22 cells challenged with toxic extracellular 1. Dixon SJ et al., Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012, glutamate concentrations. Following glutamate exposure, we analyzed 149(5): 1060-72 cell viability by real-time cellular impedance measurements, evaluated 2. Vandenabeele P et al., Molecular mechanisms of necroptosis: an ordered cellular explosion. Nature Rev. Mol. Cell Biol. 2010, 11: 700-714 mitochondrial morphology, and investigated mitochondrial parameters 3. Vanden Berghe T et al., Regulated necrosis: the expanding network of non-apoptotic cell such as mitochondrial ROS formation or mitochondrial calcium overload death pathways. Nat Rev Mol Cell Biol. 2014, 15 (2): 135-147 using specific fluorescent dyes for flow cytometric analysis. We show 4. Hitomi J et al., Identification of a molecular signaling network that regulates a cellular necrotic that siRNA-mediated knockdown fully preserved cell viability and cell death pathway. Cell. 2008, 135: 1311-1323 blocked major hallmarks of glutamate-induced mitochondrial damage such as mitochondrial fragmentation, the formation of mitochondrial ROS and mitochondrial calcium overload. Our results indicate a distinct role for Grp75 in the response of HT22 cells to glutamate-induced oxidative stress and highlight its neuroprotective potential.

References: 1. Burbulla, L. et al.: J. Hum. Mol. Gen., 2010, 19(22): 4437-4452. 2. Hsu, W. et al.: J. Clinical Cancer Res., 2008, 14(19): 6237-6245. 3. Ran, Q. et al.: Biochem. Biophys. Res. Comm., 2000, 1: 174-179. 4. Burbulla, L. et al.: Cell death & disease, 2014, 5:e1180. 5. Jin, J. et al.: Mol Cell Proteom, 2006, 5(7): 1193-1204 6. Kawai, A. et al.: J Cell Sc, 2001, 114: 3656-3574. 7. Liu, F.-T. et al.: Brain Res, 2015, 1604: 52-61.

POS.218/SL.08 Noradrenergic and serotonergic compounds to target narcoleptic episodes in a mouse model

Schmidt, C.; Leibiger, J.; Fendt, M.

For abstract see Short Lecture SL.08

POS.219 Oxidative stress activates mechanisms of regulated necrosis via CYLD

Eisenbach, I.1; Hoffmann, L.1; Ganjam, G. K.1; Culmsee, C.1 1 Institute for Pharmacology and Clinical Pharmacy, Biochemical-Pharmacological Center Marburg, University of Marburg, Karl-von-Frisch Straße 1, 35032 Marburg, Germany

Oxidative stress is regarded as a major trigger for neuronal dysfunction and death in the ageing brain and in multiple neurodegenerative disorders. How oxidative stress mediates neuronal death and whether the associated mechanisms are accessible for therapeutic intervention strategies is not clarified. Increasing evidence suggests, however, that

174 • DPhG Annual Meeting 2016 Conference Book DRUG SCREENING

Binding assays are an important tool in the early drug development 3.15 Drug Screening process to characterize the affinity of a compound towards a biological target. In classic binding assays, radioligands that can be quantified by means of scintillation counting are used as reporter ligands. This POS.220 procedure offers a high sensitivity, but no selectivity as only the radioisotope is detected. Recently, MS Binding Assays, a powerful TiDEC: Enabling Chemistry for DNA-encoded Small alternative to radioligand binding assays employing unlabelled reporter Molecule Screening Libraries ligands, which are quantified by LC-MS/MS, have been established. The strategy of MS Binding Assays has already been applied Brunschweiger, A. successfully for a variety of targets, such as GPCRs and Department for Chemistry and Chemical Biology, TU Dortmund, Otto-Hahn-Str. 6, 44227 Dortmund, Germany neurotransmitter transporters [1, 2, 3]. Besides its high sensitivity, the use of mass spectrometry for the detection of reporter ligands also DNA-encoded small molecule screening libraries (DELs) have found provides an unrivalled selectivity that can be used to quantify multiple widespread use in drug research.1 DELs consist of DNA-encoded reporter ligands simultaneously. In this study, we want to present an MS molecules. These are chimeric structures composed of a drug-like Binding Assay for the simultaneous characterization of binding affinities compound connected to a PCR-amplifiable DNA strand that identifies at the three monoamine transporters hDAT, hNET, and hSERT, which the small molecule by its sequence. Encoding with DNA allows for are important targets for drugs addressing affective disorders. pooling of large numbers of molecules and to screen them efficiently for An important requirement for the simultaneous MS Binding Assays is bioactive compounds by selection. DELs are synthesized through the use of reporter ligands which label the target selectively even in the alternated organic preparative chemistry and encoding steps performed presence of related targets. Therefore, 4-(2-benzhydryloxyethyl)-1-(4- in combinatorial manner. Chemical methodology applied to the fluorobenzyl)piperidin-3-ol (D-84) [4] was chosen as a reporter ligand synthesis of DELs must be strictly DNA-compatible, and is currently for hDAT, (+)-(S,S)-Reboxetine for hNET [5], and (S)-Citalopram for restricted to mostly carbonyl reactions such as amide bond formation, hSERT [6], respectively. For these three markers an LC-ESI-MS/MS Pd-catalyzed C-C cross-coupling reactions, and “click” reactions.1,2 This quantification method was developed and validated according to the restriction defines a challenge in the field: The development of FDA guideline for bioanalytical method validation. In the binding assay, synthesis methodology for DELs is desperately needed to expand membrane fractions made from HEK cells stably expressing the targets chemical space covered by these libraries.1 Rich sources of drug-like were incubated simultaneously with the three markers and competitors heterocyclic structures are Brønsted acid-, and transition metal ion- if needed. After the incubation, the target-marker complexes were catalyzed reactions. However, strong acids cleave the glycosidic bond separated from the unbound markers by a simple filtration step. The of purine nucleosides, and also many transition metal ion catalysts bound markers were then liberated from the target and quantified with cause depurination through a tautomery-based mechanism. Thus, we the established LC-MS/MS method. developed a DEL synthesis strategy based on a hexathymidine sequence, called “hexT”, serving as an adapter oligonucleotide: TiDEC, oligothymidine-initiated DNA-Encoded Chemistry.3 This adapter tolerated a surprisingly broad spectrum of catalytic systems, and allowed for subsequent ligation of coding DNA sequences. Among the catalysts that are now available for DEL synthesis are acidic organocatalysts, and transition metal ions such as Au(I), Ag(I), and Cu(I). Our strategy opened access to substituted and functionalized heterocyclic scaffold structures as DNA-conjugates from simple, readily With this approach, we were able to carry out saturation experiments to available starting materials. For example, application of transition metal characterize the binding affinities of D-84 towards hDAT, of (+)-(S,S)- catalysts furnished DNA-heterocycle conjugates through [3+2] Reboxetine towards hNET and of (S)-Citalopram towards hSERT cycloaddition reactions. Some of the newly synthesized DNA- simultaneously in presence of all three targets. Analogously, it was conjugated heterocycles display structural motifs found in natural possible to determine the binding affinities of a competitor towards each products, while others represent core structures of clinical candidates or one of the three monoamine transporters together in a single approved drugs. All heterocyclic scaffolds were designed to enable experiment. The binding affinities of the three markers as well as the subsequent DNA-encoded combinatorial library synthesis by well- ones of test compounds employed as competitors determined in this described, robust reactions. way, were in good agreement with literature data. These results prove that MS Binding Assays enable the characterization of binding affinities at multiple targets simultaneously.

References: 1. Zepperitz, C.; Höfner, G.; Wanner, K.T.: ChemMedChem 2006, 1(2): 208 – 217. 2. Grimm, S.H.; Höfner, G.: Wanner, K.T.: ChemMedChem 2015, 10(6): 1027 – 1039. 3. Massink, A. et al.: Purinergic Signal. 2015, 11(4): 581 – 594. 4. Ghorai, S.K. et al.: J. Med. Chem. 2003, 46(7): 1220 – 1228. 5. Hajós, M. et al.: CNS Drug Rev. 2004, 10(1): 23 – 44. 6. Hyttel, J. et al.: J. Neural. Trans. 1992, 88(2): 157 – 160. The TiDEC approach to DNA-encoded libraries: a) conjugation of starting materials to the hexT adapter by amide bond formation; b) heterocycle formation by acid or coinage metal catalysis; c) cleavage, purification, and characterization of the products; d) encoding by T4 DNA ligation. POS.222 Compound screening and assay development for sirtuins References: 1. Salamon, H. et al.: ACS Chem. Biol. 2016, 11(2):296-307. Swyter, S.1; Rumpf T1; Enzensperger, C.2; Einsle, O.3; Jung, M.1 2. Klika-Skopic, M. et al.: MedChemComm, 2016, DOI: 10.1039/C6MD00243A. 1 Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität, Albertstraße 25, 3. Brunschweiger, A. et al.: Eur. Pat. Appl. 15202448.5. 79104 Freiburg, Germany 2 Institut für Organische Chemie und Makromolekulare Chemie, Friedrich-Schiller-Universität Humboldtstr. 10, 07743 Jena, Germany 3 Institut für Biochemie, Albert-Ludwigs-Universität, Albertstraße 25, 79104 Freiburg, Germany POS.221 Simultaneous MS Binding Assays for Monoamine Sirtuins are an evolutionary conserved family of NAD+-dependent Lysine deacylases (KDAC) [1]. There are seven human isotypes which Transporters differ in their subcellular localization, their enzymatic activities as well as in their deacylation substrates. The main enzymatic activity of Sirt1-3 is 1 2 1 1 Neiens, P. ; De Simone, A. ; Höfner, G. ; Wanner, K. T. the deacetylation, but also the deacylation of longer fatty acids has 1 Department Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5 - 13, 81377 Munich, Germany been observed. They deacylate a wide range of protein substrates such 2 Department for Life Quality Studies, Alma Mater Studiorum – University of Bologna, Corso as p53, α-Tubulin or Acetyl-CoA-Synthetase. A dysregulation of the D’Augusto 237, 47921 Rimini, Italy

DPhG Annual Meeting 2016 Conference Book • 175 POSTERS cellular acetylation level has been associated with human diseases e.g. cancer, neurodegerenative and metabolic diseases which makes a modulation of sirtuin activity a promising strategy for pharmaceutical intervention [2, 3], For the screening of potential modulators of sirtuins, a well functional assay system feasible for high throuput-screening (HTS) is indispensable. So far, there are some well-established assay systems for measuring the binding or activity potential of sirtuins in complex with ligands. However, all these protocols deal with labeled substrate or are challenging in the performance for HTS [4, 5]. Here we present the development of a homogeneous, label free assay bases on a peptide substrate for Sirt2 & 3. It is performable for the measurement of the deacylation activity of acetylated and myristoylated peptides. To prove the comparability of this assay we screened indole derivatives bases on EX-527 [6] in comparison to a well-known assay and show insights into the binding of a discovered analogue of EX-527.

Acknowledgments: We thank the DFG for funding (RTG 1976, http://www.cofactor-diversity.uni- freiburg.de/RTG) References: 1. Trapp J, Jung M, Curr. Drug Targets, 2006, 7(11): 1553-60 2. Hoffmann G et al, J. Biol. Chem., 2014, 289(8): 5208-16 3. Feldman JL, Baeza J, Denu JM, .J Biol. Chem., 2013, 288(43): 31350-6 4. Heltweg B, Trapp J, Jung M, Methods, 2005, 36(4): 332-7 5. Schiedel M, et al, J. Biomol. Screen., 2015, 20(1): 112-21 6. Napper A D, et al, J. Med. Chem., 2005, 48(25): 8045-54

176 • DPhG Annual Meeting 2016 Conference Book OTHER TOPICS

pharmacies, which was first managed by special magistrates, 3.16 Other Topics subsequently by physicians and finally by pharmacists. The aim of this dissertation was to investigate, compare and analyse the historical development of ‘Visitationes’, which spread in the form of POS.223 regularities for pharmacists and monitoring their compliance over Mediterranean metropolises like Venice, Marseille or Avignon to Doctoral theses of pharmacists in the 19. century German speaking territories, where mainly free imperial cities like using the example of the Ludoviciana in Gießen Nuremberg, Munich or Cologne enacted comparable instructions for pharmacists. The spreading of this supervision, its configuration and Sakkas, A.-S.1; Friedrich, C. organisation was analysed in more than 50 geographical sites, covering 1 Institute for the History of Pharmacy, Philipps-Universität Marburg, Roter Graben 10, 35032 Marburg, Germany the time between the 14th and 18th century, separated in intervals of a hundred years each. In this way a differentiated, tesselated picture of To obtain a doctoral degree was an ambitious goal for the pharmacists pharmacists and 'Visitation' legislation arose, outlining not only the of their time, not only in order to leave their craft for science but also as structure of authorities, frequency and organisation of revisions, but strive for a fulfilled life. also practical arrangement and assignment of tasks meaning controlling Not to stay away too long from their normal business, pharmacists of licence, certificate of proficiency, appropriateness of staff, trainees might as well be able to achieve the desired degree by providing a level of knowledge just as local orderliness by total inspection of handwritten or already printed treatise in absentia. For more than 80 property, check-up of instruments, dispensatories and taxes and valid years (1781–1860) the University of Gießen was exactly the centre for printed regulations for pharmacists. Above all of prime importance was this kind of doctoral degrees. the control of quality and identity of repository and the correct filling in This poster explains in short the promotion practise at the time of Justus and configuration of the visitation record. Liebig (1803–1873), who was ordinary in chemistry from 1825 to 1852 The analysis of the supervision situation of pharmacies in many in Gießen. For scholars of Liebig, it was absolutely sufficient to provide different German towns and regions until the end of the 18th century an essay printed in a scientific journal yet. For example Wilhelm was central point of the project initially, whereas with the beginning of Mettenheimer (1802-1864) who had been a participant in the the 19th century the Prussian conditions of visitation became pharmaceutical laboratory of Liebig, obtained his doctoral degree with particularly relevant. Based on archive materials of ‘Rheinprovinz’, the the already published report ‘Chemische Untersuchung der Soole zu new Prussian province after 1815, intensive research work was Theodorshalle bei Kreutznach‘. Justus Liebig himself confirmed the conducted concerning the organisation and execution of pharmacy relevance of the results, because the investigation was executed under 'Visitation' in Prussia until the end of World War Two, always applying his scientific surveillance. the earlier notified system of analyzing bit by bit the historical facts of Mettenheimer however, as free citizen of Frankfurt (Main), didn’t have this topic, which is characterised by a large range of details. to dispute, similar to Carl Leverkus (1804-1889). Karl Friedrich Oppermann (1805-1872) and Friedrich Kodweiß (1803-1866), as well as Mettenheimer, wanted to be examined in chemistry and natural sciences. While three years ago, the question was discussed, whether it POS.225 would be possible to obtain the philosophical doctoral degree with only examination subjects in the field of natural sciences, it was no longer a Rudolf Schmitz - From being a student of pharmacy to big problem with Oppermann and Kodweiß. The candidates in question having become the first full professor of the history of however, caused a stir with their wish for a mutual examination. pharmacy Synchronized examinations were approved of all professors, as neither legal custom, nor relevant rules stood against them. Löhnert, A.1; Friedrich, C.1 The early practise to doctorate on selected universities like Gießen, was 1 Institute for the History of Pharmacy, Philipps-Universität Marburg, Roter Graben 10, a good opportunity for pharmacists to get a PhD without high school 35032 Marburg, Germany graduation and partially in absentia. The poster describes the scientific career of the life of the pharmacist-

historian Rudolf Schmitz (1918–1992). References: 1. Universitätsarchiv Gießen, Phil O 18, 1827. Die Prüfung und Doctorpromotion des During the research titled “Rudolf Schmitz (1918–1992) and his Pharmaceuten J. Fr. Wilhelm Mettenheimer aus Frankfurt a. M. betr[effend]. scientific school in Marburg” it has to be made clear inhowfar Rudolf 2. Universitätsarchiv Gießen, Phil O 18, 1830. Betr[ifft] Promotionen 1830. Schmitz has succeeded in founding a scientific school of the history of 3. Mettenheimer, Friedrich von: Die Geschichte der Familie Mettenheimer. Frankfurt am Main pharmacy in Marburg. 1897. Five aspects according to the methodic analysis of schools of pharmacy 4. Friedrich, Christoph: Apotheker Carl Leverkus. In: Pharmazeutische Zeitung 149 (2004), S. by Christoph Friedrich are to be examined. These are the headmaster, 3864–3866. the scientific research programme, the pupils, the style of work as well 5. Schwinges, Rainer Christoph (Hrsg.): Examen, Titel, Promotionen. Akademisches und staatliches Qualifikationswesen vom 13. bis zum 21. Jahrhundert. Basel 2007 as the scientific and social appreciation. Among these the headmaster (Veröffentlichungen der Gesellschaft für Universitäts- und Wissenschaftsgeschichte; 7). and his scientific research programme are the most important points of this examination. Biographical data as well as the scientific progress of Rudolf Schmitz at the Philipps-University Marburg are presented here. A description of his childhood and his time at different schools in North Rhine-Westphalia POS.224 during the 1920s and 1930s, of his studies and graduation in the 1940s History of German 'Pharmacy Visitation' and 1950s up to his postdoctoral lecture qualification in the year 1957 (Apothekenvisitation) from the Mediterranean beginnings and his nomination to the first full professor for the history of pharmacy in the 13th century to the end of Second World War in 1967 should give a deeper insight. So this poster tries to give a summary of the way that Rudolf Schmitz Horstmann, R. D.1; Friedrich, C.1 had taken during his lifetime period being a student of pharmacy up to 1 Institute for the History of Pharmacy, Philipps-Universität Marburg, Roter Graben 10, 35032 becoming the first full professor of the history of pharmacy and today Marburg, Germany being one of the most important and acknowledged pharmacist- historians of the 20th century. Until nowadays, pharmacy supervisions in the form of (mostly) unexpected ‘Visitations’ are still part of German pharmacies workaday life. This special kind of control is one of the oldest sections of European government administrations and accompanied pharmacists all along ever since the ‘Constitutions of Melfi’, decreed between 1231 and 1241 by Friedrich II (1194–1250), sovereign of the Kingdom of the Two Sicilies, who initialised the legitimate separation of physicians and pharmacists. Originally intended to control their implementation, these regulatory decrees became the basis for an official supervision of

DPhG Annual Meeting 2016 Conference Book • 177 POSTERS

POS.226 POS.227 Patient experience with a Althaea officinalis L. root extract The pharmacy sector in Baden-Württemberg from 1945 to in dry cough – results from a pharmacy survey during 1961 cough season 2015/2016 Denninger, I.1; Friedrich, C.1 Fink, C.1; Elsässer, P.1; Rabini, S.1; Abdel-Aziz, H.1 1 Institute for the History of Pharmacy, Philipps-Universität Marburg, Roter Graben 10, 35032 Marburg, Germany, [email protected] 1 Pytomedicines Supply and Development Center, Innovation & Development, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany The end of World War II and the division of Germany into different Measuring the customer experience and satisfaction is the key factor to occupation zones meant serious consequences for the German customer knowledge and understanding. Especially in the area of non- pharmacy sector. The legislation for the pharmacy sector passed on to prescribed over-the-counter (OTC) medication, the experiences and the federal states which were initially dependent in their legislative needs of customers are the key resource for information. measures on the respective occupying powers. The introduction of A cough syrup with aqueous extracts of Althaea officinalis (STW42) is unlimited freedom of establishment in the American occupation zone available on the German market for more than 15 years. spread great unrest amongst the German pharmacists. This issue After a successful survey on consumer experience with a solid dosage remained at the center of intensive discussions for many years. The form (lozenges) containing Althea officinalis extract [1], the aim of this reaction of the French military government on the decree of the freedom project is to collect data on the liquid syrup. of establishment in the American zone was intently observed by the Cough patients were recruited directly at the point of sales. If patients pharmacists in Baden. were willing to participate in the survey, a questionnaire for the The problems of pharmacies in postwar Germany were very different acquisition of data was handed over by the pharmacist. The data ones, depending on the location. In the cities they suffered heavily from collected were demographic data, usage patterns, customer the war-related destruction, while in rural areas the supplies of finished recommendation and satisfaction, general assessment. Additionally, medical products and raw materials for the recipes were difficult to effectiveness in treating cough and cough related symptoms as well as maintain. The biggest challenge for the owner of the International tolerability were evaluated. Pharmacy in Karlsruhe was the reconstruction of completely destroyed Acceptance of the survey by the pharmacists was good: Patients were pharmacy. the operation initially continued in a neighboring building as recruited in 152 German pharmacies during the cough and cold season a emergency pharmacy. The required medicines, serums, vaccines, 2015/2016. 561 patients returned the questionnaire. More than 90% of baby food, sugar, oil and alcohol could immediately be procured from the patients complained about cough during daily life and work. Most the close manufacturers and wholesalers. The Rats-Apotheke in subjects started the therapy with STW42 after duration of cough of 3-6 Waldshut, located far off any pharmaceutical wholesaler had always days (45.8%). been producing a major part of their medicines. For this purpose, the Nearly 44% of the patients were repurchasers and 68% used the pharmacy owned a large stock of raw materials owned and operated its Althaea officinalis extract as only medication to treat the cough. own medicinal plant garden. Most subjects (78.5 %, n = 405) got knowledge of STW42 from their pharmacist. 17.8 % (n= 92) of the subjects used STW42 syrup due to References: previous good experience. 67.3 % (n = 346) of the subjects were Literature is available from the author on request. female. The average age was 40.7 ± 16.3 years. The duration of treatment was recorded for up to 7 days according to the questionnaire. The highest proportion of subjects (34%, n = 165) took the syrup for a period of 7 days. 24.3% (n= 118) took the syrup for 4 days. The POS.228 average dose per day was 31.9 mL. 70.3% of the subjects used the Continuous exposure to fine particulate matter (PM2.5) Althaea officinalis extract as only medication to treat their dry cough. 91.7% of the subjects discontinued the therapy with STW42 because from biomass combustion induces stress response, they were free of symptoms. 81.4% (n= 394) of the subjects reported an autophagy and cell death in human bronchial epithelial improvement of the symptom "irritative cough“ until the end of their BEAS-2B cells therapy with STW42. The symptoms "scratching in the throat" and "pain in the throat" improved by 74.4% and 68.3%, respectively. 305 (72.4%) Dornhof, R.1; Maschowski, C.2; Osipova, A.1; Gieré, R.3; Merfort, I.1; subjects reported that the feeling of dryness in the throat improved, too. Humar, M. 1 58.6 % of subjects perceived an alleviation of their symptoms within 10 1 Institute of Pharmaceutical Sciences, Pharmaceutical Biology and Biotechnology, Albert- Ludwigs University Freiburg, 79104 Freiburg, Germany minutes after taking STW42. Most subjects (42%) reported duration of 2 Institute of Earth and Environmental Sciences, Albert-Ludwigs University Freiburg, 79104 action between 2-4 hours. Freiburg, Germany Most subjects assessed the global effectiveness of the syrup as either 3 Department of Earth and Environmental Science, University of Pennsylvania, 19104-6316 Philadelphia, USA “very good” (29.1 %, n = 150) or “good” (54.6 %, n = 281). The majority of subjects assessed the tolerability of STW42 as either “very good” Fine particulate matter (PM2.5) from biomass combustion can (56,9 %, n = 285) or “good” (41,9 %, n = 210), and only 2 subject adversely affect human health [1,2]. However, cellular effects have assessed the tolerability as “bad”, no subject as “very bad”. According remained elusive. In the present study potential molecular mechanisms to these results more than 84.9% of the subjects assessed their global were investigated after intermediate- and long-term exposure of human satisfaction with STW42 as either "very pleased" or “pleased". Before bronchial epithelial BEAS-2B cells to PM2.5. We observed a three- treatment with STW42 the condition of the subjects due to the disease phase response: in the initial phase PM2.5 did not affect cellular was assessed as "good" by 8.4% and after the therapy by 64.2%. survival or proliferation. However, it triggered an activation of the stress 87.1% (n= 446) would buy the syrup again. response p38 MAPK which, along with RhoA GTPase and HSP27, Collecting consumer data via pharmacy surveys occurs as a suitable mediated morphological changes in epithelial cell monolayers, including tool for receiving important insights in customer experience with OTC actin cytoskeletal rearrangements and paracellular gap formation. The products. The results reveal a very good acceptance and evaluation of p38 inhibitor SB203580 prevented phosphorylation of HSP27 and STW42: All cough and cough related symptoms improved well over the ameliorated morphological changes. During an intermediate phase, treatment period of 7 days. The outcome of this study well reflects PM2.5 triggered proliferative regression and activation of an adaptive application of STW42 and indicates its high value in the treatment of dry stress response necessary to maintain energy homeostasis, including cough. AMPK, repression of translational elongation, and autophagy. In the

final phase of exposure, accumulation of intracellular PM2.5 promoted References: 1. Rabini, S. et al.: Planta med. 2015, 81: 1424. lysosomal destabilisation and cell death, which was dependent on lysosomal hydrolases, p38 MAPK and included caspase-3 activation, but not the inflammasome and pyroptosis. All these events may impair epithelial barriers and contribute to PM-associated lung and systemic diseases.

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Acknowledgments: The presented work is part of an interdisciplinary EU-funded research project (see http://www.biocombust.eu), supported in part through the Interreg IV Program POS.231 “Oberrhein” (project C35 BIOCOMBUST). New tools for pharmacy-specific literature search References: 1. Lelieveld, J. et al.: Nature 2015, 525(7569): 367-371. developed by PubPharm - the Specialised Information 2. Smith, K.R. et al.: Annu Rev Public Health 2014, 35: 185-206. Service Pharmacy

Krüger, A. T.1; Keßler, K.1; Ghammad, Y.2; Wulle, S.1; Balke, W.-T.2; Stump, K.1 1 Universitätsbibliothek, Technische Universität Braunschweig, Pockelsstraße 13, POS.229 38106 Braunschweig, Germany 2 Institut für Informationssysteme, Technische Universität Braunschweig, Mühlenpfordtstraße Oxycodon – a hundred year’s history 23, 38106 Braunschweig, Germany

Unger, M.; Helmstädter, A. The Specialised Information Service (SIS) Pharmacy Institute of Pharmaceutical Chemistry, Goethe University, Frankfurt, Germany (Fachinformationsdienst Pharmazie) aims at sustainably improving the supply of literature for academic pharmaceutical research in Germany. Exactly a hundred years ago, in 1916, the opioid derivative oxycodon Therefore new search tools are being developed which are providing was synthesized by the Jewish chemists Martin Freund (1863-1920) full text access to pharmaceutical journals free of charge. The project is and Edmund Jakob Speyer (1878-1942) at the University of Frankfurt. being funded by the German Research Foundation (Deutsche Freund, head of the chemical laboratory of the “Physikalischer Verein” Forschungsgemeinschaft) since January 1st 2015. A special feature of in Frankfurt since 1895, became professor of chemistry at the newly the project is the collaboration between Braunschweig University founded Goethe University in 1914. Speyer who did his PhD in 1901 at Library and the Institute for Information Systems at Braunschweig the University of Heidelberg under the supervision of Emil Knoevenagel, University, through which cutting edge informatics research is being qualified as ‘Privatdozent’ in Frankfurt (1915, habilitation thesis Beiträge incorporated into search tools. zur Kenntnis des Thebains und seiner Derivate) and worked together A core aspect is the user-centric implementation of an extensible and with Freund very closely. However, he became Professor not before customizable research platform. The PubPharm discovery system was 1932. In 1933 already, he lost this position due to his Jewish faith and developed by the SIS Pharmacy. It is a search engine that allows eventually he died in the Lodz Ghetto where he had been deported to, pharmacy-specific literature search. The underlying database index in 1942. goes well beyond Medline (PubMed) data to provide a comprehensive From April 1917 onwards, oxycodon was marketed as a substitute for set of publications for pharmaceutical research. Hence, using one morphine under the trade name Eukodal by the company E. Merck, single query, many different literature resources can be found, for Darmstadt. The product became immediately successful despite its example, interesting Medline articles, articles in chemical journals (that great potential to induce addiction. In was marketed until 1990. are not contained in Medline), pharmaceutical e-books and PhD theses. Interestingly, in recent years the substance was re-introduced into the The PubPharm discovery system contains an availability check which, German marked as an analgesic, also in combination with naloxone for for users from universities in Germany, is personalized based on the prevention of opioid-induced obstipation. After 100 years, the location. Through this, the full text of many publications can be directly substance is still a corner-stone of pain treatment as being listed in the accessed. The database also contains authority data, which allows WHO treatment guidelines. query expansion and thereby retrieval of additionally relevant documents. For example, if the search query is the brand name of a Acknowlegements: We sincerely thank the Division Corporate History, E. Merck, Darmstadt (Head: Dr. Sabine Bernschneider-Reif) for supporting this study. pharmaceutical drug, also publications containing only the INN or IUPAC name will be retrieved.

The PubPharm discovery system can be found here:

www.pubpharm.de

In addition to the personalized availability check, SIS Pharmacy offers POS.230 full text access to pharmaceutical journals that are not usually provided The Institute of experimental therapy by many university libraries. These more than 50 journals include, for example, “Expert Opinion on Drug Delivery” and “Current Medicinal The first research department of the Marburgian Chemistry”. Researchers from pharmaceutical institutes at universities Behringwerk in Germany can register for gaining electronic full text access free of charge. These journals provided via SIS-licences are also included in The construction work on Emil von Behring´s (1854–1917) private the PubPharm search system. laboratory began in 1896 on the Schlossberg of Marburg. Besides the The PubPharm search tools undergo a continuous development academic works, Behring dedicated his laboratory to his private process. One additional application is structure search. It will be research in particular in the field of tuberculosis. A significant part of the possible to search for a compound not only by its (textual) name but institute´s financing was drawn from a contract on Behring´s innovation also by its molecular structure using a formula editor. The structure development Diphtherieheilserum (diphtheria serum) with the search function will be implemented within the next year. Research at “Farbwerke Hoechst vorm. Meister Lucius & Brüning“. In 1903 Behring the Institute for Information Systems aims at developing innovative entered into contract with the Marburg-based company Dr. Siebert & Dr. search tools. If they proof to be beneficial, they will be incorporated into Ziegenbein on production and distribution of tetanus serum and vaccine the retrieval mechanisms of the PubPharm system. One innovative against bovine tuberculosis. A year later he and the two mentioned application is a search function based on Natural Language Processing. pharmacists, Carl Siebert (1863–1931) and Hans Ziegenbein (1867– It might provide a new way to find similar documents or compounds with 1920), founded the Behringwerk Marburg/Lahn. Within all these similar effects. Functions of other search tools are for example based contracts the Schlossberg laboratory played a key role on the progress on similar key words, citations or molecular structures. The proposed and research of the institute and later on the newly established innovative search function takes the whole context of a document into company. account and can find similar documents which do not contain the same From 1904 to 1927 it served as research department of the or similar key words, compounds or molecular structures. Behringwerk (from 1914 Behringwerke). The institute´s scientists were an interdisciplinary team and had background in human and veterinary More information about SIS Pharmacy and the PubPharm system can medicine, chemistry and pharmacy. The studies by Behring and his be found in the PubPharm blog: staff have been published in Beiträge zur experimentellen Therapie. https://blogs.tu-braunschweig.de/pubpharm/

DPhG Annual Meeting 2016 Conference Book • 179 POSTERS

POS.233 POS.234 The PILOT-Study - part of the LENA project Conjugation and labeling of bioactive peptides via Nω- A dry-run as training-concept for paediatric clinical studies carbamoylated arginines with complex sampling requirements Keller, M.1; Kuhn, K. K.1; Einsiedel, J.2; Hübner, H.2; Bernhardt, G.1; Ciplea, A. M.1; Burckhardt, B. B.1; Ablonczy, L.2; Breur, J. M. P. J.3; Cabrele, C.3; Vanderheyden, P. M. L.4; Gmeiner, P.2; Buschauer, A.1 Burch, M.4; Dalinghaus, M.5; De Wildt, S. N.5; Kleine, K.6; Klingmann, I.7; 1 Institute of Pharmacy, University of Regensburg Universitätsstr. 31, D-93053 Regensburg, Germany 8 2 1 Swoboda, V. ; Szatmári, A. ; Läer, S. 2 Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University, 1 Department of Clinical Pharmacy and Pharmacotherapy, Heinrich- Heine-University, Schuhstrasse 19, D-91052 Erlangen, Germany Universitätsstr.1 40225 Düsseldorf, Germany 3 Department of Molecular Biology, University of Salzburg, Billrothstrasse 11, A-5020 Salzburg, 2 Gottsegen György Hungarian Institute of Cardiology, Haller utca 29 1095 Budapest, Hungary Austria 3 Wilhelmina Children's Hospital/University Medical Center Utrecht, Pediatric Heart Center, 4 Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, Lundlaan 6 3584 EA Utrecht, The Netherlands B-1050 Brussels, Belgium 4 Great Ormond Street Hospital for Children NHS Trust, Great Ormond Street WC1N 3JH London, United Kingdom 5 Erasmus Universitair Medisch Centrum Rotterdam,‘s, Gravendijkwal 230 3015CE Rotterdam Conjugation of fluorophores or radionuclide-bearing moieties to and Radboud University, Comeniuslaan 4, 6525 HP Nijmegen, The Netherlands biologically active peptides is widely applied to prepare pharmacological 6 Simply Quality – Dr. Karl Kleine, Johannes-Damrich-Str.4, 82362 Weilheim i. OB, Germany tools as well as cancer diagnostics and therapeutics. Whereas lysine, 7 Pharmaplex bvba, Avenue Saint-Hubert 51, 1970 Wezembeek-Oppem, Belgium 8 Medical University of Vienna, Spitalgasse 23 1090 Vienna, Austria cysteine, and N-terminal amino acids have been mostly used for versatile peptide conjugation, arginine, bearing a strongly basic Objectives: The LENA project (Labeling of Enalapril from Neonates up guanidine group, has been neglected. We introduced a new, widely to Adolescents) aims to improve the healthcare of children with heart applicable strategy to peptide conjugation based on the nonclassical failure by an enalapril orodispersible mini-tablet. In LENA, the Renin- bioisosteric replacement of the guanidine group in arginine by a Angiotensin-Aldosterone-System (RAAS) in children will be investigated functionalized carbamoylguanidine moiety.[1] Appropriately protected to improve the understanding of RAAS maturation and ACE-inhibitor Nω-carbamoylated L-arginine derivatives (Fmoc-Arg(Boc)-OH, cf. effects in children. The drug, its metabolite and the humoral parameters Figure) can be incorporated into peptides in the position of interest investigated in serum and plasma face stability problems and thus according to Fmoc solid phase peptide synthesis. This was require an immediate sample processing and a strict adherence to a demonstrated, e.g. for peptide ligands of angiotensin II (AngII), rigorous sampling protocol. neurotensin (NT) and neuropeptide Y (NPY) receptors. Aim: To perform training and an evaluation (PILOT Study) to Synthesized precursor peptides, bearing a primary amino group at the demonstrate successful preparation and performance of sampling as modified arginine side chain, were conjugated and labeled in a versatile required in the LENA paediatric trials for the medical staff. manner by analogy with procedures established for lysine residues. The Methods: The medical staff involved in the trials was trained in a modified peptides proved to be chemically stable (buffer, pH 7.4). tutorial and hands-on workshop concerning small-volume blood Tritiated AT1, NTS1 and NPY Y4 receptor ligands (Kd values < 2 nM) sampling of drug concentration and time-critical, sensitive humoral were economically accessible by acylation of the precursor peptides parameters in paediatric patients under optimal conditions. One to four with commercially available succinimidyl [3H]propanoate. Retained high months after passing this training, the involved 5 clinical centres were receptor affinities of fluorescently labeled AngII and NT(8−13) evaluated by performing a PILOT Study as an on-site study in their analogues revealed that conjugation to space-filling moieties such as familiar environment. The participating centres conducted a procedure fluorophores via Nω-carbamoylated arginines was tolerated as well. resembling the LENA trials sampling routine for regular study-visits on This work demonstrates that the arginine position in biologically active three adult volunteers, covering the sampling procedure, sample peptides can be used to prepare radio- and fluorescence labeled preparation, storage and shipment by utilizing equipment and molecular and pharmacological tools. documentation matching the paediatric trials. The medical staff draw blood samples from three healthy volunteers to determine the drug concentration and the humoral parameters aldosterone, renin, plasma renin activity and angiotensin I. To mimic the drug concentrations without administering the drug itself, the blood collection tubes were pre-spiked with two different mixtures of enalapril and its metabolite enalaprilat, creating predefined concentrations when filled with whole blood. The obtained drug concentrations were compared to reference values, obtained under optimal conditions at the bioanalytical laboratory. Results: In the initial performance of the PILOT-Study the centres obtained 94% of the scheduled samples with sufficient volume for analysis. The humoral parameters, being especially prone to Acknowledgments: This work was funded by the Deutsche Forschungsgemeinschaft degradation where detectable in all samples, with values beyond the (Sachbeihilfe KE 1857 / 1-1, Graduiertenkolleg 1910) lower reference level of physiological concentrations. The acceptable References: values of concentrations for enalapril/enalaprilat where initially missed 1. Keller, M. et al.: J. Med. Chem. 2016, 59: 1925-1945. by one centre, resulting in a re-run which was then successful. Conclusions: As the accuracy of pre-analytical sample preparation is crucial to the projects objectives and a key element for quality and reliability of gathered data, an upfront check of the team member’s ability to obtain POS.235 these high-quality samples, is necessary. The concept of a training and Is cyanide a significant metabolite of Tofacitinib citrate? evaluation (PILOT-Study) has shown to be a suitable approach for detecting improper sample handling. Geese, H.1; Pfaffenrot, E.2; Laufer, S. A.2; Clement, B.1 1 Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-Universität, Gutenbergstraße 76, 24118 Kiel, Germany Acknowledgements:The research leading to these results has received funding from the 2 Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-University European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement Tuebingen, Auf der Morgenstelle 8, 72076 , Tuebingen, Germany n°602295 (LENA)

Tofacitinib (CP-690,550) was one of the first orally available Janus kinase (JAK) inhibitors approved by the US Food and Drug Administration (FDA) for the treatment of moderate to severe rheumatoid arthritis [1]. Metabolism studies of Tofacitinib were conducted in human subjects. It was previously shown that Tofacitinib is a P450 substrate and mainly metabolized by CYP3A4/3A5 and CYP2C19 [2]. The identified metabolites included a carboxylic acid (4) metabolite of Tofacitinib, but the mechanism of the formation of this

180 • DPhG Annual Meeting 2016 Conference Book OTHER TOPICS metabolite was not investigated [2,3]. With respect to the structure of POS.236 tofacitinib containing a cyano group with α–hydrogen atoms, a formation of cyanide by these enzymes is possible as outlined in figure Oral Turinabol. Die Entstehungsgeschichte eines 1. Anabolikums The goal of this study was to investigate a potential formation of cyanide by an initial attack of P450 enzymes (figure1). Möckel, A. To determine cyanide a modified pyridine-barbituric acid method was Institut für Geschichte der Pharmazie Marburg used [4]. Cyanide formation was induced by incubating Tofacitinib with a mixture of porcine or human liver microsomes and NADPH as a cofactor of CYP450. As a result potentially formed cyanide reacts with cholarmine-T. After adding the pyridine-barbituric acid reagent a violet POS.237 product is formed, which could be detected by spectrophotometry. By these incubations the formation of cyanide could be demonstrated. Exam behavior of newly registered candidates in the 4 Cyanide formation is time-dependent, after 15 min the amount of partial exams of the 1st phase of the pharmaceutical state cyanide decreased. So the highest concentration after incubation with exam 2006-2016: Pulling through, nonattendance, porcine liver microsomes was 40 ± 5,3 pmol CN /pmol CYP and with approved sick note human liver microsomes 23 ± 2,4 pmol CN /pmol CYP. Studies performed with recombinant isoenzymes identified CYP3A4 Spahn-Langguth, H.1; Winter, N.1; Shahla, H.1 and CYP2D6 as the main catalysts for cyanide formation. 1 Institut für medizinische und pharmazeutische Prüfungsfragen, Große Langgasse 8, Ketoconazole as an unspecific inhibitor of P450 enzymes was added to 55116 Mainz, Germany the incubation mixture in different concentrations. A decrease of the Within the pharmaceutical state exam the 1st state exam -usually- cyanide formation was detected at a concentration of 50 µM represents a series of 4 subsequent written (MCQ) exams. The ketoconazol. nationwide written state exams are taking place semiannually and By blocking an α–hydrogen atom in a Tofacitinib derivative simultaneously. Due to the broadness and depth of the respective 4 (replacement of an α–hydrogen atom with isopropyl) no cyanide was subject groups, candidates need to show continuous and persistent detected. performance on four subsequent days. In conclusion the amount of cyanide is formed by metabolic Data available in the period since 2006, i.e., after finalization of the 2- transformation of Tofacitinib is low and might be not of toxicological year-transition to the currently valid exam specifications of the German relevance. Additionally, thiocyanate synthetase converts up to 0, 5 AAppO, were the basis for the retrospective analyses. mg/kg body weight per hour cyanide into thiocyanate and the lethal Out of the group of a total of approximately 19 K newly registered dosis of cyanide in the whole blood is 2, 97 mg/l [5,6]. Nevertheless, El candidates, an average of 0.29 % did not attend any of the 4 individual Ghawabi and his group examined the chronic poisoning after a exams, while 90.11 % attended all 4 exams. And out of this subgroup, longterm cyanide exposure to industrial smoke [7]. The most frequent 64.4 % were able to pass all 4 exams at once. symptoms (78-81%) were headache, weakness and changings in taste When candidates were not able to take part on one day or skipped one and smell. Additionally, chronic cyanide toxicitiy by oral intake leads day (average total absence in at least 1/4 and up to 4/4 exams, 9.89 unequivocally to goitre, tropical ataxic neuropathy and spastic paresis %), then out of this latter subgroup 14 % (1003/7168) were approved [8]. withdrawals, 86 % (6165/7168) were non-approved = failed. The following tendencies (preferences) were detected for absence in only 1 out of 4 subject groups (total 4.95 %, i.e., appr. 1/2 out of the above given 9.89 %): Day 1 = "Chemistry", 0.67 %, Day 2 = "Pharmaceutical and human

biology", 1.91 %; Day 3 = "Physics, physical chemistry, drug formulation", 1.13 %; Day 4 = "Drug analysis", 1.24 %. When Fig. 1: Potential formation of cyanide by CYP450 candidates attended one day only and were absent in 3/4 exams (1.01 out of the 9.89 %), the total of 1.01 subdivides onto the 4 exams as References: follows: Day 1 - 0.69 % > Day 4 - 0.16 % > Days 2 & 3 - 0.08 % each. 1. West, K.: Curr Opin Investig Drugs 2009, 10(5):491-504. 2. FDA: Pharmacology Review 2011, Application Number 203214Orig1s000, 64-94. The average overall grade achieved by the candidates who passed all 4 3. Dowty, M. E. et al.: Drug Metabol and Dispos 2014, 42: 759-773. exams in phase 1 within their 1st trial was 3.13 (max grade, 1.0; min 4. Asmus, E. et al.: Fresenius´Z. Anal. Chem. 1953, 138: 414-422. grade, 4.0) with no significant positive or negative trend over the 5. Reichl, F.X.: Taschenatlas der Toxikologie 2002, 2: 134. included 10-years period. 6. Hall, A.H. et al.: Annals of emerg. Medic. 1986, 1096: 115- 122. 7. El Ghawabi, S. H. et al: British Journ of Industrial Med. 1975, 32: 215-219. 8. Okolie, N. P. et al: Food and Chemical Toxicol. 1999, 37(7): 745-750.

DPhG Annual Meeting 2016 Conference Book • 181 POSTERS

AUTHOR INDEX Becker, D...... 128 A Becker‐Pauly, C...... 117 Beckmann, A.‐M...... 117 Bednarski, P. J...... 108 Abdallah, D...... 98 Beerhues, L...... 145 Abdelaziz, A...... 147 Behr, J...... 168 Abdel‐Aziz, H...... 98, 120, 146, 167, 178 Bendas, G...... 105, 106 Abdel‐Tawab, M...... 91 Bendel, T...... 54, 146 Abdul, H. K...... 123 Bergemann, C...... 159, 161 Abebe, D...... 103 Bermudez, M...... 137, 139 Ablonczy, L...... 180 Bernat, V...... 136 Achenbach, J...... 68, 120 Bernhardt, G...... 131, 133, 180 Ackermann, T...... 94 Bertoletti, N...... 123, 125 Adamski, J...... 123 Betz, M...... 58, 125 Agoglitta, O...... 131 Bhatia, S...... 43, 109 Aigner, A...... 112, 163 Biel, M...... 40 Alban, S...... 92, 106 Bischoff, I...... 97, 107 Albold, D...... 159 Bittner, F...... 126 Al‐Gousous, J...... 75, 151 Blanchard, N...... 36 Althaus, J...... 101 Blassmann, U...... 93 Amidon, G. L...... 75, 151 Blöcher, R...... 99, 123 Anamur, C...... 38, 160 Blomme, C...... 107 Andina, D...... 164 Bödefeld, T...... 142 Andraschko, M...... 119, 168 Boehlich, G. J...... 139 Angioni, C...... 41, 97, 123 Bogdahn, M...... 27 Antoni, F...... 131 Böger, R. H...... 122 Ardelt, M. A...... 113 Boldt, K...... 167 Arnold, G. J...... 113 Bonaterra, G. A...... 98 Arnold, K...... 162 Bopp, B...... 43, 107, 109 Arntjen, A...... 154 Borek, C...... 105 Aryal, D. K...... 136 Borkhardt, A...... 43, 109 Ashtikar, M...... 145 Bosman, I...... 110 Asmari, M...... 94 Boß, M...... 123 Atzler, D...... 122 Böttcher, S...... 166 Ausbacher, D...... 172 Böttcher‐Friebertshäuser, E...... 140 Bracher, F. . 40, 89, 97, 104, 123, 124, 125, 126, 135, 136, B 139 Braig, S...... 110 Brambilla, D...... 164 Bacher, L...... 154 Brandl, F. P...... 26 Bajorath, J...... 117 Brandt, S...... 137 Balasupraminiam, S...... 88 Braun, F...... 123 Balke, W.‐T...... 179 Braun, M. D...... 143 Balsevich,G...... 77 Breiholz, S...... 118 Baltes, F...... 106 Breitsamer, M...... 160 Barocelli, E...... 138 Breur, J. M. P. J...... 180 Bartel, A...... 93 Brezesinski, G...... 57, 116 Bartel, K...... 110 Briegel, J...... 93 Bartl, N...... 123 Briel, D...... 130 Bassett, D. J. P...... 103 Brinker, C...... 134 Bauer, J...... 54, 146 Brönstrup, M...... 171 Bauer, R...... 52 Browne, E...... 108 Bauer, S. M...... 102 Brückl, L...... 148 Bäurer, S...... 90 Brüggerhoff, A...... 123 Bausch, A...... 143 Brüne, B...... 123 Bause, M...... 133 Brunschweiger, A...... 175 Bayer, T...... 134 Brusotti, G...... 48 Beck, R. C. R...... 162 Brust, P...... 130

182 • DPhG Annual Meeting 2016 Conference Book AUTHOR INDEX

Büning, H...... 46 Di Capua, A...... 166 Bunjes, H...... 155 Diedrich, D...... 43, 109 Burch, M...... 180 Diehl, O...... 123 Burckhardt, B. B...... 93, 180 Dieter, R...... 143 Burkhardt, J...... 112 Dietrich, A...... 31 Buschauer, A...... 131, 133, 138, 180 Dolga, A...... 174 Busskamp, V...... 44 Dong, Y...... 86 Butterweck, V...... 146 Dornhof, R...... 99, 178 Dorrestein, P. C...... 54, 146 Dougalis, A...... 19 C Draheim, H. J...... 19 Duan, D...... 136 Cabrele, C...... 180 Ducho, C...... 37, 172 Calderón, M...... 151 Duda, J...... 19 Calleri, E...... 48 Duerr, C...... 91 Carballo, C ...... 62, 96 Dukor, R. K...... 62, 96 Ceelen, F...... 168 Duque Escobar, J...... 166 Chaikuad, A...... 102 Chakrabarti, A...... 134 Chao, Y. K...... 40, 136 E Chaves, P. S...... 162 Chen, C. C...... 40, 136 Ebbing, L...... 118 Cheung, S.‐Y...... 124 Eberhard, J...... 94 Choe, C...... 122 Ebert, R...... 41, 97 Chytil, P...... 149 Eckl, K...... 151 Cialla‐May, D...... 161 Eder, M...... 77 Ciglia, E...... 107 Edkins, K...... 152 Ciossek, T...... 19 Efferth, T...... 51, 98 Ciplea, A. M...... 180 Ehrig, K...... 106 Clement, B...... 96, 126, 180 Eickhoff, C...... 118 Corder, G...... 136 Einsiedel, J...... 180 Cordes, A...... 98 Einsle, O...... 175 Cordts, K...... 122 Eisenbach, I...... 174 Cramer, J...... 58, 125 Eisend, S...... 118 Culmsee, C...... 68, 120, 140, 143, 174 El Deeb, S...... 88, 94 Cumbana, R...... 41, 97 Elliott, J.A...... 86 Elsässer, P...... 178 Elz, S...... 138 D Engelke, L ...... 160 Engelke, L...... 38 Dai, B...... 97 Engels, B...... 105 Dalinghaus, M...... 180 Engert, J...... 38, 115, 149, 160 Dallanoce, C...... 138, 142 Enzensperger, C...... 175 Daniels, R...... 155 Erdmann, F...... 134 Darveau, T...... 108 Ernst, J...... 162 Dauer, K...... 152 Ernst, T...... 43, 109 De Amici, M...... 138, 142 Eschenhagen, T...... 165 De Beer, T...... 84 Estevam, E. C...... 148 De Mello Martins, A. G. G...... 94 Etrych, T...... 149 De Simone, A...... 175 Ewe, A...... 112 De Vries, J...... 139 De Wildt, S. N...... 180 Decker, M...... 132, 138, 142 F Denezhkin, P...... 148 Deng, L...... 167 Fabian, J...... 101 Dengler, D...... 136 Fach, M...... 112 Denninger, I...... 178 Fallarero, A...... 172 Depke, T...... 171 Faust, A...... 130 Derendorf, H...... 70 Felberg, M...... 118 Deters, M. A...... 118 Feldmann, J...... 110

DPhG Annual Meeting 2016 Conference Book • 183 POSTERS

Fellner, C...... 38, 160 Gieré, R...... 178 Fendt, M...... 33, 174 Giguere, P. M...... 136 Feng, Y. X...... 30 Ginsel, C...... 126 Ferreirós, N...... 41, 97 Girreser, U...... 96 Fetzer, C...... 113 Glatzel, D...... 145 Fiebig, H...... 109 Glud, K...... 156 Fiebig, J...... 81, 117 Gmeiner, P...... 136, 137, 180 Fiedler, S...... 31 Göbel, T...... 123 Fink, C...... 178 Goebgen, E. B...... 121, 171, 172 Fischer, D...... 159, 161, 162, 163 Goepferich, A...... 157 Fischer, M. R...... 119 Goeres, D. M...... 172 Fischer, T...... 152 Gohlke, H...... 43, 107, 109 Fish, I...... 137 Gollos, S...... 110 Flammini, L...... 138 Gomez‐Lopez, N...... 103 Flemming, S...... 99 Göpferich, A...... 18, 157 Forget, A...... 62, 96 Gopireddy, S. R...... 158 Forster, M...... 102 Gorgus, E...... 169 Frankenreiter, S...... 165 Gregoritza, M...... 26 Franz, L...... 130 Grice, J. E...... 147 Frey, O. R...... 93 Grienke, U...... 132 Fricker, G...... 151 Griffin, S...... 148 Fridh, V...... 58, 125 Grimm, C...... 40, 136 Friebe, A...... 165 Gronauer, T...... 113 Friedrich, C...... 177, 178 Grond, S...... 54, 146 Frieg, B...... 43, 109 Gross, H...... 54, 146 Frieß, W...... 116, 154 Grundmann, M...... 105, 123 Friess, W...... 57, 91, 115 Grüttner, C...... 161 Fritsch, A. K...... 158 Guarinini, L...... 108 Fritzsche, W...... 159 Guck, J...... 110 Fröhlich, T...... 113 Gudermann, T...... 31 Froriep, D...... 126 Guilherme dos Santos, M...... 105 Fuhrmann, G...... 29, 111 Gundler, A. L...... 165 Fujiwara, T...... 103 Günther, M...... 109 Furdas, S...... 126 Günther, S...... 99 Fürst, R...... 97, 107, 145 Gütschow, M...... 117, 131 Furtmann, N...... 117 Fütterer, S...... 169 H

G Haas, V...... 157 Haeggström, J. Z...... 98 Gajer, J...... 126 Hafner, K...... 77 Galanski, M...... 110 Hahn, R...... 148 Ganjam, G. K...... 140, 143, 174 Hahne, T...... 91 Garscha, U...... 98, 99 Hake, T...... 101 Gassen, N. C...... 77 Hamacher, A...... 110 Geertz, B...... 165 Hamed, M...... 94 Geese, H...... 180 Hanekamp, W...... 101 Gegenfurtner, F. A...... 111 Hänggi, S...... 147 Geh, K. J...... 150 Hanke, T...... 124 Gehringer, M...... 102 Hansen, F. K...... 43, 107, 109 Gehrmann, S...... 155 Harder, M. J...... 100 Geisslinger, G...... 41, 97, 123 Hartmann, J...... 77 Gellrich, L. M...... 127 Hartmann, M...... 123 Genewsky, A...... 77 Hartmann, R. W...... 94 Gerloff, C...... 122 Hartung, N...... 121 Gerstmeier, J...... 99 Haryadi, B. M...... 149 Ghammad, Y...... 179 Hasan, M...... 108 Ghoreschi, K...... 102 Hauer, J...... 43, 109 Giera, M...... 104 Haunberger, A...... 157

184 • DPhG Annual Meeting 2016 Conference Book AUTHOR INDEX

Haupenthal, J...... 94 Hüther, J...... 98 Häupl, T...... 99 Hausch, F...... 76 Hauser, A. T...... 134 I Häußler, S...... 171 Havemeyer, A...... 96, 126 Ibraimi, M...... 153 He, D...... 82, 150 Ihle, F...... 168 Heck, R...... 155 Ilan, N...... 105 Hedtrich, S...... 64, 103, 151 Imanidis, G...... 147 Heering, J...... 99, 123 Imhof, A...... 143 Heilmann, J...... 95, 121 Imig, J. D...... 123 Heimburg, T...... 134 Irsheid, L...... 105 Heine, A...... 58, 123, 125 Heitel, P...... 100, 127 Held, J...... 129 J Helmstädter, A...... 146, 179 Hemmers, S...... 104 Jacob, C...... 148 Henke, S...... 38, 160 Jaehde, U...... 119, 122 Hennies, H. C...... 151 Jagodzinsk, A. I...... 122 Henrich, A...... 119 Jakobs, H. H...... 126 Hermann, S...... 155 Jelinek, A...... 140 Herrmann, J...... 53 Jin, N...... 162 Hespeler, D...... 154 Joerger, M...... 107, 119 Heßelbach, K...... 99 Johannessen, C...... 62, 96 Hildebrandt, C...... 158 Jongen, L...... 107 Hinz, S...... 166 Jose, J...... 43, 107, 109, 115 Hirsch, R...... 74 Juchum, M...... 109 Hittinger, M...... 169 Jung, M...... 124, 126, 134, 135, 175 Hochhaus, A...... 43, 109 Jung, N...... 147 Hochscherf, J...... 107 Jürgenliemk, G...... 95, 121 Hoferm, N...... 19 Just, S...... 167 Hoffmann, A...... 132 Hoffmann, K...... 137 Hoffmann, L...... 140, 174 K Hoffmann, T...... 152 Hofmann, K...... 31 Kahnt, A. S...... 41, 97, 123 Höfner, G...... 88, 94, 175 Kaiser, A...... 101, 123 Höldrich, M...... 90 Kalayda, G. V...... 110 Holl, R...... 61, 131 Kalinin, D...... 131 Holsboer, F...... 22 Kalinowsky, L...... 99, 127 Holstein, J...... 102 Kamlah, A...... 124 Höltke, C...... 130 Kandil, R...... 103 Holze, J...... 142 Kany, A. M...... 94 Holzgrabe, U...... 89, 138, 142 Karlsson, R...... 58, 125 Honrath, B...... 174 Kassack, M. U...... 43, 109, 110 Hönzke, S...... 64, 103 Kauffold, J...... 154 Hopf, Y. M...... 119 Kaysser, L...... 54, 146 Hoppe, J...... 122 Kazmaier, U...... 107, 111 Horak, J...... 90 Keck, C. M...... 148, 152, 153, 154, 161, 162 Horstmann, R. D...... 177 Keils, A...... 140 Hoß, S. G...... 105 Kelber, O...... 98, 120, 146, 167, 169 Huang, F.‐C...... 152 Keller, M...... 40, 136, 180 Huang, G...... 132 Kelter, G...... 109 Huang, X. P...... 136 Kempin, W...... 27 Hubert, M...... 150 Kessel, E...... 82, 150 Hübner, H...... 136, 137, 180 Keßler, K...... 179 Huge, V...... 93 Khalil, E...... 145 Huisinga, W...... 119 Khayyal, M. T...... 98 Humar, M...... 99, 178 Kiemer, A. K...... 79 Hummel, F. C...... 122 Kim, G.‐J...... 99

DPhG Annual Meeting 2016 Conference Book • 185 POSTERS

Kim, N. H...... 103 L Kinscherf, R...... 98 Kirchmair, J...... 132 Lächelt, U...... 82, 150 Kirmeier, T...... 78 Läer, S...... 93, 118, 180 Kisko, T. M...... 143 Lahrsen, E...... 92 Klebe, G...... 58, 123, 125 Lamers, C...... 123 Klein, P. M...... 82, 150 Lämmerhofer, M...... 90 Kleine, K...... 180 Lang, F...... 43, 109 Kleinebudde, P...... 72 Lang, M...... 130 Kleinschmidt, T. K...... 98 Langguth, P...... 75, 147, 151, 169 Kleusch, C...... 94 Laufer, S. A...... 100, 102, 109, 132, 180 Kling, R. C...... 136 Le Guiner, C...... 45 Klinger‐Strobel, M...... 162 Lee, D. J...... 82, 150 Klingmann, I...... 180 Lee, S...... 166 Klintworth, D...... 118 Lehmann, C...... 41, 97 Klitsche, F...... 95 Lehr, M...... 101 Klöckner, J...... 142 Lehr, T...... 113 Kloft, C...... 107, 119, 120, 121, 171, 172 Leibiger, J...... 33, 174 Klopp‐Schulze, L...... 107 Leipoldt, F...... 54, 146 Klymiuk, N...... 20 Lell, P...... 38, 160 Knaab, T. C...... 129 Lemcke, T...... 134, 166 Knapp, S...... 97, 99, 102 Leroux, J. C...... 164 Kneidinger, N...... 168 Leutz, S...... 137 Kniess, A...... 165 Levit, A...... 136 Knuth, S...... 121 Li, Y...... 103 Kobilka, B. K...... 136 Liebl, J...... 112, 113 Koch, O...... 66, 130 Liedl, K. R...... 132 Koch, P...... 67, 130 Liening, S...... 98 Koeberle, A...... 42, 92, 110, 145 Liewert, I...... 106 Koeberle, S. C...... 42, 92 Lilischkis, R...... 148 Koepf, E...... 116 Lin, H...... 86, 136 Köhler, J...... 131 Lindhorst, F...... 115 Köhnke, J...... 94 Lintermans, A...... 107 König, B...... 34, 131, 133 Lirk, F...... 124 Königshoff, M...... 31 Liss, B...... 19 Köpf, E...... 57 Liu, B...... 145 Körner, A...... 104 Liu, S...... 90 Köse, M...... 137 Löber, S...... 136 Kostenis, E...... 123 Loeser, K ...... 42, 92 Kraff, S...... 119 Lohmüller, T...... 110 Kraft, K...... 120 Löhnert, A...... 177 Kralisch, D...... 163 Lohrer, B...... 125 Kramer, C...... 132 Lorenz, L...... 172 Kramer, J. S...... 99 Lötters, S...... 89 Krattenmacher, D...... 167 Luber, M...... 124 Kraus, T...... 103 Lucas, H...... 149 Krauss, J...... 126 Luciani, P...... 164 Kreiss, C...... 169 Lüdeke, S...... 43, 62, 96, 109 Kressler, J...... 149, 160 Lühmann, T...... 81, 117 Krieg, T...... 165 Lukat, P...... 145 Krimmer, S. G...... 58, 125 Lukowski, R...... 143, 165, 167 Krüger, A. T...... 179 Lum, L. G...... 103 Kuhn, K. K...... 180 Lunter, D...... 63, 155, 157 Kulik, A...... 54, 146 Luong, B...... 97 Kullmann, M...... 110 Kuntsche, J...... 156 Kunze, T...... 118 Kurland, H.‐D...... 161 M Kurz, T...... 43, 107, 109, 129 Kutza, J...... 160 Mackenroth, L...... 19

186 • DPhG Annual Meeting 2016 Conference Book AUTHOR INDEX

Mäder, K...... 149, 160 Mosad, S...... 147 Magdolen, V...... 140 Moser, C...... 111 Mahringer, A...... 151 Mozafari, M...... 88 Mair, C. E...... 132 Mueller, T. D...... 117 Maison, W...... 95, 127 Müller, C...... 89, 104 Mak, T...... 166 Müller, C. E...... 110, 137, 166 Malsch, D...... 159 Müller, F. A...... 161 Mandl, M. M...... 112, 113 Müller, J...... 120 Manglik, A...... 136 Müller, M...... 35 Mangold, M...... 117 Müller, R...... 53, 59, 65, 97, 105, 110, 111, 145, 159, 161 Marchais‐Oberwinkler, S...... 123, 125 Müller, R. H...... 161, 162 Marek, M...... 134, 135 Müller, S. L...... 128 Markarewicz, O...... 162 Müller, T...... 149 Markl, D...... 86 Müller, T. D...... 81 Marschall, C...... 115 Müller, U...... 118 Martin, B...... 131 Müller‐Goymann, C...... 73 Martins, H...... 143 Marzouk, M. A...... 108 Maschowski, C...... 178 N Massolini, G...... 48 Matera, C...... 138, 142 Nadithe, V...... 103 Maul, K. J...... 91 Nafie, L. A...... 62, 96 Maurer, E...... 117 Namjoshi, S. N...... 147 Mayer‐Wrangowski, S. C...... 102 Nann, Y...... 143 Mayr, D...... 112 Nasim, M. J...... 148 McCorvy, J...... 136 Natile, G...... 108 Meinel, L...... 81, 117 Natile, M...... 108 Melesina, J...... 134 Nawroth, T...... 147 Melzer, B...... 139 Negro, G...... 19 Menche, D...... 65, 105, 110 Neiens, P...... 175 Mendel, R. R...... 126 Neitemeier, S...... 140 Merfort, I...... 99, 178 Neri, D...... 23 Merk, D...... 100, 101, 123, 127, 129 Neundorf, I...... 107 Merkel, O. M...... 103 Neurohr, C...... 168 Messerer, R...... 138, 142 Neven, P...... 107 Meßner, M...... 113 Newcomer, M. E...... 99 Metz, A...... 125 Nguyen, T. M. H...... 154 Meyer, K...... 113 Nguyen, T. N...... 137 Miceli, M...... 151 Nicke, A...... 143 Michalakis, S...... 47 Niefind, K...... 107 Michalcova, L...... 94 Niess, R...... 147 Michels, S...... 143 Mikula, M...... 96 Mingo, V...... 89 O Mirakaj, V...... 104 Mobashery, S...... 60 Obarcanin, E...... 118 Mohammadi, M...... 103 Obst, K...... 151 Mohammed, Y. H...... 147 Oetjen, E...... 165, 166 Mohr, C...... 152 Oliveira, E. G...... 162 Mohr, E...... 165 Ong, N...... 124 Mohr, K...... 142 Ortland, I...... 122 Möhwald, M...... 150 Ortner, N. J...... 19 Molina, M...... 151 Osipova, A...... 178 Möller, D...... 135 Ott, I...... 92 Möller, G...... 123 Ottl, J...... 49 Monaldi, D...... 135 Moore, B. S...... 54, 146 Mordmüller, B...... 129 P Morhenn, K...... 165 Morrison, H...... 42, 92 Parnham, M. J...... 41, 97

DPhG Annual Meeting 2016 Conference Book • 187 POSTERS

Parra‐Guillen, Z. P...... 119 Rodrigues, A. G...... 160 Paulke, B...... 153 Roehr, A. C...... 93 Pei, C...... 86 Rollinger, J. M...... 132 Pein, H...... 42, 92 Romier, C...... 134, 135 Pendl, E...... 147 Romp, E...... 99 Perfahl, S...... 108 Rossi, A...... 42, 92 Peric, N...... 127 Roth, B. L...... 136 Pernpeintner, C...... 110 Rothert, M...... 126 Pfaffenrot, E...... 102, 180 Rotter, M...... 101 Pham, N. B...... 166 Roy, A...... 62, 96 Pierce, R...... 134 Rummler, S...... 98 Pinggera, A...... 19 Rumpf, T...... 175 Piva, M. B. R...... 106 Ruth, P...... 30, 143, 165, 167 Plank, R...... 151 Rüther, A...... 43, 62, 96, 109 Plesch, E...... 40, 136 Pletz, M. W...... 162 Plitzko, B...... 126 S Pockes, S...... 138 Pogoryelov, D...... 99 Sakkas, A.‐S...... 177 Pollinger, J. C...... 127 Salah, M...... 123 Poppe, A...... 107 Sarin, N...... 110 Pötzinger, Y...... 163 Sassano, M. F...... 136 Pourasghar, M...... 150 Sautebin, L...... 42, 92 Praefke, B. A...... 100 Sayed, A...... 133 Proschak, E...... 99, 100, 101, 123, 127 Schacht, M...... 139 Przybylski, S...... 112 Schader, T...... 123 Pyo, S. M...... 161, 162 Schaefer, J...... 93 Pysniak, K...... 96 Schaeftlein, A...... 121 Schäfer, K. H...... 148 Schäfer‐Korting, M...... 64, 103 Q Scheffler, K...... 50 Scheler, S...... 148 Quinn, R. J...... 166 Scherließ, R...... 153, 158, 159 Scherrer, G...... 136 Scherübl, C...... 56 R Scheunemann, M...... 130 Schiedel, A. C...... 137 Rabel, M...... 161 Schiedel, M...... 135 Rabini, S...... 120, 178 Schiestl, M...... 55 Rach, R...... 147 Schiller, S...... 153 Rackl, M...... 158 Schirmeister, T...... 105 Rademann, J...... 128 Schlegel, L...... 91 Radi, L...... 112 Schlenk, F...... 159 Rahnfeld, L...... 163 Schlesinger, M...... 106 Rammensee, H.‐G...... 21 Schmidberger, M...... 155 Ramotowska, E...... 96 Schmidt, A...... 143 Raney, S. G...... 147 Schmidt, C...... 33, 174 Rath, S...... 140 Schmidt, C. Q...... 100 Rauh, D...... 102 Schmidt, J...... 101, 129 Rehbaum, H...... 83 Schmidt, M...... 134 Reichart, B...... 20 Schmidt, M. V...... 77 Rein, T...... 77 Schmidt, S. K ...... 88 Renner, S...... 20 Schmidtke, M...... 132 Richter, M...... 132 Schmidtko, A...... 32 Richter, T...... 107 Schmidtkunz, K...... 134 Ritzmann, M...... 38, 160 Schmoeckel, E...... 112 Robaa, D...... 134 Schneider, F...... 42, 92 Roberts, M. S...... 147 Schneider, J...... 96 Rodrigues de Sá Alves, F...... 132 Schneider, M...... 98, 150, 162 Rodrigues Moita, A. J...... 43, 109 Schnorr, J...... 169

188 • DPhG Annual Meeting 2016 Conference Book AUTHOR INDEX

Schoenfeld, A...... 92 Steinebach, C...... 131 Scholler, M...... 131, 133 Steinhilber, D...... 41, 97, 99, 123 Scholz, M. S...... 128 Steinmetzer, T...... 140 Schönhoff, M...... 122 Stelzer, D...... 168 Schorn, M...... 54, 146 Stepan, J...... 77 Schrage, R...... 142 Stevens, M. M...... 29, 111 Schramm, K. W...... 133 Stewart, P. S...... 172 Schramm, R...... 168 Stirnberg, M...... 117 Schramm, S...... 132 Stöckelhuber, M...... 89 Schratt, G...... 143 Stocklauser, R...... 143 Schreiber, F...... 104 Stoiber, K...... 110 Schreiber, J...... 128 Stolfa, D. A...... 126 Schrenk, D...... 169 Storr, M...... 120 Schröder, T...... 148 Stößel, A...... 137 Schroeder, R...... 57, 116 Stranik, O...... 159 Schubert‐Zsilavecz, M...... 91, 100, 123, 124, 127, 129 Straubinger, J...... 167 Schulz, M...... 118 Striessnig, J...... 19 Schulze, J...... 163 Strobach, D...... 69 Schumann, L...... 132 Strödke, B...... 97 Schützenmeister, N...... 139 Strøm, M. B...... 172 Schüürmann, J...... 115 Strunz, A. K...... 118 Schwab, W...... 152 Stump, K...... 179 Schwarting, R. K. W...... 143 Stumpf, F...... 153 Schwedhelm, E...... 122 Subeska, A...... 101 Schweiger, A...... 85 Swoboda, V...... 180 Schwendler, A...... 98 Swyter, S...... 124, 175 Schwenk, R...... 107 Szatmári, A...... 180 Scott, S...... 166 Scriba, G. K...... 98 Seebohm, G...... 128 T Seeger, J...... 121, 172 Seibel, J...... 105 Täuber, A...... 73 Seidlitz, A...... 27 Tauber, C...... 128 Seifert, I...... 91 Temme, L...... 138 Senger, J...... 135 Temporini, C...... 48 Sergi, M...... 148 Thakur, A...... 103 Serio, A...... 29, 111 Thamm, J...... 161 Serive, B...... 166 Theiler, S...... 130 Shahla, H ...... 181 Thon, N...... 93 Shastri, V. P...... 62, 96 Tins, J...... 93 Shen, Y. C...... 86 Titz, A...... 39, 146, 162 Sherbakova, A...... 167 Toewe, A...... 41, 97 Shoichet, B. K...... 136, 137 Torge, A...... 162 Sieber, S. A...... 113 Tränkle, C...... 142 Sievers‐Engler, A...... 90 Tuluc, P...... 19 Sikandar, A...... 94 Tyl‐Bielicka, A...... 96 Simmet, T...... 100 Simon, R. P...... 126 Sippl, W...... 126, 134 U Sonderegger, C...... 148 Sotriffer, C...... 138 Ullrich, A...... 107 Spahn‐Langguth, H...... 133, 147, 181 Ulrich, M...... 110, 112 Spieler, V...... 81, 117 Ulrich‐Merzenich, G...... 167 Stadler, J...... 38, 160 Unger, M...... 179 Stadler, M...... 124, 126 Untergehrer, M...... 121 Stahl, M...... 113 Urbanetz, N. A...... 158 Stark, S...... 131, 133 Stauber, R...... 105 Stehning, T...... 107 Stein, S...... 43, 109

DPhG Annual Meeting 2016 Conference Book • 189 POSTERS

V Williams, B. M...... 86 Windbergs, M...... 147 Windorf, M...... 160 Van Asten, K...... 107 Winter, G...... 38, 115, 116, 149, 150, 160 Van Koppen, C. J...... 123 Winter, H...... 168 Vanderheyden, P. M. L...... 180 Winter, N...... 181 Verjee, S...... 146 Winzi, M...... 110 Vetter‐Kerkhoff, C...... 93 Witt, M...... 115 Vial, M...... 166 Witt‐Enderby, P. A...... 108 Vlodavsky, I...... 105 Wittmann, S...... 99, 101 Voelkel, M...... 42, 92 Wittmann, S. K...... 123 Vögerl, K...... 135 Wöhr, M...... 143 Vogeser, M...... 168 Woiwode, U...... 90 Vollmar, A. M...... 65, 105, 110, 111, 112, 113 Wojtyniak, J.‐G...... 113 Vollrath, M...... 115 Wolber, G...... 137, 139 Volpp, M...... 89 Wolf, E...... 20 von Grafenstein, S...... 132 Wolf, F...... 54, 146 von Kügelgen, I...... 137 Wölker, J...... 92 von Schwarzenberg, K...... 65, 105, 110 Wöll, S...... 153 Vuorela, P. M...... 172 Wulle, S...... 179 Wünsch, B...... 128, 130, 138, 140 W Wurglics, M...... 123

Wadie, W...... 98 X Wagner, E...... 82, 150 Wagner, N...... 89 Xie, Y...... 103 Wagner, S...... 130, 162 Xu, Z...... 132 Wahl, J...... 89 Wahl‐Schott, C...... 40, 119 Währa, M...... 94 Y Walter, J...... 117 Walter, N. M...... 102 Yasin, A...... 115 Walther, E...... 132 Yealland, G...... 151 Wanner, K. T...... 88, 94, 175 Yordanova, Y...... 154 Warncke, P...... 159 Wätzig, H...... 88, 91 Weber, A...... 168 Z Weber, J...... 31 Wehle, S...... 138 Zahler, S...... 110, 111 Weickert, A...... 105 Weinigel, C...... 98 Zara, L...... 125 Weiser, C...... 120 Zaremba, W...... 154 Weiser, T...... 101 Zeitler, J. A...... 86 Weiss, V...... 149, 160 Zeitlinger, M...... 120 Zerfass, P...... 143 Weitschies, W...... 27 Weizel, L...... 101, 123 Zettler, J...... 54, 146 Wentsch, H. K...... 102 Zhang, J...... 143 Werner, B. P...... 116 Zhang, S...... 112 Werner, M...... 124 Zhang, Z...... 157 Zheng, Y...... 86 Werner, S...... 159 Werner, V...... 81, 117 Zhou, X. B...... 30 Wersig, T...... 160 Zimmermann, A...... 90 Werz, O...... 42, 80, 92, 98, 99, 110, 124, 145 Zimmermann, G...... 168 Wich P. R...... 112 Zlotos, D. P...... 108 Zoller, M...... 168 Wich, P. R...... 28, 117 Wicha, S. G...... 71, 171 Zöls, S...... 38, 160 Wieland, T...... 30 Zubeil, F...... 54, 146 Wiesneth, S...... 95

190 • DPhG Annual Meeting 2016 Conference Book

Geschäftsführer und Leiter der Geschäftsstelle Apotheker Dr. Michael Stein DPhG Geschäftsstelle Varrentrappstraße 40-42 60486 Frankfurt Tel.: +49-(0)69-71915960 Fax: +49-(0)69-719159629 Email: [email protected] http://www.dphg.de

Univ.-Prof. Dr. Gerhard Winter Ludwig-Maximilians-Universität München Institut für Pharmazeutische Technologie und Biopharmazie Butenandtstraße 5-13 81377 München, GERMANY Phone: +49-(0)89–218077022 Fax: +49-(0)89–218077020 Email: [email protected]

15.09.2016

DPhG Annual Meeting 2016 Conference Book • 191

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192 • DPhG Annual Meeting 2016 Conference Book

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DPhG Annual Meeting 2016 Conference Book • 193

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DPhG Annual Meeting 2016 – Conference Book www.2016.dphg.de

ISBN 978-3-9816225-3-9 Annual Meeting of the – DPhG German Pharmaceutical Society Munich, Germany October 4 – 7, 2016 at Ludwig-Maximilians-University www.2016.dphg.de