Annual Meeting of the German Pharmaceutical Society – DPhG

Pharmaceutical research: From basic research to medical applications

Heidelberg, Germany September 01 – 04, 2019 at Heidelberg University Conference Book Pharmaceutical research: From basic research to medical applications

Annual Meeting of the German Pharmaceutical Society 2019 - DPhG Heidelberg, Germany, September 2019 ISBN 978-3-9816225-6-0 Annual Meeting of the German Pharmaceutical Society – DPhG

Conference Book Pharmaceutical research: From basic research to medical applications

Heidelberg, Germany September 01 – 04, 2019 at Heidelberg University www.2019.dphg.de

Institutional Sponsors

Förderer der DPhG-Jahrestagung 2019

TABLE OF CONTENTS

CONFERENCE COMMITTEES ...... ii WELCOME ADDRESS ...... iii GENERAL INFORMATION ...... iv LOCATIONS ...... vi 1 CONFERENCE PROGRAM OVERVIEW ...... 1 2 PLENARY LECTURES ...... 11 3 SCIENTIFIC LECTURES ...... 17 3.1 New Research, New Researchers I ...... 18 3.2 Ion channels as drug targets ...... 22 3.3 Fachsymposium Dermatopharmakologie: “Predictivity of in vitro tests in pharmacology and toxicology” ...... 26 3.4 Cysteine-reactive inhibitors and probes ...... 31 3.5 Dysfunctional Cellular Signaling in Cardiovascular Diseases ...... 34 3.6 Biopharmacy, Pharmaceutical Technology ...... 37 3.7 Gold! Hunting treasures in chemistry and pharmacology ...... 41 3.8 Evidence-based value added to innovative health care by pharmacists ...... 44 3.9 Simulation in Pharmaceutic and Formulation Development ...... 47 3.10 G-Protein-coupled Receptors ...... 51 3.11 Radiopharmacy & Labeling ...... 55 3.12 Proteomics in Medicine and Pharmacy ...... 60 3.13 Microfluidics / Cell-based assays and organoids for drug discovery ...... 64 3.14 New Research, New Researchers II ...... 67 3.15 The promise of cure - recent breakthrough innovation in cell- and gene therapy ...... 72 3.16 Natural Products – From Molecular Pharmacology to Clinical Applications ...... 75 3.17 Bioanalytical Mass Spectrometry ...... 78 3.18 Challenges and opportunities in model based dose adaptation ...... 81 4 POSTERS ...... 84 4.1 Medicinal Chemistry and drug design ...... 85 4.2 Other Topics ...... 120 4.3 Analytics and toxicology ...... 123 4.4 Cancer ...... 126 4.5 Clinical Pharmacy ...... 128 4.6 Natural Compounds ...... 134 4.7 Inflammation ...... 136 4.8 Pharmaceutical Technology and biomaterials ...... 138 4.9 Pharmacology ...... 153 4.10 Poster Short Talks ...... 158 AUTHOR INDEX ...... 167

DPhG Annual Meeting 2019 Conference Book • i CONFERENCE COMMITTEES

Scientific committee: Prof. Dr. Christian Klein Prof. Dr. Charlotte Kloft Prof. Dr. Thomas Efferth Prof. Dr. Christoph Friedrich Prof. Dr. Irmgard Merfort Prof. Dr. Peter Gmeiner Prof. Dr. Ulrike Holzgrabe Prof. Dr. Jochen Klein Prof. Dr. Michael Lämmerhofer Prof. Dr. Peter Langguth Prof. Dr. Gerhard Winter Prof. Dr. Thorsten Lehr Prof. Dr. Werner Weitschies Prof. Dr. Angelika Vollmar Prof. Dr. Dr. Peter Ruth Prof. Dr. Hermann Wätzig Prof. Dr. Dr. Achim Schmidtko Prof. Dr. Christoph Friedrich Prof. Dr. Ulrich Jaehde

Organization committee: Prof. Dr. Michael Wink Prof. Dr. Christian Klein Prof. Dr. Stefan Wölfl Prof. Dr. Gert Fricker Dr. Gabriele Reich

ii • DPhG Annual Meeting 2019 Conference Book WELCOME ADDRESS

Dear colleagues, the President of the German Pharmaceutical Society (DPhG) and the Congress Chairman would like to welcome you to Heidelberg and Heidelberg University. Heidelberg as the city of sciences with a unique romantic flair is the ideal location for our international annual DPhG meeting 2019. The meeting is cosponsored by one of our most important partners, the Pharmaceutical Society of Japan (PSJ).

The title of this year’s meeting is „Pharmaceutical research: From basic research to medical applications“. A broad range of sessions for oral and poster presentations will cover important aspects of current pharmaceutical research. We are looking forward to exciting talks, presented by many high level speakers – including Nobel Prize winner Prof. Harald zur Hausen - and stimulating discussions. A particularly important part of the meeting is the interaction of younger scientists with other attendees. A dedicated session for poster short-talks will allow selected PhD students to present their work. For excellent young postdocs and group leaders we will have special sessions to give them the opportunity of sharing their research results.

Many thanks to the scientific committee headed by Prof. Ch. Klein and the local organization team, to the scientific chairs as well as to the Pharmaceutical Society of Japan (JPS) for the strong support. This abstract book provides all necessary information on the program as well as on the abstracts of the scientific contributions of plenary lectures, scientific lectures, short poster lectures and posters.

We are glad to have you here for a stimulating meeting in Heidelberg and hope you will enjoy the scientific program as well as the location.

Prof. Dr. Stefan Laufer, DPhG-President

Prof. Dr. Michael Wink, Congress Chairman

DPhG Annual Meeting 2019 Conference Book • iii GENERAL INFORMATION

The Annual DPhG Meeting 2019 takes place at the Hörsaalzentrum Chemie of the Heidelberg University (Im Neuenheimer Feld (INF) 252).

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.

CONFERENCE OFFICE The Conference office is located at the Conference building Im Neuenheimer Feld 252, 69120 Heidelberg. Opening hours: Monday, September 2nd, 2019: 11:00 – 18:00; in the foyer of the main building Tuesday, September 3rd , 2019: 8:00 – 17:00; in the foyer of the main building Wednesday, September 4th, 2019: 8:00 – 12:00; in the foyer of the main building

BAGGAGE CHECKROOM Your baggage can be stored at the conference office during the opening hours (no liability is assumed).

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.

iv • DPhG Annual Meeting 2019 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 (i.e. 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 authors index on page 167 for direct access to specific abstracts.

POSTER SESSIONS Topics: Session I: Medicinal chemistry and drug desing and Other topics Session II: Antiinfectives, Cancer, Inflammation, Analytics, Biotechnology and Biopharmaceutics, Clinical Pharmacy, Natural Compounds, Pharmaceutical technology and biomaterials and Poster Short Talks (mixed topics). Presenting authors are asked to be present at their poster during the poster sessions.

Poster session I Poster session II Tuesday, September 3rd, 2019 Monday, September 2nd, 2019, 16.00 - 16.30 and Session 19:30 – 20:30 Wednesday, September 4th, 2019 10.00 - 10.30 Wednesday, September 2nd, 2019, Tuesday, September 3rd, 2019, Set-up before 16:00 Between 11:00 – 15:00 Wednesday, September 3rd, 2019, Wednesday, September 4th, 2019, Dismantling before 10:30 until 12:00

CONFERENCE DINNER Separate registration necessary (special fee). Please refer to the Conference office for registration and details. The Conference dinner will take place at “Schloßhotel Molkenkur”, Klingenteichstraße 31, 69117 Heidelberg.

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

DPhG Annual Meeting 2019 Conference Book • v LOCATIONS

The Congress will take place at Hörsaalzentrum Chemie of the Heidelberg University (Im Neuenheimer Feld (INF) 252).

It‘s easy to get to Heidelberg University by public transportation via Tram or bus.

The line 5 takes ten minutes to get from the Main Station (Hauptbahnhof) to Technologiepark Station. Due to limited parking space, we strongly recommend taking public transport.

For directions please refer to the campus map in the inner cover at the end of the abstract book.

vi • DPhG Annual Meeting 2019 Conference Book Schloßhotel Molkenkur (Conference Dinner): The Schloßhotel Molkenkur (Klingenteichstraße 31, 69117 Heidelberg) is located above the city of Heidelberg and invites you to enjoy the wonderful view over the old town and the Neckar and the Rhine valley.

BY PUBLIC TRANSPORTATION Go from Heidelberg Main Station East to station “Universitätsplatz” by bus 32. Take the bus 30 to station “Molkenkur” (approx. 35 mins).

Alternatively you can use the Heidelberg Bergbahn from station Kornmark to station Molkenkur.

DPhG Annual Meeting 2019 Conference Book • vii

1 CONFERENCE PROGRAM OVERVIEW

Samstag, 31.08.2019

Tag der Offizinpharmazie Organisationskomitee: Kathrin Müller, Michael Hannig, Juliane Kresser, Nadine Metzger (DPhG FG Allgemeinpharmazie und Apothekerkammer Baden-Württemberg)

Veranstaltungsort: Universität Heidelberg, Campus Im Neuenheimer Feld (INF), Hörsaalzentrum Chemie (Gebäude INF-252), 69120 Heidelberg; Großer Hörsaal

Arzneimitteltherapie für Kinder

15:00 – 15:10 Uhr Begrüßung

15:10 – 15:50 Uhr Pädiatrie trifft Pharmakologie: Besonderheiten im Kindes- und Jugendalter Prof. Dr. Matthias Schwab, Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie, Stuttgart

15:50 – 16:30 Uhr Kinderarzneimittel in Rezeptur und Defektur Prof. Dr. Rolf Daniels, Universität Tübingen

16:30 – 16:45 Uhr Kaffeepause

16:45 – 17:30 Uhr Kinder sind keine kleinen Erwachsenen – Arzneimitteltherapie für Kinder Margit Schlenk, Moritz-Apotheke Nürnberg

17:30 – 18:00 Uhr Panel mit den Referenten und Publikumsbeteiligung Im Anschluß Mitgliederversammlung der DPhG-FG-Allgemeinpharmazie, u. a. mit Vorstandswahl

Wichtig! Nur angemeldete Teilnehmer/innen erhalten Zutritt. Bitte melden Sie sich online unter: www.lak-bw.de/Fortbildung/Seminarplan an.

1 • DPhG Annual Meeting 2019 Conference Book Sunday, September 1st Vorsymposium der Fachgruppe „Geschichte der Pharmazie“: Pharmazie in Heidelberg Ort: Universität Heidelberg, Campus Im Neuenheimer Feld (INF), Hörsaalzentrum Chemie (Gebäude INF-252), 69120 Heidelberg; Hörsaal West Begrüßung durch den Vorsitzenden der FG Geschichte der Pharmazie 14:00 – 14:15 Prof. Dr. Christoph Friedrich, Marburg Entwicklung des Hochschulfaches Pharmazie an der Universität Heidelberg 14:15 – 15:00 Dr. Arnt Heilmann, Hirschhorn (Neckar) Aus der Geschichte des Apothekenwesens in Heidelberg 15:00 – 15:45 Dr. Albert Borchardt, Heidelberg 15:45 – Kaffeepause 16:15 Friedrich Bergius (1884-1949) – 16:15 – Chemiker, Unternehmer und Nobelpreisträger 17:00 Prof. Dr. Wolf-Dieter Müller-Jahncke, Heidelberg Das Deutsche Apothekenmuseum im Heidelberger Schloss 17:00 – 17:45 Dr. Elisabeth Huwer, Heidelberg

DPhG Annual Meeting 2019 Conference Book • 2 Monday, September 2nd Main Symposium (Congress language English) 09:00 – Sitzung des VdPPHI (HS West) 12:00 Chair: B. Clement 13:00 – Opening of the Annual DPhG Meeting 2019 (Großer HS) 13:45 Pharmaceutical research: From basic research to medical applications 13:45 – Plenary lecture 1, G. Borchard, Critical quality attributes of nanomedicines: What matters and 14:30 why? (Großer HS) PL.1 14:30 – Coffee break 15:00

SHORT TALKS (parallel sessions I) 15:00 – SL1 (Großer HS) SL2 (HS West) SL3 (HS Ost) 16:30 New Research, Ion channels as Fachsymposium New Researchers I drug targets Dermatopharmakologie: Chair: S. Laufer Chairs: P. Ruth, M. Freichel “Predictivity of in vitro tests in pharmacology and toxicology” Chairs: G. Weindl, J. Klein 15:00 SL.01 15:00 SL.05 15:00 SL.09 K. Nakayama: A. Guse: M. Schäfer-Korting: Molecular basis for protein NAADP: a fundamental Reconstructed human skin can trafficking within cilia and modulator of Ca2+ release predict the efficacy and safety ciliopathies revealed by the channels in Lymphocytes of drugs in humans visible immunoprecipitation (VIP) assay and CRISPR/Cas9 system 15:30 SL.02 15:30 SL.06 15:30 SL.10 R. Wombacher: Y-K. Chao: R. Landsiedel: Protein Labelling and Agonist-Dependent switching of Adverse outcome pathway- Manipulation in Living Cells Ion selectivity in endolysosomal based concepts and tools for Using Bioorthogonal Chemistry TPC2 channel assessing skin sensitization 15:50 SL.03 16:00 SL.07 16:00 SL.11 A. S. Kahnt: V. Tsvilovskyy: G. Weindl: Supporting resolution? Identification of OCaR1 as a Reconstructed tissue models Biosynthesis of specialized pro- gatekeeper of lysosomal-granular with integrated immune cells – resolving mediators (SPM) in Ca2+ release and regulated Do we need advanced in vitro human leukocytes exocytosis tests for the evaluation of drugs and chemicals? 16:10 SL.04 16:15 SL.08 16:15 SL.12 M. Koziolek: J. Camacho-Londono: C. Zoschke: The application of salivary Determination of fatal ventricular Human cell-based tumor caffeine concentrations to study arrhythmias by a new regulator microenvironment models for gastric emptying of fluids and of Ca2+ signaling at the improved preclinical drug formulations in humans lysosomal-SR junction development 16:45 - Plenary lecture 2, R. Bartenschlager, Curative therapy for chronic hepatitis C: Successes and 17:30 remaining challenges (Großer HS) PL.2

3 • DPhG Annual Meeting 2019 Conference Book 17:30 – Meetings der DPhG-Fachgruppen 18:45 Fachgruppe Fachgruppe Fachgruppe Fachgruppe Fachgruppe Fachgruppe Pharm./Med. Pharm. Pharmakologie Pharm. Klinische Industrie- Chemie Biologie J. Klein Technologie Pharmazie pharmazie P. Gmeiner A. Vollmar (HS West) D. Fischer T. Lehr (Großer HS) (HS Ost) (Gebäude INF (HS Med. (Gebäude INF 327, Klinik, INF 327, Seminarraum 410) Seminarraum 3, EG) 4, EG) AG Radiopharmazie, Dr. PD Reischl (Gebäude INF 327, Seminarraum 1, EG) 19:00 – Poster Session I (19.30 – 20.30 h) and Welcome Reception 21:30

Tuesday, September 3rd SHORT TALKS (parallel sessions II) 08:30 – SL4 (Großer HS) SL5 (HS West) SL6 (HS Ost) 10:00 Cysteine-reactive Dysfunctional Biopharmacy, inhibitors and Cellular Signaling Pharmaceutical probes in Cardiovascular Technology Chair: T. Schirmeister Diseases Chair: G. Fricker Chairs: T. Wieland, R. Gilsbach 08:30 SL.13 08:30 SL.16 08:30 SL.19 P. Czodrowski: K. Lorenz: E. Wagner: Cysteine, pKa, covalent attack: Selective inhibition of nuclear Molecular Therapeutics from any correlation? ERK1/2 functions – two quite DNA to Cas9/sgRNA: Chemical distinct implications: cardiac Evolution of Nanocarriers hypertrophy and cancer?

09:00 SL.14 09:00 SL.17 09:00 SL.20 M. Gütschow: R. Gilsbach: Y. Takakura: Novel Cathepsin B Inhibitors Identification and modulation of Development of exosome- with Inversely Oriented chromatin interactions in cardiac based nucleic acid drug Warheads myocytes delivery system 09:30 SL.15 09:30 SL.18 09:30 SL.21 M. Gehringer: V. Nikolaev: U. Lächelt: Covalent Kinase Inhibition by Altered cyclic nucleotide Metal-Organic Reversible and Irreversible microdomains in cardiac disease Nanopharmaceuticals Cysteine Targeting 09:45 SL.22 C. Wölk: Development of lipoplex- loaded model surface coatings for the in situ transfection in the field of bone regeneration 10:00 – Coffee break 10:15

DPhG Annual Meeting 2019 Conference Book • 4 SHORT TALKS (parallel sessions III)

10:15 – SL7 (Großer HS) SL8 (HS West) SL9 (HS Ost) 11:45 Gold! Hunting Evidence-based value added to Simulation in treasures in innovative health care by Pharmaceutical chemistry and pharmacists and Formulation pharmacology Chairs: H. Seidling, U. Jaehde Development Chair: C. Klein Chairs: D. Fischer, P. Langguth 10:15 SL.23 10:15 SL.26 10:15 SL.29 S. Hashmi: T. Dreischulte: H. Wachtel: Gold Catalysis: Functionalized Evidence-based value added to Simulations in pharmaceutical Gold Carbenes, Unexpected innovative health care by technology Selectivities pharmacists: Ambulatory care 10:45 SL.24 10:45 SL.27 10:45 SL.30 I. Ott: T. Hoppe-Tichy: M. A. Nguyen: Gold Organometallics in Evidence-based value added to Pharmacokinetic Modelling and Medicinal Chemistry innovative health care by Simulation to Support Early pharmacists - Hospital Setting Clinical Drug Development 11:15 SL.25 11:15 SL.28 11:15 SL.31 A. Miller & N. Gunkel: U. Jaehde: A. N. Ciciliani: Homoleptic gold Evidence-based value added to Simulation of pulmonary dithiocarbamate TXNRD1 innovative health care by deposition during inhalation inhibitors prevent tumor pharmacists: Long-term care therapy recurrence in a small cell lung facilities 11:30 SL.32 cancer mouse model G. Fuhrmann: Exosome-Hydrogels as Machinery for Anti- Inflammatory Therapeutics’ Synthesis 11:45 – Poster Short Talks (Großer HS) 12:45 Chairs: D. Fischer, P. Ruth, S. Wölfl 11:45 PST.01 12:14 PST.11 K. Mäder: M. Felix: Intracochlear PLGA based implants for Proteomic Exploration of IDH-Mutant Brain dexamethasone release: challenges and solutions Tumors 11:48 PST.02 12:17 PST.12 F. Mitrach: J. Seeger: Stabilization of Calcium Phosphate nanoparticles Determinants of growth-kill behaviour of as carrier system for siRNA fluoroquinolone resistant Escherichia coli under levofloxacin exposure in a dynamic in vitro infection model 11:51 PST.03 12:20 PST.13 D. Lunter: A. Müller-Schöll: Optimization of thermogelling emulsions with Computational treatment simulation to assess enhanced substantivity for use in treatment of the risk for non-efficacy in tamoxifen treated chronic skin diseases breast cancer patients of different ethnicities 11:54 PST.04 12:23 PST.14 P. Uhl: X. Cheng: Cell penetrating liposomes enable the oral Triple-color reporter system to follow up delivery of peptide drugs directed differentiation of iPSC towards the three germ layers

5 • DPhG Annual Meeting 2019 Conference Book 11:57 PST.05 12:26 PST.15 L. Ilia: G. Treccani: Identification and quantification of microdialysis Ketamine promotes early changes in dendritic variability using a dynamic in vitro microdialysis morphology in the hippocampus of a genetic rat system and nonlinear mixed-effects modelling model displaying depressive-like behaviour 12:00 PST.06 12:29 PST.16 Y-M. Pudritz: R. Bilancia: Pharmacology meets (clinical) pharmacy – virtual Sex dimorphism in rat platelet aggregation patients for pharmacy students 12:03 PST.07 12:32 PST.17 S. Schwarthoff: N. Wössner: Addressing targets relevant in neurodegenerative Thiocyanates as a new class of selective Sirt1 disorders: synthesis and biological activity of inhibitors multifunctional heterobivalent carboline derivatives 12:06 PST.08 12:35 PST.18 M. Borchers: B. Praefke: “Gender Medicine” in the 18th century?! Small molecules as MKK4 inhibitors for the Pharmacotherapy in the hessian hospital regeneration of hepatocytes Merxhausen 12:09 PST.09 12:38 PST.19 S. Heuter: S. Andreev: Platelets induce a chemoresistance of tumor cells From JAK to GSK-3β: Synthesis and Biological by upregulating drug efflux transporters Evaluation of 7-Chloro-9H-Pyrimido[4,5-b]indoles as Inhibitors of Glycogen Synthase Kinase-3β 12:11 PST.10 12:41 PST.20 F. Almouhanna: J. Hofmann: Glycolytic Flux and p53 status influence Growth Bioisosteres of the Natural Product Taxifolin and Inhibition in response to the G6PD Inhibitor DHEA their Impact on Amyloid-β 42 Aggregation and in Colorectal Cancer Cells Intracellular Oxidative Stress 12:45 – Lunch break 13:45 Workshop PubPharm - Tools für die pharmaziespezifische Recherche, C. Draheim (HS West)

13:45 – Plenary lecture 3, W. Haefeli, Achievements, persisting challenges and unmet needs in medication 14:30 safety (Großer HS) PL.3

SHORT TALKS (parallel sessions IV) 14:30 – SL10 (Großer HS) SL11 (HS West) SL12 (HS Ost) 16:00 G-Protein-coupled Radiopharmacy & Labeling Proteomics in Receptors Chairs: W. Mier Medicine and Chairs: P. Gmeiner, B. Pharmacy Wünsch Chair: A. Sinz 14:30 SL.33 14:30 SL.37 14:30 SL.41 M. Bünemann: T. Ross: M. Bantscheff: Dynamics of GPCR - G protein Visualisation of cardiac fibrosis Drug effects on protein interactions as the key to using [68Ga]MHLL1 – A PET homeostasis understand coupling tracer for the fibroblast selectivity of GPCRs activation protein

DPhG Annual Meeting 2019 Conference Book • 6 14:50 SL.34 15:00 SL.38 15:00 SL.42 D. Weikert: G. Reischl: K. Marcus: Bivalent ligands targeting Development and validation of 2- Laser Capture heterodimers of dopamine nitroimidazole-furanoside based Microdissection/Mass receptors fluorine-18 labeled PET- spectrometry- a promising tool radiopharmaceuticals for the in- in tissue-based clinical vivo detection of hypoxic tissue in proteomics tumors 15:15 SL.35 15:20 SL.39 15:30 SL.43 M. Decker: M. Keller: A. Maurer: Photopharmacology in Functionalized Nω- GMP Radiolabeling and First-in- Alzheimer research: Tools to carbamoylated arginines give Man Imaging of an Antibody for investigate GPCRs access to labeled peptide receptor the Diagnosis of Aspergillosis ligands 15:35 SL.36 15:40 SL.40 15:45 SL.44 G. Wolber: O. Prante: M. Ardelt: In Silico Pharmacology: A Radioynthesis, in-vitro and in-vivo Inhibition of Cdk5 – A New Way Mechanistic View on GPCR studies of 177Lu-labeled to Improve the Standard Modulation neurotensin receptor-1 Therapy of Hepatocellular antagonists as radiotracers for Carcinoma the therapy of pancreatic cancer 16:00 – Poster Session II and Coffee Break 16:30 16:30 – Plenary lecture 4, S. Knapp, Strategies for selective targeting of protein kinases (Großer HS) 17:15 PL.4 17:30 – DPhG Hauptversammlung (Großer HS) 19:00 Ab Conference Dinner (Restaurant Schlosshotel Molkenkur, Klingenteichstraße 31, 69117 19:30 Heidelberg)

7 • DPhG Annual Meeting 2019 Conference Book Wednesday, September 4th SHORT TALKS (parallel sessions V) 08:30 – SL13 (Großer HS) SL14 (HS West) SL15 (HS Ost) 10:00 Microfluidics / Cell-based New Research, New The promise of cure - recent assays and organoids for Researchers II breakthrough innovation in drug discovery Chairs: A. Link, F. Hansen cell- and gene therapy Chair: S. Wölfl Chair: H. Apeler 08:30 SL.45 08:30 SL.48 08:30 SL.53 C. Merten: D. Merk: K. Cichutek: Droplet Microfluidics in Direct PPARγ activation by L- Cell and gene therapy antibody discovery and thyroxin and TETRAC links developments: it is now or never! personalized cancer therapy thyroid hormone and PPAR signaling 08:50 SL.49 09:00 SL.54 J. Gerstmeier: J. Stitz: Targeting biosynthetic networks Hybrid retrovirus-transposon of the proinflammatory and vectors proresolving lipid metabolome: a promising pharmacological strategy for intervention with inflammation 09:10 SL.46 09:10 SL.50 S. Wölfl: N. Schützenmeister: Analyzing toxicity and Natural Products as Novel Lead metabolic interactions with Structures: Method microfluidic liver-kidney-on-a- Development and Biological chip systems Testing 09:35 SL.47 09:30 SL.51 09:30 SL.55 X. Cheng: D. Kalinin: G. Schmiedeknecht: Seeking for chemical OCT4 Small-molecule inhibitors of Challenges in manufacturing and substitutes for the generation human coagulation factor XIIa: Quality Control of ATMPs of human iPSCs synthesis and anticoagulant activity 09:45 SL.52 C. Lamers: Structure-activity study and molecular insights in the mode of action of complement C3 inhibitor Cp40 10:00 – 10:30 Poster Session II and Coffee Break

DPhG Annual Meeting 2019 Conference Book • 8 SHORT TALKS (parallel sessions VI) 10:30 – SL16 (Großer HS) SL17 (HS West) SL18 (HS Ost) 12:00 Natural Products - From Bioanalytical Challenges and Molecular Pharmacology Mass opportunities in to Clinical Applications Spectrometry model based dose Chairs: M. Wink, T. Efferth Chair: M. Lämmerhofer adaptation Chairs: C. Kloft, T. Lehr 10:30 SL.56 10:30 SL.59 10:30 SL.62 T. Efferth: T. Alexandrov: O. Scherf-Clavel : Systems biology with natural Spatial metabolomics in tissues Therapeutic drug monitoring in products for individualized and single cells oncology – Case examples cancer therapy 11:05 SL.57 11:05 SL.60 11:00 SL.63 M. Wink: C. Hopf: L. Klopp-Schulze: Treatment of patients with MALDI Mass Spectrometry-Based Model-based precision dosing of cardiac AL amyloidosis with Applications in Pharmacology tamoxifen therapy epigallocatechin-3-gallate (EGCG) from green tea- results of a clinical study and transcriptomics 11:40 SL.58 11:40 SL.61 11:30 SL.64 C. Jessen-Trefzer: S. Pace: S. Wicha: Targeting the mycobacterial Sex bias in colitis yields a Methodological aspects of membrane with protective role of specialized pro- model-based dose adaptations photosensitizers resolving mediators in female mice

12:00 – Lunch Break 13:00 13:00 - Plenary lecture 5, H. zur Hausen, “Indirect“ Mode of Cancer Induction by Infections 13:45 (Großer HS ) PL.5 13:45 – Closing ceremony (Großer HS) 14:30

9 • DPhG Annual Meeting 2019 Conference Book

2 PLENARY LECTURES

11 • DPhG Annual Meeting 2019 Conference Book PLENARY LECTURES

PL.1 Critical quality attributes of nanomedicines: What matters and why?

Gerrit Borchard, PharmD, PhD School of Pharmaceutical Sciences Geneva-Lausanne, University of Geneva, Switzerland [email protected]

The application of nanotechnology to the development of advanced therapeutics has brought about a number of so-called “nanomedicines”. Such drugs, though showing high variability in terms of size, shape, materials used, etc. share complex structures, which cannot be characterized in their entirety. Being mostly of synthetic origin and their preparation often including a self-assembly step, such drugs are recently referred to as non-biological complex drugs (NBCDs) [1]. Examples include glatiramoids (Copaxone®) [2], liposomal formulations (Doxil®) [3], and nanoparticles such as iron-carbohydrate particles (Venofer®) [4, 5]. Their size and attributes at the molecular scale confer these systems certain properties that impact their interaction with their biological environment, and thus influence PK/PD and safety profiles. The former FDA commissioner Gottlieb called for an improved review process of generic drug applications (ANDAs) [6]. This must be viewed in relation to nanomedicines, as well, as several follow-on products have entered the market, including a glatiramer generic approved by FDA has now issued a guidance draft on products containing nanomaterials [7] as an answer to a request by the U.S. House of Representatives’ Committee on Energy and Commerce in 2015 to the U.S. Government Accountability Office (GAO). Even though the draft guidance is suggesting a list of properties of nanomedicines (“Nanomaterial Quality Attributes”) to be tested, it lacks true guidance on individual (groups of) nanomedicines. It presumes levels of knowledge of product behavior that are typically not available, neither for NDA nor for generic, ANDA products. Due to their inherent complex structure, nanomedicines are impossible to be fully characterized by physicochemical methods alone, in vitro as well as in vivo studies, leading up to verification in clinical trials, are required. Therefore, an effort is needed to discover these correlations between specific critical quality attributes (CQA) and their impact on biological activity, ideally to be able to predict in vivo behavior. This can only be realized through a multi-pronged analytical approach and correlation to clinical data. This presentation will discuss the current state of identification of CQAs using examples from currently marketed nanomedicines.

References: [1] Ambardekar, V. V. & Stern, S. T. in Non-Biological Complex Drugs: The Science and the Regulatory Landscape (eds. Crommelin, D. J. A. & Vlieger, J. S. B. de) 261–287 (2015). [2] Borchard, G. and Crommelin, D.J.A. Equivalence of glatiramer acetate products: challenges in assessing pharmaceutical equivalence and critical clinical performance attributes. Exp. Opin. Drug Del. 15 (2018) 247-259. [3] Wibroe, P. P., Ahmadvand, D., Oghabian, M. A., Yaghmur, A. & Moghimi, S. M. An integrated assessment of morphology, size, and complement activation of the PEGylated liposomal doxorubicin products Doxil®, Caelyx®, DOXOrubicin, and SinaDoxosome. J. Control. Release 221, 1–8 (2016). [4] Di Francesco, T., Philipp, E. and Borchard, G. Iron sucrose: assessing the similarity between the originator drug and its intended copies. Ann. NY Acad. Sci. 1407 (2017) 63–74. [5] Di Francesco, T. and Borchard, G. Development and validation of a robust and easily reproducible protocol for the determination of size and size distribution of iron sucrose using dynamic light scattering. J. Pharm. Biomed. Anal. 152 (2018) 89-93. [6] https://www.fda.gov/NewsEvents/Newsroom/Press Announcements/ucm591184.htm, accessed April 5, 2018. [7] Drug Products, Including Biological Products, that Contain Nanomaterials Guidance for Industry (Draft). https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm

DPhG Annual Meeting 2019 Conference Book • 12 PLENARY LECTURES

PL.2 Curative therapy for chronic hepatitis C: Successes and remaining challenges

Prof. Dr. Ralf Bartenschlager Zentrum für Infektiologie, Molekulare Virologie, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 344, 69120 Heidelberg Abteilung “Virus-assoziierte Karzinogenese”, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg Deutsches Zentrum für Infektionsforschung, Standort Heidelberg

Infections with the hepatitis C virus (HCV) are a global burden and a main cause for acute and especially chronic liver diseases. According to WHO, around 71 million people suffer from chronic hepatitis C worldwide, an estimated 250,000 people in Germany. Although the first molecular cloning of the HCV genome in 1989 marked the starting point to study this medically important virus, for many years it was not possible to propagate HCV in cultured cells. Only with the advent of molecular biology, genetically engineered HCV mini-genomes could be designed that allowed the selection for specific variants capable to replicate with high efficiency in a human hepatoma cell line. This break-through provided the long-sought tool to study the replication of HCV, but also laid the ground for the development of antiviral therapy. Prior to the availability of selective drugs, patients with chronic hepatitis C were treated by use of an interferon – ribavirin combination therapy. However, this therapy had many contra-indications and numerous side-effects and therefore, the overall success rate of this treatment was rather low. However, with the availability of the HCV cell culture system, highly efficacious drugs could be developed targeting three different HCV proteins: the serine-type proteinase, the RNA-dependent RNA polymerase and the multi-functional replicase factor NS5A. Drugs targeting the viral proteinase have been approved for the first time in 2013, but only in combination with interferon. However, following the development of much more potent drugs with higher genetic barrier of resistance and active against all three HCV targets and all HCV genotypes, interferon-free therapy has become possible. Nowadays, all-oral interferon-free therapy of chronic hepatitis C is well established, reaching virus elimination in more than 95% of treated patients with little side effects and only very few contraindications. This result is remarkable, given that HCV is prone to establish persistence, but is due to the peculiar replication cycle of this virus. This unprecedented efficacy has sparked great hopes to eradicate HCV globally by pure antiviral therapy. However, this aim is questionable for several reasons, including high costs of available drugs, the high number of undiagnosed infections and reinfections after successful virus elimination. For these reasons, a prophylactic vaccine is warranted, but its development is probably one of the biggest challenges towards global eradication of HCV.

13 • DPhG Annual Meeting 2019 Conference Book PLENARY LECTURES

PL.3 Achievements, persisting challenges, and unmet needs in medication safety

Walter E. Haefeli UniversitätsKlinikum Heidelberg – Medizinische Klinik Abt. Klinische Pharmakologie & Pharmakoepidemiologie Im Neuenheimer Feld 410 69120 Heidelberg

There are no drug treatments without risk, but many therapies without benefit, often because essential details of the medication process escape careful attention of either health care professionals or patients and their relatives. In the last decade, in Germany significant progress in medication safety has been made, notably in the area of information about and documentation of drug treatment, transsectoral communication and continuity of care, identification of risks associated with drugs, and the treatment of special patient populations such as children and older patients. However, despite all these efforts, little has been achieved in major areas of concern. As an example, excessive bureaucracy is still present in many essential prescription steps and digitization of the most error-prone steps of the medication process is still not a common standard, thus precluding electronic decision support. Finally, and most notably, the still omnipresent, multifaceted challenge of non-adherence and non-persistence is largely unchanged, limiting the success of many potentially effective therapies much too often.

DPhG Annual Meeting 2019 Conference Book • 14 PLENARY LECTURES

PL.4 Strategies for selective targeting of protein kinases

Stefan Knapp Johann Wolfgang Goethe-University, Institute for Pharmaceutical Chemistry, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany. Johann Wolfgang Goethe-University, Structural Genomics Consortium, Buchmann Institute for Life Sciences; Max-von-Laue-Str. 15, D- 60438 Frankfurt am Main, Germany.

Protein kinases are enzymes with remarkable domain plasticity, a property that plays a key role not only in their regulation but also in the mode of action of inhibitors and drugs targeting diverse activation states. In this presentation I will outline some of the challenges targeting conformational states of kinases as well as new opportunities for drug discovery. I will present structure based design strategies for the development of highly selective inhibitors including inhibitors binding to allosteric sites and I will discuss consequences targeting distinct activation states of kinases. Modulators of kinase scaffolding function and the potential targeting pseudokinases will also be discussed.

References: Chaikuad, A., E, M.C.T., Zimmer, J., Liang, Y., Gray, N.S., Tarsounas, M., and Knapp, S. (2014). A unique inhibitor binding site in ERK1/2 is associated with slow binding kinetics. Nature Chemical Biology 10, 853-860. Muller, S., Chaikuad, A., Gray, N.S., and Knapp, S. (2015). The ins and outs of selective kinase inhibitor development. Nature chemical biology 11, 818-821. Chaikuad, A., Koch, P., Laufer, S.A., and Knapp, S. (2018). The Cysteinome of Protein Kinases as a Target in Drug Development. Angew Chem Int Ed Engl 57, 4372-4385. Georgi, V., Schiele, F., Berger, B.T., Steffen, A., Marin Zapata, P.A., Briem, H., Menz, S., Preusse, C., Vasta, J.D., Robers, M.B., et al. (2018). Binding Kinetics Survey of the Drugged Kinome. J Am Chem Soc 140, 15774-15782. Heroven, C., Georgi, V., Ganotra, G.K., Brennan, P., Wolfreys, F., Wade, R.C., Fernandez-Montalvan, A.E., Chaikuad, A., and Knapp, S. (2018). Halogen-Aromatic pi Interactions Modulate Inhibitor Residence Times. Angew Chem Int Ed Engl 57, 7220-7224. Vasta, J.D., Corona, C.R., Wilkinson, J., Zimprich, C.A., Hartnett, J.R., Ingold, M.R., Zimmerman, K., Machleidt, T., Kirkland, T.A., Huwiler, K.G., et al. (2018). Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement. Cell Chem Biol 25, 206-214 e211.

15 • DPhG Annual Meeting 2019 Conference Book PLENARY LECTURES

PL.5 “Indirect“ Mode of Cancer Induction by Infections

Harald zur Hausen, Timo Bund, and Ethel-Michele de VIilliers, Deutsches Krebsforschungszentrum 69120 Heidelberg, Germany

Chronic inflammation induced by infections may lead to malignant proliferations, as evidenced by gastric cancers linked to Helicobacter pylori infections, by bladder cancer as the consequence of persisting schistosoma infections, and more recently by chronic hepatitis C virus infections resulting in liver cancer. In none of these cancers, persistence has been noted of the respective agents within the cancer cells. Global epidemiological studies suggested a joint risk for colon- and breast cancers after consumption of red met or milk products of Eurasian dairy cattle (zur Hausen, 2009, 2012, zur Hausen and de Villiers, 2014, 2015, zur Hausen et al., 2017, 2019). Our group cloned and sequenced a large number of small single-stranded circular DNAs (1400- 200 Nucleotides) initially from bovine sera and dairy products of Eurasian cattle. They revealed structural characteristics of specific bacterial plasmids, but also DNA elements characteristic for viruses (de Villiers et al., in print). Analyzed types revealed transcription, translation and replication in specific human cells. Monoclonal antibodies permitted the demonstration of their major replication protein in peri-tumoral tissue of colon cancer cells, regularly surrounding the Lieberkühn’s crypts, from which cancer arises. By micro-dissection the genomes of specific BMMFs were recovered and analyzed. Since sugars in human milk protect against these infections, the prime risk phase for acquiring them is the weaning and post-weaning period. Cow milk does not contain the same protective sugars. Available data point to decade-long chronic inflammations by infections as specific triggers for random mutations (characteristic for indirect carcinogenesis) via oxygen radical production, resulting in colon cancer in average 40- 70 years later. Besides colon cancers, breast and prostate cancers emerge as additional “hot” candidates. Due to the obvious plasmid origin of our isolates, we proposed to label these diseases as plasmidoses.

DPhG Annual Meeting 2019 Conference Book • 16

3 SCIENTIFIC LECTURES

17 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

3.1 New Research, New Researchers I Chair: S. Laufer

SL.01 Molecular basis for protein trafficking within cilia and ciliopathies revealed by the visible immunoprecipitation (VIP) assay and CRISPR/Cas9 system

Kazuhisa Nakayama1, Yohei Katoh1 1Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

Primary cilia are microtubule-based projections from the surface of various eukaryotic cells, and function as cellular antennae by sensing extracellular stimuli, such as fluid flow, and by receiving and transducing developmental signals, such as Hedgehog signalling. Owing to their crucial roles, defects in ciliary assembly and ciliary protein trafficking result in a wide range of hereditary diseases, collectively called the ciliopathies. The biogenesis and functions of cilia rely on bidirectional trafficking of proteins along the axonemal microtubules, which is mediated by the intraflagellar transport (IFT) machinery. The IFT machinery is composed of large multiprotein complexes, IFT- A (composed of 6 subunits), IFT-B (16 subunits), and BBSome (8 subunits), with the aid of two motor protein complexes, kinesin-2 (3 subunits) and dynein-2 (11 subunits) [1]. To reveal the molecular basis for the ciliary protein tafficking, we first tried to delineate the overall architectures of the large protein complexes. To this end, we developed a novel strategy for detection of protein–protein interactions, named the visible immunetation (VIP) assay, in which not only binary but also one-to-many and many-to-many protein interactions can be visually detected, without the need of Western blotting [2]. By utilizing the VIP assay, we revealed the overall aerchtectures of the IFT-A [3,4], IFT-B [5,6], BBSome [2,7], kinesin-2 [8], and dynein-2 [9,10] complexes. Furthermore, we determined the interaction modes among these multiprotein complexes. On the other hand, to determine the roles of individual subunits, we developed a practical strategy to disrupt specific genes using the CRISPR/Cas9 system [11]. By using this knockout strategy, we revealed the roles of individual subunits of the complexes [3,4,6–11] and the moleculer basis for the ciliopathies [4,10].

Acknowledgements: Our study was suporeted in part by grants from JSPS, MEXT, Astellas Foundation, and Uehara Memorial Foundation.

References: 1. Nakayama, K. & Katoh, Y. (2018) Ciliary protein trafficking mediated by IFT and BBSome complexes with the aid of kinesin-2 and dynein-2 motors. J. Biochem., 163, 155-164. 2. Katoh, Y., et al. (2015) Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins. J. Cell Sci., 128, 2351-2362. 3. Hirano, T., Katoh, Y. & Nakayama, K. (2017) Intraflagellar transport-A complex mediates ciliary entry and retrograde trafficking of ciliary G protein–coupled receptors. Mol. Biol. Cell, 28, 429-439. 4. Takahara, M., et al. (2018) Ciliopathy-associated mutations of IFT122 impair ciliary protein trafficking but not ciliogenesis. Hum. Mol. Genet., 27, 516-528. 5. Katoh, Y., et al. (2016) Overall architecture of the intraflagellar transport (IFT)-B complex containing Cluap1/IFT38 as an essential component of the IFT-B peripheral subcomplex. J. Biol. Chem., 291, 10962-10975. 6. Takei, R., Katoh, Y. & Nakayama, K. (2018) Robust interaction of IFT70 with IFT52–IFT88 in the IFT-B complex is required for ciliogenesis. Biol. Open, 7, bio033241. 7. Nozaki, S., et al. (2018) BBS1 is involved in retrograde trafficking of ciliary GPCRs in the context of the BBSome complex. PLoS ONE, 13, e0195005. 8. Funabashi, T., et al. (2018) Interaction of heterotrimeric kinesin-II with IFT-B-connecting tetramer is crucial for ciliogenesis. J. Cell Biol., 217, 2867-2876. 9. Hamada, Y., et al. (2018) Interaction of WDR60 intermediate chain with TCTEX1D2 light chain of the dynein-2 complex is crucial for ciliary protein trafficking. Mol. Biol. Cell, 29, 1628-1639. 10. Tsurumi, Y., et al. (2019) Interactions of the dynein-2 intermediate chain WDR34 with the light chains are required for ciliary retrograde protein trafficking. Mol. Biol. Cell, 30, 658-670. 11. Katoh, Y., et al. (2017) Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology- independent knock-in system. Mol. Biol. Cell, 28, 898-906.

DPhG Annual Meeting 2019 Conference Book • 18 NEW RESEARCH, NEW RESEARCHERS I

SL.02 Protein Labelling and Manipulation in Living Cells Using Bioorthogonal Chemistry

Richard Wombacher*, Philipp Werther, Mathis Baalmann, Michael Ziegler 1Department of Pharmaceutical Chemistry, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, INF 364, 69120 Heidelberg

The ability to visualize or rapidly perturb specific molecular interactions within cellular protein networks is of major importance to investigate the role of individual biomolecules in the temporal and spatial context of a living cell or organism. Our research is focused on the use of small molecules as probes to either manipulate the cellular localization of proteins [1-3] or to specifically label proteins in living cells [4]. Particularly small chemical fluorophore probes with tailor-made photo-physical properties have gained prime importance for modern microscopy technologies. Ideally a fluorescent probe should have excellent specificity (low background) for its target, high photo-stability and photon output as well as optimal spectral properties suited for the microscopic technology and biological question to study. Fluorogenic probes for bioorthogonal labelling chemistry are highly beneficial to improve target specificity and to reduce background signal in fluorescence microscopy imaging [5]. We recently reported a palette of fluorogenic dyes for intracellular live cell protein labelling [6]. We demonstrate the power of this fluorogenic dyes in multi-colour live cell imaging under no-wash conditions. Moreover, we adapted our concept of fluorogenicity to fluorophores that possess spontaneous-blinking based on intramolecular spirocyclization at physiological pH [7]. We successfully applied these fluorogenic spontaneously blinking fluorophores (fSBF) in live cell single-molecule localization microscopy (SMLM). With our probes we are able to visualize cellular structures with a localization precision of ~20 nm, far below the diffraction limit. Further, we utilize the probes to visualize dynamic cellular events with high temporal and spatial resolution in living cells [8, 9].

Acknowledgments: We gratefully acknowledge financial support by the DFG-SPP1623 and thank our collaboration partners Dirk Ollech and Dr. Dr. Ada Cavalcanti-Adam (MPI for Medical Research) and Klaus Yserentant, Felix Braun and Prof. Dr. Dirk-Peter Herten (Heidelberg University, Institute of Physical Chemistry).

References: [1] Schelkle, K. M. et al. Wombacher, R.: Angew. Chem. Int. Ed. 2015, 54(9): 2825-2829. [2] Baalmann, M., Wombacher, R.: Nachrichten aus der Chemie 2016, 3(64): 305-309. [3] Ollech, D. et al. Wombacher, R., Cavalcanti-Adam, A.: submitted. [4] Baalmann, M. et al. Wombacher, R.: Bioconjugate Chem. 2019, 3(5): 1405-1414. [5] Shieh, P., Bertozzi, C. R.: Org. Biomol. Chem. 2014, 46(12): 9307-9320. [6] Wieczorek, A. et al., Wombacher, R.: Chem. Sci. 2017, 2(8): 1506-1510. [7] Uno, S.N. et al.: Nat. Chem. 2014, 6(8):681-9. [8] Werther, P., Möhler, J., Wombacher, R.: Chem. Eur. J. 2017, 23(72), 18216–18224. [9] Werther, P., Yserentant, K. et al. Herten, D.-P., Wombacher, R.: submitted.

19 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.03 Supporting resolution? Biosynthesis of specialized pro-resolving mediators (SPM) in human leukocytes

Ebert R1; Cumbana R1; Spohner, K1; Kutzner L3; Ferreirós N2; Schebb NH3; Steinhilber D1; Kahnt AS1 1 Goethe-University, Institute of Pharmaceutical Chemistry, ZAFES, Max-von-Laue-Str. 9, D-60438 Frankfurt/Main, Germany 2 Goethe University, Institute of Clinical Pharmacology, Pharmazentrum Frankfurt, ZAFES, Theodor Stern Kai 7, D-60590 Frankfurt/Main, Germany 3 Chair of Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Germany

The resolution of inflammation is an active process controlled by endogenous specialized pro-resolving mediators (SPM). Derived from polyunsaturated fatty acids such as arachidonic and docosahexaenoic acid these lipid mediators are formed by various immune cells during the course of an acute inflammatory response by the concerted action of different lipoxygenases and cytochrome P450 enzymes. However, the detailed molecular mechanisms underlying SPM biosynthesis are not completely understood. Yet, this knowledge is of great importance for the development of pro-resolving pharmacotherapies as well as a better comprehension of potential resolution-toxic effects induced by already existing anti-inflammatory therapies. Therefore, we investigated lipoxygenase expression and SPM formation in primary human immune cells such as neutrophils, polarized macrophages and various cell lines. We found that enzymes known to be involved in the biosynthesis of pro- inflammatory leukotrienes such as FLAP, cPLA2alpha, LTA4 hydrolase and LTC4 synthase influence transcellular lipoxin and resolvin formation. Furthermore, macrophage polarisation and activation considerably rearranged the enzymatic layout of the cells leading to substantial changes in enzyme, lipid mediator and cytokine patterns. In addition, an outlook on possible new players involved in SPM biosynthesis will be given.

References: 1. Lehmann, C. et al. Lipoxin and resolvin biosynthesis is dependent on 5-lipoxygenase activating protein. FASEB J. 2015 Dec 29(12):5029-43.

DPhG Annual Meeting 2019 Conference Book • 20 NEW RESEARCH, NEW RESEARCHERS I

SL.04 The application of salivary caffeine concentrations to study gastric emptying of fluids and formulations in humans

Maximilian Sager1, Philipp Jedamzik1, Michael Grimm1, Werner Weitschies1, Mirko Koziolek1 1 Department of Biopharmaceutics and Pharmaceutical Technology, Institute of Pharmacy, University of Greifswald, Felix-Hausdorff-Straße 3, 17489 Greifswald, Germany

For many orally administered drugs, the kinetics of drug plasma concentrations are closely correlated with the kinetics of gastric emptying. Hence, it is essential to understand the process of gastric emptying in healthy subjects but also in different groups of patients. Typically, in vivo methods such as magnetic resonance imaging (MRI), scintigraphy or the paracetamol absorption test are used to study gastric emptying of drinks, food or oral formulations in humans. However, these methods are either not applicable on a routine basis, costly and time- consuming or require the presence of medically trained personnel. It was therefore our aim to find an in vivo method that can be used to determine gastric emptying of fluids and formulations in humans in a simple, inexpensive and non-invasive manner. Since saliva is an easily accessible body fluid that can be sampled at a high frequency, we aimed to find a marker for gastric emptying that shows a high correlation between plasma and saliva concentrations. In this regard, caffeine seemed to be well suited in humans. In a proof-of-concept study with 6 healthy subjects, a low dose of caffeine (35 mg) was administered in fasted and fed state together with 240 mL of water. To prevent the contamination of the oral cavity but still to allow the labelling of the liquid phase, caffeine was administered in form of an ice capsule. Subsequently, we compared caffeine concentrations in saliva determined by LC-MS/MS with gastric emptying kinetics determined by MRI. The results of this study confirmed the hypothesis that the caffeine concentrations in saliva are able to describe the gastric emptying kinetics of water in humans.1 In another study, hard gelatin capsules containing 50 mg of caffeine were administered in fasted state with 240 mL of water. In order to directly compare the disintegration times of the capsules, saliva sampling and MRI scans were performed simultaneously.2 The comparison of the disintegration times revealed a difference of around 4 min between the two methods. Since capsule disintegration occurred mostly in the stomach, salivary caffeine concentrations only increased after disintegration was observed in MR images. This delay was caused by gastric mixing and emptying as well as by intestinal absorption of caffeine. Nonetheless, the application of caffeine was still useful for characterizing the in vivo behavior of oral formulations and thus, was used in follow-up studies. In the future, this simple and reliable method shall be applied in different groups of patients (e.g. geriatric patients, patients with diabetes) to study gastric emptying of various fluids as well as to describe the in vivo transit and disintegration behavior of different oral formulations. These data will be directly implemented into physiologically relevant in vitro test methods and physiologically based pharmacokinetic (PBPK) models in order to predict the extent of changes associated, for instance, with age or certain diseases. This technique will therefore be very useful in optimizing oral pharmacotherapy by better understanding the behavior of oral drug products in the human gastrointestinal tract.

This work was partly performed within the OrBiTo project (http://www.orbitoproject.eu), which is funded by the Innovative Medicines Initiative Joint Undertaking (http:// www.imi.europa.eu) under Grant Agreement no. 115369, resources of which are composed of financial contribution from the European Union’s Seventh Framework Program and EFPIA companies in kind contribution.

References: 1. Sager M, Jedamzik P, Merdivan S, Grimm M, Schneider F, Kromrey M-L, Hasan M, Oswald S, Kühn J, Koziolek M, Weitschies W. 2018. Low dose caffeine as a salivary tracer for the determination of gastric water emptying in fed and fasted state: A MRI validation study. Eur J Pharm Biopharm 127: 443–452. 2. Sager M, Grimm M, Jedamzik P, Merdivan S, Kromrey M-L, Hasan M, Koziolek M, Tzvetkov M V., Weitschies W. 2019. Combined Application of MRI and the Salivary Tracer Technique to Determine the in Vivo Disintegration Time of Immediate Release Formulation Administered to Healthy, Fasted Subjects. Mol Pharm 16: 1782-1786.

21 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

3.2 Ion channels as drug targets Chairs: P. Ruth, M. Freichel

SL.05 NAADP: a fundamental modulator of Ca2+ release channels in Lymphocytes

Andreas H. Guse The Calcium Signaling Group, Dept. of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany

Nicotinamide adenine dinucleotide (NAD) has long been known as cofactor of oxidoreductases. For a long period of time this task in metabolism has been recognized as major, if not the only function of NAD. However, over the past couple of years it became clear that NAD also serves as (i) precursor for Ca2+ mobilizing second messengers, (ii) substrate for posttranslational protein modifications (mono- or poly-ADP-ribosylation), and (iii) as acceptor for acetyl groups during de-acetylation reactions. Ca2+ mobilizing second messengers play an important role in almost all cell types and in many signaling processes. In particular, Ca2+ signaling during T-lymphocyte activation is essential for the adaptive immune response. In recent years, we have analyzed the role of NAD-derived Ca2+ mobilizing second messengers during this process. Biochemical time course analysis in T cells stimulated via the T cell receptor/CD3 complex revealed nicotinic acid adenine dinucleotide phosphate (NAADP) as earliest 2nd messenger, followed by D-myo-inositol 1,4,5- trisphosphate (IP3), and cyclic ADP-ribose. On the sub-second and second timescale of T cell activation we recently characterized Ca2+ microdomains involving type 1 ryanodine receptors activated by NAADP (1,2). In addition, Ca2+ entry via ORAI1, controlled by STIM1 and STIM2, constitutes a further essential mechanism underlying Ca2+ microdomains (2). Though TRPM2 does not appear to be involved in Ca2+ microdomains, we recently identified 2’-deoxy-ADPR (2dADPR) as a significantly better TRPM2 agonist than ADPR (3). Using HPLC and mass spectrometry, endogenous 2dADPR was detected in Jurkat T-lymphocytes. Consistently, cytosolic nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) and NAD-glycohydrolase CD38 are able to sequentially catalyze synthesis of 2dADPR from nicotinamide mononucleotide and 2ʹ-deoxy-ATP in vitro. Taken together, adenine nucleotide 2nd messengers NAADP and 2dADPR play significant roles in shaping T cell Ca2+ signaling.

1. Wolf IM, …, Guse AH. Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor. Science Signal. 2015; 8: ra102. 2. Diercks B-P, …, Guse AH, ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+ microdomains upon T cell activation. Science Signal. 2018;11(561): eaat0358. 3. Fliegert R, …, Guse AH. 2ʹ-Deoxyadenosine 5ʹ-diphosphoribose is an endogenous TRPM2 superagonist, Nature Chem Biol. 2017;13(9):1036-1044.

DPhG Annual Meeting 2019 Conference Book • 22 ION CHANNELS AND DRUG TARGETS

SL.06 Agonist-Dependent switching of Ion selectivity in endolysosomal TPC2 channel

Yu-Kai Chao1*, Susanne Gerndt2*, Cheng-Chang Chen2*, Yu Yuan3*, Einar Krogsaeter1, Franz Bracher2*, Sandip Patel3*, Christian Grimm1* 1Walther Straub Institute of Pharmacology and Toxicology, Faculty of Medicine, Ludwig-Maximilians-Universität, Munich, Germany 2Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians-Universitat, Munich, Germany 3Department of Cell and Developmental Biology, University College London, London, UK.

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we probed the ion selectivity of the lysosomal ion channel TPC2 that is hotly debated. Both the ion selectivity and activating ligand(s) of TPCs are equivocal. Initial studies characterized TPCs as non-selective Ca2+-permeable channels activated by + NAADP. But other studies indicate that TPCs are highly-selective Na channels and activated directly by PI(3,5)P2. We identify two structurally distinct, cell-permeable TPC2 agonists through a high throughput screen. One of these evoked robust Ca2+ signals in live cells and non-selective cation currents in direct endo-lysosomal patch clamp recordings. In contrast, the other evoked weaker Ca2+ signals and Na+-selective currents. Physiologically, these 2+ properties were mirrored by the Ca mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Novel small molecules thus mimic disparate intracellular signalling cues in switching the ion selectivity of TPC2. A new paradigm whereby a single ion channel mediates distinct ionic signatures on demand emerges.

23 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.07 Identification of OCaR1 as a gatekeeper of lysosomal-granular Ca2+ release and regulated exocytosis

Volodymyr Tsvilovskyy1*, Ulrich Kriebs1*, Aline Bach1*, Archana Jha2, Angela Wirth1, Christian Grimm3, Ulrich Wissenbach4, Guido Wabitz7, Katrin Zimmermann5, Petra Weissgerber4, Dominik Vogt1, Dagmar Schumacher1, Juan Camacho-Londono1, Yvonne Samstag7, Christoph Dieterich8, Christian Wahl-Schott3, Oliver Strobel9, Alexander Pfeifer5, Martin Biel3, Peter Lipp6, Veit Flockerzi4, Shmuel Muallem2, Marc Freichel1 1 Pharmakologisches Institut, Ruprecht-Karls-Universitaet Heidelberg, Germany 2 National Institute of Health/NIDCR, Bethesda, USA 3 Institut für Pharmakologie, Ludwig-Maximilians-Universität München, Germany 4 Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, Homburg Germany 5 Institut für Pharmakologie und Toxikologie Friedrich-Wilhelms-Universität Bonn Germany 6 Institut für Molekulare Zellbiologie, Universität des Saarlandes, Homburg Germany 7 Institut für Immunologie, Ruprecht-Karls-Universitaet Heidelberg, Germany 8 Abteilung Bioinformatik, Universitaetsklinikum Heidelberg, Germany 9 Klinik für Allgemeinchirurgie, Universitaetsklinikum Heidelberg, Germany * shared authorship

Exocytosis triggered by agonists depends on an increase of the Ca2+ concentration in close vicinity of secretory granules and is strictly limited in resting cells. We identified a novel organellar calcium regulator we termed OCaR1, which tightly controls Ca2+ release from lysosomal and secretory granules. OCaR1 is a putative transmembrane protein that co-localises with markers of endo-lysosomal organelles. In secretory cells such as pancreatic acinar cells OCaR1 also locates to secretory granules as demonstrated using cells from OCaR1-YFP knock add on mice. Acinar cells of OCaR1-deficient mice exhibit an increase in both spontaneous and regulated exocytosis which goes along with an enlargement and globalisation of intracellular Ca2+ oscillations. Notably, exaggerated Ca2+ oscillations in OCaR1-deficient acini can be suppressed by a NAADP antagonist or deletion of two pore channel 2 (TPC2). OCaR1 controls spontaneous and agonist-evoked Ca2+-dependent exocytosis also in mast cells, another secretory cell type, suggesting a broader prevalence of this regulatory mechanism. Together, our results provide the first demonstration that OCaR1 in lysosomes and secretory granules operates as a gatekeeper of exocytosis, a process, known to contribute in the development of pancreatitis and, possibly, to pancreatic malignancies.

DPhG Annual Meeting 2019 Conference Book • 24 ION CHANNELS AND DRUG TARGETS

SL.08 Determination of fatal ventricular arrhythmias by a new regulator of Ca2+ signaling at the lysosomal-SR junction

Juan E. Camacho Londoño1, Michael Berlin1, Vladmir Kuryshev1/2, Sören Meyer3, Christoph Hofmann4, Lucas Bacmeister1, Ulrich Kriebs1, Mirko Völkers4, Florian Leuschner3, Christoph Dieterich2 and Marc Freichel1 1. Pharmakologisches Institut and Innere Medizin III: 2. Biomatik und Systemkardiologie, Klaus Tschira Institute for Computational Cardiology, 3. AG Innate Immunity in Cardiovascular Disease and 4. AG Molecular Biology of Cell Growth. Ruprecht-Karls-Universität Heidelberg, D-69120 Heidelberg, Germany. *All authors belong to the DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, Germany

Altered Ca2+ release from intracellular stores in cardiomyocytes is a trigger of ventricular arrhythmias. Recently, it was shown that Two-pore channels (TPC) are activated by nicotinic acid adenine dinucleotide phosphate (NAADP) after catecholamine stimulation in cardiomyocytes, and that TPC2 at lysosomal/sarcoplasmic reticular junctions contribute to the development of cardiac hypertrophy and arrhythmia through β-adrenergic signaling. We identified novel Organellar Ca2+ Regulator proteins (termed OCaR1 and OCaR2) contributing to Ca2+ release from acidic organelles and we aimed to determine their implication in pathological cardiac remodeling. Methods: We generated global and cardiomyocyte-specific OCaR1- and OCaR2-deficient mice (KO) by gene targeting in ES cells. Ca2+ signaling was analyzed in isolated adult cardiomyocytes under resting and pacing conditions, and using several agonists/antagonists to address NAADPsensitive Ca2+ stores including NAADP, Isoproterenol and Ned-19 among others. We induced cardiac remodeling by chronic Isoproterenol (ISO) infusion. Cardiac contractility was determined in vivo by pressure-volume (P/V) loop measurements under Isoflurane anesthesia. Telemetric ECGs were recorded in conscious freely moving mice. Whole transcriptome sequencing was done by NGS and we analyzed changes in gene expression during cardiac remodeling. Results: OCaR1 and OCaR2 transcripts are detected in isolated adult mouse cardiomyocytes. In resting cardiomyocytes from global (KO) or cardiomyocyte-specific (OCarR2 fx/fx/αMHC-Crepos) OCaR2-deficient mice, ISO and NAADP evoked spontaneous intracellular Ca2+ oscillations that were not observed in cells from corresponding control mice or from OCaR1-KO mice. Exaggerated ISO-evoked Ca2+ transients in OCaR2-deficient cardiomyocytes were blocked by the NAADPantagonist Ned-19. In beating (1Hz pacing) OCaR2-deficient myocytes, the occurrence of spontaneous diastolic Ca2+ transients was increased by more than 50% under ISO (100nM) stimulation. After chronic (7d) ISO treatment, OCarR2 cardiomyocyte-specific mice developed increased cardiac hypertrophy accompanied by significantly different gene regulation. In contrast, under basal conditions and ISO stimulation, P/V loop analysis revealed no differences in cardiac contractility parameters in cardiomyocyte-specific OCaR2-deficient mice. However, ECG telemetry revealed that the overstimulation of the β-adrenergic axis in OCarR2fx/fx/αMHC-Crepos mice evoked polymorphic ventricular tachycardia that resulted in sudden cardiac death within 20-40 min that was not observed in any of the control animals. Additionally, after a cardiac model of ischemia/reperfusion OCarR2 cardiomyocyte-specific mice presented and increased occurrence of ISO-triggered salvos accompanied by decreased cardiac function. Conclusion: We identified OCaR2 proteins in cardiomyocytes as key regulators of Ca2+ oscillations arising from NAADP-sensitive Ca2+ stores that are triggered by β-adrenorecepto stimulation. Inactivation of OCaR2 proteins in murine cardiomyocytes had no impact on basal cardiac contractility or on the inotropic response to catecholamines. In contrast, OCaR2 proteins are critical determinants of neurohumoral-induced cardiac hypertrophy as well as of the development of fatal catecholaminergic ventricular tachycardia and cardiac dysfunction.

25 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

3.3 Fachsymposium Dermatopharmakologie: “Predictivity of in vitro tests in pharmacology and toxicology” Chairs: G. Weindl, J. Klein

SL.09 Reconstructed human skin can predict the efficacy and safety of drugs in humans

Schäfer-Korting M Freie Universität Berlin, Institut für Pharmazie (Pharmakologie und Toxikologie), Königin-Luise-Str. 2+4,14195 Berlin, Germany

The poor success rate of drugs in clinical studies which have been successfully tested preclinically (3.4-33%; Wong et al, 2019) indicates the need for improved preclinical approaches. Reconstructed human tissues overcome the species gap between animal and man. Respective models of diseased tissues and introducing diversity should also restrict other limitations of animal experiments. Testing in rodents often makes use of a single strain and animals are of single sex and close in age – in order to keep data variability and thus sample size low. Yet, thereby diversity is eliminated. At Freie University Berlin we have established the technique of reconstructing full-thickness human skin (RHS) by standardized coculture of juvenile human keratinocytes and fibroblasts. Atopic dermatitis can be modeled by the knock-down of the filaggrin gen and co-culture with the proinflammatory cytokines IL-4 and interleukin-13 resulting in a less developed stratum corneum barrier and a shift of the normal skin surface pH to pH 6.4 (Hönzke et al, 2016). The efficacy of topical applied glucocorticoids can be derived from the nuclear translocation of the glucocorticoid receptor resulting in a decline in inflammatory proteins. Drug penetration into the tissue can be quantified by chromatographic approaches as well as by stimulated X-ray microscopy, the latter allows for high- end local resolution (Yamamoto et al, 2016). Dexamethasone penetration into RHS and human skin ex vivo is close (Wanjiku et al., in press). The model of atopic skin even allows to study drugs delivered by nanoparticulate carrier systems (Giulbudagian et al, 2017). Skin thinning, the dominant glucocorticoid induced adverse drug reaction following the topical use becomes obvious by impaired collagen synthesis (Weindl et al, 2011). Glycation induced clumpsy collagen characterizes the skin in old age and in diabetes mellitus. This damage can be included into RHS by collagen pre-incubation with ribose (Balansin Rigon et al, 2018). An alternative approach to aged RHS is the use of fibroblast from aged donors (Hausmann et al, 2019a). Those constructs depict the typical morphological changes and the poorer access of drugs to viable skin which is in accordance with studies on fentanyl penetration of skin obtained from aged donors (Holmgaard et al, 2013). Acceptance of the proposed preclinical approach will increase with proven predictivity. The qualification of the human-based testing can be documented by studying drug effects in the model and in human – preferably first in the model and next in human. The protocols need to confirm with the high standards of clinical studies. Using reconstructed human epidermis for the in vitro approach, we observed a major increase in filaggrin formation and less caffeine permeation following Lactococcus lysate. The improved barrier in the construct is paralleled by a decrease in transepidermal water loss in 30 female volunteers. Skin irritation was neither detected in RHE nor reported by the volunteers. Because of an enhanced betadefensin-2 formation in the construct, the antimicrobial barrier may improve, too (Hausmann et al., 2019b). Taken together the results show the possibility to introduce disease and diversity into a reconstructed human tissue as well as a first approach into the essential qualification of the human-based testing in preclinical research. Federal Ministry of Education and Research (BB3R), German Research foundation (CRC 1122), Sebapharma GmbH & Co. KG Wong CH, Siah KW, Lo AW, Biostatistics 2019: 20, 273-286 Hönzke S et al: J. Invest. Dermatol. 2016: 136, 631-9 Giulbudagian M et al: Nanoscale 2017: 10, 469-479 Yamamoto K et al: Anal. Chem. 2015: 87, 6173-9 Wanjiku B et al., Anal. Chem. in press Weindl G et al: ATLA 2011: 39, 173-187 Balansin Rigon R et al.: Int. J. Mol. Sci. 2018 19 Hausmann C et al.: Sci. Rep. 2019: 2913 Holmgaard R et al.: Skin Pharmacol. Physiol. 2013: 26, 155-9 Hausmann C et al.: Skin Pharmacol. Physiol. 2019: 32, 72-80

DPhG Annual Meeting 2019 Conference Book • 26 FACHSYMPOSIUM DERMATOPHARMAKOLOGIE

SL.10 Adverse outcome pathway-based concepts and tools for assessing skin sensitization

Kolle S1; Birk B1; Mehling A2; Landsiedel R1 1 BASF SE, Experimental Toxicology and Ecology, RB/TB Z470, Carl Bosch-Straße 38, 67056 Ludwigshafen am Rhein, Germany 2 BASF Personal Care and Nutrition GmbH, Henkelstraße 67, 40589 Düsseldorf, Germany

In general, no single non-animal method can fully cover the complexity of any given animal test; rather, data from several approaches are needed to address an in vivo toxicity endpoint. Chemicals often induce specific molecular key events that can initiate a sequence of early cellular key events (KE), which may result in an observable toxic effect in vivo. This sequence is termed an adverse outcome pathway (AOP). Integrating information from in vitro, in silico, and in chemico methods into toxicity testing strategies has been widely considered the way of providing adequate and relevant information about chemicals’ hazardous properties.

Skin sensitization test data are required or considered by chemical regulation authorities around the world. These data are used to develop product hazard labeling for the protection of consumers or workers and to assess risks from exposure to skin sensitizing chemicals [1]. There has been significant progress in recent years in the development and application of new methods for assessing the skin sensitization potential of chemicals. The KE of in skin sensitization have been described in an AOP [2]. To date, OECD test guidelines are available for non-animal test method addressing the first three KE: protein binding [direct peptide reactivity assay, DPRA, OECD 442C), keratinocyte- (LuSens and KeratinoSens, OECD 442D) and dendritic cell activation (e.g. h-CLAT, OECD 442E). Numerous prediction models have been proposed to combine the results of three assays (and other information sources); 12 of them have been listed as case studies by the OECD [3]. Among them, the “2 out of 3” integrated testing strategy (ITS) is a simple defined approach (DA) that has demonstrated very good sensitivity, specificity and overall accuracy numbers for predicting the skin sensitization potential. Chemicals with at least two positive results in tests addressing KE 1–3 are predicted sensitizers, while chemicals with none or only one positive outcome are predicted non-sensitizers. The ‘2 out of 3’ prediction model achieved accuracies of 90% or 79% when compared to human or LLNA data, respectively [4] and thereby even exceeded the predictivity of the LLNA. The methods used by the “2 out of 3” ITS prove to also recognise pre- and prohaptens [5]. A recent analysis of discordant results (1 out of 3 positive) found that most of these cases are due to borderline results of an assay [6] The problem of borderline results (i.e. assay read-outs close to the classification thresholds) has been recognised and described for skin sensitization methods in vitro and in vivo [7]. While the skin sensitization hazard of substances can already be identified by non-animal methods, the classification of potency is still challenging. A kinetic DPRA (kDPRA) method was developed to determine the reaction rate constant of haptens with model peptides [8]. In a recent ring-trial, the method proved to be robust and reproducible; chemicals’ reaction rate constants correlated well with their skin sensitization potencies. Testing for skin sensitization is regarded a vanguard of modern toxicological testing, addressing complex toxic effects by combining different assays which address KEs of an AOP. For skin sensitisation, we have sufficient methods and prediction models at hand, and we know their applicabilities and limitations. This should allow for a full replacement of animal studies, which is currently not quite completed [9].

References: Kleinstreuer, Nicole C., et al. "Non-animal methods to predict skin sensitization (II): an assessment of defined approaches." Critical reviews in toxicology 48.5 (2018): 359-374.

27 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

Kolle, Susanne N., et al. "Modern Skin Toxicity Testing Strategies." Environment and Skin. Springer, Cham, 2018. 27-40. OECD, Guidance Document on the Reporting of Defined Approaches and Individual Information Sources to be Used within Integrated Approaches to Testing and Assessment (IATA) for Skin Sensitisation, OECD Series on Testing and Assessment, No. 256, OECD Publishing, Paris, (2017) https://doi.org/10.1787/9789264279285-en Urbisch, Daniel, et al. "Assessing skin sensitization hazard in mice and men using non-animal test methods." Regulatory Toxicology and Pharmacology 71.2 (2015): 337-351. Urbisch, Daniel, et al. "Assessment of pre-and pro-haptens using nonanimal test methods for skin sensitization." Chemical research in toxicology 29.5 (2016): 901-913. Kolle, Susanne N., et al. "A review of substances found positive in 1 of 3 in vitro tests for skin sensitization." Regulatory Toxicology and Pharmacology (2019). Leontaridou, Maria, et al. "The borderline range of toxicological methods: Quantification and implications for evaluating precision." ALTEX- Alternatives to animal experimentation 34.4 (2017): 525-538. Wareing, Britta, et al. "Prediction of skin sensitization potency sub-categories using peptide reactivity data." Toxicology in Vitro 45 (2017): 134-145. Sauer, Ursula G., et al. "Local tolerance testing under REACH: accepted non-animal methods are not on equal footing with animal tests." ATLA - Altern Lab Anim 44.3 (2016): 281-99.

DPhG Annual Meeting 2019 Conference Book • 28 FACHSYMPOSIUM DERMATOPHARMAKOLOGIE

SL.11 Reconstructed tissue models with integrated immune cells – Do we need advanced in vitro tests for the evaluation of drugs and chemicals?

Weindl G1 1Universität Bonn, Pharmazeutisches Institut, Abteilung Pharmakologie und Toxikologie, Gerhard-Domagk-Str. 3, 53121 Bonn, Germany

To ensure human safety, the benefit-risk assessment of drugs and chemicals requires relevant and reliable test methods. Animal models have historically been widely used for pharmacological and toxicological studies, however, the limited biological relevance of animal models to human as well as ethical and regulatory pressure underlines the urgent need for alternative in vitro test systems. Three-dimensional multilayer skin constructs are one of the most developed and advanced in vitro engineered tissue models that eliminate species differences while at the same time saving animal experiments. Besides testing for skin irritation, corrosion and phototoxicity, reconstructed skin is increasingly playing a role in the evaluation of complex endpoints such as genotoxicity or sensitization. Furthermore, engineered in vitro skin disease models have been established in basic and preclinical research. The immune system is critically involved in most human skin diseases. Skin-resident dendritic cells, including Langerhans cells localized in the epidermis, are the most relevant subset of immune cells during the sensitization phase of allergic contact dermatitis and the initial mechanisms of skin inflammation in atopic dermatitis. Yet, commercially available models including reconstructed human epidermis and reconstructed human full-thickness skin, which is comprised of an additional dermis equivalent, largely lack immunocompetent cells. To overcome this limitation, in-house skin models with integrated immune cells have been successfully developed although the stable integration into reconstructed skin poses a particular challenge. The phenotype and functionality of the integrated immune cells in the in vitro environment must be preserved to study major processes in dendritic cells such as differentiation, antigen uptake, maturation and migration. The technically challenging and labour-intensive reconstruction of complex tissue models may be less relevant for regulatory testing which requires robust, transferable and standardised test methods. In preclinical research, advanced tissue models reflect the physiological and pathophysiological processes more reliably than cell-based assays in monoculture or simpler tissue models and allow the analysis of immune-molecular mechanisms under controlled experimental conditions.

Financial support by Federal Ministry of Education and Research (Berlin-Brandenburg research platform BB3R; project 031A262A) is gratefully acknowledged.

29 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.12 Human cell-based tumor microenvironment models for improved preclinical drug development

Christian Zoschke1, Leonie Gronbach1, Christopher Wolff1, Konrad Klinghammer2, Ingeborg Tinhofer-Keilholz2, Ulrich Keilholz3, Monika Schäfer-Korting1 1. Freie Universität Berlin, Institute of Pharmacy (Pharmacologc and Toxicology), Königin-Luise-Str. 2+4,14195 Berlin, Germany 2. Charité - Universitätsmedizin Berlin, Department of Hematology and Oncology, Hindenburgdamm 30, 12203 Berlin, Germany 3. Charité - Universitätsmedizin Berlin, Charité Comprehensive Cancer Center, Charitéplatz 1, 10117 Berlin, Germany

Epithelial carcinomas like head and neck and non-melanoma skin cancer are among the most frequent cancers in man. Thereby, squamous cell carcinomas of the head and neck metastasize more frequently than their cutaneous counterparts, contributing to the high mortality. Recent studies in various cancer entities suggest the determining role of cancer microenvironment for the tumor's response to chemotherapy together with the challenges of intra- tumor heterogenicity (Stanta et al. 2018 Front Med, Ren et al. 2018 Mol Cancer). Herein, we emulate oral and cutaneous squamous cell carcinoma in stratified tumor microenvironment models and test drug efficacies. Moreover, we investigate the effects of confounders like body-site or the glycation of the extracellular matrix on epithelial differentiation. Cancer cells, either from patient derived tumor tissue or cell lines, were grown together with normal primary keratinocytes and fibroblasts in a 3D in vitro model. Oral mucosa models with patient-derived cancer cells reliably preserved the tumor grading, while the cell lines reproducibly emulated one tumor grading. While three applications of 0.7 µg/mL docetaxel induced cell death and reduced proliferation, cetuximab (10 µg/mL or 100 µg/mL) failed to alter these parameters. Ingenol mebutate (150 µg/g) almost completely eradicated cancer cells from cutaneous cancer models following three applications of the commercial gel (Zoschke et al. 2016 J Control Rel). The body site of which fibroblasts were isolated determined the epithelial differentiation in the respective skin constructs as shown for altered expression of e.g. granulocyte-macrophage colony-stimulating factor, e-cadherin, and filaggrin (Hausmann et al. 2019 Sci Rep). Next, we replaced included a glycated extracellular matrix, being typical for aged or diabetic persons. These glycated constructs showed increased proliferation and differentiation in the epithelial layers (Rigon & Kaessmeyer et al., 2018 Int J Mol Sci). In conclusion, stratified tumor microenvironment models closely mimic epithelial cancers in vitro and capture certain drug effects. However, fibroblasts and the extracellular matrix extensively modulate epithelial differentiation in these models. Emulating heterogeneous cancers in heterogeneous tumor microenvironments might improve the currently low predictive capacity of in vitro models for anti-cancer effects in patients.

Federal Ministry of Education and Research (BB3R), German Research foundation (CRC 1122)

Stanta et al.: Front Med. 2018: 85 Ren et al.: Mol Cancer. 2018: 17 Rigon R & Kaessmeyer S et al.: Int. J. Mol. Sci. 2018: 19 Hausmann C et al.: Sci. Rep. 2019: 2913 Zoschke C et al.: J Control Rel. 2016: 233

DPhG Annual Meeting 2019 Conference Book • 30 CYSTEIN-REACTIVE INHIBITORS AND PROBES

3.4 Cysteine-reactive inhibitors and probes Chair: T. Schirmeister

SL.13 Cysteine, pKa, covalent attack: any correlation?

Czodrowski, P.

Abstract not available.

31 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.14 Novel Cathepsin B Inhibitors with Inversely Oriented Warheads

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

Cathepsin B, a papain-like cysteine protease, is involved in various physiological processes. In tumor cells, it adopts a peripheral distribution and affects the extracellular matrix either directly by extracellular proteolytic degradation of its components or indirectly via activation of other proteases. The breakdown of the extracellular matrix remodels the tumor environment, promotes tumor invasion, and enables angiogenesis and metastasis. The crucial roles of cathepsin B at multiple points of the tumor development have been established in several in vitro and in vivo models and cathepsin B was proposed to be a prognostic marker in patients with various types of cancer [1,2]. The typical feature of peptidic inhibitors of cysteine proteases includes an N-capped peptide structure bearing an electrophilic warhead (e.g. a nitrile [3-7], aldehyde, halomethyl or acyloxymethyl ketone or Michael acceptor) in place of the scissile peptide bond. A linker can direct a carboxylic group to the S’ region to allow for an advantageous salt bridging with the histidine residues of the occluding loop of cathepsin B, thus enhancing cathepsin B selectivity [8]. In E-64-derived epoxysuccinyl derivatives, a peptide part can bind along the S subsites of cathepsin B, while a dipeptide moiety is oriented towards the S’ sites [9,10]. We have designed cathepsin B inhibitors with dipeptide portions directed towards the occluding loop and equipped with fine-tuned, inversely oriented warhead structures which are cleaved upon the action of the active site cysteine leading to irreversible inhibition.

- S Cl O O N N S N O N - - H Cl H O CO2 O CO2 - O

Formation of an S-carbamoyl enzyme through the reaction of cathepsin B with a representative inhibitor.

Kinetic data at four human cathepsins obtained for an extended series of around 200 unpublished representatives of this chemotype demonstrated their selectivity for cathepsin B, supported the exciting mode of action and allowed to draw detailed structure-activity relationships. The synthesis and the biochemical characterization of these novel cysteine protease inhibitors will be presented. The tailored structure of our new inhibitors allows the design of PET ligands and (in continuation of our previous studies [11-13]) linker connection for the assembly of activity-based probes.

References: 1. Fonović, M., Turk, B.: Biochim. Biophys. Acta 2014, 1840(8): 2560–2570. 2. Reiser, J., Adair, B., Reinheckel, T.: J. Clin. Invest. 2010, 120(10): 3421–3431. 3. Frizler, J. et al.: Chem. Eur. J. 2011, 17(41): 11419–11423. 4. Frizler, J. et al.: J. Med. Chem. 2011, 54(1): 396–400. 5. Frizler, J. et al.: J. Med. Chem. 2012, 55(12): 5982–5986. 6. Schmitz, J. et al.: ACS Med. Chem. Lett. 2014, 5(10): 1076–1081. 7. Kohl, F. et al.: Org. Biomol. Chem. 2015, 13(41): 10310–10323. 8. Schmitz, J. et al.: ACS Med. Chem. Lett. 2016, 7(3): 211–216. 9. Schaschke, N. et al.: FEBS Lett. 1998, 421(1): 80–82. 10. Schaschke, N. et al.: FEBS Lett. 2000, 482(1-2): 91–96. 11. Häußler, D. et al.: Chem. Eur. J. 2017, 23(22): 5205–5209. 12. Schulz-Fincke, A.C. et al: Biochemistry 2018, 57(5): 742–752. 13. Steinebach, C. et al.: Chem. Commun. 2019, 55(12): 1821–1824.

DPhG Annual Meeting 2019 Conference Book • 32 CYSTEIN-REACTIVE INHIBITORS AND PROBES

SL.15 Covalent Kinase Inhibition by Reversible and Irreversible Cysteine Targeting

Gehringer, M.1; Forster, M. 1; Laufer, S.A.1 1Eberhard Karls Universität Tübingen, Institute of Pharmaceutical Sciences, Department of Pharmaceutical/Medicinal Chemistry, Auf der Morgenstelle 8, 72076 Tuebingen, Germany

Covalent inhibitors have a long history in medicinal chemistry and over 40 drugs relying on a covalent mechanism are currently approved. Typically, such compounds address catalytic nucleophiles (e.g. cysteine, serine or threonine residues) in the active sites of enzymes. The mode of action of most approved covalent modifiers has been discovered serendipitously while the use of reactive groups in rational drug design has long been regarded with skepticism due to potential issues arising from irreversible off-target modification, haptenization and idiosyncratic toxicity.[1] Within the last decades, however, covalent targeting resurged as a promising strategy in drug discovery and the concept of "targeted covalent inhibitors" (TCIs) evolved.[2] TCIs address non-catalytic amino acid side chains by means of covalent reactive groups (CRGs) frequently termed "warheads". The latter are supposed to react with the target amino acid upon ligand binding in a proximity-driven manner and inherent warhead reactivity should be limited to a necessary minimum. If properly designed, TCIs can have various advantages such as increased potency and specificity, limited competitivity with natural ligands or substrates (e.g. ATP in the case of kinases) and, depending on the target's turnover rate, a decoupling of pharmacodynamics from pharmacokinetics enabling prolonged therapeutic effects while minimizing exposure.[1,2] The TCI concept has been most successfully applied to kinases[3] with currently six drugs approved by the FDA. Due to its high intrinsic nucleophilicity, cysteine is the residue most amenable to TCI design. All of the approved covalent kinase inhibitors, as well as the vast majority of experimental TCIs address poorly conserved cysteines by using carboxamide-derived Michael acceptors as the electrophiles. However, with the employment of more diverse warhead types, other residues such as tyrosine or lysine are becoming increasingly ligandable.[4] Moreover, covalent-reversible targeting approaches, which might resolve some of the aforementioned liabilities associated with irreversible TCIs, have recently gained attention.[4] Our current research is focused on the identification of highly specific covalent kinases inhibitors. We employ structure-based design approaches to address particular cysteines with classical electrophiles (i.e. α,β-unsaturated amides), but also with less common warhead chemistries (e.g. SNAr-reactions). By applying covalent trapping strategies, we were able to generate irreversible and covalent-reversible inhibitors with exquisite selectivity. For example, we designed highly isoform- and kinome-selective Janus kinase (JAK) 3 inhibitors by engaging a poorly conserved cysteine absent in the other JAK family members.[5,6] These efforts, as well as recent work on covalent probes for kinases featuring a cysteine residue in the so-called "hinge region", will be presented.

References: [1] Bauer, R.: A. Drug Discovery Today 2015, 20 (9), 1061−1073. [2] Singh, J., Petter, R. C., Baillie, T. A.: Nat. Rev. Drug Discovery 2011, 10 (4), 307−317. [3] Chaikuad, A., Koch, P., Laufer, S. A., Knapp, S.: Angew. Chem. Int. Ed. 2018, 57 (16), 4372−4385. [4] Gehringer, M., Laufer, S. A.: J. Med. Chem. 2019, ASAP, 10.1021/acs.jmedchem.8b01153 [5] Gehringer, M., Forster, M., Laufer, S. A.: ACS Comb. Sci. 2015, 17 (1), 5–10. [6] Forster, M., Chaikuad, A., Dimitrov, T., Döring, E., Holstein, J., Berger, B.-T., Gehringer, M., Ghoreschi, K., Müller, S., Knapp, S., Laufer, S. A.: J. Med. Chem. 2018, 61 (12), 5350−5366.

33 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

3.5 Dysfunctional Cellular Signaling in Cardiovascular Diseases Chairs: T. Wieland, R. Gilsbach

SL.16

Selective inhibition of nuclear ERK1/2 functions – two quite distinct implications: cardiac hypertrophy and cancer?

Lorenz, K.

Abstract not available.

DPhG Annual Meeting 2019 Conference Book • 34 DYSFUNCTIONAL CELLULAR SIGNALING IN CARDIOVASCULAR DISEASES

SL.17 Identification and modulation of chromatin interactions in cardiac myocytes

Martin Schwaderer1,2, Stephan Nothjunge1,2, Joachim Wolff3, Theresa Kehl1, Björn A. Grüning3, Lutz Hein1,4, Ralf Gilsbach1 1Institute of Experimental and Clinical Pharmacology and Toxicology, Albertstrasse 25, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany 2Hermann Staudinger Graduate School, Albertstrasse 21, University of Freiburg, 79104 Freiburg, Germany 3Bioinformatics Group, Department of Computer Science, Georges-Köhler-Allee 106, University of Freiburg, 79110 Freiburg, Germany 4BIOSS Centre for Biological Signaling Studies, Schänzlestrasse 1, University of Freiburg, 79104 Freiburg, Germany

Background: Epigenome studies in cardiac myocytes revealed dynamic establishment of active regulatory sites during development and disease (Gilsbach et al., Nat. commun. 2018). The aim of this project was to identify target promoters of distal regulatory sites and to prove the functional relevance of chromatin interactions in cardiac myocytes. Methods: We performed Hi-C and Promoter Capture Hi-C experiments to analyze higher order chromatin organization and promoter interactions in cardiac myocytes. We perturbed active regulatory sites using the CRISPRi system. CRISPRi utilizes a programmable catalytic-deficient Cas9 fused to the KRAB repressor domain. ChIP-seq, RNA-seq and Western Blot analysis were carried out to access the functional consequences of promoter and enhancer perturbation. Results: Our Hi-C experiments revealed that the higher order chromatin organization, including TADs and A/B compartments, remains stable in cardiac myocyte postnatal development and disease. We furthermore generated a promoter interaction map for newborn, adult and diseased cardiac myocytes. In total we detected more than 30.000 genomic elements contacting 2432 genes. These interactions were strongly enriched for enhancer-promoter and promoter-promoter interactions. Among those were for example a spatial interaction between the NPPA and NPPB promoter and between a putative enhancer and Gata6. CRISPRi-mediated silencing of the NPPA promoter induced heterochromatin formation at the NPPA promoter and silencing of NPPA and NPPB gene expression, indicating that the NPPA promoter has enhancer function and controls NPPB gene expression. Since Gata6 has been shown to be implicated in pathological cardiac hypertrophy, we further used CRISPRi to silence a putative enhancer 320kb downstream of Gata6. This resulted in reduced gene expression, protein levels and genome-wide binding of Gata6. RNA-seq experiments revealed that enhancer-mediated silencing of Gata6 enhancer affected more than 600 genes. These genes were significantly associated with cardiovascular developmental and stress processes. Conclusion: This study unraveled promoter interactions in cardiac myocytes and showed that functional epigenetic-modulation of distal regulatory elements allows steering of gene expression programs. Furthermore, will this project show the dynamics of promoter interactions in cardiac myocyte development and disease.

35 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.18 Altered cyclic nucleotide microdomains in cardiac disease

Nikolaev, V.

Abstract not available.

DPhG Annual Meeting 2019 Conference Book • 36 BIOPHARMACY; PHARMACEUTICAL TECHNOLOGY

3.6 Biopharmacy, Pharmaceutical Technology Chair: G. Fricker

SL.19 Molecular Therapeutics from DNA to Cas9/sgRNA: Chemical Evolution of Nanocarriers

Ernst Wagner Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig-Maximilians-Universität, Munich; Nanosystems Initiative Munich (NIM); e-mail: [email protected]

It took five decades from first gene transfections to approval of gene therapies as medical drugs. Up to date, >2930 clinical gene therapy trials have been initiated, 9 gene therapy products, 1 siRNA drug, and 8 oligonucleotide drugs received market authorization. Intracellular delivery has been critical for the success of these therapeutic macromolecules. A further refinement of delivery carriers will have a tremendous impact for efficacy of future nanomedicines. Different chemical evolution approaches are pursued toward synthetic nucleic acid or protein nanocarriers. Our strategy focuses on a bioinspired sequence-defined process including (i) artificial amino acids active in specific delivery steps, (ii) precise assembly into defined sequences by solid phase-assisted synthesis, (iii) screening for a pre-defined delivery task and selection of top candidates, followed by random or educated variation for a next round of selection. The selected type of cargo (natural product, proteins, nucleic acids including pDNA, siRNA, miRNA, PMOs, mRNA, or Cas9/sgRNA) directs the optimal motifs and sequences of nanocarriers.

References Ginn S et al. Gene therapy clinical trials worldwide to 2017: An update. J Gene Medicine 2018, e3015. Lächelt U, Wagner E. Nucleic acid therapeutics using polyplexes – A journey of 50 years (and beyond). Chem. Rev. 2015, 115, 11043−11078. Zhang P et al. Enhanced intracellular protein transduction by sequence defined tetra oleoyl-oligoaminoamides targeted for cancer therapy. Advanced Funct. Materials 2015, 25, 6627–6636. Klein PM et al. Folate receptor-directed orthogonal click-functionalization of siRNA lipopolyplexes for tumor cell killing in vivo. Biomaterials 2018, 178, 630-642. Reinhard S et al. Precise enzymatic cleavage sites for improved bioactivity of siRNA lipo-polyplexes. Bioconjug Chem 2018, 29, 3649- 3657. Truebenbach I et al. Combination chemotherapy of L1210 tumors in mice with pretubulysin and methotrexate nanoparticles. Mol. Pharmaceutics 2019, 16, 2405−2417. Luo J et al. IL4-receptor-targeted dual antitumoral apoptotic peptide - siRNA conjugate lipoplexes. Advanced Funct. Materials 2019, online.

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SL.20 Development of exosome-based nucleic acid drug delivery system

Yoshinobu Takakura Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan

A variety of nucleic acid drugs have been studied as promising drug candidates for the treatment of a wide range of diseases, including cancer, viral diseases and other incurable diseases. For a successful therapy with these nucleic acid drugs, it is important to establish a rational strategy for the delivery of the nucleic acid drugs. Exosome are extracellular vesicles secreted from most types of cells. As exosomes are endogenous delivery vehicles responsible for intercellular transport of biomolecules including nucleic acids, natural and genetically engineered exosomes are expected to become potent delivery systems for nucleic acid therapeutics. We have developed novel methods of labelling exosomes with luciferase and 125I using fusion proteins to study pharmacokinetics of exogenously administered exosomes, one of the most important issues for the development of exosome-based delivery systems. Based on the findings, we have developed a novel exosome-based antigen-adjuvant co-delivery system using genetically engineered tumour cell-derived exosomes containing endogenous tumour antigens and CpG DNA, a potent adjuvant. Vaccination with CpG DNA-modified exosomes exhibited a strong antitumor immunity, indicating that our novel exosome-based antigen-adjuvant co-delivery system can be a useful approach in cancer immunotherapy.

This research was supported by a grant-in-aid by scientific research from Japan Society for the Promotion of Science.

1. Takahashi, Y. et al.: J Biotechnol, 2013, 165, 77-84. 2. Morishita, M. et al.: J Pharm Sci, 2015, 104, 705-713. 3. Imai, T. et al.: J Extracell Vesicles, 2015, 4, 26238. 4. Yamashita, T. et al.: Eur J Pharm Biopharm, 2016, 98, 1-8. 5. Morishita, M. et al.: Biomaterials, 2016, 11, 55-65. 6. Matsumoto, A. et al.: J Pharm Sci, 2017, 106, 168-175. 7. Charoenviriyakul, C. et al.: Eur J Pharm Sci, 2017, 96, 316-322. 8. Morishita, M. et al.: Mol Pharm, 2017, 536, 310-317. 9. Matsumoto, A. et al.: Cancer Sci, 2017,108, 1803-1810. 10. Charoenviriyakul, C. et al.: Mol Pharm, 2018, 15, 1073-1080.

DPhG Annual Meeting 2019 Conference Book • 38 BIOPHARMACY; PHARMACEUTICAL TECHNOLOGY

SL.21 Metal-Organic Nanopharmaceuticals

Benjamin Steinborn,1 Ruth Röder,1 Andreas Zimpel,2 Ernst Wagner,1 Stefan Wuttke,3 Ulrich Lächelt1 1 Department of Pharmacy and Center for NanoScience (CeNS), LMU Munich, 81377 Munich, Germany 2 Department of Chemistry and Center for NanoScience (CeNS), LMU Munich, 81377 Munich, Germany 3 School of Chemistry, College of Science, University of Lincoln, Lincoln LN67TS, United Kingdom

The combination of organic building units with Lewis base function and inorganic metal ions or metal oxide clusters creates a diverse chemical space of hybrid materials. Crystalline metal-organic frameworks (MOFs) with tuneable size and porosity are an interesting compound class for the utilization in biomedical applications such as imaging, photodynamic therapy or drug delivery. Analog to more established drug delivery systems, MOFs require suitable functionalization approaches to enable change and control of the behaviour in a biological environment. On the other hand, selected drug molecules can serve as organic linkers in coordination polymers themselves and enable the formation of drug-metal nanopharmaceuticals with very high drug loading capacity. Here, different concepts for the functionalization of MOF nanoparticles (Figure 1) [1-3] and the assembly of direct drug-metal nanoparticles (Figure 2) with the aim of potential utilization in biomedical applications are presented.

39 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.22 Development of lipoplex-loaded model surface coatings for the in situ transfection in the field of bone regeneration

Husteden, C.1; Groth, T.2; Wölk, C.1 1 Martin Luther University Halle-Wittenberg, Institute of Pharmacy, Department of Medicinal Chemistry, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany 2 Martin Luther University Halle-Wittenberg, Institute of Pharmacy, Biomedical Materials Group, W Heinrich-Damerow-Str. 4, 06120 Halle (Saale), Germany

The German legislation modernised and expanded the definition of medicines for new therapies by including the advanced therapy medicinal products (ATMPs) in the § 4b of the German Medicines Act (AMG). The term ATMPs covers gene therapy medicinal products, somatic cell therapy medicinal products and tissue engineered products, which are defined in the EU guideline 2009/120/EG. An interesting field of research and one application of ATMPs is the DNA-induced bone regeneration in the field of regenerative medicine. In the Western World 5 to 10% of bone fractures show insufficient healing which results in a longer leave of absence and higher costs for the health care system. Hence, numerous strategies in the regenerative medicine focus on initiating fast osteogenesis on artificial scaffolds to stimulate the healing of bone fractures. However, these strategies primary use recombinant proteins to induce osteogenesis, which may result in complex biotechnological manufacturing and therefore high therapy costs.

Figure 1: Schematic illustration of self-assembling of polyelectrolyte multi layers (PEMs) using the layer-by-layer (LbL) technique.

In our project we produce functionalized material films loaded with DNA. Functionalised material coatings are developed based on polyelectrolyte multi layers (PEMs, see Figure 1). These PEMs are composed of biocompatible polymers and were loaded with layers of DNA. The incorporated DNA is complexed by highly effective cationic lipid composites [1]. This lipid encapsulation has protective and transfection enhancing effects. The project focuses on the development of methods to achieve effective loading of the PEMs with lipid/DNA complexes (lipoplexes) and on the intensive characterization of DNA-loaded LbL-films using surface sensitive biophysical methods, namely, atomic force microscopy, raster electron microscopy, zeta potential measurements, and fluorescence microscopy. Furthermore, the interactions of cells with the functionalized PEMs were investigated using molecular biological methods to determine cell attachment, viability parameters, and effectivity of DNA transfer. Final experiments using the chorio-allantoic membrane of the chicken egg as in vivo system demonstrate the high efficacy of the DNA- loaded surface coatings in complex tissues.

Acknowledgements: Christian Wölk thanks the Deutsche Forschungsgemeinschaft (DFG) for funding (Projekt number 396823779).

References: [1] Wölk et al. Adv. Colloid Interface Sci. 2017, 248: 20-34

DPhG Annual Meeting 2019 Conference Book • 40 GOLD! HUNTING TREASURES IN CHEMISTRY AND PHARMACOLOGY

3.7 Gold! Hunting treasures in chemistry and pharmacology Chair: C. Klein

SL.23 Gold Catalysis: Functionalized Gold Carbenes, Unexpected Selectivities

A. Stephen K. Hashmi Organisch-Chemisches Institut, Heidelberg University, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany. [email protected]

Only after two papers from 2000 had demonstrated the full potential of gold catalysis for organic transformations by a high increase of molecular complexity,1,2 homogeneous gold catalysis was developed to a versatile tool for organic synthesis.3,4 For a long time the field was exclusively focusing on electrophilic and nucleophilic species, radical intermediates were not involved, but this changed in 2013.5 Apart from the synthesis of different heterocycles (Scheme 1), the use of these principles also allows a number of C-C coupling reactions, which in a formal sense can also address C,H bonds.6 Principles like dual activation, selectivity control by remote steric repulsion and the use of di- and even mononuclear gold(I) complexes for photochemical reactions will be discussed. All these results will include results from computational chemistry. R1

R2 2 1) KAuBr4 (5 mol%) R 1,2-DCE, 20 oC, 2 h 3 N + O R1 R N o + H2O 3 PG N 2) 40 C, 10 h R N PG

Scheme 1. Intermolecular reaction of an anthranil derivative with an ynamide to provide a multi-annulated heterocyclic system in only one step.

References 1. A. S. K. Hashmi, T. M. Frost, J. W. Bats, J. Am. Chem. Soc. 2000, 122, 11553. 2. A. S. K. Hashmi, L. Schwarz, J.-H. Choi, T. M. Frost, Angew. Chem. Int. Ed. Engl. 2000, 39, 2285. 3. D. Pflästerer, A. S. K. Hashmi, Chem. Soc. Rev. 2016, 45, 1331. 4. A. M. Asiri, A. S. K. Hashmi, Chem. Soc. Rev. 2016, 45, 4471. 5. G. Revol, T. McCallum, M. Morin, F. Gagosz, L. Barriault, Angew. Chem. Int. Ed. 2013, 52, 13342. 6. J. Xie, S. Shi, T. Zhang, N. Mehrkens, M. Rudolp, A. S. K. Hashmi, Angew. Chem. Int. Ed. 2015, 54, 6046.

41 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.24 Gold Organometallics in Medicinal Chemistry

Ott, I.1 1 Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, Beethovenstr. 55, 38106 Braunschweig, Germany

Organometallic gold complexes have recently attracted mayor attention in inorganic medicinal chemistry based on their strong cytotoxic activities and their multiple pharmacological effects against tumor cell proliferation.[1,2] The enhanced stability of their metal-carbon bonds has also been a driving argument regarding the design of potent gold metallodrugs with an improved stability under physiological conditions. We and other groups have recently reported on gold(I) complexes with N-heterocyclic carbene (NHC) or alkynyl + ligands and their potential as anticancer agents (see the figure for some examples of the [Au(I)(NHC)2] and alkynylAu(I)(NHC) types).[3-6] The complexes generally triggered notable cytotoxic activities, displayed an efficient cellular uptake, and were good inhibitors of the selenoenzyme thioredoxin reductase (TrxR). Other relevant pharmacological effects, such as antimitochondrial properties, could be confirmed additionally. In this presentation our most relevant findings on the medicinal chemistry of gold(I) organometallics will be summarised with a focus on important early preclinical parameters such as structure-activity-relationships, stability, protein binding, or cellular uptake.

Acknowledgments: Financial support by the DFG (Deutsche Forschungsgemeinschaft) is gratefully acknowledged.

References 1. Casini, A., Sun, R. W.-Y., Ott, I. in Metallodrugs: Development and Action of Anticancer Agents, Vol. 18 of Metal Ions in Life Sciences, Sigel, A. et al. (eds.), (Walter de Gruyter) 2018. 2. Cinellu, M. A., Ott, I., Casini, A. in Bioorganometallic Chemistry G., Jaouen, M. Salmain (eds.), (Wiley-VCH) 2015. 3. Hickey, J. L. et al.: J. Am. Chem. Soc. 2008, 130: 12570-12571 4. Rubbiani, R. et al.: J. Med. Chem. 2011, 54: 8646-8657 5. Schmidt C. et al.: Chem. Eur. J. 2017, 23: 1869-1880 6. Schmidt C. et al.: Metallomics 2019, 11: 533-545

DPhG Annual Meeting 2019 Conference Book • 42 GOLD! HUNTING TREASURES IN CHEMISTRY AND PHARMACOLOGY

SL.25 Homoleptic gold dithiocarbamate TXNRD1 inhibitors prevent tumor recurrence in a small cell lung cancer mouse model

Nikolas Gunkel1,2 and Aubry K. Miller1,2 1Cancer Drug Development Group, German Cancer Research Center (DKFZ), INF 280, Heidelberg, 69120, Germany 2German Cancer Consortium (DKTK), INF 280, Heidelberg, 69120, Germany

Disulfiram (DSF), long used as a safe treatment for alcoholism, has recently received attention for its potential to be repurposed as an anti-cancer treatment. Currently, it is believed that DSF’s cancer-killing properties are the result of a chemical reaction in vivo, wherein the copper complex of DSF (CuET, 1) is the active agent.1 We have found that the combination of DSF with the FDA-approved drug aurothiomalate is highly toxic to small cell lung cancer (SCLC) cell lines, while showing low toxicity to other cancer cell types as well as to normal cells. Like copper salts and DSF, aurothiomalate and DSF undergo a chemical reaction, which produces the dinuclear dithiocarbamate gold complex 2 (DKFZ-608). As with other gold complexes, DKFZ-608 is a thioredoxin reductase 1 (TXNRD1) inhibitor; however, unlike some other gold complexes, it is devoid of activity on the glutathione system and the proteasome at concentrations cytotoxic to cells. Targeting thioredoxin reductase 1 (TXNRD1) to kill cancer cells has long been discussed as a promising strategy for numerous reasons: (1) TXNRD1 over-expression was repeatedly identified as having a statistically significant association with poor prognosis. (2) It is suggested to be a key player in redox regulation, with a lower dependency in most normal adult cells and tissues than in cancer cells. (3) Inhibitors of TXNRD1 activity cause cytotoxicity in cancer cells. Despite this promising target profile, the success rate of TXNRD1 inhibitors has been disappointing in pre-clinical and clinical studies. One reason for this lack of success might be the physicochemical shortcomings of lead compounds, which pose liabilities in the area of bioavailability, stability, and dose-limiting side effects. We find that DKFZ-608 (and analogs) may have the potential to overcome these liabilities and finally realize the clinical potential of TXNRD1 inhibitors. We present data describing the discovery of a lead compound and its optimization leading to improved formulation, on-target and anti-tumor activity. This compound class shows superior efficacy and selectivity for TXNRD1, and binds in competition with the TXNRD1 substrate thioredoxin-1 (TXN1). Consequently, low TXN1 expressers, like small cell lung cancer cells, are hypersensitive to the drug, while cells, which express higher levels of TXN1, are resistant. After treatment of cells with our inhibitors, we observe oxidative stress only in the cytoplasm, but not in mitochondria, contributing to a low general toxicity profile. Finally, in an SCLC animal model, DKFZ-608 eradicates residual tumor cells surviving first line therapy and completely prevents tumor recurrence.

[1] Skrott et al. Nature, 2017, 552, 194–199.

43 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

3.8 Evidence-based value added to innovative health care by pharmacists Chairs: H. Seidling, U. Jaehde

SL.26 Evidence-based value added to innovative health care by pharmacists: Ambulatory care

Dreischulte T Klinikum der Universität München, Department of General Practice and Family Medicine, Ludwig-Maximilians-Universität München

Over the last two decades, numerous studies have examined the incidence and prevalence of preventable drug related morbidity internationally as a means to characterize the quality of medicines management in the community. Systematic reviews estimate that approximately 5% of emergency hospital admissions are drug related with deficiencies in all key stages of the medicines use process, namely prescribing, monitoring and patient adherence (Howard et al). Aging populations and the parallel increase in chronic conditions, multi-morbidity and polypharmacy imply the risk of a further rise in the burden of preventable drug related harm and unrealized benefit. Pharmacists are increasingly recognized as an underutilized resource to reduce this burden, and a growing body of research literature evaluates the impact of pharmacist interventions in the ambulatory care setting, which is at least partly fueled by diminishing general practitioner resources in many countries, including Germany. A recent systematic review by the Cochrane Collaboration included 111 randomized controlled trials evaluating the clinical impact of pharmacist interventions targeting patients compared to care as usual (de Barra et al). The authors found overall “weak” evidence that the evaluated pharmacist interventions improved blood pressure control and “very weak” evidence that they improved diabetes control, while there was “weak” to “moderate” evidence for the lack of impact on adverse drug reactions, hospital admissions and mortality. The authors highlight substantial heterogeneity between interventions and outcome measures and the need for further research to examine which intervention components are effective in which context. Of note is, that none of the included studies were conducted in Germany (although one multi-centre study included a German site). Nevertheless, evaluations of pharmacist interventions in the German ambulatory care setting are increasing. For example, the West Gem study demonstrated in a cluster randomized stepped wedge trial that close collaboration between a study pharmacist and GPs can significantly improve the quality of prescribing as measured by the Medication Appropriateness Index in vulnerable older patients (Rose et al). The international research literature on pharmacist interventions highlights a number of challenges to the design of interventions and to evaluating their impact on clinical outcomes. Addressing these challenges in future evaluations of pharmacist services will be important to inform their wider implementation in the German health care setting.

References 1. Howard RL et al. Br J Clin Pharmacol. 2007 Feb; 63(2): 136–147 2. de Barra M et al. Pharmacist services for non-hospitalised patients. Cochrane Database of Systematic Reviews 2018, Issue 9. Art. No.: CD013102. DOI: 10.1002/14651858.CD013102. 3. Rose O et al. PLoS ONE 2016,11(6):e0156304

DPhG Annual Meeting 2019 Conference Book • 44 EVIDENCE-BASED VALUE ADDED TO INNOVATIVE HEALTH CARE BY PHARMACISTS

SL.27 Evidence-based value added to innovative health care by pharmacists - Hospital Setting

Dr. Torsten Hoppe-Tichy, Heidelberg University Hospital and Cooperation Unit Clinical Pharmacy, School of Pharmacy, Heidelberg University

In the last decades hospital pharmacy in Germany has made a big step forward. Being primarily a logistical unit to supply drugs to the ward level, pharmacists have become an integrated part in the interprofessional group of bedside health care professionals/experts. Clinical Pharmacy as the latest addition to the 4 examination subject chemistry, biology, pharmacology and technology has been shown to be an integral part in patient care. Hospital pharmacists fulfill several important tasks in patient care. During the presentation, the way of the patient (and the patients medication) is followed and the several steps which can be supported by hospital pharmacists are highlighted and discussed in light of existing evidence in literature. Thereby, the current development in Germany will be presented in the context of international standards.

45 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.28 Evidence-based value added to innovative health care by pharmacists: Long-term care facilities

Jaehde U Institute of Pharmacy, Department of Clinical Pharmacy, University of Bonn

Several studies have shown that the incidence of adverse drug reactions is particularly high in long-term care (LTC) facilities leading to severe consequences such as falls and hospitalization. In a prospectively designed cross- section analysis in North Rhine-Westfalia the incidence of adverse drug reactions was found to be 8 per 100 resident-months with a preventability rate of 60% (Jaehde & Thürmann 2012) indicating serious deficiencies in the health care of LTC residents. Various medication safety-enhancing interventions are currently being developed that have the potential to improve the health status of LTC residents and to reduce costs. A systematic review identified 27 randomized controlled trials describing interdisciplinary interventions in LTC facilities of which 18 demonstrated a statistically positive effect. Participation of primary physicians and/or a pharmacist were identified as success factors providing first evidence that pharmacists can play a major role in such interventions (Nazir et al. 2013). Recently, we have developed an intervention in North Rhine-Westfalia involving community pharmacists supplying drugs to the LTC facilities in collaboration with a health insurance (AOK Rheinland/Hamburg) and the Pharmacists’ Association North Rhine. Based on prescription data of the AOK, the current medication and further information from the nursing home, the pharmacists performed a medication review for LTC residents aged at least 65 years and taking five or more drugs per day based on the patients’ medication only. Documented potential drug-related problems (DRPs) and the implementation rate of pharmaceutical interventions were evaluated descriptively. To assess the quality of the medication reviews, a corresponding reference system was developed based on the analysis of two experienced clinical pharmacists. 12 pharmacies performed medication reviews for 94 LTC residents. Overall, the pharmacists documented 154 potential DRPs (mean 1.6 per patient, SD 1.5) of which the most common were drug-drug interactions (40%) followed by potentially inappropriate medication (PIM) (16%) and inappropriate dosages (14%). 33% of the pharmacists’ interventions to solve DRPs were successfully implemented, mostly dosage adjustments. The identification of potentially severe drug-drug interactions and PIM showed the highest agreement (88% and 73%) with the reference system (Bitter et al. 2019). In conclusion, there is increasing evidence that community pharmacists can identify and solve relevant DRPs in the medication of LTC residents. The reference system assessing the quality of medication reviews can contribute to its transparency and reveals the potential for its improvement. The community pharmacists’ knowledge of the LTC residents and their relation to the prescribers is crucial for providing successful medication reviews and hence contributing to medication safety.

References 1. Jaehde U, Thürmann P: Z. Evid Fortbild. Qual. Gesundh. wesen. 2012, 106: 712-6 2. Nazir A et al.: J. Am. Med. Dir. Assoc. 2013, 14: 471–8 3. Bitter K et al.: BMC Geriatr. 2019, 19: 39

DPhG Annual Meeting 2019 Conference Book • 46 SIMULATION IN PHARMACEUTIC AND FORMULATION DEVELOPMENT

3.9 Simulation in Pharmaceutic and Formulation Development Chairs: D. Fischer, P. Langguth

SL.29 Simulations in pharmaceutical technology

Wachtel, H. Boehringer Ingelheim Pharma GmbH & Co. KG, Binger Str. 173, 55216 Ingelheim, Germany

Establishing and applying simulation tools in the field of pharmaceutical technology seems to be obvious in a quickly growing industry sector. However, the classical education of pharmacists and the urgent acceleration of the drug discovery and -development processes will support the investment in simulations in industry and academia only where convincing advantages exist. This presentation explains basic concepts in simulation and gives a series of examples demonstrating the gain in knowledge obtained. The industry perspective shows that investment and time required to deliver results must be carefully considered when selecting the simulation strategy. Apart from computer based numerical simulations, e.g. scaled analogue models may show sufficiently precise results. The following examples will be discussed: - Mixing of liquids in a 800 L tank - Operation of a static mixer [1] - Air flow inside an inhaler [2] - De-agglomeration of powder mixtures [3] - Generating an aerosol for pulmonary treatment of horses [4] Setting up the appropriate simulations requires a data-driven understanding of the relevant processes. In this respect the concept of “Quality by Design” is supported. Moreover, in the field of medical device development, the simulations are well suited to document and to explain design decisions in the design history file. An important step towards regulatory acceptance is research performed by FDA, especially the publication of a guidance for industry dealing with Computational Modeling [5].

Acknowledgments: Michael Becker, Deborah Bickmann, Anna Maria Ciciliani, Özgür Ertunç, Sungjin Jang, Andree Jung, Ralf Kröger, and Bernhard Schmitz contributed to different projects of the Fluid Dynamics and Simulation Team at Boehringer Ingelheim. I thank Peter Langguth (Univ. Mainz) for fruitful discussions and continuous support.

References: 1. Schmitz, B.: Numerische Simulation der Strömung und des Stofftransportes innerhalb des statischen Mischers Fluitec CSE-6 zur Untersuchung des Einflusses unterschiedlicher Zulaufgeometrien auf die Mischleistung (Master Thesis, TH Bingen) 2018. 2. Ertunç, O. et al.: Int. J. Pharm. 2011, 416(1): 25-34. 3. Kroeger, R., Wachtel, H.: Respiratory Drug Delivery 2016, Volume 1, 2016: 89-98. 4. Wachtel, H. et al., ISAM2019 (Montreux), Poster N-038 2019. 5. FDA, Reporting of Computational Modeling Studies in Medical Device Submissions 2016, web-link: https://www.fda.gov/media/87586/download (last visited July 11, 2019).

47 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.30 Pharmacokinetic Modelling and Simulation to Support Early Clinical Drug Development

Nguyen, M.A. 1Sanofi-Aventis GmbH, R&D, Translational Medicine and Early Development - PKDM (Pharmacokinetics, Pharmacodynamics and Drug Metabolism), Industriepark Höchst, 65926 Frankfurt, Germany

At the transition phase between late research and early clinical development, a strategy to efficiently explore the relationship between drug exposure and in vivo response in humans needs to be clearly outlined. This gives rise to questions on the selection of appropriate doses for the first-in-human studies and on the extrapolation of exposure/response relationships observed in vitro and in animals to the intended patient population, among others. Modelling and simulation can help to answer these questions, thereby having a direct impact on the design of early clinical studies and ultimately supporting strategic decision making. Allometric scaling and physiologically based pharmacokinetic modelling are common tools to predict human pharmacokinetics based on preclinical and in vitro ADME properties. The risk of potential pharmacokinetic drug- drug interaction and food effects can be evaluated using modelling and simulation to support risk/benefit assessment and clinical study design. In addition, combining pharmacokinetic and pharmacodynamic modelling allow the early estimation of therapeutic doses for the intended indication. The presentation will cover the general use of pharmacokinetic modelling and simulation in early drug development in a pharmaceutical industry setting. The applications and limits of pharmacokinetic modelling before first-in-human studies will be elaborated based on recent examples including efficacious dose estimation and prediction of drug- drug interaction.

Figure 1. Pharmacokinetic modelling to support early drug development. PBPK – physiologically based pharmacokinetics. PK/PD – pharmacokinetic/pharmacodynamics

Acknowledgments: Examples presented here were discussed in dedicated project teams, in particular in close collaboration with colleagues from TMED and DMPK at Sanofi Frankfurt. Project-related collaboration with colleagues from UCB S.A. is also much acknowledged.

DPhG Annual Meeting 2019 Conference Book • 48 SIMULATION IN PHARMACEUTIC AND FORMULATION DEVELOPMENT

SL.31 Simulation of pulmonary deposition during inhalation therapy Ciciliani, A.-M.1, Heussel, C. P.2,3,4 , Langguth, P.1 , Wachtel, H.5 1Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Germany 2Department of Interventional and Diagnostic Radiology with Nuclear Medicine, Thorax Clinic at University of Heidelberg, Heidelberg, Germany 3Translational Lung Center Heidelberg, Member of the German Center for Lung Research (DZL), Heidelberg, Germany 4Department of Interventional and Diagnostic Radiology, University of Heidelberg, Heidelberg, Germany 5Analytical Development, Boehringer Ingelheim Pharma GmbH & Co. KG, Ingelheim am Rhein, Germany

Background: The type and design of an inhalation device determines the deposition of the inhaled drug in the lungs. We simulated the particle deposition of five commonly used inhaler aerosols which were generated by four devices in different lung models: tiotropium bromide (Respimat®), glycopyrronium bromide (Breezhaler®), aclidinium bromide (Genuair®), vilanterol and fluticasone (both Ellipta®). Methods: Using realistic breathing patterns, we combined the output of an Alberta idealized mouth-throat model with four different virtual lung models: One self-constructed single-path model and three computed tomography- based models of the bronchial tree of a child, a middle-aged person and an elderly person to determine the amount of drug retained in the throat, deposited in the upper airways and in the periphery of the lungs. Results: Despite geometric differences between the three CT lung models, we found similar regional deposition results with all evaluated devices; however, with the Genuair® aerosol, there was a trend towards higher drug deposition in the upper airways of the child lung model compared with the adult and geriatric lung model. The single-path model showed lower deposition in the upper airways of the lung than the CT models. The Respimat® inhaler showed the lowest throat deposition and at the same time the highest deposition in the whole lung and in the different lung regions compared to the other tested inhalers. Conclusion: Human lung models that predictably simulate the amount of total drug delivered to the lungs, as employed here, could be helpful in the development of new drug formulation and devices, especially in children.

Figure 1. Simulated particle deposition patterns of tiotropium droplets in different lung models. From left to right: adult CT model, child CT model, elderly patient CT model, adult single-path model. The shown CT models were created using ITK-snap software[1].

Table 1. Particle deposition in different lung models: LD = in vitro lung dose = Alberta throat model output, UA = Deposition in the upper airways (trachea and the first 5-7 generations), P = Deposition in the peripheral lung airways (approx. generation 7 -23), SD = standard deviation. All results in [% of nominal dose] Inhaler API LD Child model Adult model Elderly Adult single- (SD) model path model UA P UA P UA P UA P Respimat Tiotropium 67 (5) 22 45 23 44 19 48 12 55 ® Genuair® Aclidinium 42 (1) 10 32 7 35 4 38 4 38

Acknowledgments Ralf Kröger (ANSYS Germany) provided help concerning CFD (computational fluid dynamics). 1. Yushkevich PA, Piven J, Hazlett HC, Smith RG, Ho S, Gee JC, Gerig G: User-guided 3D active contour segmentation of anatomical structures: significantly improved efficiency and reliability. Neuroimage 2006; 31: 1116-1128

49 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.32 Exosome-Hydrogels as Machinery for Anti-Inflammatory Therapeutics’ Synthesis

Fuhrmann G1,3; Chandrawati R2,3; Pamar P3; Keane TJ3; Bertazzo S4; Stevens MM3 1Helmholtz Institute for Pharmaceutical Research Saarland, Department of Pharmacy, Saarland University, 66123 Saarbrücken, Germany; 2UNSW School of Chemical Engineering, NSW 2052 Sydney, Australia; 3Imperial College London, Department of Bioengineering, Prince Consort Road, SW7 2AZ London, United Kingdom; 4University College London, Department of Medical Physics and Biomedical Engineering, WC1E 6BT, London, United Kingdom

Extracellular vesicles (EVs) are natural lipid-based membranous particles produced by almost any cell. They may transfer nucleic acid and protein-cargoes selectively between cells locally and at distance, which has created excitement for both fundamental biology and in the drug delivery field [1]. Although initial clinical trials are ongoing, the use of EVs for therapeutic applications may be limited due to potential “dilution effects” upon systemic administration or undesired off-target activity which may affect their ability to reach their target tissues. To tap their full therapeutic potential in a localised manner, we created a biomedical hydrogel containing EVs designed to achieve local delivery of therapeutics [2]. Due to the challenge of incorporating EVs into hydrogels without compromising their biological constitution, such approach had not been developed yet. EVs from human mesenchymal stem cells (MSC) were isolated and characterised regarding size distribution and yield, loaded with a glucuronidase enzyme using our established saponin-assisted technique [3]. Liposomes made from egg phosphocholine/cholesterol mol-60/40% were used for comparison. EVs and liposomes were incorporated into polyvinyl alcohol (PVA) hydrogel (12 wt%) and crosslinked with PEG (Figure 1). Incorporation of glucuronidase-loaded EVs or liposomes into biocompatible PVA hydrogels did not impact on hydrogel biomechanical properties and preserved the enzyme’s stability compared to free enzyme during two weeks of incubation at 37°C, as assessed using glucuronide-fluorescein.

Figure 1. Hydrogel design. (a) Schematic depiction of vesicle containing hydrogels and (b) their morphologies.

To visualise EVs within hydrogels, uranyl acetate-labelled vesicles were imaged in carefully dehydrated gels by density-dependent colour scanning electron microscopy [4]. Taking advantage of density-sensitive backscattered imaging we spatially localised EVs within 3D-hydrogels. When incubating hydrogels with curcumin-glucuronide prodrug and murine RAW264.7 macrophages, all gels showed significant anti-inflammatory activities, as assessed by cell viability measurements. Recycling of gels after 7 d showed that this effect was lost for gels containing free enzyme but not when encapsulated into vesicles, indicating that both natural and synthetic vesicles may protect enzymes in the hydrogels during enhanced periods. Interestingly, without addition of curcumin-prodrug gels containing EVs showed an inherent anti-inflammatory effect, as detected by TNF-alpha gene expression analysis in bone marrow-derived primary macrophages. Our results indicate that hydrogels with MSC EVs are anti- inflammatory per se, which has not been reported to date. Our work on incorporation of EVs into a biomedical hydrogel may create an important basis for their therapeutic application.

Acknowledgements: This work was supported by the European Union (Marie Curie Intra-European Fellowship) and the German Federal Ministry for Science and Education BMBF (NanoMatFutur programme, grant 13XP5029A). References 1. Goes A, Fuhrmann G*: ACS Infect Dis 2018, 6:881-892. 2. Fuhrmann G et al.: Adv Mater 2018, 1706616. 3. Frank J, Richter M et al.: Sci Rep 2018, 8:12377. 4. Bertazzo S et al.: Nat Mat 2013, 12:576-583.

DPhG Annual Meeting 2019 Conference Book • 50 G-PROTEIN COUPLED RECEPTORS

3.10 G-Protein-coupled Receptors Chairs: P. Gmeiner, B. Wünsch

SL.33 Dynamics of GPCR - G protein interactions as the key to understand coupling selectivity of GPCRs

Bünemann, M.

Abstract not available.

51 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.34 Bivalent ligands targeting heterodimers of dopamine receptors

Budzinski, J.1; Kaindl, J.1; Hübner, H.1; Gmeiner, P.1; Weikert D.1 1 Friedrich-Alexander University Erlangen-Nürnberg, Department of Chemistry and Pharmacy, Medicinal Chemistry, Nikolaus-Fiebiger- Straße 10, 91058 Erlangen, Germany

Besides their numerous physiological functions, dopamine D2-like receptors are associated with severe neuropsychiatric disorders such as Parkinson’s disease and schizophrenia. In addition to their signalling as isolated entities, they transiently form homo- [1] and heterodimers [2] with other G protein-coupled receptors (GPCRs). Such interactions between GPCRs are known to affect ligand binding, signal transduction and receptor trafficking. In this context, the close relation and high co-localization of D2R with the neurotensin receptor 1 (NTS1R) is of substantial interest [3]. Bivalent ligands consisting of two pharmacophores tethered by an appropriate linker represent powerful tools to selectively address GPCR heterodimers. 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 between heterodimer-expressing cells and those that express only one individual receptor [4].

Starting from the crystal structure of NTS1R and a D2R homology model based on the D3R x-ray structure, we have developed bivalent ligands consisting of two pharmacophores connected by an appropriate spacer to specifically address D2R-NTS1R heterodimers. These ligands possess subnanomolar binding affinity for the D2R-NTS1R heterodimer and exhibit over 1,000-fold selectivity over the D2R monomer. Applying methods such as enzyme fragment complementation and radioligand binding, we study the formation of receptor heterodimers and we aim to further elucidate the signal transduction profile of the ternary ligand- protein assemblies. Our results indicate that the signaling behavior of the D2R-NTS1R dimer is mainly driven by NTS1R. Importantly, bivalent ligands with appropriate spacer length strongly enhance β-arrestin-2 recruitment to the receptor dimer compared to a combination of two individual agonists, pointing towards a special mechanism of action [5].

Whether the D2R-NTS1R interaction is unique to the D2R subtype, or if other dopamine receptors, such as D3R, are able to interact with NTS1R in a similar way, is one of our central research questions. We employ our bivalent ligands to study these putative D3R-NTS1R dimers in comparison to monovalent controls, using the previously established methods complemented with bioluminescence resonance energy transfer (BRET).

Acknowledgments: This project is supported by the DFG research training group GRK 1910 “Medicinal Chemistry of Selective GPCR Ligands”.

References: 1. Guo, W. et al.: EMBO J., 2008, 27(17): 2293-2304. 2. Perreault, M.L. et al.: Neuropsychopharmacology, 2014, 39(1): 156-168. 3. Binder, E.B. et al.: Pharmacol. Rev., 2001, 53(4): 453-486. 4. Shonberg, J., Scammells,P.J., Capuano, B.: ChemMedChem, 2011, 6(6): 963-974. 5. Huebner, H. et al. Nat. Commun., 2016, 7: 12298.

DPhG Annual Meeting 2019 Conference Book • 52 G-PROTEIN COUPLED RECEPTORS

SL.35 Photopharmacology in Alzheimer research: Tools to investigate GPCRs

Michael Decker Pharmazeutische und Medizinische Chemie, Institut für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg, Am Hubland, 97074, Germany

In the last years the field of “photopharmacology” has emerged that incorporates units, such as azobenzenes and diarylethenes, into biologically active molecules, to obtain photoswitchable molecules that can reversibly change their structure and concomitantly their activity upon irradiation with light, normally UV light. Within our research efforts for anti-Alzheimer drugs targeting the human cannabinoid receptor subtype 2 (hCB2R) [1] and the human muscarinic acetylcholine receptor subtype 1 (hM1R) [2], we have incorporated azobenzene units into hCB2R agonists and both dualsteric and bivalent hM1R agonists [3], the latter simultaneously address both the orthosteric and allosteric binding sites of the receptor. Applying computational studies, such as molecular dynamics, as well as a portfolio of pharmacological assays, such as radioligand binding, functional studies, as well as FRET techniques, we were able to develop the first selective photoswitchable hCB2R “affinity on-switch” [4]. Furthermore, a dualsteric photoswitchable hM1R ligand was developed, the activity of which can be regulated by light as demonstrated in a set of FRET studies [5]. We also synthesized, characterized photophysically and pharmacologically bivalent hM1R ligands based on the agonist iperoxo and incorporated fluorine atoms to yield “red-shifted” ligands that show a considerably higher extent of photoconversion and can be switched by visible light [6]. We observed for the first time that “red-shifted” ligands can also differ significantly in their pharmacological activity [6]. Ongoing work on hM1R orthosteric agonists yielded "on- or off-switches" regarding efficacy and potency.

These findings show that photopharmacology can be successfully applied to various GPCR ligands, and the field is moving beyond the proof-of-concept, since it seems possible to specifically design GPCR ligands as “on- or off- switches”, and to compounds that are “affinity switches” and/or “efficacy switches”. This significantly expands the toolbox of GPCR investigation with specialized molecular tools supporting the investigation of the molecular basis of receptor function. The underlying principles seem generally applicable, since also a photoswitchable dualsteric hM2R ligand was developed and applied to optically control cardiac function, even in vivo [7].

Financial support of the Elite Network of Bavaria for the International Doctoral Program “Receptor Dynamics”, of the DAAD with funds of the BmBF, and of the DFG (under DE1546/10-1), is gratefully acknowledged.

1. Dolles, D. et al.: J. Med. Chem. 2018, 61(4): 1646-1663. 2. Chen, X.; et al.: J. Med. Chem. 2015, 58(2): 560-576. 3. Rodríguez-Soacha D A, Decker M: Adv. Therap. 2018, 1800037: 1-9. 4. Dolles, D.; et al.: Adv. Therap. 2018, 1700032: 1-6. 5. Agnetta, L.; et al.: Angew. Chem. Int. Ed. 2017, 56(25): 7282-7287. 6. Agnetta, L.; et al.: J. Med. Chem. 2019, 62(6): 3009–3020. 7. Riefolo, F. et al.: J. Am. Chem. Soc. 2019,141(18), 7628-7636.

53 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.36 In Silico Pharmacology: A Mechanistic View on GPCR Modulation

Marcel Bermudez, Gerhard Wolber* 1Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany

During the last decades, computational approaches have become essential for drug design and structural biology. For research on G protein-coupled receptors (GPCR), in particular, in silico methods and pharmacological experiments represent a strong and necessary combination for functional understanding being a prerequisite for rational and targeted drug design. Over the last decade, a multitude of GPCR-ligand complexes were determined by crystallography providing an indispensable structural view on this protein class, although these crystal structures only represent snapshots of a highly dynamic conformational ensemble. GPCRs are conformationally dynamic signaling machines, transferring information across membranes via multiple signaling pathways. Although about 30 % of all marketed drugs directly target GPCRs, little is known about the ligand-induced conformational changes that lead to intracellular signals. We combine molecular dynamics simulations and chemical interaction analysis (3D dynophores) to provide a more dynamic view on how binding of small organic molecules on the extracellular side of the receptor changes conformational behavior responsible for downstream signaling [1,2]. We will explain how computational approaches guided by pharmacological experiments iteratively unveil mechanisms of specific receptor functions [3,4]. A deep mechanistic understanding of GPCR functionality is essential for the rational design of tailor-made GPCR modulators. We will present case studies focusing on modulated receptor activation and biased signaling, which highlight the explanatory power of computationally- derived receptor models and their usage for prospective rational drug design.

[1] M Bermudez, TN Nguyen, C Omieczynski and G Wolber. Strategies for the discovery of biased GPCR ligands, Drug Discov Today 2019, 24(4):1031-1037 [2] M Bermudez and A Bock. Does Divergent Binding Pocket Closure Drive Ligand Bias for Class A GPCRs? Trends in Pharmacological Science 2019. 40(4):236-239 [3] L Agnetta, M Bermudez, F Riefolo, C Matera, E Claro, R Messerer, T Littmann, G Wolber, U Holzgrabe, M Decker. Fluorination of Photoswitchable Muscarinic Agonists Tunes Receptor Pharmacology and Photochromic Properties, J Med Chem 2019, 62(6):3009-3020 [4] M Bermudez, A Bock, F Krebs, U Holzgrabe, K Mohr, MJ Lohse and G Wolber. Ligand-specific restriction of extracellular conformational dynamics constrains signaling of the M2 muscarinic receptor, ACS Chem Biol 2017, 12(7):1743-1748

DPhG Annual Meeting 2019 Conference Book • 54 RADIOPHARMACY AND LABELING

3.11 Radiopharmacy & Labeling Chair: W. Mier

SL.37 Visualisation of cardiac fibrosis using [68Ga]MHLL1 – A PET tracer for the fibroblast activation protein

Langer, L. B. N.1; Hess, A.2; Korkmaz, Z.1; Reffert, L. M.1; Bankstahl, J. P.2; Thackeray, J. T.2; Bengel, F. M.2; Ross, T. L.1 1Department of Nuclear Medicine, Radiopharmaceutical Chemistry, Hannover Medical School, Carl-Neuberg- Strasse 1, 30625 Hannover, Germany. 2Department of Nuclear Medicine, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.

Objectives: The fibroblast activation protein (FAP, also named seprase) is a serine protease with the ability to cleave Pro-X peptide bonds and exhibits endopeptidase activity with a preference for Ac-Gly-Pro motifs.1 Interestingly, the expression of FAP in healthy tissue is very limited, whereas high FAP-levels can be observed in over 90% of common human epithelial malignancies, fibrotic and inflammatory processes.2, 3 This unique expression pattern predestines FAP as a potential diagnostic target for molecular imaging and therapy in various diseases. Therefore, we recently designed the gallium-68 labeled FAP-inhibitor [68Ga]MHLL14 for imaging using positron emission tomography (PET). In this project, [68Ga]MHLL1 was evaluated in vitro and in vivo in a preclinical myocardial infarction (MI) model to visualise fibrosis after tissue damage. Methods: p-NCS-benzyl-NODA-GA was coupled via thiourea formation to a primary amino function of a pyrrolidine- based FAP-warhead, followed by HPLC-purification. The non-radioactive reference compound was realized by using [natGa]gallium(III)chloride. The 68Ga-eluate was obtained from a 68Ge/68Ga-generator and purified by anionic post-processing. In an automated synthesis (GRP-module 3V, Scintomics) 60 nmol precursor was labeled in HEPES at 100 °C for 20 min. Radiochemical yields were determined using a RP18 radioHPLC. Cell uptake assays were performed with different FAP(+) cell lines (3T3, MC-38). One week prior to imaging, the MI was induced via coronary ligation in C57BL/6 mice (n = 4). A dynamic PET-scan was performed for 60 min with 15.2±0.5 MBq [68Ga]MHLL1, followed by a 10 min static scan 20 min after injection of 15.2±3.8 MBq [18F]FDG and a fast CT scan for colocalization of signals. Binding specificity was assessed by pretreatment with the natGa-labeled reference (1mg/kg, iv, 5 min prior, n =1). In a subset of animals, ex vivo autoradiography confirmed the signal localization with histologic comparison in adjacent sections. Results: After HPLC-purification the precursor MHLL1 was obtained with 28.5±16.8% chemical yield, the reference compound with 6%. The radiochemical yield was 40.0±5.4% with a radiochemical purity of 59.4±9.3%. Uptake assays proved specificity in two FAP-positive cell lines. In vivo PET-imaging of the myocardial infarction using [68Ga]MHLL1 showed accumulation in the infarct territory at ~20 min p.i., with continuous washout. Tracer uptake was elevated in the infarct region compared to remote myocardium at 30-60 min after injection (1.7±0.3 vs. 1.2±0.2%ID/g, p=0.02). Ex vivo autoradiography displayed elevated activity in the infarct territory relative to remote myocardium. Histologic workup demonstrated fibrosis and tissue derangement typical of scar in the region of elevated [68Ga]MHLL1 binding, proving tracer specificity for active fibrosis. Conclusions: In a first in vivo study, the FAP inhibitor [68Ga]MHLL1 presented specific uptake on fibrosis sites in myocardial infarct areas. Therefore, the novel tracer shows high potential as FAP-specific imaging agent, and thus, as a valuable tool in monitoring scar formation and fibrogenesis after infarct and further FAP(+) processes and diseases.

55 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

Acknowledgements: The authors would like to thank Petra Felsch and Alexander Kanwischer for their excellent technical support. The project was supported by the International Isotope Society – Central European Division.

References: 1. Aertgeerts, K. et al.: J. Biol. Chem. 2005,280(20):19441–19444. 2. Rettig, W. J. et al.: Int. J. Cancer 1994,58(3): 385–392. 3. Levy, M. T. et al.: Hepatology 1999,29(6): 1768–1778. 4. Langer, L. B. N. et al.: J. Labelled Compds. Radiopharm. 2019,62(S1): XXXX.

DPhG Annual Meeting 2019 Conference Book • 56 RADIOPHARMACY AND LABELING

SL.38 Development and validation of 2-nitroimidazole-furanoside based fluorine-18 labeled PET- radiopharmaceuticals for the in-vivo detection of hypoxic tissue in tumors

Maier, F. C.1; Schweifer, A.2; Damaraju, V. L.3; Cass, C. E.3; Ehrlichmann, W.1; Kneilling, M.1,4; Pichler, B. J.1; Hammerschmidt, F.2; Reischl, G.1 1 Department of Preclinical Imaging and Radiopharmacy, Eberhard Karls University of Tübingen, Röntgenweg 15, D-72076 Tübingen, Germany 2 Faculty of Chemistry, Institute of Organic Chemistry, University of Vienna, Währingerstraße 38, A-1090 Vienna, Austria 3 Department of Oncology, University of Alberta, Edmonton, Alberta, Canada 4 Department of Dermatology, Eberhard Karls University of Tübingen, Tübingen, Germany

Tumor hypoxia has proven to be a major negative predictive factor for therapy outcome. Positron emission tomography (PET) is a valuable tool for imaging of hypoxia, and radiolabeled 2-nitroimidazoles (azomycins) have been used as biomarkers for many years. [18F]Fluoromisonidazole ([18F]FMISO) can be seen as the most prominent example, which is incorporated into cells via passive diffusion. Another representative, [18F]fluoro-azomycin-α- arabinoside ([18F]FAZA) may be regarded as α-configuration nucleoside, but it is postulated that it also enters cells only via diffusion and is not transported by cellular nucleoside transporters. To mimic nucleosides more closely and thereby enhancing image contrast in comparison to [18F]FAZA, our objective was to 18F-radiolabel an azomycin-2´- deoxyriboside with β-configuration (β-[18F]FAZDR, [1,2]) and comparatively evaluate it versus [18F]FMISO, [18F]FAZA and α-[18F]FAZDR. First, for a deeper insight, we comparatively studied the interaction of FAZA, β-FAZA, α-FAZDR and β-FAZDR with nucleoside transporters (SLC29A1/2 and SLC28A1/2/3) in-vitro. Precursors and non-radioactive standards for [18F]FAZDR derivatives were synthesized from methyl 2-deoxy-D-ribofuranosides. The precursors were radiolabeled in an automated synthesizer. [18F]FMISO, [18F]FAZA or [18F]FAZDR were injected in BALB/c mice bearing CT26 colon carcinoma xenografts, PET scans were performed after 1, 2 and 3 h post injection (p. i.). Nucleoside transporter studies showed variable interactions of the compounds. The highest interactions being for β-FAZDR (IC50 124 ± 33 µM for SLC28A3), but also for FAZA with the non-nucleosidic α-configuration, interactions were remarkable (290 ± 44 µM {SLC28A1}; 640 ± 10 µM {SLC28A2}). α-[18F]FAZDR and β-[18F]FAZDR were synthesized in reasonable radiochemical yields (15.9 ± 9.0% (n = 3), resp. 10.9 ± 2.4% (n = 4)), with radiochemical purities > 98% and molar activities > 50 GBq/µmol. In small animal PET imaging tumor-to-muscle ratios (TMR) were significantly higher for β-[18F]FAZDR at 1 h (2.76) compared to [18F]FAZA (1.69, P < 0.001). In another set of experiments α-[18F]FAZDR was compared to β- [18F]FAZDR and [18F]FMISO. Highest TMR were again observed for β-[18F]FAZDR at 1 h p.i. (2.52 ± 0.94, n = 4) in comparison to α-[18F]FAZDR (1.93 ± 0.39, n = 4) and [18F]FMISO (1.37 ± 0.11, n = 5) with possible mediation by the involvement of nucleoside transporters. After 3 h p. i. TMR were not significantly different for all tracers (2.5 – 3.0). In conclusion, syntheses of α- and β-FAZDR could be developed (non-radioactive or 18F-labeled), allowing their further evaluation. First PET imaging results with both [18F]FAZDR anomers showed advantages over [18F]FAZA and [18F]FMISO, regarding higher tumor contrast at earlier time points p. i., indicating their potential as PET hypoxia tracers. Differences in uptake behavior may be attributed to a potential variable involvement of transport mechanisms and need further investigation.

1. Schweifer, A. et al.: Nucl. Med. Biol. 2016, 43(12): 759-769. 2. Maier, F. C. et al.: Pharmaceuticals 2019, 12(1): 31.

57 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.39 Functionalized Nω-carbamoylated arginines give access to labeled peptide receptor ligands

Keller, M. University of Regensburg, Institute of Pharmacy, Universitätsstraße 31, 93053 Regensburg

Conjugation of radiolabeled moieties or fluorophores to bioactive peptides, in particular via lysine, cysteine or the N-terminal amino acid, is a straightforward approach to obtain labeled peptidic molecular tools. However, the structural modification can impair the biological activity of the peptides and numerous bioactive peptides are devoid of lysine and cysteine. As some of those peptides contain arginine, so far neglected with respect to the preparation of peptide conjugates, we developed a widely applicable labeling strategy for peptides, based on the bioisosteric replacement of arginine by a functionalized Nω-carbamoylated arginine, the latter enabling conjugation of the peptides to various moieties such as fluorescent dyes [1]. This concept was successfully demonstrated for peptidic agonists of neurotensin, angiotensin II and neuropeptide Y Y4 receptors [1,2].

Likewise, the replacement of the guanidine group in the non-peptidic argininamide-type neuropeptide Y Y1 receptor antagonist BIBP3226 [3] by an Nω-carbamoylated guanidine moiety gave access to a radioligand ([3H]UR-MK299) with very high Y1 receptor affinity (Kd = 0.044 nM) [4]. Notably, also the ‘cold’ form of this radioligand proved to be a powerful molecular tool as it considerably promoted the crystallization of the Y1 receptor [5].

Acknowledgements: M. Keller thanks all collaborators who have contributed to the projects. In particular, long-lasting support by Prof. Dr. A. Buschauer, who died in July 2017, is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft (Research Grants KE 1857/1-1 and 1-2, Graduiertenkolleg 1910).

References: 1. Keller, M. et al.: J. Med. Chem. 2016, 59: 1925-1945. 2. Kuhn, K. K. et al.: J. Med. Chem. 2016, 59: 6045-6058. 3. Rudolf, K. et al.: Eur. J. Pharmacol. 1994, 271: R11-R13. 4. Keller, M. et al.: J. Med. Chem. 2015, 58: 8834-8849. 5. Yang, Z. et al.: Nature 2018, 556: 520-524.

DPhG Annual Meeting 2019 Conference Book • 58 RADIOPHARMACY AND LABELING

SL.40 Radioynthesis, in-vitro and in-vivo studies of 177Lu-labeled neurotensin receptor-1 antagonists as radiotracers for the therapy of pancreatic cancer

Prante O1, Reigl U1, Maschauer S1, Gmeiner P2, Kuwert T1 1 Department of Nuclear Medicine, Molecular Imaging and Radiochemistry, Friedrich-Alexander University (FAU), Erlangen, 91054, Germany 2 Department of Chemistry and Pharmacy, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, 91054, Germany

Objectives: Pancreatic adenocarcinoma is the eighth leading cause of cancer death in the world with the poorest prognosis amongst all human malignant solid tumors [1]. The neurotensin receptor-1 (NTS1) is overexpressed in pancreatic tumor tissue [2], thereby it could be a molecular target for receptor-mediated radiotherapy. In this study two non-peptide 177Lu-labeled NTS1 antagonists were evaluated in vitro and in vivo to assess their suitability for NTS1-mediated radiotherapy. Methods: 177Lu-FAUC 469 (1) and 177Lu-DOTA-CL 156 (2) were synthesized with > 98 % radiochemical yield, determined by radio-HPLC analysis of the crude product. The in-vitro evaluation was performed using AsPC-1 cells, a human pancreatic adenocarcinoma cell line. Saturation binding, internalization and efflux studies were accomplished by stability tests of the radiotracers in plasma. NMRI nude mice were used for in-vivo biodistribution studies (1.5 MBq ID/animal) and radiotherapy studies (25 MBq ID/animal, n = 6). The body weights and tumor volumes were determined five times per week. At the endpoint of therapy, the liver, spleen, kidney, heart and tumor tissue morphology were determined by HE, Masson-Goldner and PAS staining. Blood samples were analyzed for AST, ALT, creatinine, bilirubin, and gamma-GT to assess kidney and liver toxicity. A statistical permutation test using a two-sample t-statistic was used to analyze the growth curves of each therapy group. Results: In-vitro characterization demonstrated high binding affinity to NTS1 (0.37 ± 0.13 nM (1) and 0.26 ± 0.06 nM (2), n = 3), fast cell uptake (82.2 ± 2.2 % for 1 and 76.4 ± 2.1 % for 2 after 240 min, n = 3) and high stability in human plasma for at least 96 h. Biodistribution data of 1 and 2 revealed excellent tumor uptake with 22.4 ± 3.3 %ID/g (1) and 34.2 ± 2.5 %ID/g (2) at 24 h p.i., together with low kidney (0.9 ± 0.5 %ID/g) and liver (1.5 ± 0.5 %ID/g) uptake of 2. Significant tumor growth inhibition was shown between day 11 and 21 after begin of treatment with 1 compared to control animals (adjusted p=0.049, n = 6). The histology of kidney and liver sections and the clinical chemistry data from blood samples did not show any toxic effects.

Fig. 1: Chemical structures of 177Lu-FAUC 469 (1) and 177Lu-DOTA-CL 156 (2)

In summary, 177Lu-FAUC 469 (1) represents a highly promising tracer for radiotherapy of NTS1-positive pancreatic carcinoma. The GMP-compliant radiosynthesis and first in-human-studies with 177Lu-FAUC 469 are ongoing.

Acknowledgments: ITG Garching is acknowledged for financial support of this study

References: 1. Cheng H, et al.: Exp. Ther. Med 2012, 4(2): 181-187. 2. Reubi J C, et al.: Gut 1998, 42(4): 546-50.

59 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

3.12 Proteomics in Medicine and Pharmacy Chair: A. Sinz

SL.41 Drug effects on protein homeostasis

Marcus Bantscheff Cellzome, A GSK company, Heidelberg

Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. Mass spectrometry-based proteomics techniques enable the investigation of the causes and consequences of protein degradation in biological systems. We recently developed ‘multiplexed proteome dynamics profiling’, mPDP, combining dynamic-SILAC labelling with isobaric mass tagging for the multiplexed analysis of drug effects and stimuli on protein degradation and synthesis. When applied in combination with other quantitative proteomics approaches such as chemoproteomics and thermal protein profiling, mPDP provides unique insights in drug mechanism-of-action. The presentation will focus on the MoA of targeted protein degraders (PROTACs) and other compounds affecting protein stability.

DPhG Annual Meeting 2019 Conference Book • 60 PROTEOMICS IN MEDICINE AND PHARMACY

SL.42 Laser Capture Microdissection/Mass spectrometry- a promising tool in tissue-based clinical proteomics

Katrin Marcus, Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Germany

The advent of laser capture microdissection (LCM) techniques promise to overcome previous limitations in the isolation of specific cells from tissue samples. At the MPC we have successfully applied this approach in combination with mass spectrometry (MS) for specific characterization of cells and subcellular structures in different tissues such as skeletal muscle and brain. In my talk I will give an introduction about the possibilities using LCM-MS and will present our results from skeletetal muscle studies addressing e.g. myofibrillary myopathies (MFM). The use of our highly sensitive LCM-MS approach helped to discover a number of new disease-relevant proteins accumulating in abnormal muscle fibers of MFM patients. This provides new insights into pathomechanisms of aggregate formation, one of the basic phenomena in the pathogenesis of the disease. The established significant differences between subtype-specific proteomic profiles may also be helpful in differential diagnostics of patients with protein aggregation myopathies.

61 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.43 GMP Radiolabeling and First-in-Man Imaging of an Antibody for the Diagnosis of Aspergillosis

Maurer, A.1; Beziere, N.1; Schwenck, J.1,2; Seyfried, D.1; Wehrmüller, J. E.3; Spycher, P. R.3; Wild, A.-M.1; Davies, G.4; Boschetti, F.6; Wiehr, S.1; Geistlich, S.3; Gunzer, M.5; Thornton, C.4; Schibli, R.3; Reischl, G.1; Pichler, B. J.1 1Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Eberhard Karls University Tuebingen, Germany 2Department of Nuclear Medicine, Eberhard Karls University Tuebingen, Germany 3Paul Scherrer Institute, Center for Radiopharmaceutical Sciences, Switzerland 4ISCA Diagnostics and Biosciences, College of Life & Environmental Sciences, University of Exeter, Exeter, United Kingdom 5Institute for Experimental Immunology and Imaging, University Hospital, University of Duisburg-Essen, Essen, Germany 6CheMatech, Dijon, France

The ubiquitous mould Aspergillus fumigatus can cause life-threatening disease in immunocompromised patients. Current diagnostics by biopsy and subsequent culture of the fungus remains slow and carries risks for the patient, while non-invasive approaches lack specificity and sensitivity. We propose a non-invasive method to diagnose this disease in vivo with high specificity by means of positron emission tomography (PET) using a 64Cu-NODAGA- labeled Aspergillus-specific antibody (JF5). Murine JF5 [1] was humanized by grafting its complementarity-determining regions into a human IgG1 framework and produced in CHO cells according to Good Manufacturing Practice (GMP). The chelator p-NCS-Bn-NODAGA was synthesized according to GMP. We developed a cleanroom-compliant conjugation and diafiltration procedure, produced 150 mg of NODAGA-hJF5 and characterized it using validated procedures. 64Cu radiolabeling and quality control procedures of the tracer were developed and validated. Degree of chelator conjugation was quantified by mass spectrometry. High-performance size exclusion chromatography and ELISA were used to control the integrity and stability of intermediates and the final product. Repeated-dose-toxicity of NODAGA-hJF5 was assessed in rodents, crossreactivity was analyzed on human tissue arrays and peripheral blood mononuclear cells, and immunoactivation was tested in mixed lymphocyte reactions. Performance of [64Cu]Cu-NODAGA-hJF5 was assessed using small animal PET imaging in a mouse model of aspergillosis. A. fumigatus spores were deposited intra-tracheally in neutropenic C57BL/6 mice and the radiotracer (13 MBq) was injected intraveneously. Simultaneous in vivo PET/MR was performed 3, 24 and 48 h after infection using a small animal PET insert within a 7T MRI (Bruker Biospin). In vivo results were validated using ex vivo biodistribution analysis (gamma counting), autoradiography and fluorescence microscopy. hJF5 was found comparable to the murine antibody in immunoassays and was successfully produced in CHO cells under GMP. Diafiltration using a tangential flow filtration system was found to be suitable for buffer exchange and conjugation of larger antibody batches within our cleanroom environment. [64Cu]Cu-NODAGA-hJF5 was successfully prepared adhering to GMP guidelines, with chelator:antibody ratios of ~2 and minimal loss of immunoreactivity. The required specifications for radiotracers were met, and in vivo and in vitro analysis showed a low toxicity profile. The radiopharmaceutical revealed high uptake in vivo at sites of A. fumigatus infection, in particular 48 h after 64 injection, and negligible accumulation in mock-infected animals. This was not observed with [ Cu]CuCl2 nor radiolabeled isotype antibody. [64Cu]Cu-NODAGA-hJF5 was found to provide the best uptake ratio in mice 48 h after injection in the diseased lungs (17.1±2.5 %ID/cc) compared to saline-treated lungs (9.4±1.1 %ID/cc)) (2). First human PET/MR imaging indicated a favorable toxicity profile, low unspecific binding to other pathologies of the respiratory tract, and enhanced focal uptake of the tracer in the lung of patients tested positive for A. fumigatus. Taken together, [64Cu]Cu-NODAGA-hJF5 proved to be a highly specific and sensitive tracer for immunoPET diagnosis of IPA, showing very promising results in preclinical models. Having established the complete production pipeline of this tracer under GMP and obtained encouraging results, we now aim to assess safety and performance of the radiopharmaceutical in clinical trials.

Acknowledgments: The research leading to these results has received funding from European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement n°602820. References: 1. Rolle, A. M. et al.: PNAS 2016, 113(8): 1026-1033. 2. Davies, G. et al.: Theranostics 2017, 7(14): 3398–3414.

DPhG Annual Meeting 2019 Conference Book • 62 PROTEOMICS IN MEDICINE AND PHARMACY

SL.44 Inhibition of Cdk5 – A New Way to Improve the Standard Therapy of Hepatocellular Carcinoma

Maximilian A. Ardelt1,2, Thomas Fröhlich3, Emanuele Martini4, Martin Müller2, Veronika Kanitz5, Carina Atzberger2, Petra Cantonati1, Martina Meßner1,2, Laura Posselt6, Thorsten Lehr7, Jan-Georg Wojtyniak7, Melanie Ulrich2, Georg J. Arnold3, Lars König6, Dario Parazzoli4, Stefan Zahler2, Simon Rothenfußer6, Doris Mayr5, Alexander Gerbes8, Giorgio Scita4, Angelika M. Vollmar2, Johanna Pachmayr1 1 Paracelsus Medical University, Institute of Pharmacy, Strubergasse 15, 5020 Salzburg, Austria. 2 Department of Pharmacy, Pharmaceutical Biology, LMU Munich, Butenandtstr. 5, 81377 Munich, Germany. 3 Laboratory for Functional Genome Analysis, Gene Centre, LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany. 4 IFOM-FIRC Institute of Molecular Oncology, Milan, Italy, and University of Milan, Department of Oncology and Hemato-Oncology, Via Adamello, 16, 20139 Milano MI, Italy. 5 Institute of Pathology, LMU Munich, Marchioninistr. 27, 81377 Munich, Germany. 6 Center of Integrated Protein Science Munich (CIPS-M) and Division of Clinical Pharmacology, Department of Internal Medicine IV, Klinikum der Universität München, Lindwurmstr. 2a, 80337 Munich, Germany. 7 Clinical Pharmacy, Saarland University, Campus C2 2, 66123 Saarbrücken, Germany. 8 Department of Medicine 2, Liver Center Munich, University Hospital, LMU Munich, Marchioninistr. 15, 81377 Munich, Germany.

Introduction: Patients diagnosed with advanced stage hepatocellular carcinoma (HCC) face a poor prognosis with very limited chemotherapeutic treatment options. The efficacy of the only approved multi kinase inhibitors – Sorafenib, Regorafenib and Lenvatinib – is impaired by low response rates and severe side effects. In the case of Sorafenib treatment a compensatory activation of growth factor receptors (GFRs) leads to treatment escape and progression of disease [2]. However, combining Sorafenib with specific inhibitors of individual GFRs has failed to improve the overall survival of HCC patients [3]. Therefore there is an urgent need for new treatment strategies for advanced stage HCC. Here, we investigated cyclin dependent kinase 5 (Cdk5) inhibition as a promising combination strategy to improve the response of HCC to the standard of care treatments. Methods: Functional effects of the combination of Sorafenib/Regorafenib/Lenvatinib and Cdk5 inhibition were evaluated by applying genetic knockdown (siRNA/shRNA, CRISPR/Cas9) and clinically tested inhibitors of Cdk5. The mode of action of Cdk5 inhibition to improve Sorafenib response was judged by a LC-MS/MS-based proteomic approach. Intracellular receptor trafficking was investigated by confocal microscopy with live cell imaging and sophisticated analysis of vesicle properties. Results: Combining Cdk5 inhibition with Sorafenib led to a synergistic impairment of HCC progression in vitro and in vivo, by inhibiting both tumor cell proliferation and migration. In response to Sorafenib HCC cells use a compensatory upregulation of the EGFR signalling pathway to evade Sorafenib treatment, which could be prevented by Cdk5 inhibition. This effect is mediated by a novel mode of action for Cdk5: Cdk5 inhibition interferes with intracellular trafficking which is crucial for GFR signalling, thereby leading to enlarged endosomal vesicles and accumulation of respective cargo proteins. Thus, GFRs are trapped in the endocytotic system and are not available for further activation at the cell surface. First in vitro experiments show that this new, Sorafenib-independent mechanism for Cdk5 can be transferred to Regorafenib and Lenvatinib treatment of HCC cells. Conclusion: The inhibition of Cdk5 offers an effective approach to enhance the response of the standard of care chemotherapeutics and prevent treatment escape. With Dinaciclib a clinically evaluated and well-tolerated Cdk5 inhibitor is readily available and our study provides evidence for clinically evaluating the combination of standard of care multi-kinase inhibitors and Dinaciclib to improve the therapeutic situation for advanced-stage HCC patients.

Acknowledgement: Financial support by DFG (VO376/17-1), Salzburger Krebshilfe Stipendium and Eisai Co., Ltd. is gratefully acknowledged.

References: 1. Pinter M., Peck-Radosavljevic M.: Aliment Pharmacol Ther. 2018, 48(6): 598–609. 2. Blivet-Van Eggelpoel, M. J. et al.: J Hepatol 2012, 57: 108-115 3. Zhu AX, Rosmorduc O, Evans TR, et al.: J Clin Oncol 2015; 33(6):559-66.

63 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

3.13 Microfluidics / Cell-based assays and organoids for drug discovery Chair: S. Wölfl SL.45 Droplet Microfluidics in antibody discovery and personalized cancer therapy

Christoph A. Merten 1 1 European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany

We have developed fully integrated droplet-based microfluidic platforms for the screening of therapeutic antibodies1, 2. In these systems tiny aqueous droplets (picoliter volumes) surrounded by oil serve as independent assay vessels. The technology allows the direct screening of several hundred thousand primary, non-immortalized murine or even human B-cells for the secretion of antibodies that do not just bind to a drug target, but functionally inhibit it. Furthermore, the technology can be used for genotypic and phenotypic characterization of blood cells at the single cell level3. We believe this opens the way for many new approaches in drug discovery, including personalized immunotherapy or the use of antibodies to control cellular pathways at will. In parallel to this we have developed screening platforms enabling rapid identification of optimal drug cocktails for personalized cancer therapy4. Results are available within 24h after surgery at consumables costs of less than 150 US$ per screen. The power of this platform has been demonstrated using cancer cell lines, mouse models and even human tumor biopsies. We now envisage first clinical trials and assess further application fields, e.g. for the efficient stratification of patients for immunotherapies.

1. B. El Debs, R. Utharala, I. V. Balyasnikova, A. D. Griffiths and C. A. Merten, Proc Natl Acad Sci U S A, 2012, 109, 11570-11575. 2. N. Shembekar, H. Hu, D. Eustace and C. A. Merten, Cell Rep, 22, 2206-2215. 3. N. Shembekar, C. Chaipan, R. Utharala and C. A. Merten, Lab on a Chip, 2016, 16, 1314-1331. 4. F. Eduati, R. Utharala, D. Madhavan, U. P. Neumann, T. Longerich, T. Cramer, J. Saez-Rodriguez and C. A. Merten, Nat Commun, 2018, 9, 2434.

DPhG Annual Meeting 2019 Conference Book • 64 MICROFLUIDS / CELL-BASED ASSAYS AND ORGANOIDS FOR DRUG DISCOVERY

SL.46 Analyzing toxicity and metabolic interactions with microfluidic liver-kidney-on-a-chip systems

Stefan Wölfl Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany

The development of new drug compounds is a challenging and time consuming process. Compounds identified by screening of small molecule libraries for disease specific molecular targets need to be further validated for target specificity and efficacy in complex biological systems in early stages of drug development. Microfluidic tissue culture systems based on established cell lines, patient specific specialized cell types or even organoid structures can provide systems for the in vitro analysis of toxicity and metabolic interactions studies that can replace animal testing in early drug development, and open new approaches for the study of physiological and biochemical interactions occurring in humans in patient specific settings. As an alternative to the reconstruction of complex organ and organoid like structures, we use a minimal, more simple approach focusing on tissue specific key metabolic properties that underlie drug metabolism and toxicity. To visualize the cellular responses we analyze metabolic adaptation and changes of the cells in the system and detect changes in gene expression in response to drug treatment using continuous monitoring and long term cultivation. As an example of an optimized microfluidic tissue culture systems for drug testing the multi-chamber systems developed within the SysToxChip consortium [1] will be presented. This chip system can be used to explore liver dependent toxic effects of drugs in human cells [2,3,4]. In addition to drug testing in early drug development, microfluidic chip systems can be used in the clinical setting. In combination with iPSC technology these systems can be used for ex vivo drug testing and to model potential metabolic interactions between cells in the human body on an individual basis.

Funding: BMBF SysTox: FKZ 031A303, iPS-Profiler: FKZ01EK1612

1. *SystoxChip Consortium: Ralf Mrowka, Universitätsklinikum Jena, Miguel Andrade, Uni-Mainz, M. Haltmeier, Genomatix GmbH, Holger Becker, MFCS Jena, Edda Klipp, HU-Berlin, Stefan Wölfl, Uni-Heidelberg, Andreas Kurtz, Charité Berlin 2. Theobald J, Ghanem A, Wallisch P, Banaeiyan A, Andrade-Navarro M, Taškova K, Haltmeier M, Kurtz A, Becker H, Reuter S, Mrowka R, Cheng X, Wölfl S. (2018) Liver-Kidney-on-Chip To Study Toxicity of Drug Metabolites. ACS Biomater. Sci. Eng., 2018, 4 (1), pp 78–89. 3. Theobald J, Cheng X, Ghanem A, Gaitantzi H, Song G, Klipp E, Wodke J, Becker H, Mrowka R, Breitkopf-Heinlein K, Dooley S, Wölfl S. (2017) Monitoring cytochrome P450 activity in living hepatocytes by chromogenic substrates in response to drug treatment or during cell maturation. Arch Toxicol. 2017, doi: 10.1007/s00204-017-2128-1. PMID: 29209748 4. Theobald J, Abu El Maaty MA, Kusterer N, Wetterauer B, Wink M, Cheng X, Wölfl S. In vitro metabolic activation of vitamin D3 by using a multi-compartment microfluidic liver-kidney organ on chip platform. Sci Rep. 2019 Mar 15;9(1):4616. doi: 10.1038/s41598-019-40851-9. PMID: 30874583

65 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.47 Seeking for chemical OCT4 substitutes for the generation of human iPSCs

Yasamin Dabiri, Rodrigo. A. Gama-Brambila, Xinlai Cheng Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany

Induced pluripotent stem cells (iPSCs) can be reprogrammed from terminally differentiated cells by ectopic expression of a core of transcription factors (OCT4, SOX2, KLF4 and MYC), which provides a promising source for autologous organ transplantation, drug discovery and disease modeling and opens a new era in the field of regenerative medicine. Despite of the great improvement in the generation of iPSCs, reprogramming of human somatic cells is still a time-consuming and inefficient process. Genetic and epigenetic abnormalities found in the iPSCs also raised safety concerns of clinical application. In comparison to genetic manipulation, small molecules show distinct advantages in the application and control and thereby have been intensively investigated. Recently, mouse chemically induced pluripotent stem cells (mciPSCs) have been successfully generated. However, the lack of sufficiently strong chemical substitutes for OCT4 makes the generation of human chemically iPSCs difficult. To solve this problem, we performed cell-based high-throughput screenings and identified a series of OCT4 inducing compounds (O4Is). We not only improved the activity of compounds by chemical optimization, but also demonstrated the benefit from our compounds in the maintenance and generation of human iPSCs. Moreover, we combined the chemical modification with various biological assessments, including DNA microarray, RNA-seq, proteomics, ATAC-Seq and knockdown/knockin technology, and successfully elucidated the mechanism of action of our chemical OCT4 substitutes.

Acknowledgments: Thank others for any contributions. Funding This work supported by the DFG grant program (CH 1690/2-1) and the BMBF grant programs Drug-iPS (FKZ 0315398A-FKZ 0315398B) and SysToxChip (FKZ 031A303A-FKZ 031A303E).

References: Dabiri, Y.; Gama-Brambila, R. A.; Taskova, K.; Herold, K.; Reuter, S.; Adjaye, J.; Utikal, J.; Mrowka, R.; Wang, J.; Andrade-Navarro, M. A.; Cheng, X.,* Imidazopyridines as Potent KDM5 Demethylase Inhibitors Promoting Reprogramming Efficiency of Human iPSCs. iScience 2019, 12, 168-181

Cheng, X.; Yoshida, H.; Raoofi, D.; Saleh, S.; Alborzinia, H.; Wenke, F.; Gohring, A.; Reuter, S.; Mah, N.; Fuchs, H.; Andrade-Navarro, M. A.; Adjaye, J.; Gul, S.; Utikal, J.; Mrowka, R.; Wolfl, S. Ethyl 2-((4-Chlorophenyl)amino)thiazole-4-carboxylate and Derivatives Are Potent Inducers of Oct3/4. J Med Chem 2015, 58, 5742-50.

Cheng, X.; Dimou, E.; Alborzinia, H.; Wenke, F.; Gohring, A.; Reuter, S.; Mah, N.; Fuchs, H.; Andrade-Navarro, M. A.; Adjaye, J.; Gul, S.; Harms, C.; Utikal, J.; Klipp, E.; Mrowka, R.; Wolfl, S. Identification of 2-[4-[(4-Methoxyphenyl)methoxy]- phenyl]-acetonitrile and Derivatives as Potent Oct3/4 Inducers. J Med Chem 2015, 58, 4976-83.

DPhG Annual Meeting 2019 Conference Book • 66 NEW RESEARCH; NEW RESEARCHERS II

3.14 New Research, New Researchers II Chairs: A. Link, F. Hansen

SL.48 Direct PPARγ activation by L-thyroxin and TETRAC links thyroid hormone and PPAR signaling

Gellrich, L.1; Heitel, P.1; Bischoff, I.2; Heering, J.3; Kilu, W.1; Göbel, T.1; Pollinger, J.1; Paulke, A.4; Steinhilber, D.1; Proschak, E.1; Wurglics, M.1; Chaikuad, A.1; Knapp, S.1; Fürst, R.2; Merk, D.1 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany 2 Institute of Pharmaceutical Biology, Goethe University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany 3 Project Group Translational Medicine and Pharmacology TMP, Fraunhofer IME, Theodor-Stern-Kai 7, D-60596 Frankfurt, Germany 4 Institute of Forensic Medicine, Goethe University Frankfurt, Kennedyallee 104, D-60596 Frankfurt, Germany

Thyroid hormones (THs) operate numerous physiological processes on genomic level through activation of the nuclear thyroid hormone receptors (THRs). Their signaling pathways were long considered well investigated but in the last decade, several further proteins including surface receptors and transporters were characterized as molecular targets of THs.[1,2] We have discovered a direct peroxisome proliferator-activated receptor gamma (PPARγ) activation by THs as further component of TH signaling. PPARγ as the THRs constitutes a ligand-activated transcription factor and is essentially involved in lipid and glucose homeostasis, adipocyte differentiation and inflammation.[3,4] We have observed remarkable activation of this nuclear receptor by several THs including T4 in cellular and cell-free environment. Amongst THs, the T4 metabolite 3,3’,5,5’-tetraiodothyroacetic acid (TETRAC) – considered as non- classical TH – turned out as most active PPARγ agonist with nanomolar potency in cellular PPARγ activation and cell-free co-activator recruitment. According to isothermal titration calorimetry and X-ray structural analysis (Figure 1), TETRAC binds to the canonical orthosteric PPARγ ligand binding site with nanomolar affinity. Moreover, TETRAC was identified as potent agonist of PPARγ’s heterodimer partner retinoid X receptor (RXR) leading to synergistic PPARγ:RXR heterodimer activation observed by markedly enhanced co-activator recruitment by the dimer. In hepatocytes and fibroblasts, THs robustly induced PPARγ regulated gene expression and THR knockdown in hepatocytes demonstrated THR independence of TH mediated PPARγ modulation. Activation of PPARγ regulated gene expression was also observed in mice indicating biological relevance of the TH-PPARγ:RXR interaction. Our results characterize PPARγ:RXR as direct molecular target of THs and essential part of TH signaling wherein the non-classical TH TETRAC was discovered as most active endogenous PPARγ ligand known to date.

Figure 1: X-ray structure of TETRAC bound to the PPARγ ligand binding domain

References: 1. Cheng, S. et al.: Endocr. Rev. 2010, 31, 139–170. 2. Senese, R. et al.: J. Endocrinol. 2014, 221, 1–12. 3. Vamecq, J., Latruffe, N. Lancet 1999, 354, 141–148. 4. Gellrich, L., Merk, D. Nuclear Receptor Research 2017, 4, 101310.

67 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.49 Targeting biosynthetic networks of the proinflammatory and proresolving lipid metabolome: a promising pharmacological strategy for intervention with inflammation

Gerstmeier, J.1, Werner, M.1, Jordan, P.M.1, Kretzer, C.1, Bilancia, R.2, Rossi, A.2, Serhan, C.N.3, Pace, S.1, Werz, O.1 1Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich Schiller University Jena, Jena, Germany; 2Department of Pharmacy, School of Medicine, University of Naples Federico II, Via Domenico Montesano 49, 80131-Naples, Italy; 3Department of Anesthesia, Perioperative and Pain Medicine, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital–Harvard Medical School, Boston, Massachusetts, USA.

Lipid mediators (LM) encompass proinflammatory prostaglandins (PG) and leukotrienes (LT) but also specialized proresolving mediators (SPM) which display pivotal bioactivities in health and disease. Pharmacological intervention with inflammatory disorders such as asthma and rheumatoid arthritis are commonly practiced with anti- inflammatory drugs that suppress LT or PG formation, which however, possess limited effectiveness and on-target- related side effects. Among the most prescribed drugs to treat inflammation are nonsteroidal anti-inflammatory drugs (NSAIDs) that interfere with the biosynthesis of arachidonic acid-derived proinflammatory PG and LT by targeting cyclooxygenases (COXs), 5-lipoxygenase (LOX), or the 5-LOX–activating protein (FLAP). However, these and related enzymes act in conjunction with marked crosstalk within a complex LM network where also proresolving SPMs are formed, with 15-LOX-1 being the key biosynthetic enzyme. So far, LM biosynthesis inhibitors (including NSAIDs) have been evaluated in cell-based studies with limited read-out, mainly addressing inflammation- promoting PGs and LTs in proinflammatory immune cells (i.e., neutrophils, monocytes, macrophage cell lines), whereas SPMs have not yet been essentially studied because they are the newest mediators uncovered with novel proresolving functions. Here, we present how prominent LM pathways can be differentially modulated in human proinflammatory M1 and proresolving M2 macrophage phenotypes that, upon exposure to Escherichia coli, produce either abundant PGs and LTs (M1) or SPMs (M2) [1]. Targeted liquid chromatography–tandem mass spectrometry–based metabololipidomics was applied to analyse and quantify their specific LM profiles upon drug exposure. Besides expected on-target actions, we found that: 1) COX or 15-LOX-1 inhibitors elevate inflammatory LT levels, 2) FLAP and 5-LOX inhibitors reduce LT biosynthesis in M1 but less in M2 macrophages, 3) zileuton blocks resolution- initiating SPM formation whereas FLAP inhibition increases SPM levels, and 4) that the 15-LOX-1 inhibitor 3887 potently suppresses SPMs in M2 macrophages. Conclusively, interference with discrete LM biosynthetic enzymes in different macrophage phenotypes considerably affects the LM metabolomes with potential consequences for the inflammation-resolution pharmacotherapy [2]. Moreover, we discovered and characterized two novel benzoxanthene lignan structures that promote the above- described LM class switch from proinflammatory towards proresolving LM, not only in human macrophages but also in a zymosan-induced murine peritonitis model in vivo, suggesting their suitability as novel leads for pharmacotherapy of inflammatory disorders [3]. Together, our data may allow better appraisal of the therapeutic potential of these drugs to intervene with inflammatory disorders.

This work was supported by the Deutsche Forschungsgemeinschaft (SFB1127, ChemBioSys, and SFB1278, Polytarget). J.G. received a Carl-Zeiss stipend.

[1] Werz, O., J. Gerstmeier, et al.: Nat Commun, 2018. 9(1): p. 59. [2] Werner, M., . . . J. Gerstmeier, et al.: FASEB J, 2019: p. fj201802509R. [3] Gerstmeier, J., et al., Biochem Pharmacol, 2019.

DPhG Annual Meeting 2019 Conference Book • 68 NEW RESEARCH; NEW RESEARCHERS II

SL.50 Natural Products as Novel Lead Structures: Method Development and Biological Testing

N. Schützenmeister* Hamburg /Germany Universität Hamburg, Pharmaceutical and Medicinal Chemistry, Bundesstraße 45, D-20146 Hamburg,

The treatment of infectious diseases is one of the challenges in drug development due to the increase of resistances of bacteria and viruses. [1] Isolation of natural products from plants, fungi, bacteria, marine organisms et cetera often led to novel drugs, which are today still important for the treatment of infectious diseases (e.g. erythromycin) [2]. Unfortunately, some promising natural products can only be isolated in small amounts from their natural sources limiting the screening for their potential biological activities. Furthermore, some of the natural products serve as lead structure for synthetic analogues with enhanced activity (e.g. amoxicillin). [3]

Total synthesis of natural products is a powerful tool to produce natural products for broader biological evaluation. An interesting example is the natural product class of the rubrolides. This marine-derived natural product family has an interesting portfolio of different biological activities [4]. In 2014, rubrolide R and S were isolated from the fungus Aspergillus terreus (OUCMDZ 1925), which was derived from barracuda intestines [5]. Both structures have been synthesised in a, dramatically short and protecting group free, sequence of three linear steps and were further evaluated in regard to their antiviral and antibiotic activities [6].

Another promising natural product was recently isolated from the endophytic fungus Phomopsis fukushii [7] has also been synthesised. The diarylether 2 showed significant anti-MRSA activity, which was further evaluated.

The so far unprecedented natural product class of the Scleropentasides bear an interesting β-C-acyl moiety.[8] Scleropentaside A 3 has been synthesised in only four linear steps starting from d-Glucose with perfect β-selectivity. The synthetic method allows synthesising β-C-acyl carbohydrates in only a few steps without using heavy metals.[9]

1. European Center for Disease Prevention and Control Antimicrobial surveillance in Europe 2013, Annual Report of the European Resistance Surveillance Network (EARS-Net), 2014. 2. WHO Model List of Essential Medicines, 20th List (March 2017), http://www.who.int/medicines/publications/essentialmedicines/en/ 3. D. J. Newman, G. M. Cragg: J. Nat. Prod. 2016, 79, 629−661. 4. S. Miao et al.: J. Org Chem. 1991, 56, 6275-6260. 5. W. Zhu et al.: J. Antibiot. 2014, 67, 315-318. 6. M. Schacht, G.J. Boehlich, J. de Vries, S. Bertram, G. Gabriel, P. Zimmermann, P. Heisig, N. Schützenmeister.: Eur. J. Org. Chem. 2017, 1745-1748. 7. Z.-J. Li, H.-Y. Yang et al.: J. Antibiot. 2018, 71, 359-362. 8. W. Disadee, C. Mahidol, et al.: Phytochemistry 2012, 74, 115–122. 9. G. J. Boehlich, N. Schützenmeister: Angew. Chem. Int. Ed. 2019, 58, 5110-5113.

69 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.51 Small-molecule inhibitors of human coagulation factor XIIa: synthesis and anticoagulant activity

Korff, M.1;Imberg, L.1; Fluck, G.1; Kalinin, D.V.1 1Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, 48149 Münster, Germany

Thrombosis is the formation of a potentially deadly blood clot inside a vein or artery which is associated with a high risk of mortality.1,2 Hospitalization is the most important risk factor for developing thrombosis resulting in approximately 10% of all hospital deaths.3 The venous thromboembolism-related annual healthcare cost range between $7594 and $16,644 per patient, which is equal to the total annual cost of $2-$10 billion for the patients in the U.S. only.4 Therefore, urgent measures should be undertaken to reduce the mortality rate and economic losses associated with thrombosis. O H O O

CH3 NH2 N OH CH3 O NH N N HN O NH N HO N N S O O O O O Cl Warfarin (1) Dabigatran (2) Rivaroxaban (3) vitamin-K antagonist thrombin inhibitor FXa inhibitor In recent years, a number of anticoagulants (e.g. 1-3) have been developed and proved to efficiently decrease the rate of stroke and other thrombosis-related disorders. Clinically used anticoagulants, however, exhibit unavoidable bleeding risk5 as they target the vital enzymes of the coagulation cascade affecting simultaneously thrombin generation contributing to thrombosis as well as thrombin formation required for hemostasis. Therefore, novel anticoagulants with a new mechanism of action are needed to control thrombosis without the risk of bleeding.

Z Z 2 2 R R N N N N NH2 NH O O N 2 N N R3 R O N R N R R1 (5) (4) (6) X 1 2 3 Z = C=O, SO2, CH2; X=C,N; R=alkyl, phenyl; R ,R ,R = alkyl, alkoxy, phenyl, hal, NH2, etc. Recent studies have proved a fundamental role of Hageman Factor (FXII) in arterial and venous thrombosis. FXII knockout mice were protected from arterial thrombosis, ischemic stroke, and deep vein thrombosis. Despite its essential function in thrombosis, deficiency of FXII does not impair hemostasis in animals and humans.4,5 Therefore, Hageman Factor represent new, potentially safe, and promising target for thrombosis prevention. To find new inhibitors targeting FXIIa, a series of aminotriazoles possessing quinoxalin-2-yl (5) and 6-phenylpyridin- 3-yl (6) moieties were designed and synthesized from commercially available -ketoesters 4. Utilizing chromogenic substrate, synthesized series of aminotriazoles 5 and 6 were subjected to enzymatic assay to evaluate their inhibitory activity towards human FXIIa. Some of the prepared aminotriazoles were found to be nanomolar inhibitors of human coagulation FXIIa (e.g. one of N-acylated derivatives of series 5 showed Ki (β-FXIIa) = 50 nM). Moreover, synthesized compounds significantly prolonged activated partial thromboplastin time (aPTT blood test) thereby showing compounds’ ability to affect the intrinsic coagulation pathway. In contrast, prepared aminotriazoles showed little to no effect on prothrombin time (PT) indicating no influence on the extrinsic coagulation pathway. Synthesized FXIIa inhibitors represent a promising lead for further development of small-molecules targeting thrombosis.

1. Gerotziafas G, Samama M.: Curr. Opin. Pulm. Med. 2004, 10: 356–365. 2. Wendelboe, A. M., Raskob, G. E.: Circ. Res. 2016, 118: 1340–1347. 3. Baglin T.P. et al.: J. Clin. Pathol. 1997, 50(7): 609-10. 4. Beckman M.G. et al.: Am. J. Prev. Med. 2010; 38(suppl): S495–S501. 5. Hohnloser, S.H., Basic, E., Nabauer, M.: Clin. Res. Cardio. 2017, 106(08): 618-628. 6. Baeriswyl, V. et al.: ACS Chem. Biol. 2015, 10: 1861–1870. 7. Renne, T. et al.: J. Exp. Med. 2005, 202: 271–281.

DPhG Annual Meeting 2019 Conference Book • 70 NEW RESEARCH; NEW RESEARCHERS II

SL.52 Structure-activity study and molecular insights in the mode of action of complement C3 inhibitor Cp40

C. Lamers1, B. Wagner1, P. Gros2, J. D. Lambris3, D. Ricklin1 1 Molecular Pharmacy, Pharmazentrum, Klingelbergstr. 50, University of Basel, 4056 Basel, Switzerland 2 Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Sciences, Utrecht University, 3584 CH Utrecht, The Netherlands 3 Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, 422 Curie Blvd., Philadelphia, PA 19104, USA

The complement system serves in blood circulation as “first line of defense” against injurious stimuli and invaders. Upon activation, a series of cascading enzymatic reactions leads to an amplification of the response and to pathogen clearance and opsonic cell killing. Yet complement has also gained increasing interest as a potential drug target, since it may be inadvertently triggered on human cells or biomaterial surfaces, thereby contributing to clinical complications in the pathogenesis of various autoimmune, inflammatory and age-related diseases as well as transplant rejection. While the involvement of dysregulated complement activation in inflammatory and autoimmune diseases is now widely recognized1, so far only one complement-specific drug has reached the market. In this presentation we reflect on the development of the picomolar complement inhibitor Cp40, a next-generation derivative of the compstatin peptide. The C3 inhibitor compstatin was originally identified using a phage display approach2 and several of its derivatives are currently in clinical development. In particular, the presented study is focused on recent structure-activity relationship investigations. Based on a novel co-crystal structure of Cp40 in complex with its complement target C3b, we used site-specific modifications/deletions, nonproteinogenic amino acids and tailor-made building blocks to elucidate the SAR in detail and identify key interaction determinants. By employing functional SPR experiments, we were able to further elucidate the molecular mode of action of Cp40. Building on this insight, we follow various strategies (e.g. prodrug approaches or bioconjugation) to improve affinity and pharmacokinetic properties for a use of this promising inhibitor class in a broad range of disease models.

1 Ricklin D, et al.: J Immunol. 2013; 190: 3831-8. 2 Sahu et al.: J Immunol. 1996; 157: 884-91.

71 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

3.15 The promise of cure - recent breakthrough innovation in cell- and gene therapy Chair: H. Apeler SL.53 Cell and gene therapy developments: it is now or never!

André Berger, Matthias Renner and Klaus Cichutek, Paul-Ehrlich-Institut, Langen, Germany

It has now been almost 30 years since the first gene therapy trial was performed, intended for the treatment of severe combined immunodeficiency with retrovirally modified haematopoetic stem cells. Since then, as with any new medical approach, clinical translation initially led to a mix of encouraging and disappointing results. Advances in gene therapy vector systems and increasing clinical experience eventually paved the way for gene therapy to become a safe and efficacious treatment option for a variety of rare and common diseases.

To date, more than 2,000 gene therapy clinical trials have been or are being conducted with more in the planning. Currently, major clinical developments concern cancer, haemophilia, haemoglobinopathies, neurological disorders and ocular diseases. Mainly genetically modified cells and AAV-based vectors are used as the respective investigational medicinal products. Moreover, other types of viral vectors, plasmids, mRNAs as well as oncolytic viruses and genome editing tools are clinically explored.

In the recent years, special attention was given to the field of cancer immunotherapy, especially chimeric antigen receptor (CAR)- or T cell receptor (TCR)-based adoptive cell therapies. Two CAR T cell medicines, namely Kymriah and Yescarta, have received marketing authorisation in the EU in 2018. By now, six GTMPs (Glybera, Imlygic, Strimvelis, Yescarta, Kymriah, Luxturna) have been granted marketing authorization by the European Commission, since marketing authorisation of the first gene therapy medicinal product (GTMP) in 2012. In the EU, marketing authorization applications of GTMPs as well as of all other advanced therapy medicinal products (ATMPs) are processed via the centralized procedure, which includes initial assessment by experts of two assigned EU member states´ competent authorities. A positive opinion of the Committee for Advanced Therapy (CAT) and the Committee for Medicinal Products for Human Use (CHMP) at the European Medicines Agency (EMA) leads to marketing authorization by the European Commission. In contrast, clinical trial authorization is granted by the national competent authority of the EU Member State, where the trial site is located.

GTMPs are considered most complex biomedicines, often initially developed in an academic setting. Thus, several activities, such as the ATMP action plan or the priority medicines program, were initiated at EU level to support the development of GTMPs and to help developers to comply with regulatory demands. Development of GTMPs generally requires close cooperation of basic scientist, manufactures, contract research organizations (CROs), the PI of the clinical trial, and independent review of the regulatory experts. Issues in manufacturing and control are high variations in cell starting material from individual patients in case of autologous genetically modified cells, non- clinical analyses issues concern obtaining data on the safety and efficacy of the product, in particular as suitable animal models are rarely available, and the design of the clinical study, for example regarding initial dosing or selection of appropriate control groups.

The cutting edge developments in gene therapy and the lack of respective guidance for these very novel products bear challenges not only for the developers, but also the regulatory experts assessing respective clinical trial applications. Such issues as well as potential solutions will be discussed in the presentation.

DPhG Annual Meeting 2019 Conference Book • 72 THE PROMISE OF CURE – RECENT BREAKTHROUGH INNOVATION IN CELL- AND GENE THERAPY

SL.54 Hybrid retrovirus-transposon vectors

Jörn Stitz University of Applied Sciences, Cologne, Faculty of Applied Natural Sciences

A variety of different viral vector systems plays, namely derived from retroviruses such as Human Immunodeficiency Virus (HIV) and Murine Leukemia Virus (MLV) as well as Adeno-Associated Virus (AAV) a pivotal role as gene delivery devices in current somatic gene therapeutic strategies. While great progress has been achieved in the past decade to optimize these vector in regards of their safety profile and gene transfer efficiency, one of the major issues still to be solved are the high costs associated with vector production and purification currently hampering the clinical application of viral vector-based gene therapeutics at economically affordable prices. Thus, we utilized new strategies using transposon vectors to shorten the development time for the establishment of stable viral packaging cells (VPCs). MLV-based VPCs served as a proof-of-concept. The novel VPCs revealed also more than 20-fold higher productivity as compared to VPCs generated using classical approaches such as serial stable transfection with plasmid-based expression constructs. We showed that this advance productivity resulted from enhance particle count and vector fitness. These data shed a promising light on the future utility of this new hybrid-technology for vector systems derived also from other parental viruses.

73 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.55 Challenges in manufacturing and Quality Control of ATMPs

Dr. Gerno Schmiedeknecht Fraunhofer Institute for Cell Therapy and Immunology (IZI), [email protected]

Fraunhofer IZI operates, with more than 130 employees, three state-of-the-art clean room facilities for aseptic manufacturing of cell and gene therapy products. Due to the complexity of the manufacturing processes and quality controls for cell and gene therapies there are many challenges and potential pitfalls on the way to bringing such a product into the clinic. This starts with the enormous and mostly underestimated costs, which are related to the - mainly manual - labor-intensive manufacturing processes and the necessity of a high grade clean room environment. Other underestimated issues are the long timelines and intensive efforts involved in obtaining product- specific GMP-manufacturing authorization and the regulatory approval for a clinical trial. During process transfer or process development various setbacks could occur regarding selection of appropriate raw materials and the subsequent supplier qualification. This is of particular importance for materials of human, animal or recombinant origin that must comply especially with viral and TSE safety regulations. The high process complexity, the variations of the cellular starting material (especially in autologous settings) as well as the variety of different cell and gene therapy products is often related to difficulties in regard to a robust process validation. The same issue occurs for the validation of the highly sophisticated analytical methods, for which standard ICH guidelines or Pharm. Eur. are often not directly applicable. All these issues need special consideration and strategies that require frequent ongoing consultation and interaction with the regulators.

DPhG Annual Meeting 2019 Conference Book • 74 NATURAL PRODUCTS – FROM MOLECULAR PHARMACOLOGY TO CLINICAL APPLICATIONS

3.16 Natural Products – From Molecular Pharmacology to Clinical Applications Chairs: M. Wink, T. Efferth

SL.56 Systems biology with natural products for individualized cancer therapy

Thomas Efferth Johannes Gutenberg University, Mainz, Germany. E-Mail: [email protected]

To combat complex systemic diseases that harbor robust biological networks such as cancer, single target intervention is frequently ineffective. In such cases, network pharmacology approaches are highly useful, because they differ from conventional drug discovery by addressing the ability of drugs to target numerous proteins or networks involved in a disease. Pleiotropic natural products are one of the promising strategies due to their multi- targeting and due to lower side effects. Transcriptomics and proteomics became milestones to predict the response of tumors to standard chemotherapy and to identify the multifaceted modes of actions of synthetic and natural new drugs. The extraction of meaningful data from highly complex big data sets is a critical bottleneck, which requires sophisticated bioinformatical tools. The discourse between different disciplines is needed to develop strategies for effective decision making based on expression profiling and mutation analysis for precision oncology and drug discovery. Based on our own results, we discuss the application of network pharmacology for cancer drug discovery. We provide an overview of the current state of knowledge on network pharmacology, focus on different technical approaches and implications for cancer therapy (e.g. polypharmacology and synthetic lethality), and illustrate the therapeutic potential with selected examples from herbal mixtures, medicinal herbs and isolated phytochemicals. Finally, we present future perspectives on their plausible applications for diagnosis and therapy of cancer.

Selected papers: Efferth T et al. Biotechnol. Adv. 2019 [Epub ahead of print] Boulos JC et al. Cancer Lett. 2019;459:248-267. Abba ML et al. Cancer Lett. 2018;429:11-18. Efferth T, Paul NW. Lancet Planet Health 2017;1:e301-e303. Efferth T. Biotechnol. Adv. 2018;36:1730-1737. Efferth T. Semin. Cancer Biol. 2017;46:65-83.

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SL.57 Treatment of patients with cardiac AL amyloidosis with epigallocatechin-3-gallate (EGCG) from green tea- results of a clinical study and transcriptomics

Hegenbarth U.1; Yang Q.2; Sobeh M.2; Sauer-Gürth H.2; Schönland S.O.1; Wink M.2 1 Amyloidosis Center, University Hospital Heidelberg, 69120 Heidelberg, Germany 2 Institute of Pharmacy and Molecular Biotechnology, INF 364, 69120 Heidelberg, Germany

Systemic amyloid light-chain (AL) amyloidosis is a rare monoclonal B cell disorder with poor prognosis due to amyloid depositions in nearly all organs. Most patients die due to advanced heart involvement. The only evidence based therapy is cytoreduction of the underlying monoclonal gammopathy to stop the production of amyloid precursors. Promising in-vitro data show that epigallocatechin-3-gallate (EGCG), a polyphenol from green tea, can impair amyloid fibrillogenesis. Anecdotal clinical observations even suggest that EGCG has the potential to degrade amyloid from tissues in patients. In the study, 38 patients with cardiac AL amyloidosis have been randomized in a 1:1 fashion after a successful chemotherapy. Major inclusion criteria were: complete or very good partial remission of the underlying plasma cell dyscrasia and cardiac involvement defined by a septum thickness > 12 mm and exclusion of other causes for myocardial hypertrophy. The time between last cycle of chemotherapy and study inclusion had to be 6 months maximum. The oral EGCG / placebo dosage was increased (month 1-3: 400 mg, months 4-6: 800 mg, month 6-12 1200 mg. EGCG levels were measured every 3 months (before and 2 h after the morning dosage) by HPLC. Cardiac MRI was performed at start and end of the study to calculate left ventricular (LV) mass. Cardiac biomarkers as NT-ProBNP, 6 minute walk test and echocardiographic parameters were obtained every 3-6 months. Out of 35 patients 8 patients stopped preterm (median 6,5 months after randomization, range 1-11,5) due to progression of gammopathy (n=5), side effects (n=2) or newly diagnosed breast cancer (n=1). Therefore, 27 patients can be analysed per protocol. No patient died during the study period. Treatment with EGCG capsules, even at higher dosage, had no severe side effects in 36/38 patients. The first analysis of study results showed no significant difference regarding LV mass, NT-proBNP values and 6-minute walktest. Therefore the primary and main secondary endpoints of the study have not been met. EGCG levels in the blood increased significantly when the dosage was increased to 2x 600 mg between months 6-12. Blood samples were used to determine the EGCG levels. In parallel, we isolated mRNA from blood cells and generated transcriptomes by RNASeq using ILLUMINA sequencers. Substantial differences could be observed in differentially regulated genes in placebo and verum patients. After 12 months, 449 differentially expressed genes are significantly up-regulated und 445 genes are down-regulated in verum patients, while 265 genes are up- regulated and 426 genes are down-regulated in placebo patients compared to the beginning of the study. Between placebo and verum patients, 38 genes are up-regulated and 22 genes are down-regulated. Based on the gene ontology enrichment and molecular function analysis, these genes are enriched with pathways in B cell activation, B cell receptor signalling and blood coagulation. Our findings could suggest an involvement of EGCG in the regulation of amyloid fibril formation. The failure of the clinical trial might have several reasons: too low EGCG levels between months 1-6, small study population, reduced compliance of the patients to withhold from green tea for one year. The EGCG effect might be not as strong as expected. However, the anti-amyloid effects of EGCG or other catechins should be further studied in AL amyloidosis patients.

Acknowledgements: The study was supported by Taiyo GmbH (EGCG powder) and funded by the Ministry of Education and Science; BMBF (GERAMY FKZ 01GM1107A).

DPhG Annual Meeting 2019 Conference Book • 76 NATURAL PRODUCTS – FROM MOLECULAR PHARMACOLOGY TO CLINICAL APPLICATIONS

SL.58 Targeting the mycobacterial membrane with photosensitizers

Jessen-Trefzer C1 1University of Freiburg, Stefan-Meier-Str. 19, Germany

Trehalose is a non-mammalian disaccharide found in the cell wall of mycobacteria. It is actively taken up by transport systems, modified and incorporated into the cell wall as trehalose monomycolate or dimycolate. We investigate the application range of several trehalose-conjugates and take benefit of the promiscuity of the extracellular enzyme complex Ag85, which incorporates trehalose tethered photosensitizers into the mycomembrane. Irradiation triggers singlet oxygen formation, killing mycobacterial cells more efficiently, as compared to photosensitizers devoid of trehalose conjugation. Our probes are potent anti-mycobacterial agents that are per se neither affected by permeability issues nor by detoxification mechanisms via drug efflux, and serve as interesting scaffolds for photo-induced therapy.

Dutta, Amit K.; Choudhary, Eira; Wang, Xuan; Záhorszka, Monika; Forbak, Martin; Lohner, Philipp; Jessen, Henning J.; Agarwal, Nisheeth; Korduláková, Jana; Jessen-Trefzer, Claudia: ACS Central Science, 2019, 5 (4), 644–650.

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3.17 Bioanalytical Mass Spectrometry Chair: M. Lämmerhofer

SL.59 Spatial metabolomics in tissues and single cells

Theodore Alexandrov1,2 1Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany 2Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA

Recent discoveries put metabolism into spotlight. Metabolism not only fuels cells but also plays key roles in health and disease in particular in cancer, inflammation, and immunity. In parallel, emerging single-cell technologies opened a new world of heterogeneous cell types and states previously hidden beneath population averages. Yet, methods for discovering links between metabolism, cell states, metabolic plasticity and reprogramming on the single-cell level and in situ are crucially lacking. First, I will present how the emerging technology of imaging mass spectrometry can be used for spatial profiling of metabolites, lipids, and drugs in tissues (Palmer et al., 2017). Second, I will present our method SpaceM for spatial single-cell metabolomics in situ (Rappez et al., 2019). We applied SpaceM to investigate hepatocytes stimulated with fatty acids and cytokines, a model mimicking the inflammation-associated transition from the fatty liver disease NAFLD to steatohepatitis NASH. We characterized the metabolic state of steatotic hepatocytes and metabolic plasticity associated with the inflammation. We discovered that steatosis and proliferation take place in distinct cell subpopulations, each with characteristic spatial organization and metabolic signatures. Overall, such methods open novel avenues for understanding metabolism in tissues and cell cultures on the single-cell level.

Palmer, A. et al. FDR-controlled metabolite annotation for high-resolution imaging mass spectrometry. Nat. Methods 14, 57–60 (2017) Rappez, L. et al. Spatial single-cell profiling of intracellular metabolomes in situ. bioRxiv (2019)

DPhG Annual Meeting 2019 Conference Book • 78 BIOANALYTICAL MASS SPECTROMETRY

SL.60 MALDI Mass Spectrometry-Based Applications in Pharmacology

Carsten Hopf, Center for Mass Spectrometry and Optical Spectroscopy (CeMOS), Mannheim, Germany, c.hopf@hs‐mannheim.de

Very fast MALDI mass spectrometry (MS) is an emerging label-free technology for high-throughput enzyme assays that are devoid of photonic interferences. Recently, two types of cell-based MALDI MS assays have been presented: In target-agnostic phenotypic assays computational analysis of drug concentration-dependent changes of mass spectral patterns reveals lipids/metabolites such as Heme B that can be identified by ultra-high resolution MS and that can serve as response markers in drug discovery or chemical biology (1). Furthermore, cellular malonyl-CoA accumulation in response to fatty acid synthase (FASN) inhibition has been utilized for the characterization of potent FASN inhibitors in a mechanistic MALDI MS cell assay (2). MALDI MS imaging (MSI) enables the label-free, spatially resolved analysis of lipids, metabolites, drugs or proteins at cellular resolution (“spatialomics”). For each pixel, it simultaneously measures the masses of hundreds to thousands of biomolecules (3). High molecular content datasets are statistically-computationally analyzed to reveal biomolecular patterns that correlate or colocalize with histopathology or other imaging modalities. New MSI trends will be discussed: Quantitative MS drug imaging as post-surgery control of drug disposition in human clinical samples (4), molecular analysis of drug-induced phospholipidosis or crystalline deposits in rodent kidney in toxicological pathology (3) and MS Imaging of endogenous tumor protease activity in fresh-frozen tissue sections (5). Most recently, combination of fast mid-infrared (MIR) imaging of naïve tissue sections with MALDI MSI has suggested a new avenue towards fast detection and molecular analysis of tumor margins as well as focused analysis of computationally defined tissue morphologies by ultra-high resolution MSI (6).

References: 1. Weigt D, Sammour DA, Ulrich T, Munteanu B, Hopf C (2018). Automated analysis of lipid drug-response markers by combined fast and high-resolution whole cell MALDI mass spectrometry biotyping. Sci Rep. 8(1):11260. 2. Weigt D, Parrish CA, Krueger JA, Oleykowski CA, Rendina AR, Hopf C (2019). Mechanistic MALDI-TOF Cell-Based Assay for the Discovery of Potent and Specific Fatty Acid Synthase Inhibitors. Cell Chem Biol. 2019 Jul 2. pii: S2451-9456(19)30205-3. doi: 10.1016/j.chembiol.2019.06.004. [Epub ahead of print] 3. Schulz S, Becker M, Groseclose MR, Schadt S, Hopf C (2019). Advanced MALDI mass spectrometry imaging in pharmaceutical research and drug development. Curr Opin Biotechnol. 55:51-59. 4. Abu-Sammour D, Marsching C et al (2019). Quantitative Mass Spectrometry Imaging Reveals Mutation Status-independent Lack of Imatinib Penetration into Liver Metastases of Gastrointestinal Stromal Tumors. Sci. Rep., in press. 5. Erich K et al. (2019). Spatial Distribution of Endogenous Tissue Protease Activity in Gastric Carcinoma Mapped by MALDI Mass Spectrometry Imaging. Mol Cell Proteomics. 18(1):151-161. 6. Rabe JH et al. (2018). Fourier Transform Infrared Microscopy Enables Guidance of Automated Mass Spectrometry Imaging to Predefined Tissue Morphologies. Sci Rep. 8(1):313.

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SL.61 Sex bias in colitis yields a protective role of specialized pro-resolving mediators in female mice

Pace S.1, Troisi F.1, Bilancia R.1,2, Gerstmeier J.1, Schädel P.1, Ialenti A.2, Rossi A.2, Serhan, C.N.3, Borrelli F.2, Werz O.1 1Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich Schiller University Jena, Jena, Germany; 2Department of Pharmacy, School of Medicine, University of Naples Federico II, Via Domenico Montesano 49, 80131-Naples, Italy; 3Department of Anesthesia, Perioperative and Pain Medicine, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital–Harvard Medical School, Boston, Massachusetts, USA.

The incidence, prevalence and severity of various chronic immune-mediated diseases with unresolved inflammatory component are sex-biased and regulated by sex hormones. Crohn’s disease and ulcerative colitis, the most common forms of inflammatory bowel diseases (IBD), are characterized by chronic intestinal inflammation caused by inflammatory signals including cytokines, chemokines, proteases, and lipid mediators (LM), associated to mucosal injury, increased epithelial permeability, and marked neutrophil recruitment. Accumulating evidence implicate sex differences in the onset and progression of IBD and sex hormones affect the susceptibility to IBD as well as disease severity and progression. Here, we demonstrate that the sex dimorphism in experimental colonic inflammation correlates to different levels of specialized pro-resolving mediators (SPM) in the colon of male versus female mice. Oral dextran sodium sulfate (DSS) administration caused more severe colon inflammation in male versus female mice along with higher colonic myeloperoxidase activity and plasma chemokine levels during the acute phase. Targeted liquid chromatography-tandem mass spectrometry-based metabololipidomics (LC-MS/MS) of colonic tissues revealed superior amounts of SPM (i.e., resolvin (Rv)D2 and RvD5, protectins, and maresin-1) and their biosynthetic lipoxygenases in healthy female versus male mice. However, SPM levels were similar in the acute inflammatory phase between the sexes and even inverted in the resolution phase. Gonadectomization of male mice elevated colonic SPM without alterations in females. Intrarectal application of RvD2, RvD5 and maresin-1 prevented DNBS-induced colon inflammation and chemokine production in male mice, and thus abolished the sex bias. Taken together, the biosynthetic pathway of SPM is sex-biased in the colon of mice with superior colonic SPM levels in female versus male animals. Our data indicate that the lower susceptibility of female mice to develop colitis versus males in experimental IBD models coincides with higher colonic SPM levels in females under healthy conditions that eventually protect against the inflammatory disease. Since resolution pharmacology is emerging as an alternative and attractive strategy to intervene with excessive and chronic inflammation, our findings may help to improve established pharmacotherapeutic concepts of IBD, in a gender-tailored manner.

DPhG Annual Meeting 2019 Conference Book • 80 CHALLENGES AND OPPORTUNITIES IN MODEL BASED DOSE ADAPTION

3.18 Challenges and opportunities in model based dose adaptation Chairs: C. Kloft, T. Lehr

SL.62 Therapeutic drug monitoring in oncology – Case examples

Scherf-Clavel, O.1, Kurlbaum, M.2, Kroiss, M.2, Fassnacht, M.2, Isberner, N.3, Klinker, H.3 1University of Würzburg, Institute for Pharmacy and Food Chemistry, Am Hubland, 97074 Würzburg, Germany 2University Hospital Würzburg, Department of Internal Medicine I, Oberdürrbacher Straße 6, 97080 Würzburg 3University Hospital Würzburg, Department of Medicine II, Oberdürrbacher Straße 6, 97080 Würzburg

Kinase inhibitors (KI) play a pivotal role in the molecular targeted therapy of a variety of malignancies. Unfortunately, therapeutic success is often associated with relevant adverse effects leading to a substantial loss of quality of life or even to discontinuation of treatment. Furthermore, a relapse of the disease is regularly observed in patients treated with kinase inhibitors after initial therapeutic response. Supra- or subtherapeutic drug exposure is a potential cause for these observations. Many KIs are characterized by a large interindividual variability in pharmacokinetics (PK) and several studies have already depicted a correlation between drug exposition on the one hand and adverse effects and/or therapeutic effectiveness on the other hand for some substances. These findings can for instance be attributed to oral administration (dependent on concomitant food uptake, gastrointestinal absorption and intragastric pH-value), metabolization by cytochrome P450 enzymes, extensive comedication, albumin dependant distribution, and adherence [1]. Many KIs are substrates of CYP3A4, induction or inhibition of this isoenzyme might be of clinical relevance. An example for a potent CYP inductor is mitotane. The orphan drug is used for the treatment of adrenocortical carcinoma (ACC) in adjuvant setting and metastatic disease. Due to the limited response rate, there are many efforts trying to replace mitotane treatment by modern KIs and new cytotoxic regimens. Small studies and case series investigating erlotinib, gefitinib and imatinib, all substrates of CYP3A4, yielded disappointing results [2, 3, 4]. It is worth noting, that mitotane exhibits very unfavourable pharmacokinetics with elimination half-lives ranging from weeks to months. Clinically relevant enzyme induction might still be observable months after cessation of drug intake. This could have led to subtherapeutic exposure in those studies. Thus, therapeutic drug monitoring (TDM) will be crucial in future trials investigating CYP3A4 substrates for the treatment of ACC when the patients were pre- treated with mitotane. TDM of KIs is associated with several obstacles: Sample collection is difficult, due to the fact, that most patients receiving treatment are outpatients. For many substances, there is a lack of a validated PK target or proposed PK- targets refer to area under the concentration-time curve (AUC) which is not easily assessable in routine drug monitoring, due to sparse sampling. Very limited data on the pharmacokinetics of KIs in patients with special conditions e.g. dialysis or hepatic impairment are available and there are substances with a daytime dependent bioavailability (e.g. Nilotinib) complicating the interpretation of through levels. Model supported therapeutic drug monitoring could remove the blind folds and even allow for having a glimpse at the future by incorporation of prior knowledge and simulations. Through levels could be derived from a sample collected at any, well-documented time point and an estimated AUC based on the pharmacokinetic model could be used to evaluate target attainment. Furthermore, new techniques enabling the patients to collect TDM samples themselves could be crucial to obtaining useful and enough data for the model supported TDM of orally administered KI.

Acknowledgments: The Hector Stiftung II gGmbH is gratefully acknowledged for funding parts of this work.

References: 1. Groenland, S., et al.: Eur. J. Clin. Pharmacol. 2019, (Epup ahead of print): 10.1007/s00228-019-02704-2 2. Samnotra, V, et al.: J. Clin. Oncol. 2007, 25(18_suppl): 15527-15527. 3. Quinkler, M, et al.: J. Clin. Endocrinol. Metab. 2008, 93(6): 2057-2062. 4. Gross, DJ, et al.: Endocr. Relat. Cancer. 2006 13(2): 535-540.

81 • DPhG Annual Meeting 2019 Conference Book SCIENTIFIC LECTURES

SL.63 Model-based precision dosing of tamoxifen therapy

Klopp-Schulze L.1, Joerger M.2, Kloft C.1 1Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Germany 2Medical Oncology and Clinical Pharmacology, Dept. of Internal Medicine, Cantonal Hospital St. Gallen, Switzerland

Background & Objectives: Patients diagnosed with an oestrogen receptor (ER)-positive breast cancer are commonly treated with the selective-oestrogen modulator tamoxifen. This oral anticancer drug is administered at a standard dose of 20 mg once daily (QD), with long treatment durations in the adjuvant setting (>5 years). Since the discovery of the 100-fold more active metabolite endoxifen, tamoxifen is considered a prodrug. Large variabilities in tamoxifen and endoxifen pharmacokinetics (PK) have been observed between patients treated with 20 mg tamoxifen daily, which has been partly attributed to variations in patients’ CYP2D6 enzyme activity. A large clinical study identified an association between endoxifen and treatment success suggesting a therapeutic target concentration (6 ng/mL [1]). Whether and how to individualise tamoxifen treatment is in focus of ongoing intense discussions. Here we compare three different dosing strategies and propose a more appropriate one which potentially maximises clinical efficacy in the individual patient applying clinical trial simulations. Methods: A large clinical target population was simulated (n=10,000; CYP2D6 poor (PM): intermediate (IM): normal/ultrarapid (NM) metaboliser: 5%:15%:80%). Using a population PK model [2] three dosing strategies were investigated: (1) Standard dosing (20 mg/day) (2) CYP2D6-adapted dosing (3) Model-informed therapeutic drug monitoring dosing For strategy (3) initial dosing was based on (2) and was revised after two weeks QD dosing considering a “virtual” blood sample using Bayesian forecasting. The lowest dose needed to achieve target concentrations was used as dose decision criterion. The strategies were compared with respect to PK target attainment (%patients at risk) and remaining variability in exposure at steady-state in the CYP2D6 subgroups and the population after QD dosing. Results: 97% PM and 52% IM were at risk of subtherapeutic concentrations after standard treatment (strategy 1). Daily doses of 40 and 80 mg for IM and PM were appropriate to reduce the risk to <10% and to obtain similar concentrations to those of NM (20 mg) (strategy 2). However, the variability in endoxifen concentrations across (64% vs. 53% CV in strategy 1 vs. 2) and within the subgroups (both >40% CV) remained large. With strategy (3) the target concentration was met with doses from 5-120 mg/day showing a narrower endoxifen concentration range within the sub/-population. Moreover, target concentrations were achieved with lower doses (as compared to strategy 2) in a considerable number of NM (5 or 10 mg: 38%), IM (10 or 20 mg: 42%) and PM (40 or 60 mg: 79%). Conclusions: With the model-informed dosing strategy (3) the PK target was met in almost all patients with the lowest individually required dose, significantly decreasing the variability in endoxifen concentrations across patients. Strategy (3) has further advantages: (i) In the long-term therapy (adjuvant or palliative setting) it might be beneficial to avoid unnecessary high concentrations reducing the risk for adverse events. (ii) By adapting the dose early in treatment the time to target attainment was reduced, which might be of importance in the neoadjuvant and metastatic setting. Prerequisites to perform strategy (3) comprise: CYP phenotype and plasma concentration determinations as well as an experienced interdisciplinary team of pharmacists/clinicians to predict individual doses. Model-based precision dosing might be the best strategy for the individual patient, however, for feasibility in practice a quick and easy-to-use tamoxifen dose decision tools ought to be available.

References: 1. Madlensky L., et al. Clin. Pharmacol. Ther. 2011 89: 718–25 2. Dahmane EBA. Thèse de doctorat: Univ. Genève 2013 no. Sc. 46172013

DPhG Annual Meeting 2019 Conference Book • 82 CHALLENGES AND OPPORTUNITIES IN MODEL BASED DOSE ADAPTION

SL.64 Methodological aspects of model-based dose adaptations

Sebastian G. Wicha Dept. of Clinical Pharmacy, Institute of Pharmacy, University of Hamburg, Germany

Precision dosing software using pharmacometric models to integrate prior information on the pharmacokinetics of a drug has the merit to streamline the therapeutic drug monitoring (TDM) process. Advantages of model-based dose adaptation include the possibility to derive individualized dosing regimens already before reaching steady state and even before the first dose through probabilistic simulation. Moreover, less reliance on fixed sampling times, a lower number of total required TDM samples and potentially increased precision in the derived individual PK estimates and calculated doses make precision dosing software appealing for clinical implementation. On the other hand, a number of scientific and implementation challenges remain to be addressed in order to benefit most from the merits of model-based TDM, of which the following will be discussed: How is the impact of uncertainty in sampling time in model-based TDM? When is TDM sampling most informative? A simulation study with the antibiotic meropenem, for which TDM is increasingly done, will be presented in which the impact of typically clinically observed uncertain recording of infusion duration (varied from ±7.5 min to ±22.5 min, standard deviation [SD]) and sampling times (varied from ±5 min to ±30 min, SD) on determined the population and individual parameters will be shown. This case study demonstrated that undocumented uncertainty of only ±5 min (SD) could substantially increase the imprecision of pharmacokinetic parameters on both the population as well as individual level of short half-life drugs such as meropenem. Moreover, if an erroneous dataset was used for the development of a pharmacometric model, more TDM samples were required to determine the individual profile accurately due to inflation of the residual variance. Which pharmacometric model shall be used as prior information in model-based dose adaptation? Case studies with vancomycin and coagulation factor VIII will be presented, in which strategies for a fit-for-purpose evaluation of pharmacometric models in model-based dose adaptation will be outlined. However, in some situations when patients are unstable or not directly assignable to a specific population, the a priori choice of a single underlying pharmacometric model remains challenging. For this purpose, a recently developed ‘learning’ model-averaging algorithm will be presented that continuously evaluates a number of candidate models during the TDM process and selects the most suitable model for an individual patient over the therapeutic course. Using a simulation study, it was shown for the antibiotic vancomycin that the algorithm was in particular useful to discriminate the pharmacokinetic profile from patients originating from substantially different populations, e.g. critically ill or obese patients and improve the predictive performance in these ‘outlier’ patients.

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4 POSTERS

DPhG Annual Meeting 2019 Conference Book • 84 MEDICINAL CHEMISTRY AND DRUG DESIGN

(multidrug resistant Gram-negative bacteria, e.g. Acenitobacter 4.1 Medicinal Chemistry and Baumanii) can be spread via those1. Because the percentage of resistant germs is rising2 and lack of new innovative antibiotics, there is a need for new methods to fight bacteria3. Soluble cationic antiseptics stands out drug design against classic antibiotics, because they have a differing mode of action POS.1 and there are little to no resistances known against them4. The downside of the soluble cations is that the only provide a temporary protection of Synthesis and structure-activity relationship studies on the surface. By attaching them permanently to a surface, a more indolylalkyl-substituted piperidine-1-carboxylates as permanent solution is attainable. The biocidal effect of simple ammonium inhibitors of fatty acid amide hydrolase and cations (e.g. trimethylalkylammonium) has been shown in the past5. As monoacylglycerol lipase anchoring group, phosphonic acids have good capabilities to form strong bonds to different metals6. Herein we present the synthesis of other more complex cationic Barth,M.1; Lehr,M.1 compound. A modular synthetic approach is used to allow easy 1 Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstraße 48, adaptation to increase coating capabilities and antimicrobial properties. 48149 Münster, Germany Synthesized molecules were coated on titanium nanoparticles to evaluate the coating properties of the compound. Afterwards the Anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are the main molecules were coated on titanium disk to evaluate the antibiotic activity. endogenic agonists of the cannabinoid receptors CB1 and CB2 in the mammalian organism. The biological effects of these so-called endocannabinoids include analgesic and anti-inflammatory activities. Effector: Antimicrobial roperties AEA and 2-AG are produced on demand and are rapidly inactivated, AEA Effector e.g.: ammonium, phosphonium here: predominantly by fatty acid amide hydrolase (FAAH) and 2-AG mainly by quaternary ammonium salts monoacylglycerol lipase (MAGL). Because inhibition of the degradation of the endocannabinoids prolongs their beneficial effects, inhibitors of Linker: Alkyl- or Aryl chains to optimize the FAAH and MAGL are regarded as new agents for the treatment of pain coating density and inflammatory diseases.

Variation of the Replacement by chaing length Anchor: Binding Metals (e.g.: Ti, Fe, Mg, Al) Here: phosphonic acids CF3CHCF3

N Introduction of O N substituents O

1

Recently, we have found that certain indolylalkyl-substituted phenyl piperidine-1-carboxylates, such as compound 1, are potent inhibitors of [References] FAAH that do not inhibit MAGL [1]. In literature, it has been reported that 1. G. D. Bixler, B. Bhushan, Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 2012, 370, 2381-2417. replacing the aryl group of aryl pyrrolidine-1-carboxylate inhibitors of 2. Annual report of the European Antimicrobial Resistance Surveillance Network (EARS-Net) FAAH and MAGL by an 1,1,1,3,3,3-hexafluoropropan-2-yl substituent 2017 shifts activity from FAAH towards MAGL inhibition [2]. In order to also 3. G. Karam, J. Chastre, M. H. Wilcox, J. L. Vincent, Critical care (London, England) 2016, 20, obtain compounds with MAGL inhibitory potency, we have started 136. investigations on indolylalkylpiperidine-1-carboxylate derivatives, which 4. P. Gilbert, L. E. Moore, J Appl Microbiol 2005, 99, 703-715. 5. E.-R. Kenawy, S. D. Worley, R. Broughton, Biomacromolecules 2007, 8, 1359-1384. contain this particular group. In the course of structure-activity 6 relationship studies among other variations the length of the alkyl spacer . C. Queffélec, M. Petit, P. Janvier, D. A. Knight, B. Bujoli, Chem. Rev. (Washington, DC, U. S.) 2012, 112, 3777-3807. was altered and various substituents were introduced into the indole heterocycle. These structural variations led to potent dual FAAH/MAGL as well as selective MAGL inhibitors. POS.3 [References] The orphan nuclear receptor Nurr1 is responsive to non- 1. Dahlhaus H., Hanekamp W., Lehr, M.: Med. Chem. Commun. 2017, 8(3), 616-620. steroidal anti-inflammatory drugs 2. McAllister, L. et al.: J. Med. Chem. 2018, 61(7), 3008-3026.

Willems, S.1; Kilu, W.1; Heering, J.2; Chaikuad, A.1; Merk, D.1 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt, Germany POS.2 2 Project Group Translational Medicine and Pharmacology TMP, Fraunhofer IME, Theodor- Stern-Kai 7, D-60596 Frankfurt, Germany Synthesis and evaluation of pharmaceutical relevant surface modifiers Nurr1, as a member of the nerve growth factor-induced  subfamily of orphan receptors [1], is a neuroprotective transcription factor mainly Naundorf, T.1; Maison, W.1 found in dopaminergic neurons. Levels of Nurr1 are diminished in 1 Universität Hamburg, Institut für Pharmazie, Bundesstraße 45, 20146 Hamburg, Germany Parkinson’s Disease (PD) patients and dopamine neuron development in midbrain is Nurr1 dependent. Moreover, Nurr1 knock-out in mature dopamine neurons resembled the progressive pathology in early stage The antimicrobial functionalization of surfaces plays a decisive role in the of PD [2]. Therefore, Nurr1 appears as promising target in PD treatment. improvement of hygiene in the personal and clinical environment. Much Originally, Nurr1 was considered as ligand-independent nuclear receptor used surfaces for instance handrails, balustrades or doorknobs present due to its closed ligand-free conformation and its high constitutive activity a big risk potential, because multi drug resistant germs for instance [1]. To date, no endogenous ligand has been clearly identified but MRSA (Methicillin-resistant Staphylococcus aureus) or 4-MRGN recently, crystal structures of Nurr1 ligand binding domain in complex

85 • DPhG Annual Meeting 2019 Conference Book POSTERS with two different prostaglandin A (PGA) forms were published [3,4]. Both Recently, we identified a series of thiol-containing inhibitors of highly structures reveal a binding site entirely different from nuclear receptors prevalent metallo-β lactamases that restore the function of the β-lactam canonical binding site. Compared to the Nurr1 ligand binding domain antibiotics [3]. Encouraged by these results, we screened our in-house (LBD) apo crystal structure [1], intermolecular stabilization of activation thiol-containing compound library and found a highly potent broad- function 2 located in the C-terminal helix 12 seems to be responsible for spectrum inhibitor of class B1 type β-lactamases, NDM-1, VIM-1 and Nurr1’s intrinsically active conformation. As ligand binding causes IMP-7 (IC50: 0.08-1 µM). outward movement of helix 12 these interactions disappear and space In order to further investigate the structure-activity relationship, a series for a new pocket is created suggesting a possibility for bidirectional of derivatives were synthesized which led to an improvement in the in modulation of Nurr1 activity (Figure 1). vitro activity against these MBL´s. In view of overcoming potential solubility and permeability issues, various chemical groups were introduced on the core structure. In addition to this, co-crystallization studies are ongoing to obtain a complex structure, providing the basis for structure-based optimization.

[1] Büttner, D. et al.: ACS Infectious Diseases. 2018 4(3): 360-372. [2] Fast, W. et al.: Curr Drug Targets. 2016, 17(9): 1029–1050. [3] Proschak, E. et al.: J. Med. Chem. 2015, 58(8): 3626-3630

POS.5 Helix 12 Synthesis and structural activity relationships of novel dihydroisoindol-based anti-infective agents against Trypanosoma brucei rhodesiense

Figure 1: X-ray structures of Nurr1 LBD in apo (PDB: 1OVL [1], grey) and Silva, D. G.1; Emery, F. da Silva2; Maes, L.3; Kratz, J. D.4; Junker, A.1. PGA1 (purple) (PDB: 5Y41 [3], petrol) bound state. 1 European Institute for Molecular Imaging (EIMI), Westphalian Wilhelms-University Münster-D- 48149 Münster, Germany. 2 Inspired by PGA bound Nurr1, non-steroidal anti-inflammatory drugs School of Pharmaceutical Sciences of Ribeirão Preto - University of São Paulo, Ribeirão Preto, São Paulo 14040-903, Brazil. (NSAIDs) as drugs interfering with prostaglandin metabolism were 3 Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Universiteitsplein screened for Nurr1 modulatory activity. In our Gal4 hybrid based cellular 1 (S7), Wilrijk B-2610, Belgium. 4 test system, Nurr1 responded to diverse NSAIDs with agonistic and Drugs for Neglected Diseases initiative - Latin America, Rio de Janeiro, RJ 20010-020, Brazil. inverse agonistic behavior. Isothermal titration calorimetry (ITC) confirmed binding of the active hits to recombinant Nurr1 protein and we Sleeping sickness (Human African Trypanosomiasis - HAT) remains one observed marked effects of NSAIDs on nuclear receptor corepressor 1 of the most public health problems in Africa. The disease is caused by (NCor1) recruitment status to Nurr1 in cell-free environment. The most two subspecies of trypanosoma brucei: gambiense and rhodesiense. active NSAIDs concerning Nurr1 modulation were selected for structural While T. b. gambiense causes a chronic illness with the onset of optimization where we discovered optimization potential (EC/IC50 5 - symptoms after a prolonged incubation period; T. b. rhodesiense causes 39 µM) in early SAR evaluation supporting the suitability of NSAIDs as a more acute illness with much more aggressive effects, with the onset leads for Nurr1 ligand discovery. Our results clearly indicate that Nurr1 is within a few days or weeks after the bite of infected tsetse flies. Existing druggable and provide NSAIDs as promising tool compounds for Nurr1’s medicines for the treatment of these diseases are insufficient, and activity understanding as well as starting points for ligand development. without treatment the disease is invariably fatal. The need for new anti- HAT drugs continues to persist and aiming into this issue; we have prepared a large number of new heterocyclic compounds having different References: 1. Wang, Z. et al.: Nature 2003, 423: 555–560 central rings [1,2]; that were further assayed against different 2. Decressac, M. et al.: Nat. Rev. Neurol. 2013, 9(11): 629–636 trypanosoma subtypes. Among the most promising was the 3. Rajan, S. et al.: PDB ID: 5Y41 2018, doi:10.2210/PDB5Y41/PDB dihydroisoindol core ring. Herein we report the optimization of a novel 4. Rajan, S. et al.: PDB ID: 5YD6 2019, doi:10.2210/PDB5YD6/PDB class of dihydroisoindol ureas, which showed selectively in vitro potency against one species of trypanosoma: T. b. rhodesiense. Employing traditional medicinal chemistry approaches we have modified two portions of the central core based on suitable modifications of R1 and R2 POS.4 (Figure 1). The compounds are fused ring ureas bearing various Identification of a new class of broad spectrum Metallo-β- substituents at the 2-position of the isoindolone portion of the backbone lactamase inhibitors (R2) and we evaluated the SAR based on the effect of halogenation, methylation as well as the introduction of various other substituents at the aromatic ring. In addition, the internal core of the molecule was Kaya C.1; Yahiaoui S.1; Kany A.M.1; Haupenthal J.1; Kramer J.S.2; modified by insertion of a nitrogen atom at the X- and Y-positions. Several Brunst S.2; Proschak E.2; Wichelhaus T.A.3; Hartmann R.W.1,4; Hirsch compounds of this series exhibited an in vitro EC50 ≤ 1µM against T. b. A.K. H.1,4 rhodesiense and no detectable activity against other parasites, like. T. b. 1Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research gambiense, T. cruzi amastigotes or L. infantum. All potent compounds Saarland, Campus E8.1, 66123 Saarbrücken, Germany were furthermore tested for toxicity against MRC-5 and PMM cell lines. 2Institute of Pharmaceutical Chemistry and Institute of Biochemistry, Goethe University Frankfurt, Max-von-Laue-Straße 9, 60438 Frankfurt, Germany The compounds exhibited no significant toxicity to both cell lines.The 3Institute of Medical Microbiology and Infection Control, Paul-Ehrlich-Straße 40, 60596 solubility studies were performed on selected compounds and they Frankfurt, Germany showed excellent solubility in the tested conditions. A relatively facile 4Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123 Saarbrücken, Germany synthetic pathway has been amenable to a large number of functional groups, giving rise to a structurally diverse set of analogs. This family of compounds thus shows significant promise for trypanosomiasis drug Increasing number of resistant pathogens generate a great risk to public discovery. health as they cause diseases that are impossible to cure. Metallo-β- lactamases, the most common cause of bacterial resistance, are a diverse set of zinc-containing enzymes that catalyze the hydrolysis of β- lactam drugs [1, 2]. The major defense mechanism against these multi- resistant enzymes is to develop β-lactamase inhibitors, which have no bactericidal effect but can maintain the usefulness of existing β-lactam antibiotics.

DPhG Annual Meeting 2019 Conference Book • 86 MEDICINAL CHEMISTRY AND DRUG DESIGN

Kloevekorn, P.1; Selig, R.2, Albrecht, W.2, Laufer, S.1 1Department of Pharmaceutical and Medicinal Chemistry, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tübingen 2HepaRegeniX GmbH, Alfred-Mendler-Weg 25/1, 89075 Ulm

The liver harbors an enormous regenerative capacity as answer to injury, but after a certain degree of serious illness this regenerative ability can collapse with mostly a fatal outcome for the patient.1 The Mitogen- Figure 1. Set of modifications of the core ring and some examples of activated Protein (MAP)-Kinase 4 (MKK4) was found to be a major substituent groups attached in portions R1, R2 and positions X and Y. regulator of liver regeneration and could be a valuable drug target helping to reawake this intrinsic capability to regenerate.2,3 Vin et al. underlined the effect of PLX4720 (Vemurafenib) as a

paradoxical-activator of ERK-signaling by inhibition of multiple off-target 1 Silva, D. G. et. al: ACS Medicinal Chemistry Letters 2017, 8, 766-770. 2 Silva, D. G. et al.: WO/2017/127627. kinases. One of these off-target kinases was determined to be MKK4. Inhibition of MKK4 could be useful for the treatment of liver failure and/or 4- inducing hepatocyte regeneration and preventing hepatocyte apoptosis. 6 POS.6 Herein, we report on the synthesis and optimization of Vemurafenib, a Design and Synthesis of fluorescently labeled analogs of FDA-approved BRAFV600E inhibitor, for the implementation as a MKK4 Vemurafenib inhibitor. The optimization process yielded in high affine and selective inhibitors, which showed a promising performance in biochemical environment sparing important off-targets maintain the desired Kircher, T.1, Laufer S.1 downstream-effects within the JNK yard. We suggest that our 1Department of Medicinal Chemistry, Eberhard-Karls-University Tuebingen, Auf der compounds could be the start for first-in-class drug candidates employed Morgenstelle 8, 72076 Tuebingen in chronic and acute liver failure.

In 2013 Wüstefeld et al. proved that silencing of Mitogen-Activated Acknowledgment: We would like to gratefully thank HepaRegeniX GmbH for the financial Protein Kinase Kinase 4 (MKK4) in mice is substantially involved in liver support and dynamic collaboration. regeneration. [1] At the same time, FDA-approved BRAF-inhibitor Vemurafenib showed an off-target-effect on MKK4. [2] With this 1. Michalopoulos, G. K.; DeFrances, M. C.: Science 1997, 276(5309): 60-66. knowledge analogues of Vemurafenib could be used as a tool for 2. Wüstefeld, T. et al.: Cell 2013, 153(2): 389-401. fluorescence-based assay systems. Finding new small molecules that 3. Willenbring, H.; Grompe, H.: Cell 2013, 153(2): 283-284. 4. Vin et al.: eLife 2013;2:e00969. inhibit MKK4 may represent a possible starting point of treating acute and 5. Albrecht, W.; Laufer, S.; Selig, R.; Kloevekorn, P.; Praefke, B. Preparation of 1H-pyrrolo[2,3- chronic liver diseases. b]pyridines as selective MKK4 kinase inhibitors for promoting liver regeneration or reducing Fluorescence polarization provides fast details about inhibitor-protein or preventing hepatocyte death. WO2018/134254A1 (2018). interaction and can be adopted for high-throughput screenings of 6. Zender, L.; Wüstefeld, T. Medicament for liver regeneration and for treatment of liver failure. compound libraries. Structure analysis of the binding mode of WO2012/136859A1 (2012). Vemurafenib to BRAF showed, that the p-chloro substituent is solvents exposed and not necessary for binding at the ATP-site. [3] The same should apply for the binding side of MKK4. Thus, the attachment of the POS.8 bulky fluorophore 5-Carboxytetramethylrhodamin (5-Tamra) through an alkyl linkage is possible without losing binding affinity. Chemical variation Synthesis and evaluation of DOTA-based fluorophores and optimization led to a selection of derivatives with different linker lengths, which possess higher binding affinities than Vemurafenib itself Silke Schmidt1, Wolfgang Maison1 and promising IC50 values. 1 Department of Chemistry, Pharmaceutical and Medicinal Chemistry, University of Hamburg, Bundesstr. 45, 20146 Hamburg, Germany

In the past years, Fluorescence imaging has become an important tool for full-body preclinical imaging, providing spatial resolutions of cellular processes in the range of nanometers. Especially the development of fluorescent systems absorbing and emitting fluorescent radiation in the visible to near-infrared electromagnetic spectrum are of interest, due to reduced scattering coefficients of biological tissues at wavelengths in the 650 nm - 1600 nm region and the absence of autofluorescence in 1200 nm - 1800 nm range enhancing the signal-to-noise ratio [1,2]. In this context quantum dots represent favorable fluorophores since their absorbance and emission properties can easily be tuned by their size and shape. Additionally, they combine desirable optical properties such as high quantum yields and high resistance to photo-bleaching [3]. Fluorescence quantum yield can further be enhanced by the establishment of a Förster resonance energy transfer system (FRET- system), incorporating the quantum dot as donor which absorbs light at Therefore, the optimized tracers meet the demands of a Fluorescence a defined wavelength and subsequently transfers energy in a non- Polarisation assay to identify new small-molecule inhibitors of MKK4. radiative manner onto a suitable acceptor such as lanthanides. The transfer depends on the distance between both chromophores, which led to the development of stable lanthanide-chelates immobilised onto 1. T. Wuestefeld et al., Cell 2013, 153: 389–401 nanoparticles [4,5]. With regard to its excellent chelating properties, the 2. H. Vin et al., eLife 2013, 2: 1-25 macrocyclic chelator 1,4,7,10-Tetra-azacyclododecan-1,4,7,10-tetra- 3. G. Bollag et al., Nature 2010, 467: 596–599 acetic acid, referred to as DOTA, is suitable for nanoparticle surface modification and can easily be tuned via its analogue 1,4,7,10-Tetra- azacyclododecan-1,4,7,10-tetra-azidoethylacetic acid (DOTAZA) comprising side-chain azide-groups, which can be functionalised in a Cu- mediated Click reaction [6,7]. Since phosphonates possess good metal

POS.7 surface binding properties, the modification of DOTAZA with alkyne Vemurafenib as a role model for novel inhibitors targeting phosphonates represents a promising approach for the establishment of MKK4 efficient DOTA-based FRET-systems (figure 1) [8]. In this work, the

87 • DPhG Annual Meeting 2019 Conference Book POSTERS synthesis and analysis of DOTAZA-phosphonate based lanthanide- POS.10 chelates immobilised onto nanoparticle is carried out. Alkyne-based cysteine cathepsin inhibitors as basis for PET tracer development

Behring, L.1,2; Trapp, C.1; Morales, M.1; Wodtke, R.1; Kuhne, K.1; Belter, B.1; Pietzsch, J.1,2; Löser, R.1,2 1Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstrasse 400, 01328 Dresden, Germany 2Technische Universität Dresden, 01069 Dresden, Germany

Among the intertwined processes leading to cancer progression, Figure 1: DOTAZA based FRET-system. protease activity plays an important role. Various attempts to develop molecular imaging probes have been made, as such probes can allow functional imaging and thus improve the understanding of tumour progression mechanisms and enable personalised cancer treatment. References: PET and SPECT tracers are particularly suitable for such applications. 1. Leblond, F. et. al.: J. Photochem. Photobiol. B 2010, 98(1): 77-94. However, novel tracers have to overcome challenges such as stability, 2. He, S. et al.: Chem. Soc. Rev. 2018, 47(12): 4258-4278. 3. Ding, F. et al.: Chem. Si. 2018, 9: 4370-4380. target efficiency and off-target effects. 4. Förster, T.: Compr. Biochem. 1967, 22: 61-80. Multiple members of the cathepsin family have been demonstrated to be 5. Clegg, R. M.: Curr. Opin. Biotechnol. 1995, 6(1): 103-110. involved in tumour invasion, metastasis, and angiogenesis. Especially 6. Kriemen, E. et al.: Eur. J. Inorg. Chem. 2015, 2015: 5368-5378. high expression levels of the cysteine cathepsins B, K, L, S, and X are 7. Kriemen, E. et al.: Chem. Asian J. 2014, 9(8): 2197-2204. correlated with an increased metastatic potential and poor prognosis [1]. 8. Mutin, P. H.; Guerrero, G.; Vioux, A.: J. Mater. Chem. 2005, 15(35-36): 3761–3768. Due to their high expression in a multitude of tumours, those enzymes

represent promising targets for the therapy and imaging of tumours.

Despite being virtually chemically inert, alkynes were shown to be able

to irreversibly inhibit cysteine proteases: Both EKKEBUS et al. and

SOMMER et al. independently described the unexpected inactivation of POS.9 de-ubiquitinating enzymes by ubiquitin or ubiquitin-like modifiers bearing Exploration of fragments as starting points for multi-target propargylamine in place of C-terminal glycine [2, 3]. We aimed to take drugs advantage of those findings for designing alkyne-based cysteine cathepsin inhibitors suitable for radiolabelling with PET nuclides. The

probes thus obtained would irreversibly bind to the target molecule S. Brunst1, J.S. Kramer1, S.George1, J.Pollinger1, W. Kilu1, J. Heering2, without showing indiscriminate thiol reactivity. D. Steinhilber1, D. Merk1, E. Proschak1 1Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue Str. 9, D-60439, Frankfurt, Germany 2Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology TMP, Theodor-Stern-Kai 7, D-60596, Frankfurt am Main, Germany

Introduction: Multi-target drugs are in the focus of pharmaceutical research and often exhibit superior efficacy and safety profiles. However, Based on a potent, highly selective dipeptidyl nitrile-based cathepsin B the identification of starting points for multi-target drug discovery is inhibitor reported by GREENSPAN et al. (left structure) [4], we designed tedious. Fragments were proposed to be ideal starting points for multi- dipeptide alkynes by isoelectronic replacement of the nitrile nitrogen target drug design [1], but prospective applications of this technique are atom by a methine group. To avoid partial enantiomerisation during the rare [2, 3]. In this study we systematically explore the possibilities to formation of the C-C triple bond as observed for the open-chain serine- identify fragments as starting point for multi-target drug design. derived alkyne, the synthesis was performed via Garner’s aldehyde. This Material and Methods: Five proteins, the soluble epoxide hydrolase ensured high stereochemical purity of the final compounds. The inhibitory (sEH), the 5-lipoxygenase (5-LO), the leukotriene A4 hydrolase (LTA4H), potential was investigated against cathepsin B, S, L and K. To optimise the retinoid X receptor (RXR), and the farnesoid X receptor (FXR) were the inhibitory potential and selectivity, we consecutively varied all recombinantly expressed. To identify structures that bind these proteins moieties attached to the dipeptidic scaffold. a differential scanning fluorimetry (DSF) screening was used. In this We identified potent alkyne-based inhibitors for all tested cathepsins, with method a shift in the melting point of the protein indicates a binding. As inactivation constants (kinact/KI) up to 10133 M-1s-1 and distinct selectivity fragments the Prestwick Library, containing 480 fragments of well-known profiles. We demonstrated irreversibility in a “jump-dilution” experiment and characterized drugs, was used. The fragments that showed a 1°C or and inhibitor reactivity in cell lysates and on living cells was exemplarily higher shift in the melting point, indicating a binding, on more than one verified for cathepsin B. During our research, MONS et al. successfully protein were then cross validated using orthogonal activity assays. demonstrated irreversible cathepsin K inhibition by alkyne-based small Results: The DSF prescreen led to the identification of 22 fragments that molecule inhibitors with no indiscriminate thiol reactivity [5], which hit on at least two of the evaluated proteins. From the 22 hits four showed indicates the viability of our concept. a shift of the sEH as well as the LTA4H. After cross validation of sEH and Among the tested inhibitors we identified two promising radiotracer LTA4H two interesting fragments remained. This early evaluation already candidates which are selective for cathepsin S and L. We successfully indicated that it is possible to identify dual acting fragments with DSF radiolabelled the cathepsin S-selective inhibitor with N-succinimidyl 4- even when using a small library of 480 fragments. [18F]fluorobenzoate ([18F]SFB). Radiopharmacological characterisation of Outlook: This study demonstrates the opportunities and limitations of a the activity-based probe obtained by that approach is in progress. fragment based approach to identify novel starting points for multi-target compounds. Further studies are required to demonstrate fragment-to- lead optimization considering potency and efficacy. We acknowledge the Graduiertenakademie TU Dresden for financial support.

[1] Löser, R; Pietzsch, J.: Front. Chem., 2015, 3: 37. References [2] Ekkebus et al.: J. Am. Chem. Soc., 2013, 135(8): 2867-2870. 1. Morphy, R., Rankovic, Z.: J. Med. Chem. 2005, 48(21): 6523–6543. [3] Sommer et al.: Bioorg. Med. Chem., 2013, 21(9): 2511-2517. 2. Achenbach, J. et al.: ACS Med. Chem. Lett. 2013, 4(12): 1169–1172. [4] Greenspan et al.: J. Med. Chem., 2001, 44(26): 4524-4534. 3. Artis, D. R. et al.: Proc. Natl. Acad. Sci. U. S. A. 2009, 109(1): 262–267. [5] Mons et al.: J. Am. Soc. 2019, 141(8): 3507-3514.

DPhG Annual Meeting 2019 Conference Book • 88 MEDICINAL CHEMISTRY AND DRUG DESIGN

POS.11 With a slow but steady increase of resistance in bacteria against current antibiotics there is a strong demand to identify new modes of action and Solid Phase Supported Parallel Synthesis of Histone to develop new and unconventional antibacterial agents.[1] Deacetylase Inhibitors (HDACi) with DNA-Alkylating Organometallic gold compounds have demonstrated promising Properties properties as anticancer and antibacterial agents. Recently, organometallic complexes consisting of gold(I) and a N-heterocyclic carbene (NHC) ligand have been reported to exhibit high activity against Sinatra, L.1; Roatsch, M.1; Hamacher, A.2; Schöler, A.1; Kassack, M. U.2; the enzyme thioredoxin reductase (TrxR), both in its bacterial and Hansen, F. K.1 mammalian form.[2,3] The inhibition of bacterial TrxR is in particular 1Institute of Pharmacy, Faculty of Medicine, Leipzig University, Brüderstr. 34, 04103 Leipzig, detrimental for many Gram-positive bacteria based on the lack of Germany 2Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, glutathione, which can compensate for TrxR inhibition.[4] Universitätsstr. 1, 40225 Düsseldorf, Germany

Overexpression of histone deacetylases (HDACs) is frequently observed in a wide variety of tumor diseases. Due to their repressive effect on gene transcription and their essential influence on drug resistance mechanisms of tumor cells, HDACs advanced to become interesting Figure 1: General structure of the studied gold NHC compounds. targets for the epigenetic therapy of cancer.[1] Glioblastoma multiforme (GBM) represents one of the most common and aggressive primary brain Based on these results, we are aiming to further explore the potential of tumors, with high resistances against standard therapies. The use of gold NHC complexes as possible antibacterial agents, to establish alkylating agents, such as temozolomide (TMZ), in combination with important structure-activity-relationships, and to combine these radiotherapy represents the current standard treatment of GBM after organometallics with other functional molecular units for the purpose of surgical resection of the tumor.[2] Recently, the first-in-class hybrid targeting (see Figure 1). This could lead to new efficient antibiotics with alkylating histone deacetylase inhibitor, tinostamustine, has been activity against resistant pathogenic bacteria.[5] The current results of reported. Tinostamustine showed superior in vivo activity compared to this ongoing project will be presented on the poster. bendamustine, temozolomide and radiotherapy as well as the potential to overcome drug resistance.[3]

In this study, we designed a scaffold containing a DNA-damaging cap References: group, different linker types and a hydroxamic acid moiety as warhead to [1]: Klahn P., Brönstrup M.: Curr Top Microbiol Immunol. 2016, 398, 365-417. [2]: Schmidt, C. et al.: Chem. Eur. J. 2017, 23(8), 1869-1880. aim at dual targeting. Notably, our parallel synthesis approach offers a [3]: Schmidt, C. et al.: Med. Chem. Commun. 2017, 8, 1681-1689. straightforward and efficient procedure for the synthesis of HDACi-based [4]: Harbut, M. B. et al.: P. Natl. Acad. Sci. USA 2015, 112(14), 4453-4458. multi-target drug libraries. Our preloaded resins are easy to handle and [5]: Butler, M. M. et al.: Antimicrob. Agents Chemother. 2007, 51(1), 119- 127. stable at least for six months stored at 4 °C. Furthermore, this approach circumvents the common difficulties of handling hydroxamic acids through the protection with our functionalized resin. The subsequent testing for in vitro activity against different HDAC isoforms showed the great potential of several compounds as HDAC1 and/or HDAC6 inhibitors POS.13 with promising anticancer activities. Notably, our solid phase supported Bivalent Ligands for the D1-H3-Receptor Heteromer: A method yielded a hit compound with promising pan-HDAC activity and Target with Neuroprotective Potential improved cytotoxicity in comparison to TMZ, mitozolomide (MTZ) and chlorambucil. To the best of our knowledge, no hybrid DNA-alkylating N. Rosier1, M. Nagl1, L. Grätz1, H. Schihada2,3, R. Franco4, S. Pockes1 HDAC inhibitors containing TMZ or MTZ have been reported so far. Thus, 1University of Regensburg, Regensburg, 93053, Germany, 2University of Würzburg, Würzburg, this series of compounds represents a solid basis for further evaluation 97074, Germany, 3Karolinska Institutet, Solna, 17177, Sweden, 4University of Barcelona, of DNA-alkylating HDAC inhibitors and for gaining deeper knowledge of Barcelona, 08007, Spain. their anticancer properties. The formation of class-A rhodopsin-like G-Protein coupled receptor (GPCR) di- and oligomers has been proven after a long controversy.1 The existence of the D1-H3-receptor heteromer was published by Rafael Franco and his group. They could prove its existence in artificial cell systems as well as in the rat striatum and cortex, using different pharmacological techniques like co-immunoprecipitation, BRET-assays and bimolecular fluorescent complementation (BiFC). Furthermore, they found a heteromeric complex consisting of the D1-, H3-receptor and subunits of the NMDA-receptor.2,3 By inhibiting this complex with the H3R antagonist thioperamide the dopamine, NMDA and β1-42-Amyloid induced cell death could be reduced.3 These results show the neuroprotective Acknowledgments: We thank the Alexander von Humboldt Stiftung for financial support. potential of this complex. Therefore highly selective bivalent D1-H3- receptor ligands could be useful in the therapy and for the comprehension References: of neurodegenerative diseases such as Alzheimers Disease (AD). [1] Haberland, M., Montgomery, R. L., Olson, E. N., Nat. Rev. Genet., 2009, 10, 32-34. For the design of the first bivalent ligands we used the known H3R [2] Lee, C. Y., Onco Targets Ther., 2017, 10, 265-270. antagonist JNJ-5207852 and the D1R antagonist SCH-23390 as lead [3] Festuccia, C. et al., J. Hematol. Oncol., 2018, doi: 10.1186/s13045-018-0576-6. structures (Fig. 1).4,5 In a three-step synthesis derivatives of JNJ-5207852 with an additional terminal amino group or carboxylic acid were synthesized. A seven-step synthesis was established to yield derivatives of SCH-23390 with a variety of functional groups added. These additional POS.12 functional groups allow the coupling of a linker, which is a crucial part of Gold(I/III) complexes with N-heterocyclic carbene ligands the bivalent ligand, because it must possess the correct properties in length, flexibility and solubility to allow both pharmacophores to bind to as antibacterial agents their respective binding pocket simultaneously. Starting from commercially available polyethylene glycol derivatives of different Büssing, R.1; Ott, I.1 lengths, highly flexible and soluble linkers were synthesized. 1[Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, Furthermore, we prepared structures, that are rigidized by multiple Beethovenstraße 55, 38106 Braunschweig, Germany] aromatic and non-aromatic ring-structures. These linkers are coupled via amide coupling reactions or click chemistry to the pharmacophores to give the final bivalent ligands.

89 • DPhG Annual Meeting 2019 Conference Book POSTERS

assays will characterize the first heterobivalent ligands, which will soon be synthesized after linking the two pharmacophores previously mentioned by conducting click chemistry.

1. Rico, J. A. et al.: Brain Struct Funct 2017, 222: 1767–1784. 2. Ferrada, C. et al.: Neuropharmacology 2008, 55: 190–197. 3. Ellenbroek, B. A.: British Journal of Pharmacology 2013, 170: 46–57. 4. Vangveravong, W. et al.: Bioorg Med Chem. 2010, 18(14): 5291–5300. Fig.1 Structures of SCH-23390 and JNJ-5207852 5. Apodaca, R et al.: J. Med. Chem. 2003, 46: 3938-3944.

Using already established radioligand binding assays on Sf9 cells, as well as functional BRET-based (e.g. Nano-Luc/HaloTag) assays on HEK- POS.15 cells the bivalent ligands will be pharmacologically characterized on all Preparation of carbamoylated guanidines and their receptors of the histamine and dopamine family, as well as on cells co- biological activity at the human and guinea pig histamine expressing the D1 and H3 receptors. Since no selective assay has been published to date to test the binding of a bivalent ligand to a receptor H2 receptor heterodimer, a possible bivalent binding mode must be demonstrated in co-expressed cells. M. Bresinsky1, B. Sadek2, S. Pockes1 1 Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany; 2 Department of Pharmacology and Therapeutics, United Arab Emirates University, The project and the PhD position of N. Rosier is supported from the International Doctoral PO Box 17666, Al-Alin/UAE; Program “Receptor Dynamics” of the Elite Network of Bavaria and is gratefully acknowledged.

1. Gomes, I. et al.; Annu. Rev. Pharmacol. Toxicol. 2016, 56: 403-25. It is believed, that the H2R is involved in the transmission of histamine 2. Moreno, E. et al.; J. Biol. Chem. 2011, 286: 5846-5854. and other neurotransmitters (e.g. acetylcholine) in the central nervous 3. Rodríguez-Ruiz, M. et al.; Mol Neurobiol. 2017, 54: 4537-4550. system (CNS). Due to this assumption the development of CNS- 4. Apodaca, R. et al.; J. Med. Chem. 2003, 46(18): 3938-3944. penetrating H2R agonists is of great interest as they are probably going 5. Iorio, L.C. et al.; J. Pharm. Exp. Ther. 1983, 226(2): 462-468. to stimulate postsynaptic H2Rs, which could have similar effects to treat cognitive disorders (e.g. Alzheimer diseases) as already reported H3R antagonists. POS.14 The prototype of highly potent H2R ligands of the guanidine class is the Synthesis and pharmacological characterization of bivalent weak partial agonist 3-(1H-imidazol-4-yl)propylguanide (SK&F-91486).1 ligands for the D2-H3-receptor heteromer Further development of this structure led to the highly potent ligands impromidine and arpromidine.2,3 However, they showed a lack of receptor subtype selectivity and a poor bioavailability. In order to increase receptor 1 1 1 2,3 4 1 M.Nagl , N. Rosier , L. Grätz , H. Schihada R. Franco , S. Pockes subtype selectivity, especially towards the H3R and H4R, the imidazolyl 1University of Regensburg, Regensburg, 93053, Germany, 2University of Würzburg, Würzburg, 97074, Germany, 3Karolinska Institutet, Solna, 17177, Sweden, 4University of Barcelona, head group was bioisosterically replaced by a 2-amino-4-methylthiazolyl Barcelona, 08007, Spain. group. The alteration of alkylated guanidines via acylated guanidines to carbamoylated guanidines was a successful approach to increase For many years the ability of rhodopsin-like class A G-Protein-coupled bioavailability, stability and to improve properties for the penetration of receptors (GPCRs) to form homo- or heterodimers and oligomers has the blood-brain barrier (BBB).4,5,6 The carbamoylguanidines were been doubted. Yet their existence has been proven and many preferably prepared in a 3-step synthesis starting with the treatment of heterodimers (Hets) have been found and investigated in recent years.1 primary amines with phosgene. Subsequently the isocyanates were The group of Rafael Franco detected a strong and selective D2-H3 transformed with N-Boc-S-methylisothiourea yielding a carbamoylated receptor interaction at the membrane level of reserpinized mice and N-Boc-S-methylisothiourea. The next step was the aminolysis of the S- could also provide evidence for D2-H3-Hets in co-transfected HEK-293 methylisothiourea partial structure with Boc-protected 3-(2-Amino-4- 2 cells. However, the functional consequences of this intricate methylthiazol-5-yl)propanamine in presence of HgCl2. Finally, the heteromerization are not fully understood but seem to play a crucial role carbamoylguanidines were deprotected under the treatment of TFA. concerning drug addiction. Therefore, the development of heterobivalent A recent project of our lab is the development of CNS-penetrating H2R ligands is important to further explore the importance of receptor- agonists. This led to UR-Po563, an enantiopure (ee> 99%) highly potent heteromerization. Furthermore, it could also turn out to be a promising and selective agonist (pKi (hH2R) = 7.60; pEC50 = 8.12, Emax = 0,95 3 alternative for treating drug addiction. (gpH2R-atrium)). At the moment UR-Po 563 is tested in vivo on several Synthesis started with the derivatization of the known D2R antagonist L- behavioral models including anxiety-related tests. Fortunately, we can 741,626 using 5 steps (Fig. 1).4 After alkylation of 5-hydroxyindole with announce that preliminary results indicate promising effects on mice 1-bromo-3-chloropropane followed by chloride substitution with azide the regarding to their anxiety behavior. Taking this compound as a lead pharmacophore was connected to the centre part of the molecule. A structure, we tried to further improve pharmacological properties at the derivative of the non-imidazole H3R antagonist JNJ- 5207852 was H2R. We started with the halogenation and alkylation of the phenyl obtained in a nine-step synthesis (Fig. 1).5 An aminofunctionality in para- nucleus in different positions. In addition, we performed a bioisosteric position of the piperidine moiety was appended and coupled with exchange of the phenyl group by a furan and thiophene residue. Most chloroacetylchloride. The next step was another chloride/azide promising was UR-MB13 with a methyl group in para position (pKi(hH2R) exchange. The centre part was synthesized from isophthalic acid which = 7,31; pEC50 = 7,65, Emax = 0,85 (gpH2R-atrium)), possessing at least was converted to isophthaloyl dichloride and propargylamine and will be 100-fold selectivity relative to the H1R, H3R and H4R. Another issue is the linked to the pharmacophores by forming triazole moieties. specificity towards other receptor family’s (e.g. dopamine). Preliminary data gave us a hint, that there also exists a certain affinity to the dopamine D3 receptors. This is attributed in particular to the 2- aminothiazolyl partial structure, which also occurs in the well-known dopamine agonist pramipexole. In order to achieve a higher specificity towards the dopamine receptors, we are also performing a bioisosteric replacement of the 2-amino-4-methylthiazol headgroup by different heteroaromatic groups.

Fig.1 Structures of L-741,626 and JNJ-5207852 1. Parsons, M.E. et al.; Agents Actions 1975, 5: 464. Pharmacological characterization of the bivalent ligands will be 2. Durant, G. J. et al.; Nature 1978, 276: 403-405. performed by radioligand binding assays on Sf9 cell membranes for all 3. Buschauer, A.; J. Med. Chem. 1989, 32: 1963-1970. histamine receptor-subtypes which are already established. Moreover, 4. Kraus, A. et al.; ChemMedChem 2009, 4: 232-240. 5. Ghorai, P. et al.; J. Med. Chem. 2008, 51: 7193-7204. functional assays for histamine and for the dopamine receptors using 6. Kagermeier, N. et al.; Bioorg. Med. Chem. 2015, 23: 3957−3969. biosensors (e.g. HaloTag and Nanoluc as well as split luciferase complementation techniques) are designed und will also be used. These

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POS.16 To achieve target-specificity, pharmacologically inactive and nontoxic forms of LSD1 inhibitor 1a with a nitro- aromatic system, so-called DOCKING AND SYNTHESIS OF GluN2A SELECTIVE NMDA bioreductive prodrugs, are designed, synthesized and tested against RECEPTOR ANTAGONISTS LSD1 activity in-vitro and on cultured AML THP1 cells. As prodrug- activating enzyme, the E. coli Nitroreductase NfsB (NTR) was selected, that is introduced in leukemic cells using a virus-mediated transfection. Rajan. R.; ¹², Wünsch. B. ¹² By reduction of the nitro-aryl bioreductive system by the NTR, the active ¹ Institute of Pharmaceutical and Medicinal Chemistry, Corrensstraße 48, 48149, WWU Münster, Germany. tranylcypromine-based LSD1 inhibitor is subsequently released and ² CiM-IMPRS Graduate School, Schlossplatz 5, WWU, 48149, Münster, Germany. forms a covalent adduct with the cofactor FAD leading to an irreversible inhibition of LSD1.

NMDA Receptors are heterotetrameric ion-channel glutamate receptors We identified promising prodrug/drug pairs by measuring the expression that require glutamate and glycine for activation. When activated it allows of CD86 surface marker and by performing colony-forming unit assays the positively charged ions to flow through the cell membrane. It controls with THP1 cells. [2] Several prodrugs are converted into the active parent synaptic plasticity and memory function. GluN2A containing NMDA drug by the NTR, which is solely expressed in transfected tumour cells. receptors have been found to be involved in anxiety, depression and Depending on the nitro-aryl system, different activation patterns can be schizophrenia. The exact role of the NMDA receptor with GluN2A subunit observed both in vitro and in vivo. By applying different targeting in the disease process is still unclear. To better understand this role, techniques such as antibody-directed enzyme-prodrug therapy (ADEPT) potent and selective GluN2A inhibitors were designed and synthesized, and gene-directed enzyme-prodrug therapy (GDEPT), these prodrugs hereby the ring B of TCN-201 was replaced by electron-rich 1,3-thiazole provide a direction for more selective anti-cancer drugs. [3] ring and 1,3-oxazole ring. In 2016, the X-ray crystal structure of the GluN2A subunit containing NMDA receptor with TCN-201 was solved. TCN-201 binds to the receptor at the interface between the GluN1 and GluN2A subunits by adopting a hair-pin or U-shaped conformation. To lock TCN-201 in this conformation, the rings A and B were replaced by a [2.2]Paracyclophane system. Docking studies suggested that compounds 1 and 2 should highly interact with the GluN2A receptor. The synthesized molecules will be tested soon. O2 Cl S N O A H B H N F N H C 1 O TCN-201

H N N A S B A O H 2 S N B O C 4 O We thank the DFG for funding within CRC992 Medical Epigenetics "MEDEP“ and the N Germany's Excellence Strategy (CIBSS –EXC-2189 –Project ID390939984). O2 O R C N Cl S A N B 2 A H H O S N 1. Harris, W. J. et al.: Cancer Cell 2012, 21(4): 473–487. F B 5 N 2. Schulz-Fincke, J. et al.: Eur. J. Med. Chem. 2018, 144: 52–67. O H C 3. Rautio, J. et al.: Nat. Rev. Drug Discov. 2008, 7(3): 255–270. O N N O2 N C Cl S N 3 N B A H H O O N F 6 N O H C POS.18 References Is a selective virtual screening possible? Wyllie et. al., Neuropharmacology, 2013, 74, 4-17. Yi et al., Neuron, 2016, 91, 1316–1329. 1 2 1 1 (a) Koerber-Ple and Massiot, J. Heterocyclic Chem., 1995, 32, 1309. Painer, C. , Mazurek, A. , Martens, M. , Lemcke, T. 1 (b) Holger et. al., Eur. J. Org. Chem., 2002, 14, 2298-2307. Institute of Pharmacy, University of Hamburg, Bundesstr. 45, 20146 Hamburg, Germany 2Faculty of Pharmacy, Medical University of Warsaw, Banacha Street 1, 02-097 Warsaw, Poland

The Dual Leucine Zipper Kinase (DLK), also known as MAP3K12, is

POS.17 expressed in the peripheral and central nervous system and is Targeting LSD1 in Cancer Cells by Nitroreductase- associated with neurodegeneration and axon regeneration. Additionally, mediated Prodrug Activation DLK is inhibiting insuline-gene transcription and secretion in beta cells. Therefore, inhibiting DLK represents an interesting concept for the treatment of diabetes.[1] Herrlinger, E.M.1; Hau, M.1,2; Redhaber, D.3; Miething, C.3; Schüle, R.4; In our project, we strive for finding diverse potential ATP competitive Manfred, J.1 1 Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 25, 79104 Freiburg inhibitors with novel scaffolds by using structure-based virtual screening. im Breisgau, Germany, www.jungm.de In this context, the question arose, whether our developed virtual 2 CIBSS –Centre for Integrative Biological Signalling Studies, University of Freiburg screening protocol was able to find truly selective DLK inhibitors. To 3 Department of Internal Medicine, University of Freiburg Medical Center, Hugstetter Str. 55, 79106 Freiburg, Germany investigate this question, we used our screening protocol on another 4 Department of Urology and Center for Clinical Research, University Freiburg Medical Center, diabetes relevant kinase (GSK-3) as the (anti)target.[2] Breisacherstraße 66, 79106 Freiburg, Germany Therefore, all screening steps were done with six DLK- and four GSK-3 conformations. A datasest of 216 known active DLK inhibitors and about The lysine-specific demethylase 1 (LSD1 or KDM1A) has emerged as a 16000 decoys (potential inactive compounds) were first docked by HTvS highly promising therapeutic target in human malignancies and several (High Throughput virtual Screening) and subsequently by the more drugs are already under current investigation in clinical trials. LSD1 is accurate SP mode in Schrödinger’s Glide module.[3] The poses were also established as a key effector of the differentiation block in acute rescored by four different scoring methods: dockingscore[4], HYDE myeloid leukaemia (AML), which may be selectively targeted by score[5], relative free binding energy (MM-GBSA)[6] and a ligand based inhibitors, leading to a pronounced therapeutic effect. [1] To increase QSAR model[7]. A consensus score was generated and enrichments were therapeutic effectiveness and decrease side effects during treatment, we calculated for both datasets. aimed to develop a specific targeting of leukemic cells with irreversible LSD1 inhibitors.

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Comparison of the results revealed, that the known DLK inhibitors were more different kinases (FGFR4, TTK, MAPKAPK2 and 3), [4] covalent highly enriched by using the DLK target structures, while using the GSK- engagement of this moiety by an electrophilic residue, a so-called 3 target structures, no enrichment could be observed (Fig. 1). "warhead" [5] might not only be exploited to achieve selectivity against To validate these findings, a dataset of known selective GSK-3 p70S6Kα, but also within the kinome. inhibitors were screened against both targets. We found, that GSK-3 Amongst the aforementioned kinases, only FGFR4 has been covalently selective inhibitors were not enriched in the DLK, but only in the GSK-3 targeted so far. The irreversible FGFR4 inhibitor BLU9931 [6] makes use target structures. of an ortho-phenylenediamine-linked acrylamide warhead to trap the GK+2 cysteine. In order to selectively address p70S6Kβ, we sought to combine the scaffold of the p70S6Kα inhibitor PF-4708671 [3] with the linker and warhead moiety of BLU9931.

For the preparation of reactive PF-4708671 analogs, we started with different 2,4-dichloropyrmidine derivatives and introduced N-protected piperazine in the 4-position via nucleophilic aromatic substitution (SNAr). The ortho-phenylenediamine linker was installed as the (substituted) 2- nitroaniline precursor by means of Buchwald-Hartwig-arylamination. After reduction of the nitro group, acylation of the primary amine with acryloyl chloride was used to generate the acrylamide warhead. Finally, deprotection of the piperazine followed by the introduction of the N- substituents by nucleophilic substitution furnished a series of putatively Fig. 1: Frequency distribution of the ligands in DLK (left) and GSK-3 covalent p70S6Kβ inhibitors. (right); the consensus score is plotted on the x-axis with low values meaning high scored compounds; frequency of DLK- and GSK3- References: inhibitors are shown enlarged 10 times. [1] Saxton, R. A., Sabatini, D. M.: Cell. 2017, 168(6), 960–976. [2] Olivier E. Pardo, O. E., Seckl, M. J.: Front. Oncol. 2013, 3, 191. [3] Pearce, L. R., Alton, G. R., Richter, D. T., Kath, J. C., Lingardo, L., Chapman, J., Hwang, C., Alessi D.R.: Biochem J. 2010, 431(2), 245−255. [1] M.-J. Stahnke et. al.: Cellular Signalling 2014, 26(9): 1792. [4] Chaikuad, A., Koch, P., Laufer, S. A., Knapp, S.: Angew. Chem. Int. Ed. 2018, 57 (16), [2] K. MacAulay, J. R. Woodgett: Expert Opinion on Therapeutic Targets 2008, 12(10): 1265. 4372−4385. [3] Schrödinger Release 2017-3, Prime. User Manual, Schrödinger, LLC, New York, NY, 2017. [5] Gehringer, M., Laufer, S. A.: J. Med. Chem. 2019, ASAP, 10.1021/acs.jmedchem.8b01153 [4] R. A. Friesner et al.: J. Med. Chem. 2004, 47(7): 1739. [6] Hagel, M., Miduturu, C., Sheets, M., Rubin, N., Weng, W., Stransky, N., Bifulco, N., Kim, J. [5] N. Schneider et. al.: J Comput Aided Mol Des 2013, 27(1): 15. L., Hodous, B., Brooijmans, N., Shutes, A., Winter, C., Lengauer, C., Kohl, N. E., Guzi, T.: [6] a) D. Bashford, D. A. Case: Annual Review of Physical Chemistry 2000, 51: 129; Cancer Discovery, 2015, 5 (4), 424−437. b) P. D. Lyne, M. L. Lamb, J. C. Saeh, J. Med. Chem. 2006, 49(16): 4805; c) S. Genheden, U. Ryde, Expert Opinion on Drug Discovery 2015, 10(5): 449. [7] a) R. D. Cramer, D. E. Patterson, J. D. Bunce: Journal of the American Chemical Society 1988, 110(18): 5959; b) G. Klebe, U. Abraham, T. Mietzner: J. Med. Chem. 1994, 37(24): 4130.

POS.20

Design, Synthesis and Optimization of Novel Tricyclic Janus Kinase 3 (JAK3) Inhibitors POS.19

Design and Synthesis of Potential Covalent Inhibitors of 1 1 1 1 1 the Protein Kinase p70S6Kβ Pfaffenrot, E. ; Weißhaupt, P. ; Forster, M. ; Gehringer, M. ; Bauer, S. ; Chaikuad, A.2; Berger, B.T.2; Knapp, S.2; Laufer, S.1 1Department of Medicinal Chemistry, Eberhard-Karls-University Tuebingen, Auf der Haarer, L. 1; Laufer, S.A. 1 and Gehringer, M.1 Morgenstelle 8, 72076 Tuebingen, Germany 1Eberhard Karls Universität Tübingen, Institute of Pharmaceutical Sciences, Department of 2Institute for Pharmaceutical Chemistry, Goethe-University and Buchmann Institute for Pharmaceutical/Medicinal Chemistry, Auf der Morgenstelle 8, 72076 Tuebingen, Germany Molecular Life Sciences, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany

The mTOR (mechanistic target of rapamycin) signaling pathway is a key regulator of cell proliferation and growth, and inhibition of this pathway The four-membered family of Janus kinases (JAK1, JAK2, JAK3 and has been used in the treatment of cancer and immune disorders. [1] TYK2) plays a vital role in immune function, inflammation and However, some parts of this signaling network remain poorly elucidated. hematopoiesis. This enzyme family belongs to the class of cytosolic The protein kinases p70S6Kα and β belong to the downstream effectors tyrosine kinases, that are involved in various signalling cascades initiated of the mTOR-complex mTORC1. While the physiological role of by cytokines and growth factors mediating immune response and 1 p70S6Kα is comparably well characterized, the specific functions of proliferation via DNA transcription in the nucleus. While the expression of JAK1, JAK2 and TYK2 is ubiquitous, JAK3 is limited to hematopoietic p70S6Kβ are less clear. A redundant functional profile of p70S6Kα and tissues and has a key function in the maturation of lymphocytes. β was initially assumed, however, it is becoming increasingly apparent Therefore, JAK3 represents an interesting target for the development of that these enzymes play distinct roles in mTOR signaling. [2] immunosuppressive drugs. Tofacitinib, Pfizer´s pan-JAK inhibitor was So far, the characterization of p70S6K -specific functions has been β approved for the treatment of rheumatoid arthritis by the FDA in 2012.2 hampered by the lack of suitable chemical probes. While specific protein kinase inhibitors (PKIs) targeting p70S6Kα have already been developed, [3] no such tool compounds are available for p70S6Kβ. Achieving specificity for p70S6Kβ constitutes a considerable challenge since the latter kinases share a virtually identical ATP binding site differing only in a single amino acid. As a distinctive feature, p70S6Kα possesses a tyrosine (Tyr-151) at the gatekeeper+2 (GK+2) position in Tofacitinib competitively binds to the ATP pocket of JAK3 forming two the so called "hinge region" and this moiety is replaced by cysteine (Cys- hydrogen bonds from its heterocycle towards the hinge region. The 150) in p70S6Kα enabling covalent targeting approaches. Since an aliphatic side chain fills the ATP pocket towards the p-loop, while the equivalently positioned cysteine is present in only four other, structurally exocyclic chiral methyl group of the piperidine side chain binds to a small

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JAK-specific binding pocket representing a selectivity vector. Recently, POS.22 our group reported a new class of tricyclic inhibitors by following a rigidization approach via anellation of another five-membered ring to the Synthesis of novel bicyclic KOR ligands with piperidine heterocycle of the Tofacitinib lead structure to fix the side chain in its substructure active syn-conformation. In addition, the impact on the inhibitory activity by derivatization of the Tofacitinib side chain was investigated.3 4 Several new synthetic routes of the new scaffold have been developed Jonas, H.1,2, Wünsch, B.1, Diana, P.2 1 Institute of Pharmaceutical and Medicinal Chemistry, Corrensstraße 48, 48149, Germany to enable derivatization of different residues that allow a more detailed 2 Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Via Archirafi 32, SAR exploration. Derivatives with substituents at the C-3 position of the 90123, Italy tricyclic core (R1) addressing the hydrophobic region II of JAK3 as well as hydrophilic substituents in order to improve solubility have been prepared. Also, the influence of the chiral methyl group at the C-2 position Strong analgesics are used to reduce the perception of pain by activation (R2) of the aliphatic side chain (X = C, N) on the inhibitory activity and of classical opioid receptors MOR, KOR and DOR. Since centuries alkaloids in poppy seeds, which mainly activate MOR are used as pain selectivity in presence of small substituents at the C-3 position of the tricyclic core (R1) has been investigated. In our attempt to characterize killers and anaesthetics. However, they can cause severe side effects the new set of compounds as JAK3 inhibitors cellular data of chosen like euphoria, physical dependency, respiratory depression and inhibition compounds was generated. In addition, an X-ray crystallography of gastrointestinal motility. Therefore, in the last decades KOR agonists structure of JAK3 in complex with a nitrile-substituted phenyl derivative were developed as alternative analgesics. Besides their analgesic effect, could be achieved. KOR agonists are potent neuroprotective and antihyperalgesic agents. They do not interfere with the respiratory system, however, depression, diuresis and sedation can occur.1 Since KOR is not only located in the CNS but also in the periphery, activation of peripherally localized 1. Cornejo, M. G.; Boggon, T. J.; Mercher, T: Int J Biochem Cell B 2009, 41 (12): 2376-2379. 2. Van der Heijde, D, et al.: Arthritis. Rheum. 2013, 65: 559-570. receptors can be used to treat visceral pain, itching skin and inflammatory 2 3. Gehringer, M. et al.: ChemMedChem 2014, 9: 277-281. diseases. 4. Gehringer, M. et al.: ChemMedChem 2014, 9: 2516-2527.

POS.21 In vitro studies on TLX druggability assessment and early ligand discovery

G. Faudone1, J. Heering2 and D. Merk1 1Institute of Pharmaceutical Chemistry, Max von Laue Straße 9, 60438 Frankfurt am Main, Germany 2Project Group Translational Medicine and Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Theodor-Stern-Kai 7, 60438 Frankfurt am Main, Germany An interesting class of KOR agonists are compounds with an ethylenediamine substructure. U-50,488 was firstly synthesized in 1981 and is a highly selective KOR agonist (Ki = 0.31 nM).3 It consists of a Recent reports suggest that the nuclear receptor (NR) TLX (homologue tertiary amine embedded in a pyrrolidine ring and a second N-atom of the Drosophila tailless gene, NR2E1), exclusively expressed in small acylated with a 2-(3,4-dichlorophenyl)acetyl moiety. Both N-atoms are areas of the hippocampus, might act as a master regulator of attached to a cyclohexane ring in trans-configuration ((1S,2S)- neurogenesis[1]. Disrupted TLX expression caused aggressiveness[2], configuration). Further studies showed that besides the cyclohexane impaired cognitive function[3], mental illness[4] and brain tumor scaffold, a piperidine or piperazine structure can be used in which the formation[5] in mice. Thus, TLX may hold promise as target to treat ethylenediamine structure is partly embedded. In our work group, neurodegenerative diseases and brain tumor formation. piperazine containing compound 1 was synthesized and the (S,S)-isomer showed the highest KOR affinity (Ki = 0.31 nM).4 In order to investigate To date, no endogenous ligands are known for the orphan receptor TLX the bioactive conformation, a bicyclic ethylene-diamine derivative 2 with and the limited data on few putative synthetic TLX modulators is a bridgehead N-atom was synthesized.5 By comparing the dihedral inconsistent. To assess the druggability of TLX, we have studied the angles (N-C-C-N) of the endo- and exo-configured isomers, the endo- receptors molecular function with several assay setups in vitro. We isomer with a dihedral angle of 58.3° was quite potent (Ki = 73 nM), while observed a remarkable inhibitory activity of TLX on several other NRs the exo-isomer with an angle of 168° was nearly inactive (Ki ≥ 1 µM). suggesting that TLX acts as transcriptional repressor. Still, with a Ki-value of 73 nM, endo-2 is less active than (S,S)-1 (Ki = To reflect this characteristic in an in vitro test system for TLX ligand 0.31 nM), we postulate that a dihedral angle of 58.3° is not the preferred discovery, we established a hybrid reporter gene assay involving VP16- angle of (S,S)-1 in its bioactive conformation.5 Our next goal is to change Gal4 as a potent inducer of reporter transcription and TLX as repressor the dihedral angle by substituting the bridgehead nitrogen atom with a to enable a bidirectional modulation of TLX activity. Screening of a carbon atom and changing the ring size of the second ring. Moreover, commercial fragment library revealed TLX ligands which exhibit positive the polarity can be changed by using different residues in 3-position of (~ 3 fold activation) as well as a negative modulation (~ 6 fold repression) the bicyclic system. of TLX activity. By expanding these results to structurally more intricate natural products and approved drugs, we identified TLX modulators with References: low micromolar potency that appear suitable for structural optimization 1. (a) Scorpes, D. I. C. et all.: Drugs Future, 1993, 18, 933 – 947; (b) Holzgrube, U. et all.: towards potent TLX ligands. Pharmazie, 1997, 52, 4 – 22. 2. Riviére, P. J.-M.: Br. J. Pharmacol., 2004, 141, 1331 – 1334. Our preliminary results characterize TLX as repressor of various ligand- 3. Von Voigtlander, P. F.: Pharmacol, 1981, 23, 134. 4. Soukara, S. et all.: J. Med. Chem., 2001, 44, 2814 – 2826. activated transcription factors in vitro and suggest that the receptor is 5. Kracht, D. et all.: Org. Biomol. Chem., 2010, 8, 212 – 225. druggable with small molecules either promoting or disturbing its transcriptional repressor activity.

References: [1] C.-L. Zhang et al., Biochim Biophys Acta., 2014, 1849, 1–23. POS.23 [2] B. S. Abrahams, J. Neurosci. , 2005, 25, 6263–6270. Design, Synthesis and Characterization of Novel [3] J. D. O’Leary et al. Behav. Brain Res., 2016, 306, 36–47. [4] R. A. Kumar et al., Am. J. Med. Genet. Part B Neuropsychiatr. Genet., 2008, 147, 880–889. Irreversible JAK3-Inhibitors with High Isoform Selectivity [5] H. Kim et al., Mol. Cells, 2010, 30, 403–408. Based on a Tricyclic Hinge-Binding-Motif Dimitrov, T.T.; Nahidino, P.A.; Forster, M.; Pfaffenrot, E., Gehringer, M.; Laufer, S.A.

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Department of Medicinal Chemistry, Eberhard-Karls-University Tuebingen, Auf der Morgenstelle Janus kinases (JAKs). This kinase family, consisting of four closely 8, 72076 Tuebingen, Germany related isoforms (JAK1, JAK2, JAK3 and TYK2), is a key player in the

regulation and homeostasis of the immune system.[1] Since JAK3 is the The Janus kinase (JAK) family includes four cytosolic non-receptor only family member, which is exclusively operating in immunocompetent tyrosine kinases that play important roles in crucial physiological cells, it earned a special attention in the last years, as a promising target processes like cell proliferation, differentiation, migration, hematopoiesis for the development of a new class of immunosuppressants.[2] and regulation of the immune system. While three of the four family Due to the high structural similarity of the four JAKs, the development of members (JAK1, JAK2 and TYK2) are ubiquitously expressed, JAK3 isoform specific inhibitors is a challenging task. However, another unique fulfils its important tasks only in cells of the immune system like B-, T- feature of JAK3 is the presence of a non-conserved cysteine residue and NK cells.[1] (C909) nearby the ATP binding site, which is replaced by a serine in the Recently approved unselective JAK-inhibiting drugs such as Tofacitinib, three other family members. A high JAK3 selectivity can be therefore Ruxolitinib, Baricitinib or Peficitinib were used for immune-mediated achieved by the targeting of C909 with electrophilic inhibitors, a strategy diseases, like rheumatoid arthritis, inflammatory bowel diseases, that has already been successfully applied by our group as well as autoimmune skin disorders or transplant rejection. However, these drugs others.[3-5] suffers from frequent side effects resulting from unselective targeting of multiple JAK-pathways, which include increased infection rates, anemia and a risk of cardiovascular diseases and malignancies.[2, 3] In order to avoid this therapeutic difficulty, highly selective JAK3-inhibition is considered as a valuable strategy to reduce these adverse side effects.

O O R O HN R O N R N H H N HN N N N N N N In a new approach to develop irreversible JAK3 inhibitors, we postulated SAR exploration N N N N N N N N H H H H that an alteration of the commonly observed hinge-binding pattern of A B C IC50 = 274 ± 53 IC50 = 79 ± 9 IC50 = 3.3 purine isosteres would favor the 3-position of the five-membered pyrrol ring as possible attachment point for electrophilic warheads to target Within our medicinal chemistry program for developing novel JAK3- C909. We developed a modular synthetic route which allowed us to selective inhibitors, we identified a tricyclic core scaffold with favourable prepare a small set of 7-Azaindoles carrying an electrophilic warhead in properties for further optimization. In a small sub-project, we tried to 3-position. Some of the new inhibitors showed high potency on JAK3 address a non-conserved cysteine nearby the ATP binding pocket of together with a highly favorable isoform selectivity. The proof-of-concept JAK3, that cannot be found in other members of the JAK-family. Different was verified by XRay crystallography showing both, the alternative substitution patterns were investigated to identify a suitable position of a orientation of the hinge binding motif as well as the covalent bond phenylacrylamide warhead to access the cysteine in an optimal angle for formation between the inhibitor and C909 of JAK3. A moderate activity covalent bond formation. and target engagement in a cellular setting was also shown for these While the first derivates A and B only showed moderate inhibition of early compounds, which can be further optimized in ongoing optimization JAK3, compound C exhibited a high potency at low nanomolar cycles. The low molecular weight of this novel core scaffold offers concentrations. In stark contrast, a close analogue of C containing a non- possibilities for further modifications in order to modulate reactive propionamide displayed an about 2500-fold weaker potency on physicochemical properties or even further increase potency and JAK3 and therefore strongly supports the hypothesis of covalent selectivity. Therefore, these compounds can serve as a promising inhibition by C.[4, 5] Further derivatisation of this lead structure allowed starting point for the development of a novel class of covalent JAK3 to explore the SARs of this inhibitor class and even resulted in additional inhibitors. selectivity within the JAK-family. Two key compounds were tested for their selectivity within a set of kinases, that are carrying cysteines in similar positions in the hinge binding pocket. Furthermore, an in-silico References: model was created to show a possible binding mode of structure C. 1. Ghoreschi, K., Laurence, A., O’Shea, J.J.: Immunol. Rev. 2009, 228(1): 273-287 2. Schwartz, D.M. et al.: Nat. Rev. Drug. Discov. 2017, 16: 843-862 3. Forster, M. et al.: Cell Chem. Biol. 2016, 23(11): 1335–1340 4. Forster, M. et al.: J. Med. Chem. 2018, 61(12): 5350-5366 1. Pesu, M., et al., Therapeutic targeting of Janus kinases. Immunological Reviews, 2008. 5. Forster, M. Gehringer, M., Laufer, S.A.: Bioorg. Med. Chem. Lett. 2017, 27(18): 4229-4237 223(1): p. 132-142. 2. Schwartz, D.M., et al., JAK inhibition as a therapeutic strategy for immune and inflammatory diseases. Nature reviews. Drug discovery, 2017. 17(1): p. 78-78. 3. Markham, A. and S.J. Keam, Peficitinib: First Global Approval. Drugs, 2019. 79(8): p. 887- 891. POS.25 4. Pfaffenrot, E., Design, Synthese und Optimierung neuer trizyklischer Januskinase 3 Inhibitoren, E.K.U. Tübingen, Editor. 2016. Dual Inhibition of STS and 17β-HSD1: A Novel Drug- 5. Forster, M., Scaffold-Hopping-Strategie zur Entwicklung neuer Prodrug Approach for the Treatment of Endometriosis kovalenter Januskinase 3 Inhibitoren mit hoher Isoform Selektivität, E.K.U. Tübingen, Editor. 2018. Mohamed, A.M. *1; Salah, M.1; Tahoun, M.1; Abdelsamie, A.S.1,2;

1 Frotscher, M. 1Department of Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C23, POS.24 66123 Saarbrücken, Germany. 2Chemistry of Natural and Microbial Products Department, National Research Centre, Dokki, Flipping the Hinge-Binding-Motif: 3,5-disubstituted 7- 12622 Cairo, Egypt. * Azaindoles as Promising Scaffold for the Development of [email protected] Covalent JAK3 Inhibitors

Estrogens, especially the most active one estradiol (E2), are well known Forster, M.1; Chaikuad, A.2; Berger, B.T.2; Müller-Knapp, S.2; to play a crucial role in the development of estrogen-dependent diseases Knapp, S.2; Laufer, S.A.1 like breast cancer [1] and endometriosis [2]. Local E2 biosynthesis in 1 Department of Medicinal Chemistry, Eberhard-Karls-University Tuebingen, Auf der endometriosis is essentially affected by the key enzymes steroid Morgenstelle 8, 72076 Tuebingen, Germany 2 Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University and Buchmann sulfatase (STS) and 17β-hydroxysteroid dehydrogenase type1 (17β- Institute for Molecular Life Sciences, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, HSD1) (Fig. 1). Both enzymes are overexpressed in endometriotic tissue Germany and lead to a strong local E2-production and thus push the progression of the disease. Consequently, inhibition of STS and 17β-HSD1 is an The physiologically important processes of proliferation, differentiation attractive concept for the treatment of endometriosis [3,4]. 17β-HSD2 is and maturation of haematologic cells are mainly driven by cytokines like the physiological counterpart of the type 1 enzyme and should not be interleukins (ILs), interferons (IFNs), and growth factors. The signal inhibited. Recently our group published the first dual STS/17β-HSD1 pathways initiated by these extracellular stimuli are often mediated by the inhibitors (DSHIs) [5] which show strong inhibition of both enzymes.

DPhG Annual Meeting 2019 Conference Book • 94 MEDICINAL CHEMISTRY AND DRUG DESIGN

These compounds bear a sulfamate group for STS inhibition and a RPA inhibition represents a promising therapeutic target for the treatment phenolic OH group entailing 17β-HSD1 inhibition (Fig. 2, A). The fact that of proliferative disorders [2] (unpublished data). the sulfamate group is cleaved upon interaction with STS and/or non- Structurally, RPA is a heterotrimeric complex consisting of the three enzymatic hydrolysis releasing the corresponding phenol gives rise to an subunits RPA1 (70kDa), RPA2 (32kDa) and RPA3 (14kDa). Henricksen intriguing design concept for inhibition of the two target enzymes (drug- et al. showed that the subcomplex of RPA2 and RPA3 is a precursor in prodrug approach). We aimed at synthesizing compounds in which the the assembly of the complete RPA complex [3]. Inhibition of the OH group is masked as a sulfamate group (Fig. 2, B). These compounds subcomplex formation can consequently disrupt the RPA complex’s should be drugs for inhibition of STS and, at the same time, prodrugs for overall function. 17β-HSD1 inhibition. Eight sulfamate compounds were synthesized, of which five showed strong inhibition of STS in a cellular assay (IC50 =13- 28 nM). The corresponding phenols were also synthesized and displayed potent 17β-HSD1 inhibition in cellulo (IC50= 5-20 nM) as well as high selectivity over 17β-HSD2. Stability measurements in buffer and DMEM revealed that the sulfamate compounds (STS inhibitors) hydrolyzed to the corresponding phenols (17β-HSD1 inhibitors), with half-life times ranging from minutes to hours depending on the substitution pattern of the compounds. In an enzymatic assay which in a time dependent manner allows monitoring of both hydrolysis of the sulfamates to phenols and increase in 17β-HSD1 inhibition, we found a strong correlation between phenol formation and enzyme inactivation. These results show that the drug-prodrug concept was successfully implemented. Thus, the novel compounds are potential therapeutics for the treatment of Fig. 1. Proposed interaction of a leading compound (RPAi) within the endometriosis. RPA’s hot spot

O O OH 17-HSD1, NADPH STS We identified a hot spot in the RPA2-RPA3 subcomplex crystal structure, 17 -HSD2, NAD+  HO which we exploited for in silico small molecule screening for new O3SO HO inhibitors (Fig. 1). We were able to identify a hit compound with a low µM Estrone-3-sulfate (E1S) Estrone (E1) 17-estradiol (E2) activity in in vitro EMSA assays. Following a classical structure highly estrogenic no estrogenic potency weakly estrogenic optimization workflow, supported by molecular modelling, we developed a series of highly potent compounds. These inhibitors showed good Figure 1. The sulfatase pathway of local E2 biosynthesis. efficacy against pancreatic cancer cell lines with IC50 values in the double- digit nM range and favorable on-target activity shown by EMSA assays. Additional target engagement evidence was further corroborated by 2D- Responsible for STS inhibition NMR protein binding studies [4] with the best suitable compound of the Sulfamate group is converted to OH group by STS and/or series (unpublished data). OSO NH non-enzymatic hydrolysis HO OSO2NH2 2 2 OH Since the target activity is established, the further development of our Drugs for STS inhibition and Drugs for 17-HSD1 lead compound focusses on the optimization of the ADME-properties to Prodrugs for 17-HSD1 inhibition inhibition Responsible for 17-HSD1 inhibition finally create an initial clinical candidate for the treatment of pancreatic cancer. A B Figure 2. Design rationale (drug-prodrug approach). Acknowledgements: The authors would like to thank Khalil Samarah for the support in chemical synthesis and characterization of final compounds during his master thesis.

References References: [1] Travis, R.C.,T.J. Key.: Breast Cancer Res. 2003, 5(5): 239. 1. Zou et al.: Journal of Cellular Physiology 2006, 208(2): 267-273 [2] Dizerega, G.S. et al.: Fertil. Steril. 1980, 33(6): 649-653. 2. Prof. L. Zender et al.: Internal Medicine VIII, University Hospital of Tübingen [3] Saloniemi, T. et al.: Am. J. Pathol. 2010, 176 (3), 1443–1451. 3. Henricksen et al.: The Journal of Biological Chemistry 1994, 11121-11132 [4] Colette, S. et al.: Hum. Reprod. 2011, 26 (6), 1362–1370. 4. SARomics Biostructures AB, Sweden [5] Salah, M. et al.: J. Med. Chem. 2017, 60(9): 4086-4092. [6] Stanway, S.J. et al.: Clin. Cancer Res. 2006, 12(5):1585-1592.

POS.27

Targeting the Osimertinib-Resistant L858R/T790M/C797S POS.26 EGFR Mutant: Design, Synthesis, Biochemical and Development of RPA Protein-Protein Interaction Inhibitors Crystallographic Evaluation of Low Nanomolar Inhibitors. as Novel Therapeutic Agents for the Treatment of Pancreatic Cancer Guenther, M.1; Wittlinger, F.1; Heppner, D.4,5, Juchum, M.1, Döring, E.1, Kelter, G.2, Fiebig, H.2, Lategahn, J 3, Keul, M., Tumbrink, H.3, Engel, 3 3 4,5 1 Schmidberger, G.1, Haase, G.-S.1, Zwirner, S.2, Moschopoulou, A.2, J. , Rauh, D. , Eck M. J. , Laufer, S. A. 1University of Tuebingen, Faculty of Science, Pharmaceutical and Medicinal Chemistry, Auf der 2 2 2 2 1 Kronenberger, T. , Dauch, D. ,Poso, A. , Zender, L. , Laufer, S. Morgenstelle 8, 72076 Tuebingen, Germany 1Department of Medicinal Chemistry, Eberhard-Karls-University Tübingen, Auf der Morgenstelle 2Cell Biology & Compound Screening Oncotest GmbH, Am Flughafen 12 – 14, 79108 Freiburg, 8, 72076 Tübingen, Germany Germany 2Internal Medicine VIII, Medical Oncology and Pneumology, University Hospital Tübingen, 3Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Straße 4a, Otfried-Müller-St. 14, 72076 Tübingen, Germany 44227 Dortmund, Germany 4Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave, Boston, MA, 02115, USA The replication protein A (RPA) is an essential protein, which is able to 5Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215 USA bind to ssDNA in human cells. It is involved in diverse DNA metabolism pathways such as replication, repair, recombination, cell cycle regulation The treatment of non-small-cell lung cancer (NSCLC) with epidermal and DNA damage checkpoints [1]. The inhibition of RPA leads to genome growth factor receptor (EGFR) inhibitors is made challenging by acquired instability and, in case of high proliferating cell lines such as cancer cells, resistance caused by somatic mutations [1]. Third generation EGFR to replication catastrophe followed by cell death. On the other hand, in inhibitors (WZ4002, Osimertinib) have been designed to overcome healthy transgenic mice shRNA mediated suppression of RPA is well resistance, mediated by the T790M mutation, through covalent binding tolerated and opens a therapeutic window up to nine days. Therefore, to the Cys797 residue of the enzyme. These inhibitors are effective against most clinically relevant EGFR mutations, however their high

95 • DPhG Annual Meeting 2019 Conference Book POSTERS dependence on this particular interaction means that additional mutation compounds led to significant H3K36 hypermethylation indicating on- of Cys797 results in poor inhibitory activity, which leads to tumour relapse target activity by in-vivo inhibition of KDM4. in initially responding patients [2,3].

F

R N 1 N R2 N OH N H H N N R HN HN 3

Based on a selectivity screening of a highly potent reversible p38 inhibitor [4], we identified EGFR inhibition as an off-target effect of this compound. Figure 1 Deferasirox High potency, as well as moderate physicochemical properties and docked to KDM4A cellular activity against p38, led us to pick this compound as a lead structure for further improvements in terms of mutant EGFR inhibition. With this concept, we have successfully developed highly potent We thank the DFG (CRC992) for funding reversible and irreversible T790M EGFR inhibitors that showed picomolar IC50-values in an enzyme assay and down to 14 nM EC50 in a [1] Rüger N. et al.: ChemMedChem, 2015, 10(11): 1875. double mutant (L858R/T790M) cellular assay. In contrast to classic third [2] Morera L. et al.: ChemMedChem, 2016, 11(18): 2063. generation EGFR inhibitors, some of these compounds showed high [3] Roatsch, M. et al.: ChemRxiv, 2019, preprint: doi: 10.26434/chemrxiv.7699853.v2 inhibitory activity in the low nanomolar range against the therapy- resistant L858R/T790M/C797S EGFR triple mutant [5-8]. Moreover, to further support the concept of the binding modes and enlighten specific interactions we determined two X-ray crystal structures for both a reversible and an irreversible compound. POS.29 Synthesis and evaluation of phosphonic acids for the BET 1. M. Juchum, M. Günther et al., Drug Resist. Updat. 2015, 20, 12 – 18. bromodomain ligands: Probing the WPF shelf to improve 2. Z. Piotrowska, et al., Cancer Discov. 2015, 5, 713-722. BRD4 bromodomain affinity and metabolic stability 3. K. S. Thress, et al., Nat. Med. 2015, 21, 560-562. 4. R. Selig, et al., J. Med. Chem. 2012, 55, 8429-8439. 5. M. Günther, et al., Angew. Chem. Int. Ed., 2016, 55, 10890 –10894 Schiedel, M.1,2; Jennings, L. E.2; Hewings, D. S.2; Picaud, S.3; Laurin, C. 6. M. Günther et al., J. Med. Chem., 2017, 60, 5613–5637 2 4 2 5 4 7. M. Juchum et al., J. Med. Chem., 2017, 60, 4636–4656 M. C. ; Bruno, P. A. ; Bluck, J. P. ; Mistry, I. N. ; Mapp, A. K. ; 5 3 2 8. M. Günther et al., Eur. J. Pharm. Sci, 2019, 128, 91-96 Hammond, E. M. ; Filippakopoulos, P. ; Conway S. J. 1 Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Nikolaus-Fiebiger-Straße 10, 91058, Erlangen, Germany; 2 Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, United Kingdom; 3 Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of POS.28 Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 3TA, United Kingdom; Synthesis and Biological Evaluation of derivatives of 4 Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109-2216, United States; Deferasirox as Inhibitors for JumonjiC-domain containing 5 CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, United Kingdom Histone Demethylases

Ligands for the bromodomain and extra-terminal domain (BET) family of Wenzler, S.1; Barthes, N. P. F. 1; Spiske M. 1; Schmidtkunz, K.1; Ngo V. bromodomains are promising therapeutic agents for treating a range of 2; Robaa, D.3; Schüle, R.4; Sippl, W.3; Hein, L.2; Jung, M.1 cancers and inflammation [1,2]. We were able to show that our previously 1 Institut für Pharmazeutische Wissenschaften, Pharmazeutische und Medizinische Chemie, Albert-Ludwigs-Universität Freiburg, Albertstraße 25, 79104 Freiburg, Germany developed 3,5-dimethylisoxazole-based BET bromodomain ligand 2 Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albertstraße 25, (OXFBD02) [3] inhibits the interactions of BRD4(1) with the RelA subunit 79104 Freiburg, Germany of NF-κB, in addition to histone H4. This ligand showed a promising 3 Martin-Luther Universität Halle-Wittenberg, Abteilung Medizinische Chemie, profile in a screen of the NCI-60 panel but was rapidly metabolised (t½ = Langenbeckstraße 4, 06120 Halle/Saale, Germany 4 Urologische Klinik und Zentrale Klinische Forschung, Klinikum der Universität Freiburg, 39.8 min). Structure-guided optimisation of compound properties led to Breisacherstraße 66, 79106 Freiburg, Germany the development of the 3-pyridyl-derived OXFBD04. Co-crystallization of OXFBD04 with BRD4(1) in combination with molecular dynamics (MD) Fe(II)- and 2-oxoglutarate-dependent Jumonji C domain-containing simulations and NMR titration experiments assisted our understanding of histone demethylases (JmjC KDMs) play an important role in the the role played by an internal hydrogen bond in altering the affinity of this regulation of epigenetic processes by oxidatively removing methyl marks series of molecules for BRD4(1). OXFBD04 shows improved BRD4(1) from distinct lysine residues. As their aberrant expression is implicated in affinity (Kd = 126 nM), optimised physicochemical properties (LE = 0.43; numerous diseases, particularly cancer, KDM-inhibitors possess a LLE = 5.74; SFI = 5.96), and greater metabolic stability (t½ = 388 min) promising therapeutic potential.[1, 2] Aiming for the development of new [4]. potent inhibitors of KDM4A, we chose the clinically approved iron- chelator Deferasirox as the lead structure. Deferasirox is able to chelate the active-site iron of JmjC KDMs and displays an inhibitory activity on Me Me N N these enzymes in the micromolar range.[3] Based on molecular modeling OH OH O O we synthesized and tested derivatives of Deferasirox by varying the N substitution pattern of the aromatic rings, the iron binding moieties as well Me Me as the core heterocycle. Our approach yielded various compounds exhibiting a low micromolar enzyme inhibition and allowing the OH OH establishment of structure-activity relationships. The cellular activity of selected compounds was assessed in an MTS-assay on the OXFBD02 OXFBD04 oesophageal cancer cell line KYSE-150. For example an imidazole, that Kd = 425 nM Kd = 126 nM showed an in-vitro IC50 of 2 µM against KDM4A, resulted in a GI50 of 3 t½ = 39.8 min t½ = 388 min µM, whereas a structurally similar, but inactive compound showed no cellular effects. Treatment of KYSE-150 cells with the most potent

DPhG Annual Meeting 2019 Conference Book • 96 MEDICINAL CHEMISTRY AND DRUG DESIGN

References:

1. Schiedel, M., Conway, S. J. Curr. Opin. Chem.Biol. 2018, 45: 166-178. 2. Schiedel, M., Moroglu, M. et al. Angew. Chem. Int. Ed. 2019, doi: 10.1002/anie.201812164 [Epub ahead of print]. 3. Hewings, D. S., Fedorov, O. et al. J. Med. Chem. 2013, 56(8): 3217-3227. 4. Jennings, L. E., Schiedel, M. et al. Bioorg. Med. Chem. 2018, 26(11): 2937-2957.

POS.30 Improved high-yield synthetic strategy of the The Human African Trypanosomiasis (HAT) is a neglected tropical disease caused by unicellular parasites, which are transmitted by the bite dibenzosuberone scaffold for p38α MAP kinase inhibitors of the Tse-Tse-fly. [1] Without proper treatment, the sickness leads unescapably to the host's death. Unfortunately, the therapy currently depends on drugs that only work in the early stages of the disease and/or Haller, V.1; Wentsch, H. K.1; Fischer, S.1; Pantsar, T.1,2; Forster, M.1; show serious side effects. Laufer, S.1 The parasite’s life cycle strongly relies on cysteine proteases of the 1Institute of Pharmacy, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, cathepsin family. Among them, TbCathepsin L (TbCatL, Rhodesain) is Germany 2School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70211 Kuopio, the main protease of Trypanosoma brucei rhodesiense. In combination Finland with its localization in the lysosome, Rhodesain most likely plays an important role in protein turnover, e.g. in digestion of nutrients and immunoglobulins. [2,3,4] Even though first RNAi studies implied that The p38α MAP kinase (MAPK) plays a crucial role in the regulation of the Rhodesain is not essential for the survival of the parasite, more recent biosynthesis of pro-inflammatory cytokines like TNF-α, IL-1β and ATF-2. studies suggest that a complete knock-down was never reached and that These cytokines are involved in the pathology of several diseases, such Rhodesain is indeed crucial for cell growth and the parasite viability. as cancer or autoimmune disorder. Hence, p38α MAPK inhibitors are [5,6,7] potential candidate for the treatment of these severe diseases. Our group Our group has developed new compounds with antitrypanosomal activity have previously developed the potent and highly selective p38α MAPK which inhibit the parasite´s major cysteine protease TbCatL in a covalent- inhibitor Skepinone-L (fig.1), which is currently in use as high-quality reversible manner. [8,9] chemical probe and clinical candidate [1,2,3]. For further optimization of these inhibitors, it is essential to get insights into drug-target interactions regarding thermodynamics, kinetics and structure-activity-relationship. Therefore, we developed an optimized method for recombinant expression of catalytically active Rhodesain. In this method, the enzyme is expressed as an inactive precursor containing a N-terminal pro-domain. Besides protein trafficking and proper folding, this pro-domain works as active-site directed inhibitor. The crystal

Figure 1: Based on the dibenzosuberone-scaffold, decorations moved structure of pro-Rhodesain was recently solved by our group. By taking away from ethers to esters and amides. advantage of the inactive Rhodesain pro-form that occurs during the protein purification, we were able to selectively label an additionally The latest published synthesis of the dibenzosuberone scaffold consists introduced cysteine residue of the protease without addressing the much more reactive active site cysteine. The thus biotinylated enzyme was of six steps with a limited yield of 37% [4]. In addition, this synthetic route has a drawback of unstable reagents, which is especially problematic for immobilized and used in SPR experiments, giving insights into the a larger scale synthesis. To address this, an improved synthesis is binding kinetics of our compounds. Furthermore, we used ITC experiments to investigate the thermodynamic parameters of the binding required. In our contribution we describe a new synthetic route with events. reduced steps starting from commercially available starting material and improved overall yield. 1. https://www.who.int/trypanosomiasis_african/en/ Our initial trials to facilitate the dibenzosuberone synthesis consisted of 2. Caffrey, C. R., et al.: Mol Biochem Parasitol., 2001, 118(1): 61-73. a three-step process involving Heck coupling, reduction of the double 3. Engstler, M., et al.: Cell., 2007,131(3): 505-515. bond and consequently cyclization via Eaton’s reagent resulting in an 4. Mbawa Z.R., Gumm I.D., Shaw E.: Eur J Biochem., 1992, 204(1): 371-379. overall yield of 66%. Further improvements led to a two-step synthesis 5. Koeller, C. M.; Bangs, James D.: Cell Microbiol., 2019, 21(4): 1–16. by utilizing a Suzuki cross coupling instead of Heck reaction to avoid the 6. Mott, B. T. et al.: J. Med. Chem., 2010, 53(1): 52-60. formation of the undesired double bond. This enhanced synthetic 7. Steverding, D., et al.: Int J Parasitol, 2012, 42(5): 481-488. 8. Schirmeister T., et al.: Am. Chem. Soc., 2016, 138(27): 8332–8335. strategy further improved the yield from 66% to 82%. 9. Latorre A., et al.: ACS Med Chem Lett., 2016, 7(12): 1073–1076 With the established new synthetic strategy, we were able to improve the initial unoptimized yield of 37% to 82% that aids us in our ongoing efforts in the design of novel dibenzosuberone based p38α MAPK inhibitors.

(1) Koeberle, S. C. et al.: Nat. Chem. Biol. 2012, 8: 141−143. POS.32 (2) Knapp, S. et al.: Nat. Chem. Biol. 2013, 9: 3–6. (3) Rudalska, R. et al.: Nature medicine, 2014, 20: 1038-1046. Structure-activity relationships of novel PAR2 ligands (4) Wentsch, H. K. et al. Angewandte Chemie 2017, 56: 5363-5367

Klösel, I.; Gmeiner, P. Friedrich-Alexander University Erlangen, Department of Chemistry and Pharmacy, Emil Fischer Center, Nikolaus-Fiebiger-Str. 10, D-91058 Erlangen POS.31 The protease activated receptor 2 (PAR2) belongs to a four-membered Insights into the binding kinetics and thermodynamics of family of G protein-coupled receptors (GPCRs) with an unusual Rhodesain with vinylsulfone-based Inhibitors by taking mechanism of activation and complex signaling pattern. In contrast to advantage of its inactive pro-form common GPCRs, activation of PARs is initiated endogenously by proteolytic cleavage of a passive sequence located at the extracellular N-terminus of the receptor. This exposes an active sequence acting as a Johe, P.1; Kersten, C. F.1; Fuchs, N.1; Hellmich U. A.2; Schirmeister, T1. tethered ligand which then folds into the binding site of the receptor and 1 Johannes Gutenberg-Universität Mainz, Institut for Biochemistry and Pharmacy, Staudinger elicits signal transduction.[1] PAR2 activation regulates different signaling Weg 5, 55128 Mainz. 2Johannes Gutenberg-Universität Mainz, Institut for Biochemistry and Pharmacy, Johann- pathways which include both G protein dependent pathways and Joachim Becherweg 30, 55128 Mainz. G protein independent pathways such as recruitment of ß-arrestin. (Fig. 1)

97 • DPhG Annual Meeting 2019 Conference Book POSTERS

POS.33 Design and synthesis of a radiotracer for ecto-5’- nucleotidase (CD73) – a novel target for the immunotherapy of cancer

Schmies, C.C.; Renn, C.; R. M. Idris; Müller, C.E. PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany

Ecto-5′-nucleotidase (CD73) is a member of the ecto-nucleotidase family, which catalyzes the hydrolysis of nucleoside monophosphates, mainly AMP, producing the signaling molecule adenosine. Further ecto- Figure 1. PAR2 activation by trypsin: The tethered ligand nucleotidases include the nucleoside triphosphate diphosphohydrolases SLIGKV (human) is exposed, and leads to signaling in (NTPDases; subtypes 1, 2, 3 and 8), the nucleotide different pathways pyrophosphatases/phosphodiesterases (NPP1-4) and the alkaline phosphatases (APs).[1] CD73 is often co-localized with adenosine receptors. CD73 inhibitors reduce extracellular adenosine levels, which results in an indirect blockade of adenosine receptor activation. Many tumor cells over-express ecto-nucleotidases, which metabolize pro- inflammatory ATP into anti-inflammatory, immunosuppressive, tumor growth-stimulating, and angiogenic adenosine.[2] Therefore, ecto- nucleotidases possess great potential as novel drugs for the (immuno)therapy of cancer and infections. The ADP analogue α,β-methylene-ADP (AOPCP, Ki = 88.4 nM, human CD73) has recently been used by our group as a lead structure for the development of potent and selective competitive CD73 inhibitors.[3-5] One of these compounds, PSB-12651 (Ki = 1.23 nM, rat CD73), was selected as a lead structure to develop a tritium-labelled radioligand for CD73. [3H]PSB-17230 represents a high-affinity tracer which is anticipated to become a useful tool for biological studies, drug screening, and diagnostic applications.

References: 1. Zimmermann, H.; Zebisch, M.; Sträter, N.: Purinerg.Signal. 2012, 8: 437-502. 2. Vitiello, L. et al.: Blood 2012, 120: 511-518. 3. Bhattarai, S. et al.: J. Med. Chem. 2015, 58: 6248−6263. Figure 2. Ligand based approach to develop novel non-peptidic PAR2 4. Junker, A. et al.: J. Med. Chem. 2019, 62: 3677-3695 ligands 5. Bhattarai, et.al.: Adv. Therap. 2019, in revision

POS.34 PAR2-promoted signaling is reported to be involved in several inflammatory diseases.[2] To better understand the signaling patterns of MALDI-TOF Mass Spectrometry Profiling of Bacteria PAR2 to cause inflammation, functionally selective ligands which only Coupled with Machine Learning Techniques Identifies activate one pathway while not affecting any other play a key role. Mode of Action of Antibacterial Drugs Moreover they also can lead to the development of highly effective drugs with a minimum set of side effects.[3] However, the development of PAR2 Luuk N. van Oosten1, Christian D. Klein1 ligands showing functional selectivity towards a distinctive pathway is still 1Medicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology, Heidelberg a rather unexplored field. University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany In this project new PAR2 ligands with potential functional selectivity were developed, applying a ligand-based approach (Fig. 2). Based on the There is a growing unmet medical need for new antibiotics to battle the sequence of the in human PAR2 naturally exposed tethered ligand increased frequency of resistance among bacterial pathogens (1). SLIGKV, shortened peptidomimetic ligands already have been Despite years of target-oriented approaches, few new drug classes or described.[4] Replacement of the N-terminal serine with heteroaromatic drugs with novel modes of action have entered the antibiotics market. moieties has been reported to be beneficial for ligand potency while the Meanwhile, the main antibiotic targets remain cell wall synthesis proteins, unnatural amino acid L-cyclohexylalanine is important to obtain PAR2 bacterial DNA gyrase, and the bacterial ribosome (2). The main problem selectivity over PAR1.[5] Moreover, a lipophilic pocket which is addressed associated with the failure of biochemical assays to deliver new drugs is by the C-terminal region has been reported.[4] These findings have been that they do not take membrane permeability into account. This is applied to design novel non-peptidic ligands for PAR2 by replacing the especially a problem in the case of Gram negative pathogens. (3) In C-terminal amino acid L-cyclohexylglycine with different substituted recent years, the shortcomings of biochemical target-based assays led benzamide derivatives and homologous structures thereof. A library of to a renewed interest in phenotypic screening procedures (4, 5). For compounds has been synthesized and evaluated biologically using antibacterial activity, growth inhibition of bacterial cell cultures provides a assays to measure IP-One accumulation and ß-arrestin recruitment, straightforward readout, but offers no further information with respect to respectively, leading to the identification of several structure activity which target sites are engaged and is limited to compounds with relatively relationships. high activity (2, 5). We present a protein mass fingerprinting method using MALDI-TOF MS in combination with data-dependent, supervised machine learning [1] Ossovskaya, V.S.; Bunnett, N.W.: Physiol. Rev. 2004, 84, 579-621. methods, to identify specific phenotypic response signatures generated [2] Bunnett, N.W.: Semin. Thromb. Hemostasis 2006, 32. [3] Adams, M.N.; Ramachandran, R.; Hooper, J.D.: Pharmacology & Therapeutics 2011, 130, in bacterial cell cultures upon exposure to well-characterized antibiotics 248-282. at sub-lethal concentrations. The approach, denoted PhenoMS-ML, [4] Yau, M. K et al.: ACS Med. Chem. Lett. 2016, 7 (1), 105-110. proves useful in the elucidation of mechanism of action in a drug [5] Barry, G. D. et al.: J. Med. Chem .2010, 53, 7428-7440. screening setting. We show that the classification models generated with the method identify and classify the mode of action of unknown antibacterial agents in wild-type Escherichia coli and Staphylococcus aureus with over 90% accuracy. The method allows for the sensitive,

DPhG Annual Meeting 2019 Conference Book • 98 MEDICINAL CHEMISTRY AND DRUG DESIGN label-free, and high-throughput identification of drug target mechanisms receptors (GlyRs). GlyRs are chloride channels composed of five at sub-lethal concentrations in a biologically relevant, phenotypic setting. subunits (α or β) and linked to hyperpolarisation and inhibition of neuronal firing.1 The crystal structure of human α3 GlyR in complex with strychnine revealed five equivalent strychnine binding sites located at the This work was funded by the basic governmental funding of Heidelberg University (Baden- Württemberg, Germany). interfaces of the subunits.2 Recently, our groups have become interested in a bivalent ligand approach for targeting GlyRs. In particular, an (E)- (1) O'Neill: Review on Antimicrobial Resistance (May 14th 2015) oxime ether group at C-11 of strychnine has been identified as a possible (2) L. L. Silver: Clin. Microbiol. Rev. 24, 71-109 (2011). linker to be incorporated into strychnine-based bivalent ligands.3 Oxime (3) D. J. Payne et al.: Nat. Rev. Drug Discov. 6, 29-40 (2007). ethers of (E)-11-isonitrosostrychnine –C=N-OR with R = Me, nPr, nBu, (4) D. C. Swinney, J. Anthony: Nat. Rev. Drug Discov. 10, 507-519 (2011). and n-pentyl were only up to 3-fold less potent antagonists compared to (5) Y. Feng et al.: Nat. Rev. Drug Discov. 8, 567-578 (2009). strychnine at homomeric α1 and heteromeric α1β GlyRs.3 Unfortunately, numerous attempts to isolate a first dimeric ligand from a reaction of (E)- 11-isonitrosostrychnine with 1,6-dibromohexane in different ratios, using different solvents and bases failed. Here, we describe an alternative POS.35 approach towards dimeric strychnine oxime ethers, Briefly, (E)-11- Conformational states of the flaviviral protease isonitrosostrychnine was O-alkylated using Br-(CH2)2-NHBoc and, after Boc-deprotection, the resulting amines were coupled with dicarboxylic acids HO2C-(CH2)n-CO2H. The final compounds were pharmacologically Mira A. M. Behnam and Christian D. P. Klein evaluated on homomeric (α1)5 and heteromeric (α1)2β3 GlyRs in a Institute of Pharmacy and Molecular Biotechnology IPMB, Heidelberg University, Im functional fluorescence-based and a whole-cell patch-clamp assay, and Neuenheimer Feld 364, 69120 Heidelberg, Germany their antagonist potencies were compared to those of strychnine.

Drug development is often complicated in the case of multiple enzyme

N conformations that affect the binding site, as it is for the flaviviral N N protease. Infections by flaviviruses, such as dengue, Zika, West Nile, and H H H 1 H H H Japanese encephalitis viruses, represent a global health burden, with no N H H N H H H H N O O drugs currently available. The protease is an attractive target, since it is O O H O H H O 11 N O (CH ) NH (CH n NH(CH2)2 N responsible for the processing of the viral genome during replication into 2 2 2) O 2 structural and non-structural proteins (NS). The enzyme is a trypsin-like O O serine protease, composed of NS2B and NS3.3 The first X-ray structures strychnine dimeric strychnine oxime ethers (n = 3, 20) released showed two states, open and close based on the relative position of NS2B with respect to NS3.4 We perform an analysis covering Acknowledgments: Deutscher Akademischer Austauschdienst, Bundesministerium für Bildung the entire repertoire of 45 crystal structures in the protein data bank for und Forschung flaviviral proteases, focusing on structures released in the past three years.5,6 The study reveals an array of conformations for this protein, beyond the reported open and close forms. We pinpoint the main criteria References: to distinguish different conformations such as the position of NS2B and 1. Lynch, J.W.: Physiol. Rev. 2004, 84, 1051-1095. the orientation of the oxyanion hole, and identify the impact of changes 2. Huang, X. et al.: Nature 2015, 526, 277-280. in the folding of NS3 on the spatial distribution in the active site. The array 3. Mohsen, A. et. al.: J. Nat. Prod., 2016, 79, 2997-3005. of conformations of the protease appears as different stages in a dynamic movement of the NS3 conserved residues 151-153. We show in one conformation a subpocket extending from the catalytic aspartate 75 to a pocket at the back of the protein, where inhibitors show an unusual POS.37 binding mode. The finding provides a different view of the flaviviral Exploiting Synergies in NASH protease. It is particularly of value for researchers concerned with the study of the protein mechanism of action, drug optimization by molecular modeling and docking studies, and strategies for inhibitor design and Heitel, P.1; Faudone, G. 1; Schmidt, J. 1; Merk, D1. synthesis in medicinal chemistry. 1Goethe University Frankfurt, Max-von-Laue-Strasse 9, 60435 Frankfurt am Main, Germany

Non-alcoholic fatty liver disease (NAFLD) occurs as first manifestation of Acknowledgments: The project was sponsored by the Deutsche Forschungsgemeinschaft, KL- the metabolic syndrome in the liver with an alarming prevalence of 14 – 1356/3-2 27% of the industrialized world’s population.[1] Even worse, 5 – 20% of the patients develop an irreversible steatohepatitis (NASH) that can References 1. Boldescu et al.: Nat. Rev. Drug Discov. 2017, 16 (8): 565-586. progress to fibrosis, cirrhosis and hepatocellular carcinoma if untreated. 2. Behnam et al.: J. Med. Chem. 2016, 59 (12), 5622-5649. Hence, there is a strong demand for novel anti-steatotic and anti- 3. Yusof et al.: J. Biol. Chem. 2000, 275 (14): 9963-9969. inflammatory drugs to treat the condition at early stage. 4. Erbel et al.: Nat. Struct. Mol. Biol. 2006, 13 (4): 372-373. The nuclear peroxisome proliferator-activated receptors (PPAR, NR1C1- 5. Lei et al.: Science 2016, 353 (6298): 503-505. 3) and the farnesoid X receptor (FXR, NR1H4) modulate lipid metabolism 6. Yao et al.: J. Am. Chem. Soc. 2019, 141 (17): 6832-6836. as well as inflammation and their therapeutic efficacy as targets in NASH has been demonstrated in clinical trials.[2-5] While FXR agonist obeticholic acid (ocaliva®) demonstrates anti-steatotic and anti-fibrotic POS.36 effects in liver,[6] PPAR (e.g. activated by elafibranor) regulates fatty Dimeric Strychnine Oxime Ethers as Bivalent Ligands acid oxidation in key metabolic tissues such as skeletal muscle thereby Targeting Glycine Receptors improving liver health indirectly.[7-8] Furthermore, PPAR activation has anti-inflammatory properties. The extrahepatic effects of PPAR and the hepatic FXR activation promise synergistic efficacy. The validity of dual Abdelmalek, C. M1; Breitinger, H-G1; Breitinger, U1; Holzgrabe, U2; pharmacotherapeutic approaches has recently been evidenced in mouse Jensen, A. A.3;Zlotos, D. P.1 1 The German University in Cairo, Faculty of Pharmacy and Biotechnology, New Cairo City, models.[9,10] We have identified anthranilic acid derivatives as excellent 11835 Cairo, Egypt lead structures for dual PPAR/FXR modulator development (pEC50 2 Institut für Pharmazie und Lebensmittelchemie, Universität Würzburg, Am Hubland, 97074 (PPAR) = 5.9, pEC50 (FXR) = 5.6).[11] Würzburg, Germany 3 Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, In the present study, we desire to yield nanomolar dual PPAR/FXR University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark agonists that will be evaluated in pre-clinical in vivo studies for their ability to cure NASH. The structure-activity relationship of the acidic head, the anthranilic acid core, and the lipophilic tail revealed several moieties The most pronounced pharmacological action of strychnine, the major boosting the potency independently for either PPAR (pEC50 up to 7.2) alkaloid from Strychnos nux vomica, is an antagonistic activity at glycine or FXR (pEC50 up to 6.6) and introducing selectivity over PPAR and

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PPAR. These structural motifs can now be recombined in one molecule investigate and possibly treat peripheral side effects on the heart and to come up with a balanced, nanomolar dual PPAR/FXR agonist with stomach. enhanced solubility and might evolve as a clinical candidate for NASH treatment. 1. Parsons, M.E. et al.; Agents Actions 1975, 5(5): 464. Acknowledgements: P. Heitel gratefully acknowledges financial support by the Else Kröner- 2. Durant, G. J. et al.; Nature 1978, 276(5686): 403-405. Fresenius-Stiftung, Translational Research Innovation - Pharma (TRIP). 3. Buschauer, A.; J. Med. Chem. 1989, 32(8): 1963-1970. 4. Kraus, A. et al.; ChemMedChem 2009, 4(2): 232-240. 5. Ghorai, P. et al.; J. Med. Chem. 2008, 51(22): 7193-7204. 6. Kagermeier, N. et al.; Bioorg. Med. Chem. 2015, 23(14): 3957−3969. References: 7. Pockes, S. et al.; ACS Omega 2018, 3(3): 2865-2882. 1. Weiß et al.: Dtsch. Arztebl. Int. 2014, 111(26): 447-452. 8. Pockes, S. et al.; ChemistryOpen 2019, 8(3): 285-297. 2. Kuipers et al.: Nat. Rev. Endocrinology 2014, 10(8): 448-498. 3. Düfer et al.: Diabetes 2012, 61(6): 1479-1489. 4. Fang et al.: Nat. Med. 2015, 21(2): 159-165. 5. Derosa et al.: J. Cell Physiol. 2018, 233: 153-161. POS.39 6. Mudaliar et al.: Gastroenterology 2013, 145(3): 547-582. Abstract withdrawn. 7. Neels, Grimaldi: Physiol. Rev. 2014, 94: 795-858. 8. Ratziu et al.: Gastroenterology 2016, 150: 1147-1159. 9. Hye Khan et al.: Biochem. Pharmacol. 2019, 166: 212-221. 10. Roth et al.: Sci. Rep. 2019, 9: 9046. POS.40 11. Merk et al.: Bioorg. Med. Chem. 2015, 23(3): 499-514. Pyridinylimidazole-based inhibitors of CNS kinases.

Pierre Koch1,2 POS.38 1Eberhard Karls Universität Tübingen, Institute of Pharmaceutical Sciences, Department of Medicinal Chemistry, Auf der Morgenstelle 8, 72076 Tübingen, Germany. Carbamoylguanidines as CNS-penetrating H2R agonists 2Department of Pharmaceutical/Medicinal Chemistry II, Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany and their physiological role in the brain: Potential drugs for neurodegenerative diseases? Protein kinases can be considered as the drug targets of 21st century. Since the approval of imatinib in 2001, more than 40 other protein kinase 1 S. Pockes inhibitors have been launched to the market. The main focus of 1University of Regensburg, Regensburg, 93053, Germany pharmaceutical research, however, is the development of protein kinase inhibitors for the treatment of cancer (> 85% of approved drugs). In the last four decades the development of histamine H2 receptor (H2R) ligands led to various classes of potent agonists. Coming from 3-(1H- Although there is strong evidence in the literature that inhibition of certain imidazol-4-yl)propylguanidine (SK&F 91486)1, a weak partial agonist, protein kinases expressed in the brain may result in neuroprotective imidazole-containing dialkylated guanidines like impromidine and effects and slow down the progression of the neurodegenerative disease, the development of CNS-penetrant kinase inhibitors has only been arpromidine turned out to be highly potent full agonists at the H2R.2,3 Beside their poor characteristics with respect to bioavailability, both promoted in recent years. Using rational approaches, we developed novel, potent, metabolically compounds showed up a lack of selectivity towards the H1,3,4R. 1-3 Bioisosteric exchange of the imidazole by 2-amino-4-methylthiazole stable (and brain penetrant) inhibitors of different CNS kinases based 4,5 eliminated the selectivity drawbacks, while amendment of the alkylated on our pyridinylimidazole-derived p38α MAP kinase inhibitor LN950 guanidine group to acylated guanidines was a successful approach to (Fig. 1). improve bioavailability and blood-brain barrier (BBB) penetration.4,5 As the acylguanidines showed up with stability problems due to hydrolytic cleavage further development to carbamoylguanidines brought highly stable, selective and potent H2R ligands.6

Figure 1: Structures of the carbamoylated guanidines targeting the histamine H2 receptor.

Most recently around one hundred compounds, including bivalent ligands, were synthesized in our group to further investigate structure- activity relationships for the histamine H2 receptor.7,8 In current projects we focussed on the development of CNS-penetrating H2R agonists (Figure 1), to get more insight about the physiological role of the H2R in the brain. Since the H2R is also described to be involved in the brain histaminergic neurotransmission and other central neurotransmitters (e.g. acetylcholine), H2R agonist could have similar effects to treat cognitive disorders (e.g. Alzheimer’s disease) as already reported H3R antagonists. The polarity of known H2R agonists is the major challenge in the search of candidates with suitable properties to cross the BBB, Fig. 1. Inhibitors of CNS kinases derived from lead compound LN950. while maintaining selectivity towards the H1,3,4R subtypes. Our approach resulted in UR-Po563, an enantiopure (ee > 99%) highly potent and selective agonist at the H2R (pKi (hH2R) = 7.60; pEC50 = 8.12, Emax = 0.95 References (gpH2R-atrium)), which is currently tested in vivo on several behavioral 1. Muth, F., El-Gokha, A. et al.: J. Med. Chem. 2017, 60, 594-607. models including anxiety-related tests at mice. As of now, preliminary 2. Ansideri, F. et al.: ACS Omega 2018, 3, 7809-7831. data exhibit very promising effects of UR-Po563 in the “Tail Suspension 3. Heider, F. et al.: Eur. J. Med. Chem. 2019, 157, 309-329. Test”. We are currently testing the abrogative effect of multiple 4. Koch, P. et al.: J. Med. Chem. 2009, 52, 72-95. 5. El-Gokha, A. et al.: Org. Biomol. Chem. 2015, 13, 10699-10704. antagonists (CNS-penetrant H1R and H2R antagonists) on the effects provided by UR-Po563. In addition, experiments are underway to

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POS.41 Despite the fact that the drug flupirtine has been on the market for many years (1986-2018), there is still incomplete understanding concerning its Basal Histamine H4 Receptor Activation: Agonist Mimicry metabolism. It was approved as a non-opioid analgesic with a unique by the Diphenylalanine Motif mechanism of action, namely the opening of voltage-gated potassium channels (Kv7.2/3) in the central nervous system. However, the reoccurrence of rare but severe liver injuries associated with the drug’s Wifling, D.1; Pfleger, C.3; Kaindl, J.2; Ibrahim, P.2; Kling, R. C.2; intake led to the recent withdrawal of flupirtine. It has been postulated Buschauer, A.1; Gohlke, H.3; Clark, T.2 that a quinone diimine, a reactive metabolite formed via oxidative 1 Institute of Pharmacy, Univ. Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany 1,2 2 Computer Chemistry Center, Univ. Erlangen-Nürnberg, Nägelsbachstraße 25, 91052 metabolism of flupirtine, is responsible for the hepatotoxicity. After oral Erlangen, Germany administration of flupirtine, the main metabolites found in human urine 3 Institute of Pharm./Med. Chemistry, Univ. Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, were the N-acetylated analog D13223, 4-fluorohippuric acid, mercapturic Germany acid derivatives and unchanged drug. For D13223 the metabolic pathway has been elucidated whereas the pathway for the biological inactive 4-

Histamine H4 receptor (H4R) orthologs exhibit species-dependent basal fluorohippuric acid is still unclear. We have initiated in vitro investigations (constitutive) activity: In contrast to mouse H4R (mH4R), human H4R by using HPLC to clarify if 4-fluorohippuric acid can be formed by the (hH4R) shows an extraordinarily high degree of basal activity. In a proposed metabolic pathway described below. First, the carbamate previous molecular-pharmacological study, the mutation F169V group of flupirtine (1) is cleaved by hepatic esterases. Next, facile significantly decreased the high basal activity of hH4R, and the basal oxidation of the product (2) to quinone diimines (3 and 4) is proposed. activities of the hH4R-F168A and hH4R-F169V+S179M mutants were Through hydrolysis of the imine (4), produced by tautomerization of even comparable to that of mH4R [1,2]. In contrast, the basal activity of quinone diimine, 4-fluorobenzaldehyde (5) could be formed. The latter is hH4R was maintained in the S179M mutation. Driven by such promising further oxidized by aldehyde dehydrogenase (ALDH), resulting in the results, we aimed at further investigating the molecular mechanism of formation of 4-fluorobenzoic acid (6), which can be enzymatically coupled basal H4R activation by performing 2 µs molecular-dynamics simulations with glycine to yield 4-fluorohippuric acid (7). This poster will present of six H4R variants (hH4R, hH4R variants S179M, F169V, F169V+S179M, results of these studies, which indicate that the proposed pathway is F168A, and mH4R), each revealing a different degree of basal activity. possible. Most interestingly, during the MD simulations, F169ECL2.55 dips into the orthosteric binding pocket only in the case of hH4R, thus adopting the role of an agonist and contributing to the stabilization of the active state. 3.32 Strikingly, the overall distances between the Cα atoms of D94 and Q3477.42, a measure of binding-pocket contraction, increased from hH4R to the S179M, F169V, F169V+S179M, F168A mutants, and were highest 3.50 for mH4R. Remarkably, the overall Cα-Cα distances between R112 and A2986.30, a measure of TM VI outward movement and GPCR activation, decreased in this order. Furthermore, based on the respective structural stabilities of TM VI, rigidity analysis was able to correctly predict the basal activities of the investigated H4R variants. Hence, these results, along with both information about additional motifs fundamental for GPCR activation and from rigidity analysis provide a molecular explanation for differential basal activities of H4R variants. Moreover, this study has provided novel insights into molecular mechanisms of basal H4R activation that are also of importance for other GPCRs.

1. Methling, K. et al.: Drug. Metab. Dispos. 2009, 37(3):479-493. 2. Siegmund, W. et al.: Br. J. Clin. Pharmacol. 2015, 79(3):501-513.

POS.43 HPLC-DAD-MS Stability Tests of Alkynylgold(I)(NHC) Complexes in Solution 1. D. Wifling, et al., Br. J. Pharmacol., 2015, 172: 785-798. 2. D. Wifling, et al., PLoS One, 2015, 10: e0117185. Hoffmeister, H.1; Ott, I.1 1 Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany

Gold organometallic compounds have been extensively investigated as POS.42 potential anticancer metallodrugs and have shown a high potential regarding antiproliferative effects [1,2,3,4]. Enhanced stability is a driving Investigations of the metabolic pathway of flupirtine argument for the design of gold(I)NHC based drugs, the more surprising metabolite 4-fluorohippuric acid in vitro by HPLC is the lack of methods for pharmaceutical analytics about their stability and solution chemistry. Such analytical methods are important key Beirow, K.; Jedamzik, J.; Bednarski, P. J. elements for future metabolomic investigations and will help to ensure a University of Greifswald, Department of Pharmaceutical/ Medicinal Chemistry, Friedrich-Ludwig- better understanding of their biological behavior [5]. Jahn-Str. 17, 17489 Greifswald, Germany We selected complexes of the type of alkynylgold(I)(NHC) for detailed stability studies by HPLC-DAD-MS, in comparison to the well-known antirheumatic agent Auranofin. A RP-based chromatographic method

101 • DPhG Annual Meeting 2019 Conference Book POSTERS was established to separate possible degradation products of alkynylgold(I)(NHC) complexes. The stability studies were performed at 37°C over 24h using dimethylformamide (DMF), dimethyl sulfoxide (DMSO), water and Dulbecco`s modified eagle medium (DMEM) solutions of each compound. Furthermore, interaction experiments of alkynylgold(I)(NHC) complexes with acetylcysteine are under way with the same set-up as the stability tests [6]. ESI (+) and (-) ionisation with a quadrupole analyser was used for mass spectrometry. The first results indicate that alkynylgold(I)(NHC) complexes are stable in the analysed solvents with no significant changes in their AUCs [Fig 1].

Figure 1: Co-crystal structure of ColH with an inhibitor [4]

Figure 1: Chromatogram of a selected complex in DMEM after 24h. The (un)functionalised R2 R3 R4 insert shows the ESI (+) mass spectrum of the main peak. side chain(s) N different SH R1 ZBGs 1. Hickey, J. L. et al.: J. Am. Chem. Soc. 2008, 130(38): 12570–12571. O 2. Andermark, V. et al.: J. Inorg. Biochem. 2016, 160: 140–148. 3. Meyer, A. et al.: Angew. Chem. Int. Ed. 2012, 51(35): 8895–8899. 4. Schmidt, C. et al.: Metallomics 2019, 11(3): 533–545. aryl linker 5. Kostiainen, R. et al.: J. Mass Spectrom. 2003, 38(4): 357–372. variations variations 6. Albert. A. et al.: J. Anal. At. Spectrom. 2012, 27(6): 975–981.

Figure 2: Structure of ColH inhibitors

POS.44 New pathoblockers: highly potent and selective inhibitors It is possible to dissect the hit compound into four parts (see Fig. 2): an aryl moiety, pointing into a ColH-specific binding pocket that is crucial for of Clostridial collagenases selectivity, a linker with possible side-chain attachments, and a zinc- binding group (ZBG), being pivotal for high binding affinity. According Voos, K.1; Kany, A. M.2; Schönauer, E.3; Haupenthal, J.2; Yahiaoui, S.2; synthetic variations have been performed to give a detailed structure- Brandstetter, H.3; Hartmann, R. W.2; Hirsch, A. H. K.2; Ducho, C.1 activity relationship (SAR), and first promising results from these studies 1Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2 3, 66123 will be presented. Saarbrücken, Germany 2Helmholtz Institute for Pharmaceutical Research Saarland, Department of Drug Design and Optimization, Campus E8 1, 66123 Saarbrücken, Germany References: 3Department of Molecular Biology, University of Salzburg, Billrothstr. 11, 5020 Salzburg, Austria 1. Silver, L. L.; Clin. Microbiol. Rev. 2011, 24(1): 71-109. 2. Clatworthy, A. E.; Pierson, E.; Hung, D. T.; Nat. Chem. Biol. 2007, 3(9): 541-548. 3. Eckhard et al.; J. Proteomics 2014, 100: 102-114. Microbial infections are still a constant threat to public health and since 4. Schönauer et al.; J. Am. Chem. Soc. 2017, 139(36): 12696-12703. daptomycin (a cyclic lipopeptide), which was first reported in 1987 and introduced as a drug in 2003, no new classes of antibiotics have been approved.[1] Even towards this new compound, first resistances POS.45 occurred just a few years after introduction onto the market, which shows the urgent need for more antibiotics with new modes of action.[2] Synthesis of muraymycin analogues with variations in the Virulence factors are known to play a major role in infections and are nucleoside moiety therefore an attractive new target. So-called ‘pathoblockers’ address those virulence factors instead of the survival factors of bacteria. As the Heib, A.1; Niro, G.1; Leyerer, K.1; Ducho, C.1 bacteria are not killed but “disarmed”, a pathoblocker exerts reduced 1 Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, selection pressure; accordingly, bacteria are then less prone to the Campus C.2.3, 66123 Saarbrücken, Germany development of resistances.[2] The extracellular zinc metalloprotease collagenase H (ColH) from the Muraymycins are a class of naturally occurring nucleoside antibiotics myonecrotic Clostridium histolyticum is such a prominent virulence factor showing promising antibacterial activity against many Gram-positive and for the progression of Clostridia-associated diseases. This protease few Gram-negative bacteria. [1,2] Their mode of action is based on the destroys the host’s connective tissue, thus leading to improved host inhibition of the bacterial enzyme MraY, thereby blocking an essential invasion and colonisation. Also, nutrition supply as well as the spread of step in the early stages of bacterial peptidoglycan synthesis. [1,2] With toxins into the damaged tissue are promoted.[3] Besides the major role respect to their structural complexity, simplified muraymycin analogues of ColH in disease development, its extracellular localisation makes it a were designed based on the results of SAR studies of the natural highly attractive target for the development of new antibiotics, as the products and semisynthetic derivatives. Their facilitated synthetic difficulties of penetrating the bacterial cell wall do not have to be taken accessibility allows further structural variations for more detailed SAR into account. insights. [1,3,4] In a screening of a focused protease inhibitor library, thiocarbamate- based compounds, that are hydrolytically cleavable prodrugs for thiols, O were found to be remarkably potent (low nanomolar range) on Clostridial R1 collagenases while still being highly selective over related human matrix HO O NH 1 2 O O N O reference: R = H R = OH metalloproteases (MMPs). An X-ray co-crystal structure of the hit H H 1 2 N N HN N O 1: R = F R = OH compound with the enzyme (see Fig. 1) clarifies this selectivity by giving HO N H H 2: R1 = CH R2 = H O O 3 further insights into the compound’s binding mode and serves as a OH R2 3: R1 = H R2 = H starting point for further optimisation.[4] NH2

DPhG Annual Meeting 2019 Conference Book • 102 MEDICINAL CHEMISTRY AND DRUG DESIGN

5’-defunctionalized muraymycin analogues with variations in the nucleoside moiety were synthesized and evaluated for their in vitro inhibition of MraY. A 5’-defunctionalized muraymycin analogue simplified in the urea dipeptide moiety, that had been previously synthesized in our group, was used as reference compound. [4] The structure of the nucleobase was then modified via synthesis of the 5-fluorouridine-based derivative 1 (see figure), thereby altering the electron density of the heteroaromatic system. The thymidine-containing analogue 2 was synthesized to evaluate the effect of increased steric demand of the nucleobase. To investigate the influence of the 2’-hydroxy group, the 2’- deoxy analogue 3 was prepared. The synthetic route followed a modular approach. The synthesis of the 5’-deoxy nucleosyl amino-acid building blocks had previously been developed in our group [5] and was adjusted for the synthesis of the modified analogues. The urea dipeptide building block was prepared via a solid phase-supported method. Evaluation of in [1] Courvalin P: Clin Infect Dis 2006; 42(1): 25-34. vitro MraY inhibition was performed via a fluorescence-based assay. [6,7] [2] Okano A, Isley NA, Boger DL: J. Am. Chem. Soc 2011; 133(35): 13946-13949. The synthetic work and results for in vitro MraY inhibition of 1-3 will be presented.

References: POS.47 1. Wiegmann, D. et al.: Beilstein J. Org. Chem. 2016, 12: 769-795. Hepatitis B and D: The importance of accessory domains in 2. McDonald, L. et al.: J. Am. Chem. Soc. 2002, 124(35): 10260-10261. 3. Spork, A. P. et al.: Chem. Eur. J. 2014, 20(47): 15292-15297. peptidic entry-inhibitors 4. Spork, A. P. et al.: Molecules 2018, 23: 2868. 5. Spork, A. P. et al.: J. Org. Chem. 2011, 76(24): 10083-10098. 1 1 2 6. Wohnig, S. et al.: Chem. Eur. J. 2016, 22(49): 17813-17819. Andreas Krause , Dennis Friedel , Katrin Schöneweis , Franziska 7. Koppermann, S. et al.: ChemMedChem. 2018, 13(8): 779-784. Schlund2, Uwe Haberkorn1, Stephan Urban2, Walter Mier1 1Department of Nuclear Medicine, Heidelberg University Hospital, Heidelberg, Germany; 2Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Heidelberg, Germany

POS.46 Today, approximately 15 million chronic hepatitis B (HBV) patients are Cationic peptide conjugation to vancomycin leads to superinfected with the hepatitis D virus (HDV) and have no specific improved pharmacokinetics and overcomes all types of treatment options [1]. As HDV is the most aggressive form of viral vancomycin resistance hepatitis, there is a high need for novel drugs. Since the discovery of the entry receptor (NTCP) of HBV/HDV, the transporter has become a major target for a whole group of upcoming F. Umstätter1, C. Domhan2, P. Uhl1, T. Hertlein3, B. Beijer1, C. Kleist1, E. drug candidates. The most prominent one is Myrcludex B, a peptide Mühlberg1, K. Leotta1, S. Zimmermann4, K.D. Klika5, M. Wink2, U. consisting of 47 amino acids and a myristoylated N-terminus. The amino Haberkorn1, K. Ohlsen3, W. Mier1 acids 9-15 (NPLGFFP) of Myrcludex B represent the essential inhibitory 1Department of Nuclear Medicine, Heidelberg University Hospital, Germany; 2Institute of sequence. The amino acids between position 30 and the C-terminus form Pharmacy and Molecular Biotechnology, Heidelberg University, Germany; 3Institute for an accessory domain that contributes to the binding efficiency [2]. While Molecular Infection Biology, University of Würzburg, Germany; 4Department of Infectious Diseases, Heidelberg University Hospital, Germany; 5Core Facility Molecular Structure Analysis, major changes in the essential site lead to abrogation of the inhibitory German Cancer Research Center, Heidelberg, Germany capability of the peptide [2], the exact role of the accessory domain is yet to be fully determined. In this study derivatives of Myrcludex B were synthesized with solid The increasing number of drug resistant bacterial strains is a major phase peptide synthesis employing the Fmoc/tBu strategy and tested for problem in modern medicine. It is perceived by the WHO in a list of “high HDV infection inhibition in cell culture models. The derivatives have priority pathogens” such as vancomycin-resistant enterococci or alanine exchanges in distinct positions: replacement of positions 32 and staphylococci. The limitation of the treatment of those pathogens by 33, positions 32-37 and 16-21 in combination with positions 34-39. All common drugs is a tremendous problem in clinics today. The resistance derivatives show profoundly reduced inhibitory efficacy against HDV against vancomycin can be differed in three main types in accordance infection. with their resistance gene called vanA-, vanB- or vanC-type [1]. The To further analyze the binding affinity of the derivative with alanine vancomycin-resistance can be explained by mechanisms that are related substitutions in positions 16-21 and 34-39 in comparison to Myrcludex B, to the cell wall components. Resistant bacteria change the binding both peptides were labelled with the fluorescent dye atto565 via an NHS- epitope resulting in a massive loss in affinity of vancomycin. Strategies ester to a lysine side chain. The alanine derivative showed a significantly of modifying vancomycin specifically for this structural change were weaker and unspecific binding, which could be confirmed in a FACS successful but chemically extremely complex [2]. assay. In this study we were able to overcome all common vancomycin- In conclusion, it could be shown that changes in the accessory domain resistances by a new technology of conjugation of cationic peptides to can result in severe reduction of inhibitory efficacy and binding affinity. vancomycin (Fig. 1A). The conjugates were not only able to overcome the resistance by an up to 1000-fold decrease of the Minimal Inhibitory 1. WHO: Hepatitis D Fact Sheet 2018, [cited 2019 22.02.]; Available from: Concentration but also attain improved pharmacokinetics in comparison http://www.who.int/mediacentre/factsheets/hepatitis-d/en/. to vancomycin as they redirect the nephrotoxic vancomycin to the liver. 2. Schulze, A., et al.: J Virol 2010, 84(4): 1989-2000. FU002, the actual lead candidate furthermore shows no cytotoxic effect in vitro and in vivo. The good in vivo tolerability and the good in vitro activity allowed testing the compound in a murine infection model. The drugability was proven by a significant reduction of colony-forming units POS.48 of a vancomycin-sensitive Staphylococcus aureus strain in the liver (Fig. Amino Acid-based allosteric Inhibitors of Zika- and 1B). Dengue-Virus NS2B/NS3-Proteases

Millies, B.1, Immerheiser, M.2, Zimmer, C.1, Kersten, C.1, Bodem, J.2, Hellmich, U. A.1, Schirmeister, T.1 1Johannes Gutenberg-University Mainz, Institute of Pharmacy und Biochemistry, Staudinger Weg 5, 55128 Mainz, Germany 2Julius-Maximilians-University Würzburg, Institute for Virology and Immunobiology, Versbacher Straße 7, 97078 Würzburg, Germany

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H N N O The potencies of the investigated D2 receptor agonists (e.g. dopamine

HO S O and pramipexole), determined in the DMR assay, were higher compared

HO OH to D2 receptor affinities obtained from competition binding studies with 3 O [ H]N-methylspiperone at genetically engineered HEK293T cells. O Inhibition of the Gi-dependent pathway with pertussis toxin led to a O N O O marked decrease in the DMR signal obtained upon quinpirole-mediated S N 2 O D receptor activation. This suggested that the holistic response, O O S measured by DMR, is mainly mediated by Gi/o and not by Gs or Gq. HN O O R Inhibition of the interaction of Gβγ-subunits with its downstream effectors HO S AA NH O O O by pre-treatment of the cells with gallein resulted only in a slight decrease NH S HN HO N NH in the DMR response compared to the control experiment in the absence O O of gallein indicating a minor contribution of Gβγ-subunits to the signal O O HN OH S detected by DMR. O O O S O HN O O O O Acknowledgments: The authors thank Harald Hübner and Peter Gmeiner (Friedrich Alexander University, Erlangen, Germany) for providing CHO-K1-hD2longR cells. HN N OH HN N Since the 1950s, the Zika-virus spread through Africa and South Asia, References: but during the last years, it emerged into the Pacific region and came to 1. Grandy, D. K. et al.: Proc. Natl. Acad. Sci. U S A 1989, 86: 9762-9766. the Americas. After being transmitted by mosquitoes like Aedes †Died July 18, 2017 albopticus, the virus releases its positive, single-stranded RNA inside the cell, from which a polyprotein is translated containing the whole virus proteome. To cleave this polyprotein into its functional parts the viral NS2B/NS3-protease is essential. Therefore, this protease is a valid target POS.50 for the development of drugs.[1] Elucidation of the Mechanism of Action of allosteric In previous work we developed inhibitors of the Dengue virus NS2B/NS3- protease.[2] Due to the high sequence and structure similarity of flaviviral Dengue-and Zika-Virus NS2B/NS3 Protease Inhibitors proteases these inhibitors also display similar affinities to the Zika virus 1 1 1 2 protease. The inhibitors show a non-competitive behaviour at both Hammerschmid, S. ; Millies, B. ; Kersten, C. F. ; Hellmich, U. A. ; 1 targets, but unfortunately lack sufficient water solubility. To obtain Schirmeister, T. 1Johannes Gutenberg-University Mainz, Institute of Pharmacy and Biochemistry, Staudinger inhibitors with better logD-values while keeping the overall compound Weg 5, 55128 Mainz, Germany geometry, the ortho-substituted aromatic ring system was exchanged to 2Johannes Gutenberg-University Mainz, Institute of Pharmacy and Biochemistry, Johann- a proline residue and other proteinogenic and non-proteinogenic amino Joachim Becher Weg 30, 55128 Mainz, Germany acids. This led to a new and potent generation of inhibitors of flaviviral proteases. Structure-activity relationship was explored and inhibitors with In recent years, the Dengue virus, a mosquito born member of the further structural variations were synthesized. The compounds were also flaviviridae family, has emerged a serious health issue as its main tested in cell-based assays, revealing cell-permeable prodrugs. vectors, namely Aedes albopticus and Aedes aegypti have spread due Metabolism and metabolites of these prodrugs were studied in in-vitro to climate change and increasing globalisation from their tropical origins assays supported by LC-MS. to more tempered climate zones such as Mediterranean and even middle European countries. [1,2,3] The Dengue virus is a positive, single

stranded sense RNA virus, whose genome consists of three structural

References: (capsid, membrane precursor, envelope) and seven non-structural (NS-) 1. Kang, C., Keller, T.H., Luo, D., Trends Microbiol. 2017, 25, 797-808. proteins. All of them are expressed as one precursor polyprotein. [4] For 2. Wu, H. et al., Antimicrob. Agents. Ch. 2015, 59, 1100-1109. virus maturation, correct processing of the polyprotein to the functional proteins is crucial. This is conducted by the hosts proteases signalase and furase and by the viral NS2B/NS3 protease complex (figure 1). [4] Inhibition of this protease suppresses viral maturation and leads to loss of pathogeny. [5] The NS2B/NS3 protease comprises two different POS.49 conformations: an inactive “open” and an active “closed” form where the Dopamine D2 receptor signaling: toward deconvolution of NS2B cofactor domain is wrapped around NS3 and partially forms the S3 the holistic readout obtained by dynamic mass pocket of the active site. [6] redistribution

L. Forster, A. Buschauer†, G. Bernhardt Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany

Dopamine receptors, belonging to the super family of G-protein coupled receptors (GPCRs), are classified according to their preferred G-protein coupling, i.e. the Gs-coupled receptors D1 and D5 (D1 family) and the Gi- coupled subtypes D2, D3 and D4 (D2 family). Since the first cloning of the human D2short and D2long receptor in 1989 [1], various conventional functional assays, such as cAMP and arrestin assays, as well as label- free ‘non-invasive’ methods (dynamic mass redistribution (DMR)) have Figure 1: Transmembrane precursor polyprotein encoded by the Dengue virus. Black and red arrows indicate cleavage sites for the hosts been used for the investigation of D2 receptor-mediated cellular signaling. However, it is still unknown to which extent distinct signaling pathways proteases and the NS2B/NS3 protease, respectively. contribute to the holistic response (cytoskeletal and cell morphological changes) measured by DMR. Previously, potent allosteric inhibitors of the NS2B/NS3 proteases were developed. [7] While molecular docking studies indicate binding in the Aiming at a deconvolution of D2 receptor-mediated holistic responses, we open conformation of the protease, the detailed mechanism of action applied the DMR technique to the human dopamine D2long receptor using genetically engineered CHO-K1 cells stably expressing the long isoform remains largely unresolved. Therefore, we applied various methods to achieve deeper insights into of the dopamine D2 receptor. The contribution of individual signaling pathways to the optical DMR signals was explored by measuring DMR the mechanism of inhibition. In contrast to our enzymatic cleavage assay traces upon agonist stimulation in the absence and in the presence of and MST measurements which showed low micromolar IC50 and KD pharmacological tools accounting for a specific silencing of signaling values, ITC experiments did not show enthalpy-driven binding. MST measurements and DRX2 SwitchSENSE sizing experiments indicated pathways (e.g. pertussis toxin: inhibition of Gi; gallein: inhibition of Gβγ). conformational changes upon inhibitor binding. These findings point to a

DPhG Annual Meeting 2019 Conference Book • 104 MEDICINAL CHEMISTRY AND DRUG DESIGN mostly entropic-driven binding of the inhibitors. Herein we report different the rapid emergence of resistance against existing treatments approaches based on cysteine conformational traps, DRX2 demonstrates the necessity for novel therapeutic approaches. [1,2] thermodynamic measurements and competitive ITC experiments to In previous publications and preliminary work, it was shown, that overcome these – so far – apparent contradictory results. transient systemic MYC inhibition serves as a suitable treatment option for HCC. [3] However, due to its lack of binding cavities MYC cannot be targeted directly by small molecules. By using a direct in vivo shRNA 1 Berg, H. van den, Velayudhan, R, Ejov, M.: WHO Regional Office for Europe 2013. screen, recent findings showed that liver cancer cells bearing mutations 2 WHO Regional Office for Europe: Fact sheet 2013, 117. 3 Wilder-Smith, A., Schwartz, E.: New Engl. J. Med. 2005, 353: 924–932. in the gene encoding tumor suppressor protein p53 (Trp53 in mice and 4 Lindenbach, BD., Thiel, H-J., Rice, CM..: Fields virology 2007, 5th ed, vol 1: 1101-1152. TP53 in humans) and that are driven by the oncoprotein NRAS become 5 Kang, C., Keller, T. H., Luo, D.: Trends Microbiol 2017, 25: 797. addicted to MYC stabilization via a mechanism mediated by aurora 6 Kim, Y. M. et al.: J Biol Chem 2013, 288(18): 12891–12900. kinase A (AURKA). [4] Therefore, an indirect MYC targeting becomes 7 Wu, H. et al.: Antimicrob Agents Ch 2015, 59: 1100. possible. Our work focusses on the design and synthesis of small molecules that prevent MYC/AURKA interaction by inducing a conformational shift in the kinase. In silico predictions propose that the crucial interaction for POS.51 complex formation is located in close proximity to the kinases hydrophobic spine. Hence we make use of the already in our group The Novel Histone Methyltransferase KMT9: an Assay successfully applied concept of type I ½ inhibitors. Guided by in silico Platform for Inhibitor Screening approaches and further mechanistic studies we aim to develop tool compounds providing structural features that are able to prevent MYC/AURKA complex formation and ultimately degrade MYC. Bacher, J.1; Wang, S.2; Barthes N.P.F.1; Metzger E.2; Schüle, R.2; Jung, M.1; 1 Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstr. 25, 79104 1. Ferlay et al. (2015), Cancer incidence and mortality worldwide: sources, methods and major Freiburg, Germany patterns in GLOBOCAN 2012, Int J Cancer, 136(5), E359-386. 2 Department of Urology, Center for Clinical Research, University Freiburg Medical Center, 2. Llovet et al. (2008), Sorafenib in advanced hepatocellular carcinoma, N Engl J Med, 359(4), Breisacherstr. 66, 79106 Freiburg, Germany 378-390. 3. Soucek et al., (2013), Inhibition of Myc family proteins eradicates KRas-driven lung cancer in Epigenetic marks established on histone tails by specific enzymes are mice. Genes Dev, 27(5), 504-513. 4. Dauch et al. (2016), A MYC–aurora kinase A protein complex represents an actionable drug important regulators of gene expression. There are three classes of these target in p53-altered liver cancer, Nat. Med., 22, 744-753. so-called epigenetic proteins, firstly writer enzymes setting a specific mark, secondly erasers which are removing it and thirdly reader proteins, which are mediating a specific effect based on the post-translational modification. Recently the spectrum of histone modifying enzymes was expanded by the discovery of the novel, heterodimeric seven-β-strand POS.53 histone methyltransferase KMT9, which is capable of monomethylating Alkynylgold(I) NHC Complexes as Thioredoxin Reductase 1 lysine 12 of histone H4 (H4K12me1) . The H4K12me1 mark is mostly Inhibiting Potential Antitumor Agents located at promoter sites of cell cycle regulating genes. Prostate cancer cells, both hormone-sensitive and castration-resistant, are sensitive to the depletion of KMT9 in-vitro as well as in xenograft models, resulting in Andre Prause1,2; Ingo Ott1,2 severely reduced proliferation. These findings make KMT9 a very 1 Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, promising drug-target for prostate cancer treatment, especially for multi- Beethovenstraße 55, 38106 Braunschweig, Germany. 2 Center of Pharmaceutical Engineering (PVZ), Technische Universität Baunschweig, Franz- drug and castration-resistant tumors. Liszt-Straße 35a, 38106 Braunschweig, Germany. In order to be able to screen for potential KMT9 inhibitors, an assay platform consisting of biochemical activity- and binding-assays, as well as biophysical methods, was established in our lab. To assess the In consequence of spread and severity of cancer there is need for novel inhibitory activity of the compounds, the platform includes a radioactivity- drugs. Thioredoxin reductase (TrxR) has been identified as a potential based filter-binding assay, which is used for initial compound screening. molecular target for the treatment of cancer. With its chemical property Furthermore, the hits retrieved in this assay are followed up in a as a soft Lewis acid, gold(I) has a high affinity to thiols and selenols, biophysical thermal shift assay to determine potential protein-stabilizing which are present in the active site of TrxR. [1,2] properties of the compounds. Because of a unique binding mode of Inspired by the disease-modifying antirheumatic drug Auranofin, several KMT9 to its substrate, it was also possible to establish an AlphaLISA® types of ligands have been used to form gold(I) complexes as TrxR based binding assay. This assay is based upon depletion of the histone inhibitors [2]. Beside halide, thiolate and phosphane based ligands, the substrate by potential inhibitors. Sinefungin as well as the natural focus has more recently shifted towards organometallic complexes due byproduct SAH were used to validate the assay platform. to their higher chemical stability. Recent published results show that alkynes and N-heterocyclic carbenes (NHCs) as ligands to gold(I) display both described effects (forming stable complexes and good activity We thank the DFG (SFB992-MEDEP) for funding. against TrxR). [1-3] NHC-gold(I) complexes demonstrated their potential as strong inhibitors 1. Metzger, E. et al.: Nat. Struct. Mol. Biol. 2019, 26(5): 361-71. of TrxR in combination with cytotoxic activity against several tumor cell lines, such as MCF-7 (breast adenocarcinoma) and HT-29 (colon adenocarcinoma) cells [3,4]. Alkynyl-gold(I)-phosphanes have shown a similar strong activity against the same cell lines with additional anti- POS.52 angiogenetic effects in zebrafish embryos [5]. Indirect MYC targeting via conformation changing Aurora A synthesis and characterization procedure for complexes with an alkynyl and a NHC ligand was developed [Figure 1]. Ongoing biological tests Kinase A inhibitors deal with the cytotoxicity and TrxR inhibition of this type of gold organometallics. First experiments confirmed strong cytotoxicity against 1 1 2 3 2,3 Flötgen, D. and Reiner, J. ; Henning, M. ; Pantsar, T. ; Poso, A. ; several tumor cell lines and efficient inhibition of TrxR. The current results 2 1 Zender, L. ; Laufer, S. of this ongoing project will be presented on the poster. 1 Department of Pharmaceutical Chemistry, University of Tuebingen, 72076 Tuebingen, Germany. 2 Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, University of Tuebingen, Tuebingen, Germany 3 School of Pharmacy, University of Eastern Finland, Kuopio, Finland.

Hepatocellular Carcinoma (HCC) – the second most frequent cause of Figure 1: Synthesis procedure for an example of an alkynyl-gold(I)- cancer-related death - has a great need for better therapeutic strategies NHC-complex. a) alkylation with ethyliodide, b) reaction with Ag2O and since the treatment options for advanced HCC are limited. Furthermore, transmetallation with SMe2AuCl, c) reaction with phenylacetylene under basic conditions

105 • DPhG Annual Meeting 2019 Conference Book POSTERS

References: Since the approval of cisplatin in 1978 by the U.S. FDA, relatively few 1. Cheng, Y. et al.: Anti-Cancer Agents in Medical Chemistry, 2017, 17: 1046-1069. platinum complexes have been approved for the treatment of cancer 2. Cinellu, M. A. et al.: Bioorganometallic Chemistry, 2015: 117-139. worldwide. Thus, the design of new Pt cytostatics is a challenging task 3. Rubbiani, R. et al.: Journal of Medicinal Chemistry, 2010, 53: 8608-8618. for medicinal chemists. On the one hand many types of cancers are not 4. Schmidt, C. et al.: Chemistry a European Journal, 2017, 23: 1869-1880. 5. Meyer, A. et al.: Angewandte Chemie Int. Ed., 2012, 124: 9025-9030. sensitive to Pt anticancer agents (e.g. breast cancer) while on the other hand treatment with cisplatin often leads to acquired resistant, which can be cross-resistance to other platinum complex e.g. Carboplatin. Over the years it has become clear that the nature of the diamine ligand strongly

influences both the innate selectivity towards different types of cancer as POS.54 well as the development of resistance to Pt(II) complexes. Surprisingly, Novel Synthetic Pentathiepins Mediate the Inhibition of the biological activities of the few known Pt(II) complexes with the 2,2´- diaminobiphenyl ligands had never been tested. These ligands are of GPx1, Cell Viability, Cell Proliferation and Increase interest due to the possibility of atropisomerism when the phenyl rings Intracellular ROS Levels are further substituted in the 6 positions with bulky groups. In our previous work we studied the cytotoxic effects of (R)-(+)- and (S)- Wolff L., Bandaru S., Napierkowski M., Schulzke C., Bednarski P.J., (-)-DABN-PtCl2 (fig. 1) on human cancer cells and investigated their 1 Institute of Pharmacy, University of Greifswald, F.-L.-Jahn-Str. 17, 17489 Greifswald, cellular uptake and the DNA binding. We found that the (R)- Pt(II) Germany complex is taken up by cells better than the (S)- Pt(II) complex but that 2 Institute of Biochemistry, University of Greifswald, F.-Hausdorff-Str. 4, 17489 Greifswald, the (S)- Pt(II) complex is more potent as a cytotoxic agent. In addition, Germany both complexes had greater potency than cisplatin on human cancer cells[1]. The discovery of the natural product pentathiepin Varacin brought the focus of research onto the wide range of biological activity that these interesting compounds possess, including antifungal, cytotoxic and DNA- damaging activity [1, 2]. Studies suggest that these properties are mainly mediated by the pentathiepin ring system and the generation of reactive oxygen species (ROS) in the presence of thiols [3, 4]. Interestingly, we have recently discovered that some synthetic pentathiepins are potent inhibitors of the glutathione peroxidase 1 (GPx1) [5], an important enzyme for maintaining the intracellular redox balance. In this project, we analysed five novel pentathiepins with regards to their potential to inhibit the bovine erythrocyte GPx1 as well as the viability and proliferation of Fig. 1: Structures of (R)-(+)- and (S)-(-)-DABN-PtCl2 nine different human cancer cell lines in vitro. In selected cell lines the influence of the compounds on the intracellular ROS level was evaluated. On the basis of this previous work, we have synthesised seven new Chemical reactions between various pentathiepins and thiols were also platinum(II) complexes with various 6,6’-disubstituted, 2,2´-diamino- investigated. To address these aims, enzyme- and cell-based microplate biphenyl ligands (fig. 2) and studied the cytotoxic effect on four different assays were performed, half maximal inhibitory concentrations (IC50) human cancer cell lines. A cisplatin resistant cell line was also used to calculated and the ROS sensor H2-DCFDA analysed via flow cytometry. evaluate if there is any cross-resistance of the new complexes with All tested pentathiepins were potent and specific GPx1 inhibitors, as well cisplatin. Due to their structural similarity we further investigated for SAR, as capable of inhibiting the viability and proliferation of all nine cancer cell which could be useful for further analogue design and a rational drug lines at very low micromolar concentrations. Moreover, four out of five design. compounds significantly increased the intracellular ROS levels. Differences between the IC50 values of the pentathiepins and their varying capabilities to generate ROS suggest that not only the pentathiepin ring mediates these biological effects but also that the distinct ring substituents can have a modulating influence.

X = C, N Fig. 2: General structure of the tested Pt(II) complexes

References: 1. Sørensen B.H., Werth P., Lambert I.H., Bednarski P.J.: Metallomics, 2018, 10, 323-326 Varacin tested structures

References: 1. Chatterji, T. and Gates, K. S.: Bioorg Med Chem Lett. 1998, 8(5): 535-538. 2. Mahendran, A. et al.: Chem. Phys. Lipids. 2016, 194: 165-170. POS.56 3. Greer, A.: J Am Chem Soc. 2001, 123(42): 10379-10386. Synthesis of new folate analogues for prostate cancer 4. Chatterji, T. and Gates, K. S.: Bioorg Med Chem Lett. 2003, 13(7): 1349-1352. 5. Behnisch, S.: Dissertation. University of Greifswald. 2017 diagnosis

Chandralingam S.1, Peric N.1, Frangioni J.2, Maison W.1 1 Pharmaceutical and Medicinal Chemistry, University of Hamburg, Bundesstraße 45, 20146 Hamburg, Germany POS.55 2Memorial Sloan Kettering Cancer Center, 1275 York Avenue, 10065 New York, United States Synthesis and biological evaluation of platinum(II) complexes with 2,2´-diaminobiphenyl ligands Folates are essential cofactors in the de novo biosynthesis of pyridine and pyrimidine [1]. Moreover, antifolates are key components in cancer Werth P.; Wulf, J.; Bednarski P.J. therapy [2]. Targeting tumor specific cell surface epitopes (tumor Institute of Pharmacy, University Greifswald, Friedrich-Ludwig-Jahn-Straße 17, 17489 markers) with small molecules can lead to improved tools for cancer Greifswald, Germany diagnosis and therapy. Elevated levels of prostate specific membrane antigen (PSMA) are used as a tumor marker for prostate cancer [3].

DPhG Annual Meeting 2019 Conference Book • 106 MEDICINAL CHEMISTRY AND DRUG DESIGN

PSMA is a glycosylated type-II membrane protein that is present in high leads to accumulation of misfolded proteins resulting in apoptosis of fast- density on the surface of malignant prostate cancer cells. Its expression growing malignant cells.[1] In consequence, the combination of increases with clinical stage, thus making it an extremely useful tumor panobinostat, a non-selective HDAC inhibitor (HDACi), and bortezomib, marker [4]. Pseudopeptides like GPI can be used as modular ligands for a proteasome inhibitor (PI), has already been approved for the treatment the targeting of prostate cancer [5]. GPI binds with nanomolar affinity to of multiple myeloma while further combinations of HDACi and PI are PSMA and permits conjugation of effector molecules (e.g. fluorophores, currently being investigated at different clinical stages. chelates) without altering the binding properties [6]. However, GPI has Due to the distinct shapes of the enzymatic targets, HDAC6 inhibitors suboptimal binding properties in vivo and needs to be improved for and proteasome inhibitors also qualify for the polypharmacological imaging applications in animals or humans. GPI has been developed as approach that uses one drug to interact with multiple targets. In 2018, our a transition state analogue of the native PSMA substrate N-acetyl- group reported the discovery of RTS-V5 as the first-in-class dual HDACi aspartylglutamate (NAAG). In addition, PSMA has been found to act as and PI.[3] The binding modes of RTS-V5 were confirmed by X-ray crystal a folate hydrolase and does thus recognize folylpolyglutamates in the structures of RTS-V5 in complex with both HDAC6 and the 20S same binding pocket as NAAG [7]. We have combined properties of the proteasome. Additional biological assays further indicated blockage of known ligand GPI with structural elements of folates to design improved the cell cycle, proliferation, colony formation and aggresome modular PSMA ligands. These ligands should be attached with a accumulation as well as the induction of apoptosis, thus proving the conjugation site for effector molecules (in our case DOTA). We have anticipated synergistic effect resulting from the inhibition of both targets. established an interesting compound library with which we would like to In comparison to the HDAC6-preferential drug candidate ricolinostat, evaluate the impact of different aromatic and hydrophobic moieties as RTS-V5 also showed promising anticancer activities against selected pteridine analogues, their spacing from GPI and the role of geometry hematological cancer cell lines as well as therapy refractory primary aromaticity of the spacer via in vitro assays using LNCaP (PSMA patient-derived leukemia cells.[3] positive) and PC3 (PSMA negative) cell lines [8]. In summary, these initial results suggest RTS-V5 as a good candidate for further optimisation. However, it became clear that the HDAC6 inhibitory modular ligand activity needed to be increased. In this study, we therefore focused on modifying the HDACi part of the drug scaffold (Figure 1) by introducing tumour cell different linker moieties and zinc binding groups. As a result, we present new RTS-V5 analogues with increased inhibitory activities against HDAC6. cell specific receptor effector molecule O H O N OH COOH O N NH HN O OH N N NH2 O H  O N P OH Folic acid O OH OH O COOH O OH * H2N P OH O O OH N HetAr conjugation site n GPI O OH H O N OH N O H O Design of improved PSMA ligand O HO NAAG

Acknowledgments: We want to gratefully acknowledge the financial support from the NIH (PCQ5 grant). Figure 1. RTS-V5. the first-in-class dual HDAC and proteasome inhibitor.

References: 1. Stokstad E.L.R., Wiley-Liss: New York, 1990, 13: 1-21. References 2. Visentin M., Zhao R., Goldman I.D., Hematol. Oncol. Clin. North Am. 2012, 26(3): 629−648. 1 Suraweera, A. et al.: Front. Oncol. 2018, 8, 92. 3. Hilgenfeld R. et al.: EMBO J. 2006, 25(6): 1375-1384. 2 Witt, O. et al.: Cancer Lett. 2009, 277(1): 8−21. 4. Heston W. D. et al.: Urology 2001, 57(6): 1179-1113. 3 Bhatia, S. et al.: J. Med. Chem. 2018, 61(22), 10299-10399. 5. Coward J.K. et al.: J. Org. Chem. 2005, 70(17): 6757-6774. 6. Maison W. et al.: J. Med. Chem. 2009, 52(2): 544-550. 7. Carter, R. E., Feldman, A. R., Coyle, J. T.: PNAS 1996, 93(2): 749-753. 8. Benesova M. et al: J. Med. Chem. 2016, 59: 1761-1775.

POS.58

Balancing HDAC Inhibition and Drug-likeness - Biological and Physicochemical Evaluation of Class I Selective POS.57 Histone Deacetylase Inhibitors Design and synthesis of dual histone deacetylase- proteasome inhibitors Schäker-Hübner, L.1; Hansen, F.K.1 1Institut für Pharmazie, Medizinische Fakultät, Universität Leipzig, Brüderstraße 34, D-04103 Reßing, N. 1; Sönnichsen, M.2; Schöler, A. 1; Borkhardt, A2.; Hauer, J. 2, Leipzig, Germany 3; Bhatia, S. 2; Hansen, F. K. 1 1Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Medical Faculty, Leipzig University, Brüderstraße 34, 04103 Leipzig, Epigenetic modifications and their crucial role in the regulation of gene Germany expression have been widely studied and many enzymes have been 2Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, identified that interact with the chromatin structure. For instance, via Heinrich Heine University Düsseldorf, Moorenstrasse 5, 40225 Düsseldorf, Germany histone lysine acetylation and deacetylation epigenetic marks are either 3Department of Pediatric Oncology and Hematology, Medical Faculty, University Hospital Carl attached resulting in open, transcriptionally active chromatin or removed Gustav Carus Dresden, Fetscherstraße 74, 01307 Dresden, Germany resulting in condensed, transcriptionally repressed chromatin. [1] Histone deacetylases (HDACs) remove acetyl groups from histone and non- Among other epigenetic enzymes, histone deacetylases (HDACs) have histone proteins and hereby influence cell cycle progression, cell been established as valuable drug targets for single-target and proliferation and differentiation. HDACs are over-expressed in several combination therapies against non-solid cancers.[3] Apart from their cancer types and inhibition of HDAC function can result in various anti- potential to modify the chromatin structure via histone modifications, cancer effects. [1] As a consequence, some HDAC inhibitors (HDACi) several of the eleven HDAC isoforms and particularly HDAC6 possess a like vorinostat, romidepsin, belinostat and panobinostat are now broader substrate spectrum, including α-tubulin and the chaperone established, FDA-approved anti-cancer drugs for the treatment of T-cell protein Hsp90.[2] HDAC6 further regulates the aggresome formation as lymphoma or multiple myeloma. the second major cellular protein degradation pathway alongside the 20S The HDAC family encompasses eleven zinc-dependent enzymes proteasome. Simultaneous blockage of both synergistic targets therefore (HDAC1-11) classified in 4 groups: class I, class IIa, class IIb, and class

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IV. Class I consists of the nuclear enzymes HDAC1-3 and HDAC8. [1] was shown in the NCI-60 DTP human tumor cell line screening (GI50/60: HDAC1 and HDAC2 are among others associated with prostate, gastric, 49 nM) and in in vitro and in vivo studies on gynecological tumors [3,4]. breast and hematopoietic cancers. [1,2] Additionally HDAC2 seems to Methods: The potential of P8-D6 treatment to reduce proliferation and be critically involved in cognitive processes like learning and memory [3] induce apoptosis in various established ovarian cancer cell lines, breast and HDAC3 was shown to play an important role in inflammation and cancer celllines and ex vivo primary cancer cells were analysed by neurodegenerative diseases. [4] From a structural point of view, the emission spectroscopy, flow cytometry and microscopy. For better differences within class I isoforms are mainly based on an internal cavity comparison, cells were treated with different concentrations of P8-D6 and the so called “foot-pocket”. Ortho-aminoanilides like tacedinaline (CI-994) standard therapeutics for 48h. Likewise, the effects on non-cancer cells are HDAC1-3 selective inhibitors. When the aminoanilide group were analysed and hepatotoxicity of P8-D6 was investigated. The additionally bears a bulky substituent such as phenyl ring to occupy the expression levels of topoisomerase I/II were measured by western blot. “foot-pocket”, the HDAC1/2 binding affinity and selectivity is strongly Fluorescence microscopy showed whether P8-D6 gets into the nucleus increased. [1] However, introducing bulky, lipophilic substituents to its target. Moreover, P8-D6 was tested in an ovarian cancer xenograft increases the molecular weight and lipophilicity considerably and mouse model as an in vivo study to determine the therapeutic efficiency. worsens solubility. As a consequence, HDACi that target the HDAC1/2 Results: The results indicate a significantly increase of apoptosis in foot-pocket are not necessarily drug-like substances. gynecological cancer cells by P8-D6 than its references. Non-cancer cells were slightly effected. No hepatotoxic effect in in vitro studies was seen. By staining, P8-D6 was detected in the nuclei. In the in vivo study P8-D6 showed promising results. Conclusion: P8-D6 has promising antitumor properties in in vitro studies on ovarian cancer and breast cancer. It has fewer side effects on normal cells than references and no hepatotoxic effect. P8-D6 is a strong and rapid inductor of apoptosis.

References: 1. Bray, F. et al.: A Cancer Journal for Clinicians. 2018, 68 (6):. 394–424 2. Robert Koch-Institut, RKI-Bib1. 2017 3. Meier C. et al.: ChemMedChem 2017, 12(5): 347-352 4. National Cancer Institute, NCI-60 DTP Human Tumor Cell Line Screen. http://dtp.nci.nih.gov/branches/btb/ivclsp.html.

POS.60 Synthesis of Gallic Acid Esters and Their Evaluation as Anticancer Drug Candidates

Figure 1: CI-994-like fragments were synthesized and their solubility, Heinz, C.1; Saifudin, A.2, Ritmaleni3, Purnomo, H.3, Kuswandi3, lipophilicity and HDAC inhibition was evaluated. Based on our results Holzgrabe, U.1 we selected drug-like candidates for further investigation. 1Institut für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg, Germany 2Faculty of Pharmacy, Muhammadiyah Surakarta University, Indonesia To balance the clear advantage of increased HDAC inhibitition and 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gadjah Mada University, selectivity via targeting the “foot-pocket” and the disadvantages of these Indonesia substances, we synthesized a small set of HDACi fragments closely related to CI-994 (Fig. 1) and evaluated their solubility, lipophilicity (log Gallic acid is a naturally occurring, non-toxic polyphenolic compound D7.4) and inhibition of selected HDAC isoforms. In this work, we report known to have various pharmacological activities. Besides antifungal, the structure-activity and structure-physicochemical properties antibacterial, antiulcer and other properties, gallic acid can prevent relationships of a series of novel class I selective ortho-aminoanilides. carcinogenesis by its antioxidant behaviour as well as induce apoptosis of tumour cells due to prooxidative properties, which increase oxidative Acknowledgments: Experimental support from A. Schöler is gratefully acknowledged. stress within the cell.1 Other antioxidants with anticancer properties can be found among References: phenolic structures such as eugenol, vanillin or carvacrol, but also [1] Roche, J.; Bertrand, P.: Eur. J. Med. Chem. 2016, 121: 451–483. molecules like cinnamaldehyde are known to prevent cancer and have a [2] Conte, M. et al.: Oncotarget 2015, 6(2), 886–901. potential for use in the chemotherapy of different cancer types.2 [3] Wagner, F. F. et al.: Chem. Sci. 2015, 6: 804–815. [4] Fangyuan, F.; Zwinderman, M. R. H.; Dekker, F. J.: Molecules 2018, 23(3): 551. With the intent to combine the beneficial properties of gallic acid with those of other (phenolic) anticancer agents, we aimed to combine gallic acid with each of the other compounds in one molecule. The different ester compounds were obtained in a five-step synthetic pathway3. The pharmacological properties of the gallic acid esters have been tested POS.59 in an MTT assay using HER-2 breast cancer cells showing promising IC50 Targeting gynaecological cancer with the new dual values. topoisomerase inhibitor P8-D6 HO

1,2 1 2 1 O O O Flörkemeier I. , Steinhauer T. N. , Bauerschlag D. O. , Clement B. O O O O 1 Pharmaceutical Institute, Department of Pharmaceutical and Medicinal Chemistry, Christian- Albrechts-University Kiel, Gutenbergstraße 76, 24118 Kiel, Germany O O OH 2 Department of Gynecology and Obstetrics, University Hospital Schleswig-Holstein, Campus O OH HO OH HO OH Kiel, Arnold-Heller-Straße 3, 24105 Kiel, Germany OH eugenol vanillin OH OH + HO OH O OH

Introduction: The development of innovative cytotoxic compounds with gallic acid suitable physicochemical properties has a high clinical need in cancer OH O O O O therapy. In gynecological oncology, breast cancer is the most common and ovarian cancer the most aggressive tumor [1, 2]. Resistance carvacrol cinnamaldehyde HO OH HO OH developments and severe side effects are the reasons for lack of OH OH treatment success. P8-D6 is an effective inductor of apoptosis by acting as a dual topoisomerase inhibitor. Its outstanding antitumoral poroperty

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References 1. Chatterjee, S. et al., Mol. Imaging, 2017, 16, 1−5. Acknowledgments: Ministry of Research, Technology and Higher Education of the Republic of 2. Mayer, A. T. et al., J. Nuc. Med., 2018, 59, 1174−1182. Indonesia, Deutscher Akademischer Austauschdienst. 3. Niemeijer, A. N. et al., Nature Commun. 2018, 9:4664, doi: 10.1038/s41467-018-07131-y 4. a) Chatterjee, S. et al. Biochem. Biophys. Res. Commun. 2017, 483, 258–263; b) De Silva, References: R. A. et al. Mol. Pharm. 2018, 15, 3946−3952. 1. a) Badhani, B., Sharma, N., Kakkar, R.: RSC Advances 2015, 5(35): 27540-27557; b) 5. Caldwell Jr, C. et al. Sci Rep. 2017, 7, 13682. Kosuru, R. Y. et al., Molecular Nutrition & Food Research 2018, 62(1): 1700699; c) 6. Miller, M. M.; Mapelli C.; Allen, M. P. et al. Macrocyclic Inhibitors of the pd-1/pd-l1 and cd80 Subramanian, A. P. et al., RSC Advances 2015, 5(45): 35608-35621. (b7-1)/pd-li Protein/Protein Interactions. WIPO (WO2014151634), USA: Bristol Myers Squibb 2. a) Bezerra, D. P. et al.: Nutrients 2017, 9(12): 1367; b) Friedman, M.: J. Agric. Food. Chem. Co. 20161093pp, WO2016039749A1; 2016. 2014, 62(31): 7652-7670; c) Hong, S.-H. et al.: Phytotherapy Research 2016, 30(5): 754-767; d) 7. Minazzi, P. et al. Org. Biomol. Chem.,2014, 12, 6915−6921. Jiankang, L., Akitane, M.: Neuropharmacology 1993, 32(7): 659-669; e) Sharifi-Rad, M. et al.: Phytotherapy Research 2018, 32(9): 1675-1687. 3. Dhingra, M. S. et al.: Med. Chem. Res. 2014, 23(11): 4771-4788. POS.62 Studies on histone deacetylase protein expression profiles POS.61 of various cancer cells and the effect of histone Imaging of PD-L1 for immunotherapy monitoring deacetylase inhibition on anticancer drug cytotoxicity

Stadlbauer S1; Roscher M1; Schaefer M1; Bauder-Wuest U1; Kopka K1 Grathwol, C.; Link, A.; Bednarski, P. J.; Behnisch-Cornwell, S. Universität Greifswald, Institute of Pharmacy, Friedrich-Ludwig-Jahn-Straße 17, 17489 1 German Cancer Research Center, Division of Radiopharmaceutical Chemistry, Im Greifswald, Germany Neuenheimer Feld 223, 69126 Heidelberg, Germany

Immunotherapy addressing the PD-L1 receptor with checkpoint inhibitors Background: Epigenetic modifications in cancer cells can cause poor is a promising therapy strategy for cancer patients. However, in average prognosis, development of cancer relapse and malfunction of anticancer only 30% of patients respond to checkpoint inhibitor immunotherapy. therapy. Thereby gene expression with regard to epigenetic modulations Therefore, it is important to select those patients prior to therapy who will can be affected by histone acetylation, –methylation and – likely respond. Molecular imaging techniques such as PET and SPECT phosphorylation just to name but a few. These modifications are have the advantage of providing a whole body scan and are able to fully reversible and cause an activation or repression of DNA transcription. address the issue of heterogeneous PD-L1 expression.[1] Low-molecular Histone deacetylases (HDACs) are one of the key mediators of weight compounds such as peptides are favourable as imaging agents, epigenetic regulation, and are classified into four groups. The classes – because they exhibit short clearance times, produce higher imaging I, –II and –VI containing a zinc ion in the active site whereas the class– + 1, contrasts within minutes to hours and are synthetically easily accessible. III, so called sirtuins, are NAD -dependent protein lysine deacetylases 2 However, so far only one cyclic 14mer peptide named WL12 was . modified with DOTAGA and labelled with 64Cu and 68Ga for studies in Results and conclusion: The focus of this current research is to determine tumour-bearing mice.[4] the protein expression profiles of various HDACs in a set of 17 human We are addressing the issue of PD-L1 imaging with the aim to design cancer cell lines from different tumor sources. First results indicate modified peptides as well as small-molecule-based tracers. In this study, correlations between the expression profiles of various HDACs such as a linear 25mer peptide called RK-10 [5] reported to exhibit high binding Sirt1 with Sirt3 and HDAC4. Furthermore we found indications for affinity for PD-L1, was modified with DOTA (1) to allow for radiolabelling correlations between HDAC isoenzyme expression and the sensitivity of with 68Ga. In addition, the cyclic peptide WL12 [6] was modified with a cell lines towards certain anticancer drugs, such as HDAC4 with DOTA (2) for the same purpose. MDA-MB231 cells as PD-L1 podophyllotoxin, methotrexate and 5-fluorouracil or Sirt5 with colchicine. overexpressing cell line and HT29 cells as PD-L1 negative cell line were In combination studies of HDAC inhibitors with anticancer drugs, used for in vitro studies. Flow cytometry experiments showed that MDA- interesting effects on the proliferation of cancer cells were identified. The MB231 cells express a significant higher level of PD-L1 compared to IC50 values of anticancer drugs were determined in combination with HDAC inhibitors at non-toxic concentrations and compared with single HT29 cells without induction. After stimulation with INF upregulation of agent cytotoxicity. Therefor unspecific HDAC inhibitors such as PD-L1 was observed in both cell lines. The levels in MDA-MB231 cells, vorinostat and trichostatin-A as well as newer azo-based Sirt2 inhibitors however, are about three times higher than in HT29 cells. Both cell lines were tested. A highly significant enhancement of cisplatin and lomustine were induced with 20 ng/ml of INF 24 h prior to the experiments to cytotoxicity in some cell lines was found when combined with an HDAC stimulate PD-L1 expression. Binding affinity studies revealed specific inhibitor, indicating a potential synergistic effect in anticancer therapy. binding for both peptides to MDA-MB231 cells whereby uptake in HT29 cells was low. The cyclic peptide 2 labelled with 68Ga exhibits a significantly higher binding affinity compared to the 68Ga labelled linear References: peptide 1. Internalization experiments corroborated these results. 2 1 Bubna, A. K.: Indian J. Dermatol. 2015, 60(4): 419. 2 exhibits a much higher internalization fraction at both 37 °C and 4 °C Yoon, S.; Eom, G. H.: Chonnam Med. J. 2016, 52(1): 1-11. compared to the linear peptide. Blocking experiments confirmed the specificity of the cyclic peptide. PD-L2, exhibiting high structural similarity to PD-L1, is a potential off-target. However, flow cytometry experiments POS.63 proved the high specificity of WL12 toward PD-L1 over PD-L2. Preliminary studies of fluorescence- and 68Ga-labeled anti- Distribution coefficient analysis shows that 2 is relatively hydrophilic miR-21 (logD = –1.80). Taken together, the cyclic peptide 68Ga-2 shows higher binding potential and specificity towards the PD-L1 receptor compared to the linear peptide 68Ga-1. Further modification with a bifunctional AAZTA Janssen, E. H. 1; Langer, L. B. N.1; Fiedler, J.2; Dräger, G.3; Thackeray, [7] (3) would allow labelling at room temperature. Therefore, peptide J.1; Bankstahl, J.P.1; Thum, T.2; Bengel F. M.1; Ross, T. L.1 WL12 will serve as a basis for the development of PET imaging probes 1 Department of Nuclear Medicine, Hannover Medical School, 30625 Hannover, Germany to visualize and quantify PD-L1 upregulation in cancer tissue. 2 Institute of Molecular and Translational Therapeutic Strategies, Hannover Medical School, 30625 Hannover, Germany 3 Institute of Organic Chemistry, Leibniz University of Hannover, 30167 Hannover, Germany

Objectives MicroRNAs (miRNAs, miRs) are short, non-coding RNAs that post- transcriptionally regulate gene expression by binding to mRNA and therefore play a significant role in many biological processes and diseases, in which they represent a promising new biomarker in the diagnosis. Using radiolabeled anti-miRNA oligonucleotides, which bind highly specifically and with high affinity to the complementary miRNAs, have the potential to assess miRNA levels in vivo. Molecular imaging can

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be performed by positron emission tomography (PET) using positron might be filled by measuring the angiotensin II type 1 receptor (AT1R) emitting radionuclides such as 68Ga and 18F. miR-21 is involved in the expression in vivo by positron emission tomography (PET). development of cardiac fibrosis, which leads to myocardial dysfunction In this study, we present novel 18F-labeled derivatives of the drugs 1,2. An anti-miR-21 oligonucleotide labeled with short-lived irbesartan and valsartan, two broadly prescribed AT1R antagonists, with radionuclides, 68Ga or 18F, would be valuable for the diagnosis of the least structural changes following described structure-activity pathogenic changes in miR-21 levels by non-invasive molecular imaging, relationships. Thereby, a fluorine was introduced into different positions especially in the early stage of a disease. The aim of this project is to of the aliphatic side chains of these drugs yielding α-F-irbesartan (IC50 = develop radiolabeled oligonucleotides targeted to miR-21 to evaluate 6.6 nM, irbesartan IC50 = 1.6 nM at hAT1R) and ẟ-F-valsartan (Ki = 14.6 potential as diagnostic tools in cardiac fibrosis. nM, valsartan Ki = 11.8 nM at hAT1R).[2,3] These tracers were successfully radiolabeled and injected into rats and pigs for PET studies. Distinct Materials and Methods kidney uptake could be observed with specificity proven by sartan Anti-miR-21 was functionalized with a hexyl-disulfide moiety and locked blocking studies. [2-4] nucleic acids (LNA). The disulfide group was reduced with dithiothreitol and coupled with the fluorescent dye ATTO 647N, the complexing agent THP and NODAGA by maleimide thiol coupling to yield ATTO647N-anti- miR-21, NODAGA-anti-miR-21 and THP-anti-miR-21, respectively. Analytics and mass confirmation were performed by HPLC-ESI-MS (TOF) and purification by Sephadex G-25 in PD MidiTrap and PD-10 desalting columns. ATTO647N-anti-miR-21 was incubated in mouse 3T3 fibroblasts for 24 h. qPCR was used to measure antisense effects and the transfection efficiency (LNA) was determined by FACS. NODAGA- anti-miR-21 and THP-anti-miR-21 were labeled with gallium-68 in first test runs. Approximately 200 MBq of gallium-68 eluate was added to the corresponding precursor in water at room temperature for THP and at 95 °C for NODAGA. The pH was adjusted to 6 for THP and to 4 for These results demonstrate the strategy of deriving novel AT1R PET NODAGA. Radiolabeling was monitored and analytics were performed tracers directly from clinically used sartans with the least structural by radioHPLC. modification. Both 18F-irbesartan and 18F-valsartan may serve as tracers for renal imaging, inflammatory assessment, and cancer diagnosis by Results providing information regarding the distribution of AT1R and RAAS Three novel anti-miR-21 derivatives were successfully synthesized: function under pathophysiological conditions. ATTO647N-anti-miR-21, NODAGA-anti-miR-21 and THP-anti-miR-21. Based on the qPCR (miR-21 knockdown) and FACS data (LNA Gratitude is expressed to the International Doctorate Program “Receptor Dynamics” of the Elite transfection efficiency) we see that ATTO647N-anti-miR-21 (100nM) is Network of Bavaria. The German Academic Merit Foundation is gratefully acknowledged for absorbed by mouse 3T3 fibroblasts with a prolonged inhibitory effect over awarding Matthias Hoffmann a PhD scholarship. This project was supported by the German 24h. Initial radiolabeling demonstrated feasibility, though low Research Council (Deutsche Forschungsgemeinschaft (DFG) grants CH 1516/2-1 and HI 1789/3-3). radiochemical yield may reflect steric hindrance by the oligonucleotide. Further optimization of the reaction time, temperature, the pH value and analytics is ongoing. [1] Ghazi, L.; Drawz, P.: F1000Res. 2017, 6(297): 297. [2] Hoffmann, M.; et al.: ChemMedChem 2018, 13(23): 2546-2557. [3] Chen, X.; et al.: ACS Omega 2018, 3(9): 10460-10470. Conclusions [4] Chen, X.; et al.: PCT/EP2018/077897 (Patent) 2018. ATTO647N-anti-miR-21 displays selective reduction of as well as binding to miR-21 in mouse 3T3 fibroblasts. The precursor NODAGA-anti-miR- 21 and THP-anti-miR-21 were labeled with gallium-68. These studies form the foundation for non-invasive imaging of miR-21 levels in cardiovascular disease. In further studies the radiolabeled derivatives will POS.65 be evaluated in vivo by µPET imaging. Novel diflapolin derivatives with benzimidazole subunit as sEH/FLAP inhibitors 1. Gupta, S.K. et al.: miR-21 promotes fibrosis in an acute cardiac allograft transplantation model. Cardiovascular Research 2016, 110: 215-226. Schoenthaler, M.1; Vieider, L.1; Zöller, E.1; Schütz, S.1; Hörmann, N.1; 2. Thum, T. et al.: MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase Mihajlović U.1; Harm, M.1; Romp, E.2; Kretzer, C.2; Temml ,V3; Garscha, signalling in fibroblasts. Nature 2008, 456: 980-984. U.2; Schuster, D.1,4; Werz, O.2; Matuszczak, B.1 1 Institute of Pharmacy, Department of Pharmaceutical Chemistry, Center for Chemistry and Biomedicine, University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria. 2. Chair of Pharmaceutical / Medicinal Chemistry, University of Jena, Philosophenweg 14, D- 07743 Jena, Germany. 3. Institute of Pharmacy, Department of Pharmacognosy, CCB,University of Innsbruck, Center POS.64 for Chemistry and Biomedicine, University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, 18F-Labeled PET Tracers Derivatived from Angiotensin II Austria. 4. Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Paracelsus Receptor Antagonists Irbesartan and Valsartan Medical University Salzburg, Strubergasse 21, A-5020 Salzburg, Austria

Matthias Hoffmann1#, Xinyu Chen2#, Mitsuru Hirano3, Rudolph A. Werner2, Kenji Arimitsu4, Hiroyuki Kimura4, Takahiro Higuchi2,3, Diflapolin (A) is an anti-inflammatory compound that was discovered in a Michael Decker1 pharmacophore-based virtual screening approach at the University of 1 Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, Julius Innsbruck. It represents the first dual inhibitor of 5-lipoxygenase- Maximilian University Würzburg, Am Hubland, D-97074 Würzburg, Germany activating protein (FLAP) and soluble epoxide hydrolase (sEH), both 2 Department of Nuclear Medicine and Comprehensive Heart Failure Centre, University Hospital targets within the arachidonic acid cascade. The compound decreases of Würzburg, Oberdürrbacherstraße 6, D-97080 Würzburg, Germany 3 Department of Bio-Medical Imaging, National Cerebral and Cardiovascular Centre, 5-7-1 the formation of pro-inflammatory leukotrienes by inhibiting FLAP (IC50 = Fujishiro-dai, Suita, Osaka 565-8565, Japan 0.2 µM), a protein, that transports arachidonic acid form the membrane 4 Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, 5 to the 5-lipoxygenase. Simultaneously, it increases the concentration of Nakauchi-Cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan # These authors contributed equally anti-inflammatory epoxyeicosatrienoic acids by avoiding their sEH- catalyzed degradation (IC50 = 20 nM). Diflapolin shows high target specificity in biological in vitro assays and in vivo experiments confirmed Renin-angiotensin-aldosterone system (RAAS) is a key player in the the anti-inflammatory properties. [1, 2] regulation of blood pressure and electrolyte balance. Therefore, it is However, a major disadvantage of diflapolin is the very low water targeted in the treatment of various diseases including hypertension, solubility. Therefore, structural optimizations are needed to increase heart failure, and nephropathy. Its adaption processes under solubility and bioavailability, while retaining or improving the dual activity. pathophysiological conditions are not yet fully understood.[1] This gap

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bolus injection of 5 to 8 MBq 68Ga-DOTA-Pur followed by a spoiled GRE 3D MRI sequence. Use of pre-concentrated 68Ga resulted in high labeling yields (68 ± 2.6 %) and RCP (≥ 98% after Strata-X purification). MicroPET/MRI images (figure 1) clearly visualized in both modalities widespread infection on the right upper torso originating from the infection induced in the armpit. BCG foci in lung (2nd infection site) and liver (systemic infection as result of lung infection) also could be clearly identified. High signals in kidneys and bladder confirmed prior findings on main excretion pathway via renal clearance. PET/MRI findings were confirmed by ex vivo histological analysis of harvested tissues. We demonstrated for the first time microPET/MRI-imaging of BCG infection in mice with a puromycin based radiopharmaceutical. Based on these results we conclude that 68Ga-DOTA-Pur is a promising tracer for imaging of bacterial infections.

Figure 1. Structure of diflapolin (A) and general structure of synthesized Figure 1: 68Ga-DOTA-Pur of BCG infected mouse; A) BCG infection in diflapolin analogues (B). armpit; B) prefunded heart; C) BCG foci in liver

Diflapolin can be divided into five parts, the heteroaromatic core (I), the spacer unit (II), the methyl substituted para-phenylene (III), the urea moiety (IV), and the 3,4-dichlorophenyl substituent (V). In a previous SAR study, derivatives with benzothiazole core have been synthesized and biologically evaluated. Results show that small modifications of subunits II, III and V have a remarkable effect on inhibitory activity on both targets. [3] Here, we present the multi-step synthesis and biological activities of a novel series of compounds (i.e. compounds of type B) that harbours a benzimidazole as the heteroaromatic core. Additional modifications were conducted on the urea substructure (IV), on the substitution pattern of the phenylene spacer (III) as well as on the phenyl ring (V). The results of the in vitro tests indicate that this bioisosteric replacement led to References: bioactive compounds. 1. Eigner, S. et al.: Mol Imaging Biol 2013, 15(1)1: 79–86

Acknowledgement: Vieider, L. and Schoenthaler, M. would like to acknowledge support by the University of Innsbruck (‘Doktoratsstipendium aus der Nachwuchsförderung’) POS.67 References: 11-Aminostrychnine and (Strychnine-11yl) propionamide: 1. Temml, V. et al.: Sci. Rep. 2017, 7: 42751. 2. Garscha, U. et al.: Sci. Rep. 2017, 7: 9398. Synthesis, Stereochemistry, and Pharmacological 3. Vieider L. et al.: ACS Med Chem Lett 2019, 10(1): 62 – 66. Evaluation at Glycine Receptors

Zlotos, D. P1; Mohsen, A. Y.1; Mandour, Y. M. 1; Marzouk, M. A2, POS.66 Breitinger, U.1; Villmann, C.3;Breitinger, H-G1; Sotriffer, C2; Jensen, A. 4 2 68 A. ;Holzgrabe, U. Ga-DOTA-PUROMYCIN: In Vivo Imaging of Protein 1 The German University in Cairo, Faculty of Pharmacy and Biotechnology, New Cairo City, Synthesis and Bacterial Infections 11835 Cairo, Egypt 2 Institut für Pharmazie and Lebensmittelchemie, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany 3 1 2 3 Institut für Klinische Neurobiologie, Universität Würzburg, , 97078 Würzburg, Germany Eigner, S. , Beckford Vera, D. , Eigner Henke, K. 4 Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, 1SKS Biotech s.r.o., Zitenicka 1894/5, 412 01 Litomerice, Czech Republic University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark 2University of California San Francisco (UCSF), Department of Radiology & Biomedical Imaging, 505 Parnassus Ave, San Francisco, CA 94143, USA 3Universitätsklinikum der RWTH Aachen, Klinik für Nuklearmedizin, Pauwelsstraße 30, 52057 Aachen, Germany Strychnine, the major alkaloid from Strychnos nux vomica, is a highly potent antagonist of glycine receptors (GlyRs). GlyRs are chloride channels composed of five subunits (α or β), which are linked to Bacterial infection is one of the major causes of morbidity and mortality hyperpolarisation and inhibition of neuronal firing.1 The crystal structure globally. Tuberculosis and multi-drug resistant bacteria are increasing of human α3 GlyR in complex with strychnine revealed five equivalent and challenge the diagnosis and treatment of infection. To prevent strychnine binding sites located at the interfaces of the subunits.2 complications, it is important to reliably distinguish between bacterial Recently, our groups have become interested in a bivalent ligand infection and sterile inflammation. Puromycin is an inhibitor of eukaryotic approach for targeting GlyRs.3 In order to examine whether a C-11 amide as well as prokaryotic protein synthesis. Therefore, its derivatives, e.g. linkage is suitable for strychnine-based bivalent ligands for GlyRs, (11S)- 68Ga-DOTA-Puromycin, are potential tracers for imaging of infections. 11-aminostrychnine 1, and the corresponding propionamide 2 were We recently proved the possibility to image protein synthesis of tumor synthesized and characterized as antagonists of homomeric α1 and cells in rat tumors using 44Sc-DOTA-Puromycin [1]. heteromeric α1β GlyRs in a functional fluorescence-based and a whole 68 We aimed at evaluation of Ga-DOTA-Puromycin for imaging of bacterial cell patch-clamp assay, and in [3H]strychnine binding studies. The infection by probing protein synthesis of bacterial colonies in BCG absolute configuration at C-11 of 1 was determined based on vicinal infected mice using a sequential microPET/MRI. coupling constants and NOESY experiment. Docking studies to the DOTA-Pur was purchased from Purimex, Germany and used without strychnine binding site from the crystal structure of the α3 glycine 68 further purification. Pre-concentrated Ga [Zhernosekov et al, 2007] for receptor showed a strychnine-analogous binding mode of compound 2, labeling of DOTA-Pur precursor was produced on a commercially explaining its high antagonistic potency. The findings provide a valuable 68 68 available Ge/ Ga-generator (ITM AG, Garching, Germany). The extension of SAR of strychnine at GlyRs and identify the C-11 amide product was purified on reverse phase C-18 cartridge and resolved in 0.9 linkage to be suitable for future development of strychnine-based bivalent % saline solution. 20 minutes static PET images of BCG infected (3 ligands targeting glycine receptors. months prior to study) mice were acquired from 30 to 50 minutes after i.v.

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The continuously growing threat from multi-resistant bacterial strains N makes a renewed focus on antibiotic development more urgent than ever before. There are many different pathways that can be targeted by H antibiotics, for example bacterial protein biosynthesis. Amongst others, RNA polymerase (RNAP) as an essential enzyme of bacterial protein biosynthesis is considered as a promising target. [1,2] In 2017 MAFFIOLI H 1; X= -NH N H H 2 et al. extracted and discovered pseudouridimycin (PUM) 1 which showed 2: X = -NHCOEt inhibitory activity against RNAP of Gram-negative and Gram-positive O bacteria. [3] They described the novel natural product with this new mode O H of action and a promising structure together with some first semi- X synthetic modifications.

Acknowledgments: Deutscher Akademischer Austauschdienst, Bundesministerium für Bildung und Forschung

References: 1. Lynch, J.W.: Physiol. Rev. 2004, 84, 1051-1095. 2. Huang, X. et al.: Nature 2015, 526, 277-280. 3. Mohsen, A. et. al.: J. Nat. Prod., 2016, 79, 2997-3005.

POS.68 In our work we plan to conduct a detailed SAR study, in order to Synthesis of acyl-β-C-glycosides synthesize new antibiotically effective derivatives of PUM. First, the exchange of pseudouridine with uridine at the deoxy-pseudouridimycin structure 2 was aspired, thus leading to target structure 3. Furthermore, G. J. Boehlich1, N. Schützenmeister*1 we intend to investigate the contribution of the guanidine moiety and 1Institut für Pharmazie, Universität Hamburg, Pharmaceutical and Medicinal Chemistry, therefore designed additional candidate structures 4 and 5. Some of the Bundestrasse 45, 20146 Hamburg, Germany corresponding synthetic work as well as first results on RNAP inhibition will be presented. In 2012, Scleropentasides have been isolated from the leaves and twigs of Scleropyrum pentandrum. Scleropentasides are β-C-glycosidic furan- Acknowledgments: Landesforschungsförderprogramm Saarland. 2-carbonyl compounds of D-glucose. These kind of C-glycosidic compounds have not been isolated from natural sources before and are unique in terms of their structural pattern so far. In terms of biological References: activities only the antioxidative properties of Scleropentasides have been 1. Turecka, K.; Waleron, K.: Curr. Pharm. Biotechnol. 2013, 14(15): 1275-86. investigated so far.[1] A synthetic approach to the Scleropentaside natural 2. Ma, C.; Yang, X.; Lewis, P. J.: Microbiol. Mol. Biol. Rev. 2016, 80(1): 139–160. product family would help to obtain more material for further biological 3. Maffioli, S. I. et al.: Cell 2017, 169(7): 1240-1248. evaluation which is necessary to evaluate this interesting natural product class and derivatives thereof in detail.

POS.70 Towards novel hybrid nucleoside antibiotics: muraymycin- streptomycin conjugates

Rohrbacher, C.1; Ducho, C.1 1 Saarland University, Pharmaceutical and Medicinal Chemistry, 66123 Saarbrücken, Germany

A β-selective type of C-glycosylation to yield acyl C-glycosidic The worldwide emerging antibiotic resistance has become an imminent compounds 1 was achieved. The reaction of lithiated dithianes 2, which problem of our time. Therefore it is necessary to develop new potential were firstly introduced by Corey and Seebach as nucleophilic carbonyl antibacterial drug candidates. A new promising class of antibacterial synthons[2]; with glycosyl halides 3 as electrophiles yielded C-glycosidic substances are the naturally occurring muraymycins. These nucleoside compounds 4 in good yields with excellent β-selectivity. Scleropentaside antibiotics are capable of inhibiting the translocase I (MraY) enzyme, A (Ar = 2-furyl) 1 was synthesized by this approach in just three linear which is part of bacterial peptidoglycan biosynthesis. They display a good steps starting from D-glucose. Also a number of derivatives with different target interaction, but on the downside show deficient bacterial cellular glycosyl or aryl units were synthesized by this method.[3] uptake.[1] In our approach we aim to conjugate muraymycin analogues to other antibiotics which already show an intrinsic ability to permeate the [1] Disadee, W. et al.: Phytochemistry 2012, 74, 115–122. bacterial cell wall. These new muraymycin conjugates should be able to [2] Corey, E. J., Seebach, D.: Angew. Chem. 1965, 77, 1134–1135. utilize the transport mechanisms of the conjugated molecules and [3] G. J. Boehlich, N. Schützenmeister, Angew. Chem. Int. Ed. 2019, 58, 5110–5113 furthermore act as dual inhibitors. As a first model substance to mediate cellular uptake streptomycin was chosen. It was previously reported that after a modification at the aldehyde function the activity of streptomycin is partially retained.[2] POS.69 Pseudouridimycin (PUM) and its derivatives as potential new antibiotics

Lauterbach, S.1; Haupenthal, J.2; Hirsch, A. K. H.2; Ducho, C.1 1 Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123 Saarbrücken, Germany 2 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Drug Design and Optimization, Campus E8.1, 66123 Saarbrücken, Germany

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Two general types of linkers will be used for these conjugates: a stable Reference: triazole conjugation achieved via CuAAC and an intracellularly cleavable 1. Miao, S., Andersen, R. J.: J. Org. Chem. 1991, 56, 6275–6280. disulfide, which releases the two parent drugs.[3] The synthesis of the 2. Zhu T. et al.: J. Antibiot. 2014, 67, 315–318. muraymycin analogues is carried out using a solid phase-supported 3. Schacht, M. et al.: Eur. J. Org. Chem. 2017, 2017, 1745–1748. 4. Li, X.-M. et al.: Heterocycles 2014, 89, 2177–2184. synthesis, as well as a synthesis in solution. The modification of 5. de Vries, J., Assmann, M., Schützenmeister, N., manuskript in preparation. streptomycin with the azide or the thiol linker, respectively, was achieved via an oxime ligation. First results from these studies will be presented.

References: 1. Ducho C., et al., Beilstein J. Org. Chem. 2016, 12: 769-795. POS.72 2. Abad J. P., Amils R., Anitmicrob. Agents. Chemother. 1990, 34: 1908-1914. 3. Latorre A., et al., Eur. J. Med. Chem. 2014, 82: 355-362. Synthesis of a modular system for adenosine receptor probes

Klapschinski T. A.1; Brockmann A.1; Hinz S.1; Vielmuth C.1; Müller C. E.1 POS.71 1PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany Biological Evaluation of halogenated Rubrolid Analogs deVries J.1, Assmann M.1, Schützenmeister N.1 G protein-coupled receptors (GPCRs) represent the largest family of 1Institut für Pharmazie, Universität Hamburg, Phamaceutical and Medicinal Chemistry, integral membrane proteins.[1] Adenosine receptors (AR) are GPCRs Bundesstraße 45, D-20146 Hamburg, Germany activated by the nucleoside adenosine. They represent novel drug targets, e.g. in immuno-oncology and for neurodegenerative diseases.[2] The labeling of drug targets in native tissues is required for research and In 1991, the first rubrolides have been isolated from the marine fungus diagnostic applications.[3,4] Antibodies are frequently used for this Ritterella rubra. This natural product class has shown various purpose, however, specific antibodies for membrane proteins such as pharmaceutical relevant activities such as moderate inhibition of protein GPCRs are difficult to obtain.[5] phosphatases 1 and 2A, antibacterial, and antiviral effects.[1] In 2014, The aim of our study has been the synthesis of a modular system for AR Rubrolide R 1 and S 2 have been isolated[2] and in 2017, the total probes attaching a variety of tags via a linker to functionalized selective syntheses of both compounds have been published.[3] These two natural high-affinity ligands. For this purpose, a highly selective antagonist was Rubrolides revealed biological activity against pH1N1 and H3N5 and selected for each AR subtype, and derivatives were synthesized Rubrolide S 2 has also shown activity against the tobacco mosaic virus.[4] containing a functional group located at the part of the binding site that is oriented towards the extracellular receptor domains. The designed compounds were synthesized in multi-step procedures, and linkers of different lengths were subsequently attached. In a next step, the AR affinities and selectivities were determined. An A2AAR antagonist modified in such a way displayed high affinity (Ki value of 0.6 nm at the human A2AAR) and high selectivity (Ki value > 1000 nm at human A1, A2B and A3ARs). The compound was consequently coupled with a BODIPY 493-503 derivative as a first tag. In the future, different fluorophores, DNAs for proximity ligation assays, and antibodies for antibody-drug conjugates (ADCs) will be considered for attachment to this and further AR ligands.

In order to increase the biological activity we investigated different halogenation patterns of the Rubrolide scaffold. Promising results were observed in the relation between substitution patterns of the synthesized Rubrolide analogs and the ability of virus inhibition. A trend towards higher brominated Rubrolides with a chloride atom at the furanone core was observed.[5]

10

8

6

4 References: 1. Dorsam, R. T.; Gutkind, J. S.: Nat. Rev. Cancer 2007, 7, 79–94.

Virus titer titer log p.f.u-/mL Virus 2 2. Hinz, S. et al.: Oncotarget 2018, 9, 13593–13611. Nigg, E. A. et al.: Proc. Nat. Acad. Sci. 1982, 79, 5322–5326. 0 Gullberg, M. et al.: Proc. Nat. Acad. Sci. 2004, 101, 8420–8424. Michel, M. C.; Wieland, T.; Tsujimoto, G. Naunyn-Schmiedebergs Arch. Pharmacol. 2009, 379, 1 µM 1 µM 1 µM 1 µM Ctrl 1 Ctrl 2

10 µM 10 µM 10 µM 10 µM 385–388. 100 µM 100 µM 100 µM 100 µM

Ribavirin 4 5 6

Figure 1: Plaque Inhibitions Assay of Rubrolide Analogs 4 – 6 und Ribavirin with influenza A virus H3N2 on MDCK cells. Sample POS.73 concentrations: 100 µM, 10 µM und 1 µM. Positive control: Ctrl 1 (no substance) und Ctrl 2 (0.1 % DMSO). Fixation of cells with 4 % PFA and Synthesis and evaluation of phosphonic acids for the visualization of viable cells with crystal violet. Results of three antimicrobial coating of metal surfaces independent experiments. n.d. – no plaques detectable.[5]

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Ruf, E.1; Maison, W.1 e.g. damaged tissues and dying cells promotes activation of P2X ion 1Universität Hamburg, Institut für Pharmazie, Bundesstraße 45, 20146 Hamburg, Germany channels and causes the influx of extracellular cations, including calcium and sodium ions [2]. The homotrimeric P2X7 receptors (P2X7Rs) are involved in the pathologies of various diseases, including autoimmune The increasing emergence of multiple drug resistance (MDR) in bacteria diseases such as rheumatoid arthritis, neurodegenerative diseases, demands the development of new antimicrobial strategies [1]. As a matrix neuropathic pain and cancer [3]. Therefore, P2X7Rs represent for the proliferation of bacteria, surfaces in hospitals or other public interesting and promising targets for the development of new therapeutic places pose a huge infection risk [2]. Bacterial growth often occurs via agents. The aim of this project is the pharmacological evaluation of the formation of a protective biofilm within the bacterial colony, which known and novel compounds targeting P2X7Rs with different functional renders treatment other than mechanical removal futile [3]. A promising assays. The compounds are characterized in three different functional step to combat this problem is the coating of metal surfaces with P2X7R assays (influx of Ca2+-ions, the uptake of YO-PRO-1 and the molecules that prevent the formation of biofilms. This approach seems release of interleukin 1ß (IL-1ß)) for their activation or inhibition of particularly appealing when it comes to the biofilm formation on invasive particular intracellular signaling pathways. The derived structure-activity tools, such as surgery equipment, catheters or implants [4]. relationships are used to guide the development and optimization of new Passive and active antifouling strategies differ depending on the point of P2X7R ligands as future therapeutics for cancer and inflammatory interaction within the biofouling process. While passive approaches diseases. suppress the reversible binding of biological matter to the surface, thereby impeding bacterial growth due to a lack of nutrients, active A approaches aim at directly killing microbes [5]. Self assembled monolayers of sulfobetaine zwitterions have already shown a drastical reduction of the unspecific protein adsorption in human blood serum [6]. Quaternary alkyl ammonium salts are effective contact biocides destroying the bacterial membrane through various ionic interactions and replacement of cations [7]. They present a direct method of killing bacteria, interacting before the protective matrix of the colony can be formed which can increase their efficacy compared to antibiotics administered after the biofilm formation. As anchoring group, phosphonic acids exhibit many favourable properties such as robust synthetic approaches, formation of strong bonds to numerous metals, pH independency as well as high stability [8]. B Herein we present the synthesis of cationic as well as zwitterionic phosphonic acids. The modular synthetic approach allows easy modification and access to a library of different substances in a low number of steps from cheap and easily available starting materials. The synthesized molecules are coated on different medicinally relevant materials such as titanium or stainless steel. Afterwards, we present the results of the biological testing of the antimicrobial activity against different strains of bacteria. C

1. Karam, G. K. et al.: Crit. Care 2016, 20(1):136-144. 2. Bixler, G. D.; Bhushan, B.: Phil. Trans. R. Soc. A 2012, 370: 2381-2417. 3. Lynch, A. S.; Robertson, G. T.: Ann. Rev. Med. 2008, 59: 415-428. 4. Lo Vetri, K. et al.: Biofouling: Types, Impact and Anti-Fouling (Nova Science Publishers) 2010. 5. Q. Shao, S. Jiang, Adv. Mater. 2015, 27, 15–26. 6. Banerjee, I.; Pangule R. C.: Kane, R. S.: Adv. Mater. 2011, 23(6): 690-718. 7. Milovic, n. M. et al.: Biotechnol. Bioeng. 2005, 90(6): 715-722. 8. Queffélec, C. et al.: Chem. Rev. 2012, 112(7): 3777-3807. Fig. 1: B) Crystal structure of a panda P2X7 receptor [4]. A) Representative dose-respose curves of functional P2X7R YO-PRO-1 uptake assay of agonist BzBzATP (EC50 = 152 ± 23 nM), two novel agonists 1 (EC50 = 53 ± 22 nM) and 2 (EC50 = 1460 ± 243 nM) and C) POS.74 antagonists AZD9056 (IC50 = 14 ± 0.7 nM), JNJ47965567 (IC50 = 13 ± Pharmacological Evaluation of Novel Compounds for their 2.1 nM) and two novel antagonists 3 (IC50 = 16 ± 1.4 nM) and 4 (IC50 = Activity at P2X Receptors 134 ± 9.6nM), n ≥ 5.

Isaak, A.; Patberg, M.; Dobelmann, C.; Pica, A. and Junker, A. References: Westfälische Wilhelms-Universität Münster, European Institute for Molecular Imaging (EIMI), 1. Kawate et al.: JGP 2011, 137: 579-590. Waldeyerstr. 15, 48149 Münster, Germany. 2. Khakh, B.S.; North, R.A.: Nature 2006, 422: 527-532. 3. Park, J-H.; Kim, Y-C.: J. Expt. Opin. Ther. Pat. 2016, 27, 257-267 Adenosine triphosphate (ATP) is known as the molecule that provides 4. Karasawa, A.; Kawate, T.: eLife 2016, 5: e22153. the energy for many different processes in living cells, but ATP also acts as an extracellular ligand on receptors in the field of purinergic signaling such as P2Y and P2X receptors [1]. The P2X receptors are non-selective trimeric ion channels activated through the binding of extracellular ATP. POS.75 The molecular architecture of this ion channel protein family is build up Insertion of NanoLuc into extracellular loops enables from homo- or heterotrimeric complexes of seven different subunits BRET-based binding assays at the neuropeptide Y Y1 and (P2X1-7). Each subunit stoichiometry determines particular functional properties of the P2X receptor subtype. An increasing ATP release by Y2 receptor

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In conclusion, we present a novel approach to establish BRET-based binding assays for GPCRs, which are incompatible with an N-terminal Grätz, L.; Littmann, T.; Müller, C.; Buschmann, J.; Keller, M.; luciferase tagging in terms of BRET measurement. Buschauer, A.†; Bernhardt, G. Institute of Pharmacy, University of Regensburg, Universitätsstr.31, 93053 Regensburg, Germany Financial support by the GRK1910 is gratefully acknowledged.

1. Stoddart, L. A., et al., Nat Methods 2015, 12(7), 661-663 Recently, real-time BRET-based binding assays were reported for 2. Yang, Z., et al., Nature 2018, 556, 520-524. several G-protein coupled receptors (GPCRs), which were N-terminally 3. Keller, M., et al., J Med Chem 2015, 58, 8834-8849 fused to the very brightly blue-emitting NanoLuciferase (NanoLuc) (λmax 4. Doods, H., et al., Eur. J. Pharmacol. 1999, 2-3, R3-R5 ≈ 460 nM) [1]. However, our efforts to apply this strategy to GPCRs with †Died July 18, 2017 comparatively long N-termini (> 25 amino acids), e.g. members of the neuropeptide Y (NPY) receptor family, did not give concentration- dependent BRET. Therefore, we intended to explore whether insertion of POS.76 the NanoLuc into extracellular loops of NPY receptors represents a 18 strategy enabling BRET-based binding assays for such GPCRs. Novel F-labeled D4-receptor ligands Based on the recently resolved crystal structure of the NPY Y1 receptor [2], we inserted the NanoLuc in an unstructured region in ECL2 of the Y1 Willmann M1, Ermert J1, Neumaier B1,2 receptor (intraNLucY192) and the Y2 receptor (intraNLucD197), and 1Forschungszentrum Jülich GmbH, Institute of Neuroscience and Medicine, INM-5, Nuclear stably expressed these constructs in HEK293T cells. As the Chemistry, Germany 2University of Cologne, Faculty of Medicine and University Hospital Cologne, Institute of pyrylium/pyridinium dye Py-5 shows ideal spectral properties ( max (ex.) λ Radiochemistry and Experimental Molecular Imaging, Cologne, Germany ≈ 470 nm, λmax (em.) ≈ 650 nm) for a combination with NanoLuc bioluminescence, we synthesized a Py-5-labelled fluorescent Y1 receptor ligand (UR-CM138), derived from UR-MK299 [2, 3], and a Py-5-labelled The role of the dopamine D4-receptor subtype in the development of Y2 receptor ligand (UR-JB264), derived from the standard Y2 receptor neurodegenerative diseases such as schizophrenia has been discussed antagonist BIIE0246 [4], as molecular tools for our BRET assays. for several decades. Specific radiotracers for more detailed preclinical and clinical investigations are still missing so far. The objective of this study was to develop D4-selective radioligands for positron emission tomography (PET). The selected lead structures exhibit high D4R subtype selectivity and a suitable LogP values, rendering them attractive for the development of a D4-selective radioligand for PET-imaging [1]. Two D4R- ligands, know from literature [2,3], were selected for labeling with fluorine-18 (t1/2=108 min), the most ideal positron emitting nuclide. D4R-radioligand [18F]I was synthesized from 2- [18F]fluorophenylpiperazine 2, obtained by an alcohol enhanced Cu(II)- mediated radiofluorination [4] of the Boc-protected precursor 1 followed by deprotection of the radiolabeled intermediate with TFA. Subsequently, [18F]I was obtained by reductive amination with 3 in a total isolated radiochemical yield of 7 % within 2 h. Three independent auto radiographic studies with molar activities up to 90 GBq/µmol showed high content of non-specific binding that covers any possible specific UR-CMCM138 binding. The chiral D4R-radioligand [18F]II was also obtained by alcohol enhanced Cu(II)-mediated radiofluorination [4] of the boronic acid precursor 4 in a RCY of 66±5 % within 60 min after isolation by semi-preparative HPLC. Preliminary in vitro autoradiographic study indicates specific binding of [18F]II in areas with D4-expression, consistent with results published earlier [5].

UR-JB264

Our strategy resulted in a concentration-dependent increase in BRET References: ratio for both receptors, i.e. binding of UR-CM138 at the Y1 receptor and 1. Lindsley, C. W. et al.: J. Med. Chem. 2017, 60: 7233-7243. of UR-JB264 at the Y2 receptor was saturable. Both fluorescent ligands 2. Witt J. O. et al.: Bioorg. Med Chem. Lett. 2016, 26: 2481-2488. exhibited high receptor affinities with equilibrium dissociation constants 3. Oh S. J. et al.: Bioorg. Med. Chem. 2004, 12: 5505-5513. (Kd) of 0.24 nM (UR-CM138, Y1R) and 20 nM (UR-JB264, Y2R), which 4. Zischler J. et al.: Chem. Eur. J. 2017, 23: 3251-3256. were in good agreement with binding data from radioligand (Ki) and flow 5. F. Kügler et al.: J. Med. Chem. 2011, 54: 8343-8352. cytometric (Kd) binding assays as well as with functional data (Kb) from β-arrestin recruitment assays. We also performed NanoBRET competition binding experiments with standard Y1 and Y2 receptor POS.77 ligands, yielding Ki values being in accordance with reported data from radioligand competition binding experiments. Determination of inhibitory potency and target residence time of p38 α MAPK inhibitors

115 • DPhG Annual Meeting 2019 Conference Book POSTERS

Kudolo, M; Dieckmann, S. M.; Laufer, S. pretreatment periods of 15 min to 2 h. However, an extended Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, preincubation time of 5 h, impaired the efficiency of free BRP-187, while Eberhard-Karls-Universitaet Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany AcDex- and PLGA-based NP containing BRP-187 retained its potent inhibitory effect, indicating that encapsulation improves the availability of p38 α mitogen-activated protein kinase (MAPK) is a serine/threonine BRP-187 in PMNL. Cellular uptake studies were designed using kinase that plays a crucial role in regulating cellular responses to external fluorescent NPs containing BRP-187, which showed temporal stress stimuli, making it a promising target for treatment of inflammatory enrichment of the drug in PMNL, M1 or M2 macrophages when analyzed diseases. Up to date no p38 α inhibitor has been launched to the market. by confocal microscopy. Finally, we will provide first evidences that BRP- The development of novel p38 α inhibitors requires appropriate testing 187 NPs are operative in human whole blood assays. systems to identify and characterize potential inhibitor candidates. Selected compounds from an in house p38 α MAPK inhibitor library were subjected to a sequential screening approach. Acknowledgments: This work was supported by the DFG-funded Collaborative Research Centre Firstly, inhibitory potency was determined by a direct ELISA assay PolyTarget (SFB 1278, project A04). previously reported by Goettert et al [1]. This ELISA assay had to be adapted due to stockout of the initially used [1] M. Werner, P.M. Jordan, E. Romp, A. Czapka, Z. Rao, C. Kretzer, A. Koeberle, U. Garscha, detection antibody resulting in the challenge of choosing an equivalent S. Pace, H.E. Claesson, C.N. Serhan, et al., FASEB J. 2019, 33, 6140-6153. antibody. As a result, the previously used antibody directed against the [2] U. Garscha, S. Voelker, S. Pace, J. Gerstmeier, B. Emini, S. Liening, A. Rossi, C. Weinigel, S. Rummler, U. S. Schubert, et al., Biochem. Pharmacol. 2016, 119, 17-26. dually phosphorylated (pThr69/71) ATF-2 protein was replaced by an anti- ATF-2 pThr71 antibody. The suitability of the newly introduced antibody was proven by conventional assay validation. With this new setup IC50 values of the selected compounds were determined. POS.79 Secondly, these compounds were analysed regarding their target Studies on the cellular uptake of nucleoside antibiotics residence time in a commercially available fluorescence polarization assay kit as reported by Kumar and Lowery [2]. Target residence time was calculated based on the recovery of kinase activity after formation of Weck, S. C.1; Niro, G.1; Meiers, J.2; Ducho, C.1 an inhibitor kinase complex at saturating inhibitor concentrations and 1 Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, subsequent dilution. Campus C2.3, 66123 Saarbrücken, Germany Results were compared to already existing data obtained by surface 2 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Drug Design and plasmon resonance. The rationale behind this comparison was to find a Optimization, Campus E8.1, 66123 Saarbrücken, Germany technique more suited for high throughput screenings, featuring a better cost efficiency and higher convenience for application as a standard Nucleoside antibiotics are representatives of antibacterially active natural laboratory procedure. products with versatile structures and mode of action. They include, amongst others, the family of muraymycins which were first isolated in References: 2002 from Streptomyces sp. [1,2] Members of the antimicrobially active Goettert, M. et al.: Anal Biochem. 2010, 406(2), 233-234. muraymycin family contain a uridine-derived core structure and can be Kumar, M. et al.: SLAS Discovery, 2017, 22(7), 915-922. divided into four groups (A-D) due to different peptide structures. [1] For instance, members of series A such as 1 comprise lipophilic side chains which are ω-functionalised with a guanidino or hydroxyguanidino- function, whereas muraymycins of series C such as 2 include an POS.78 unfunctionalised L-3-hydroxyleucine. The antibiotic potency of muraymycins is based on the inhibition of the essential bacterial Encapsulation of the dual FLAP/mPGES-1 inhibitor BRP187 membrane protein translocase I (MraY). [1] As key enzyme, MraY is in biodegradable polymers improves its bioavailability in involved in the intracellular section of peptidoglycan biosynthesis. human whole blood Therefore, muraymycins are attractive candidate structures for antimicrobial drug development and thus, several artificial analogues of Kretzer, C.1; Shkodra-Pula, B.2; Garscha, U. 1; Klemm, P 2; Vollrath, A. muraymycins have been synthesized in our group. 2; Schubert. S 2,4; Schubert, U.S. 2,3; Banoglu, E. 5; Werz, O.1 1Institute of Pharmacy, Friedrich Schiller University Jena, Philosophenweg 14, D-7743 Jena, Germany; 2Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstraße 10, 07743 Jena, Germany; 3Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany; 4Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy, Friedrich Schiller University, Lessingstraße 8, 07743 Jena, Germany; 5Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Etiler, 06330 Yenimahalle, Ankara, Turkey. Due to the location of the active site of MraY at the cytosolic side of the cellular membrane, inhibitors of MraY are required to cross the bacterial Leukotrienes (LTs) and prostaglandin (PG) E2 play pivotal roles in the cell wall. [1,3] In Gram-positive bacteria, such as Staphylococcus aureus, initiation and maintenance of inflammation [1]. To prevent their antibiotics only have to pass through one membrane whereas Gram- production from the substrate arachidonic acid (AA), a new negative bacteria like Escherichia coli are composed of two membranes, pharmacological approach was designed, that is, the dual inhibition of one inner and one outer membrane. An essential challenge for the microsomal prostaglandin E2 synthase-1 (mPGES-1) and the 5- development of muraymycins towards drug candidates is their limited lipoxygenase-activating protein (FLAP) [2]. Such dual FLAP/mPGES-1 uptake into bacterial cells. With the objective to investigate the cellular inhibitors are proposed to exhibit lower risk of side effects versus non- uptake in Gram-positive and Gram-negative bacteria, an appropriate steroidal anti-inflammatory drugs (NSAIDs). The lipophilic and acidic assay was established in our group. This assay is based on the BRP-187 ((4-(4-chlorophenyl)-5-[4-(quinolin-2- incubation of a defined amount of bacterial cells with the candidate ylmethoxy)phenyl]isoxazol-3-carboxylic acid) has a high tendency to bind compound. The cellular uptake was then quantified by LC-MS after cell plasma proteins and therefore displays low bioactivity in human whole lysis. For Gram-negative bacteria a differentiation between periplasmic blood (HWB) and in vivo experiments. To overcome this problem, we and cytosolic uptake has also been achieved. Some first results encapsulated BRP-187 in biocompatible polymers (i.e., acetylated regarding the cellular uptake of several synthetic muraymycin derivatives dextran (AcDex) and poly(lactic-co-glycolic acid) (PLGA) via in different types of bacteria will be presented. nanoprecipitation. Dynamic light scattering (DLS) showed that the produced particles were spherical and have a hydrodynamic diameter (dh) around 200 nm. The encapsulation efficiency was determined by UV- Acknowledgments: Deutsche Forschungsgemeinschaft VIS spectroscopy to 60-80% and the different particles with and without drug were tested on their ability to inhibit the 5-lipoxygenase product References: formation in polymorphonuclear leukocytes (PMNL). The potency of free 1. Wiegmann, D. et al.: Beilstein J. Org. Chem. 2016, 12: 769–795. 2. McDonald, L. A. et al.: J. Am. Chem. Soc. 2002, 124(35): 10260–10261. BRP-187 and drug-loaded nanoparticles (NPs) was comparable in 3. Tanino, T. et al.: J. Med. Chem. 2011, 54(24): 8421–8439.

DPhG Annual Meeting 2019 Conference Book • 116 MEDICINAL CHEMISTRY AND DRUG DESIGN

HO3SO O POS.80 OH O H O O N CH O O 3 NH2 NH HN SO H Dynamic pharmacophore models unveil binding mode HO OH 3 N N HN O N O NH HO N S ensembles of partial GPCR agonists O N O O OSO3H O O Cl Heparin (1) Dabigatran (2) Rivaroxaban (3) antithrombin III activator thrombin inhibitor FXa inhibitor Bermudez, M.1; Wolber, G.1 1Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2, 14195 Berlin, Germany To disrupt the intrinsic coagulation pathway and prevent thrombosis (without affecting extrinsic pathway), FXIIa proteolytic activity should be blocked by an appropriate inhibitor. For this purpose, after careful Given the essential role of G protein coupled receptors (GPCRs) in many analysis of FXIIa’s crystal structure, two series of small molecule (patho)physiological processes, more than 30 % of currently marketed inhibitors (6-7) possessing aminotriazole scaffold were designed and drugs deploy their therapeutic effect by targeting GPCRs. However, the synthesized starting from commercially available carboxylates 4 and molecular understanding of how ligands translate their chemically anhydrides 5. encoded information to intracellular signalling is still incomplete, which R1 renders novel methodologies for studying GPCRs highly necessary for O 1,2 Z O O rational drug design . R X N N N X X N X OH R1 In this talk we will focus on a novel computational approach, which R R O 3 4 R N N NR R X N X 2 X R R5 6 combines classical three-dimensional pharmacophores with MD-based X O R R2 sampling in a fully automated fashion. The resulting dynamic (6) (4) (5) (7) 1 6 pharmacophores (dynophores) provide information about the interaction X = N, C; Z = C=O, SO2, CH2; R, R -R = alkyl, alkoxy, phenyl, hal, NH2, etc. pattern in space and time 3,4. This enables us to mechanistically Synthetically obtained compounds were tested for their ability to inhibit understand protein-ligand complexes as dynamic entities. the whole blood and plasma coagulation using in vitro coagulation assays We will demonstrate the descriptive power of dynamic pharmacophores (activated partial thromboplastin time (aPTT), prothrombin time (PT)) and on a challenging task in GPCR research, the mechanistic basis for partial compared to the activity of anticoagulants 1-3. Some of the prepared receptor activation. Using muscarinic receptors as a model system we aminotriazoles 6-7 significantly prolonged coagulation time in aPTT tests show the existence of binding mode ensembles for classical partial showing compounds’ ability to affect the intrinsic coagulation pathway, muscarinic agonists like pilocarpine or arecoline 5. By a comparison with which is initiated by FXIIa. In contrast, synthesized compounds were the full agonist iperoxo and the inverse agonist QNB, we can link distinct found to have little effect on PT showing thereby little to no influence on binding modes with receptor activation and deactivation. the extrinsic coagulation pathway. This is an indirect indication of their Furthermore, we discuss the concept of binding mode ensembles in the selectivity towards the serine proteases from the intrinsic coagulation context of crystallographic data and recent NMR studies, which indicate pathway. Based on the biological activity data structure-activity that binding mode ensembles might play an important, but relationships were established. Currently, we study specific inhibitory underestimated role for ligand-dependent pharmacological effects at activity and selectivity of synthesized aminotriazoles 6 and 7 towards GPCRs and other drug target classes. FXIIa.

1. Gerotziafas G, Samama M.: Curr. Opin. Pulm. Med. 2004, 10: 356–365. Acknowledgements: We thank the Deutsche Forschungsgemeinschaft (German Research 2. Wendelboe, A. M., Raskob, G. E.: Circ. Res. 2016, 118: 1340–1347. Foundation – DFG 407626949) for financial support of Marcel Bermudez. 3. Hohnloser, S.H., Basic, E., Nabauer, M.: Clin. Res. Cardio. 2017, 106(08): 618-628. 4. Baeriswyl, V. et al.: ACS Chem. Biol. 2015, 10: 1861–1870. References 5. Renne, T. et al.: J. Exp. Med. 2005, 202: 271–281. 1. M. Bermudez et al.: Drug Discov. Today, 2019 2. M. Bermudez and A. Bock: Trends Pharmacol. Sci., 2019 3. A. Bock et al.: J. Biol. Chem., 2016 4. M. Bermudez et al.: ACS Chem. Biol., 2017 5. M. Bermudez et al.: manuscript in preparation POS.82 Improvement of bioanalytical sensitivity and COX-2- selective antitumor activity of cobalt alkyne complexes due to ligand fluorination POS.81 Synthesis and biological activity of aminotriazoles Baecker D1; Obermoser V1; Kirchner E A1; Kircher B2,3; Gust R1 targeting human coagulation factor XIIa 1 Department of Pharmaceutical Chemistry, Institute of Pharmacy, CMBI – Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria 2 Immunobiology and Stem Cell Laboratory, Department of Internal Medicine V (Hematology Imberg, L.1; Fluck, G.1; Korff, M.1; Kalinin, D.V.1 and Oncology), Medical University of Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria 3 Tyrolean Cancer Research Institute, Innrain 66, 6020 Innsbruck, Austria 1Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, 48149 Münster, Germany [(Prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl (Co-ASS) is a Thromboembolic conditions are one of the leading reason for mortality metal complex that demonstrated promising growth-inhibitory potency worldwide claiming the lives of more people than AIDS, breast cancer, against certain tumor cell lines [1]. The inhibition of both cyclooxygenase and car accidents combined. The high death toll of thrombosis is isoenzymes (COX-1 and COX-2) is assumed as probable mode of action. explained by its connection to the top three cardiovascular killers: heart However, the selective inhibition of COX-2 is aimed because it plays a attack, stroke, and venous thromboembolism.1,2 Considering a high significant role in carcinogenesis, while COX-1 mainly regulates thrombosis-related mortality rate, urgent measures should be undertaken homeostatic functions [2]. to reduce death rate, disability, and economic losses caused by this With the objective of generating COX-2-selective compounds, the disorder. approach to introduce a fluorine substituent within the aromatic moiety of Anticoagulants are life-saving drugs, which are used to counteract Co-ASS was followed as a frequently applied strategy in medicinal thrombosis. However, all currently used anticoagulants (e.g. 1-3) exhibit chemistry [3]. Moreover, it was intended to exploit the fluorination for the a life-threatening side effect of internal bleeding3 as they target vital purpose of bioanalytical labeling. enzymes of the coagulation cascade. Whereas mainstream research in The influence of the fluorine substituent on the cytotoxic and the this field is focused on “well-established” drug targets (thrombin and antimetabolic properties against colon (HT-29) and breast cancer cell factor Xa), we strive to develop novel inhibitors of Hageman Factor lines (MDA-MB-231, MCF-7) was determined. The inhibition of both (FXIIa, one of a key player in thrombosis development) causing no cellular and isolated COX as well as the induction of the apoptosis-related internal bleeding risk.4,5 caspases 3/7 were evaluated. Additionally, cellular uptake studies were conducted based on the sequential analysis of both cobalt and fluorine performing high-resolution continuum-source atomic respectively molecular absorption spectrometry (HR CS AAS / MAS). The compounds exhibited a remarkably less activity in the COX-1/2- negative MCF 7 cell line, while they reduced the cell biomass in the COX-

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1/2-positive HT-29 and MDA-MB-231 cells in the low micromolar range POS.84 and showed a concentration-dependent antimetabolic activity. Fluorination effected a preferential inhibition of COX-2 compared to Co- Polyamine-Based Activity Assay for the Histone ASS. Furthermore, it was demonstrated in cellular uptake studies that an Deacetylase 10 (HDAC10) about 5-fold higher bioanalytical sensitivity (HR CS MAS technique) can be achieved when using fluorine as tracer instead of cobalt. 1 2 2 1 Fluorinated cobalt alkyne complexes derived from Co-ASS were Daniel Herp , Stephen Shinsky , David Christianson , Manfred Jung 1 Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 25, 79104 Freiburg synthesized and characterized as promising antitumor active agents. The im Breisgau, Germany outcomes confirm that the interaction with the COX cascade can be 2 Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, assumed as mode of action and fluorination shifted the selectivity 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, USA Email: [email protected] towards COX-2. Moreover, the measurement of fluorine using HR CS MAS was proved as a highly sensitive method for the quantification of fluorinated cobalt complexes in biological samples. While histone deacetylases (HDACs) as epigenetic enzymes are related to tumorigenesis, developmental disorders and neurodegenerative Financial support by the “Tiroler Wissenschaftsfonds (TWF)” (GZ: UNI-0404/1982) is gratefully diseases, they are also involved in physiological developmental acknowledged. processes.1 All HDAC inhibitors approved up to now by the FDA, also the new generation like Panobinostat (Farydac®), are non-selective 1. Ott, I. et al.: J. Med. Chem. 2005, 48: 622-629 . inhibitors hitting several HDAC subtypes. The lacking specificity causes 2. Goradel, N. H. et al.: J. Cell. Physiol. 2019, 234: 5683-5699. a wide range of undesirable side effects. Reducing these side effects is 3. Gillis, E. P. et al.: J. Med. Chem. 2015, 58: 8315-8359. a main goal of drug discovery. Therefore, specific test systems for the different HDAC subtypes are required. Recently the substrate spectrum of HDAC10 was determined. Due to the singularity in the amino acid structure containing a glutamic acid as POS.83 gatekeeper residue, HDAC10 was described as a polyamine Fragment screening with STD-NMR to identify new deacetylase.2 According to this data we developed a selective assay inhibitors of the anti-infective target DXS system for HDAC10. A polyamine-based AMC-substrate (1) was synthesised. Adapted from an established assay platform a suitable fluorescence assay system for HDAC10 was designed and validated.3 1,2 1,3 1 1,2,3 Johannsen, S. ; Gierse, R. M. ; Haupenthal, J. ; Hirsch, A. K. H. Fluorescence of the deacetylated product (2) is quenched by 1Helmholtz-Institute for Pharmaceutical Research Saarland, Campus E 8.1, 66123 Saarbrücken, Germany derivatization with naphthalene-2,3-dialdehyde (NDA). Measuring the 2Department of Pharmacy, Saarland University, Campus C 2.2, 66123 Saarbrücken, Germany remaining fluorescence allows to determine the HDAC10 activity. 3Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, A strong effect on HDAC10 was recently shown for the “HDAC6 The Netherlands selective” inhibitor Tubastatin A.4 Using the newly developed assay system we were able to confirm HDAC10 inhibition of Tubastatin A and The WHO reports that tuberculosis is among the ten top causes of death other common HDAC inhibitors. This data implicates new chances for worldwide with 1.6 million estimated deaths and 10.0 million new inhibitor design. infections in 2017. An alarming number of 558 000 people developed a rifampicin-resistant form of tuberculosis (82% of those were also multi- drug resistant).[1] In this dangerous situation, identification of new drug targets with an unprecedented mode of action and novel inhibitors are urgently required for the advancement of drug discovery against multi- drug resistant Mycobacterium tuberculosis. One of the promising and underexplored targets is 1-deoxy-D-xylulose- 5-phosphate synthase (DXS), an enzyme from the methylerythritol phosphate (MEP)-pathway that is entirely absent in humans but is essential for medically relevant pathogens (e.g. Mycobacterium tuberculosis, Plasmodium falciparum).[2] DXS not only catalyses the first and rate-limiting step of the MEP-pathway but its product is an important precursor for the biosynthesis of essential vitamins (B1 and B6) in bacteria. Several inhibitors of DXS have been published to date. While they are very potent in vitro, they often show only weak activity in cell based assays.[3] To improve selectivity and potency, more research into the MEP-pathway in general and DXS in particular is urgently needed. Funding by Deutsche Forschungsgesellschaft (DFG) Ju 295/13-1. A ligand-based virtual screening afforded three structurally unrelated hit 1. Drazic, A. et al., The world of protein acetylation. BBA-Proteins Proteom 2016, 1864 (10), classes, two of which show fragment-like characteristics, combined with 1372-1401. an excellent ADMET profile and activity in the single-digit micromolar 2. Hai, Y. et al., Histone deacetylase 10 structure and molecular function as a polyamine range against Plasmodium falciparum and Mycobacterium tuberculosis deacetylase. Nat Commun 2017, 8. in cell-based assays.[4] To enhance the structural diversity of our hits, 3. Heltweg, B., Jung, M., A homogeneous nonisotopic histone deacetylase activity assay. J Biomol Screen 2003, 8 (1), 89-95. we initiated a fragment screening campaign using a halogen-enriched 4. Geraldy, M. et al., Selective Inhibition of Histone Deacetylase 10: Hydrogen Bonding to the fragment library with 148 compounds. Gatekeeper Residue is Implicated. J Med Chem 2019, 62 (9), 4426-4443. Saturation-transfer difference (STD)-NMR was chosen for the initial screening in a hope to obtain additional information about the pharmacophore. Dividing the library into 15 screening cocktails enabled us to identify the best binders. To confirm binding, we relied on microscale thermophoresis (MST) and determined Kd values of the most promising fragments. POS.85 Based on these results, virtual and synthetic fragment growing will be Synthesis and pharmacological evaluation of used to identify a potent inhibitor of DXS as a potential new agent against conformationally restricted κ-opioid receptor agonists Mycobacterium tuberculosis. Gao, D.1, Wünsch, B.1 [1] WHO: Global Tuberculosis Report 2018 (World Health Organization) 2019. 1 Institute of Pharmaceutical and Medicinal Chemistry, Corrensstraße 48, 48149, Germany [2] Wang, X.; Dowd, C. S.: ACS infectious diseases 2018, 4, 278. [3] a) Bartee, D. et al.: Acc. Chem. Res. 2018, 51, 2546; b) Masini, T. et al.: Chem. Sci. 2014, 5, 3543; c) Smith, J. M. et al.: J. Antibiot. 2014, 67, 77. In the field of κ-opioid receptors (KOR), research has been focused on [4] Manuscript under review. the analgesic activity of KOR agonists for decades. KOR are widely expressed in the central nervous system (CNS) and peripheral tissues. In contrast to μ-opioid receptor (MOR) agonists, KOR agonists do not

DPhG Annual Meeting 2019 Conference Book • 118 MEDICINAL CHEMISTRY AND DRUG DESIGN produce adverse and dangerous side effects like euphoria, respiratory depression, constipation, tolerance, and physical dependence. However, centrally active KOR agonists produce centrally mediated side effects such as sedation, dysphoria and diuresis. Peripherally restricted KOR agonists can avoid these CNS side effects and might be useful for the treatment of visceral and neuropathic pain, pruritus, and inflammation.1 Therefore, research has been focusing on KOR agonists with peripheral restriction. Figure 1. Telmisartan (left) and the general structure of the derived compounds (right).

The activation of PPARγ was analyzed in COS-7 cells employing a Dual Luciferase Assay. Drug efficacy on resistant CML cells (K562-resistant) was evaluated in combination with imatinib in an apoptosis assay. The novel compounds themselves were not cytotoxic against non- resistant and resistant cells. However, when combined with imatinib, Since KOR affinity and selectivity are strongly dependent on the three compounds (X = H, F, Br; R = NH2) resensitized the cells and a stereochemistry,2 we developed conformationally restricted KOR significant induction of apoptosis was observed. agonists derived from GR-89696. It is a high-affinity and very potent KOR In conclusion, apoptosis was caused in resistant cells treated with agonist but contains a rather flexible pyrrolidin-1-ylmethyl residue.3 In imatinib together with the non-cytotoxic compounds. Our investigations order to analyze the bioactive conformation, the ethylenediamine clearly demonstrate that this combination represents an opportunity for pharmacophore was conformationally fixed by incorporation into a resensitizing resistant CML cells. Thus, the therapeutic breakthrough in bicycle framework. CML using imatinib can be maintained efficiently and drawbacks of the On the poster the synthesis of the four stereoisomers of 1 is presented. treatment might be overcome. This approach also appears promising for The (1S,5S,6R)- stereoisomer 1a shows the highest KOR affinity of the circumventing drug resistances in other cancers, especially if TKIs are four stereoisomers. The N-benzyl moiety outside the pharmacophoric applied as therapeutics. ethylenediamine system allows further chemical modifications (e.g. 2, 3) and thus modification of pharmacokinetic properties (e.g. passage of the 1. Prost, S. et al.: Nature 2015 525: 380–383. blood brain barrier). 2. Rousselot, P. et al.: Cancer 2017 123: 1791–1799. 3. Samukawa, E. et al.: Int J Oncol 2017 51: 1674–1684.

References: 1. Vanderah, T. W.: The Clinical Journal of Pain 2010, 26: S10-S15. 2. Soukara, S. et al.: J. Med. Chem. 2001, 44 (17): 2814-2826. 3. Birch, P. J. et al.: Br. J. Pharmacol. 1991, 103(3): 1819-23.

POS.86

PPARγ modulators as add on agents to resensitize imatinib-resistant CML cells

Schoepf AM1, Salcher S2, Obexer P2,3, Gust R1 1 Department of Pharmaceutical Chemistry, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria 2 Tyrolean Cancer Research Institute, Innrain 66, 6020 Innsbruck, Austria 3 Department of Pediatrics II, Medical University of Innsbruck, Innrain 66, 6020 Innsbruck, Austria

Recent studies examined the achievement of deeper molecular response in the treatment of chronic myeloid leukemia (CML). A new strategy is based on the combination of the tyrosine kinase inhibitor imatinib with Peroxisome Proliferator-Activated Receptor gamma (PPARγ) ligands [1]. Pioglitazone, a full agonist of PPARγ, has been screened for the therapy of resistant CML and revealed the gradual elimination of the residual CML stem cell pool in combination with imatinib [2]. The partial PPARγ agonist telmisartan (Figure 1) was studied concerning its effect in cancer therapy as well [3]. It is preferable to pioglitazone due to its lower potency on PPARγ and thus having less side effects. Our group showed for the first time that telmisartan, when combined with imatinib, can circumvent the resistance of the CML cells more effectively than the full PPARγ agonist pioglitazone. These findings induced us to modify telmisartan leading to two series of compounds (Figure 1, X = H, F, Br, OH, NH2). The carboxylic acid series (R = OH) was aimed to activate PPARγ comparably to telmisartan, while the series bearing a carboxamide moiety (R = NH2) was assumed to show lower activation. Hence, the correlation between the impact on PPARγ and the potency of the compounds on resistant CML cells was intended to be understood.

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Pioneering human induced pluripotent stem cell (iPSC)-based pre- 4.2 Other Topics clinical studies have raised safety concerns and pinpointed the need for safer and more efficient approaches to generate and maintain patient- specific iPSCs. One approach is searching for compounds that influence POS.87 pluripotent stem cell reprogramming using functional screens of known PubPharm: from informatics research to search tools for drugs. Our high-throughput screening of drug-like hits showed that imidazopyridines—analogs of zolpidem, a sedative-hypnotic drug—are pharmacy-specific literature able to improve reprogramming efficiency and facilitate reprogramming of resistant human primary fibroblasts. The lead compound (O4I3) Draheim, C.1; Keßler, K.1; Kroll, H.2; Wawrzinek, J.2; Wulle, S.1; Balke, showed a remarkable OCT4 induction, which at least in part is due to the W.-T.2; Stump, K.1 inhibition of H3K4 demethylase (KDM5, also known as JARID1). 1 Technische Universität Braunschweig, Universitätsbibliothek, Universitätsplatz 1, 38106 Experiments demonstrated that KDM5A, but not its homolog KDM5B, Braunschweig, Germany serves as a reprogramming barrier by interfering with the enrichment of 2 Technische Universität Braunschweig, Institut für Informationssysteme, Mühlenpfordtstraße 23, 38106 Braunschweig, Germany H3K4Me3 at the OCT4 promoter. Thus, our results introduce a new class of KDM5 chemical inhibitors and provide further insight into the pluripotency-related properties of KDM5 family members.

PubPharm (www.pubpharm.de) is a free search platform, which provides a comprehensive set of more than 55 million pharmacology-, chemistry- and pharmacy-specific publications. Among its unique characteristics is the capability to search in many different literature resources, e.g. subject-specific journal articles, books (e-books, PhD theses), conference papers and information on clinical trials, with a single query. Through an availability check based on location, the full text of many publications can be directly accessed. One major goal of PubPharm is to address the exponential growth of scientific publications, which is making it increasingly difficult to find and access relevant information in databases, by collaborating with the Institute of Information Systems at Braunschweig University. Term-based searches usually lead to a vast number of results. A suitable means for tackling this problem is the automatic extraction of important pharmaceutical entities included in publications as well as their known Acknowledgements: and hidden relationships with each other. State-of-the-Art approaches in 1. Deutsche Forschungsgemeinschaft (DFG) grant program (CH 1690/2-1), Cheng, X.; Gama- the field of artificial intelligence provide new opportunities to learn, to Brambila, RA. recognize and in future to predict these entity-relationships. Results of 2. Landesgraduiertenförderung (LGF) fellowship program for individual doctoral training from research in this field are being applied in PubPharm as new and Universität Heidelberg, Dabiri, Y. innovative search tools [1]. Through this, related substances, diseases 3. Bundesministerium für Bildung und Forschung (BMBF) grant programs; Drug-iPS (FKZ and genes are displayed in PubPharm when searching for a drug 0315398A/B) and SysToxChip (FKZ 031A303A/E), Cheng, X. substance. PubPharm also includes structure, similarity and substructure search References: capabilities. For each search result, links to relevant sources (DrugBank, 1. Dabiri, Y. et al. Imidazopyridines as Potent KDM5 Demethylase Inhibitors Promoting PubChem, ChEMBL) and patent documents are provided. Reprogramming Efficiency of Human iPSCs: iScience. 2019, 12: 168-181. The Specialised Information Service (SIS) Pharmacy 2. Papp, B, and Plath, K. Epigenetics of reprogramming to induced pluripotency: Cell. 2013, 152(6): 1324-1343. (Fachinformationsdienst Pharmazie), which aims at sustainably improving the supply of literature for academic pharmaceutical research in Germany, is responsible for developing PubPharm [2]. The project is being funded by the German Research Foundation (Deutsche Forschungsgemeinschaft) since January 1st, 2015 and has been POS.89 approved for a second funding phase lasting until 2020. The collaboration Average performance and failure rates in the 1st pharmacy state between the University Library and the Institute for Information Systems exam: Developments in the respective 4 examination modules at Braunschweig University results in innovative retrieval tools based on following major changes in legal regulations cutting edge informatics research, such as described above, and is being conducted by involving the pharmaceutical research community. N. Winter1, J. Schubert1, A. Braun1, J. Schleißmann1, J. Jünger1, V. Schillings1, H. Spahn1, H. Shahla1 More information about SIS Pharmacy and PubPharm can be found here: 1IMPP (Institut für medizinische und pharmazeutische Prüfungsfragen), Fachbereiche https://ub.tu-braunschweig.de/fid_pharmazie/englisch/index_en.php Pharmazie und EDV, Statistik und Dokumentation, Malakoff-Passage, Rheinstr. 4, 55116 Mainz

Almost worldwide in pharmaceutical education, the fields of basic Acknowledgments: This work was funded by the Deutsche Forschungsgemeinschaft. sciences as a basis of pharmaceutical and pharm.-clinical sciences (pharmaceutical biology and biotechnology, pharmaceutics, References 1. Wawrzinek, J. et al.: International Conference on Asian Digital Libraries. 2017, 41–53. pharmacology, medicinal chemistry or clinical pharmacy), that belong to 2. Stump, K. et al.: Pharmakon. 2018, 6(4): 260-266. the undergraduate study program, have not been focusing on the patient yet, but rather on understanding natural sciences and their relevance for the pharmacy curriculum. As opposed to countries with pre-pharmacy requirements (as accomplished in college programs), in Germany natural science education is fully integrated into the first two years of studying and is supposed to be as much pharmacy-relevant as possible. POS.88 With changes in the legal regulations (2nd AAppO-ÄndV, 2000) the Imidazopyridines as Potent KDM5 Demethylase Inhibitors relevance of pharmaceutical content was enforced within this period of time (referred to as “2nd period”), for instance with the introduction of Promoting Reprogramming Efficiency of Human iPSCs additional subjects that focus on patients and on drug formulation (basic pharmaceutics, anatomy/physiology). The state examination modules Dabiri, Y.1; Gama-Brambila, RA. 1; Cheng, X.1 consist of 4 independent submodules (= 4 separate MCQ exams = 4 1Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer subject groups), (I) pharmaceutical chemistry, (II) pharmaceutical Feld 364, 69120, Heidelberg, Germany biology, biochemistry/biotechnology, (human biology in the 2ndperiod), (III) physics, physical pharmacy, (drug formulation in the 2nd period), (IV) pharmaceutical analysis and QC. This enforcement was accomplished

DPhG Annual Meeting 2019 Conference Book • 120 OTHER TOPICS via a process that was designed to be minimally irritating to the notwendigen Reparaturarbeiten an dem Haus durchgeführt. Derart candidates and that took constructive alignment and the necessary instandgesetzt konnte es 1949 (nach Neuorganisation der change management respectively into consideration. apothekerlichen Interessenverbände in Berlin) an den neu gegründeten The major outcome characteristics (average grades and failure rates) BAV übergeben werden. In den darauf folgenden Jahrzehnten war die and their consistencies have been compared between the periods before Carmerstraße 3 nicht nur Schauplatz der wechselvollen jüngeren (“1st period”) and after (“2nd period”) the implementation of the new Vereinsgeschichte des BAV, auch anderen Organisationen ‒ wie die concept. DPhG-Landesgruppe Berlin ‒ nutzten gern und oft die sich dort anbietenden und recht ansehnlichen Räumlichkeiten. Nachdem die Aim: Subject of the retrospective data analyses were the analyses of Apothekerkammer Berlin 1963 neu entstand, zog diese ebenfalls in das exam outcomes for the 4 examination modules of the 1st pharmacy state Apothekerhaus. Damit beherbergte das Gebäude eine weitere wichtige exam and the comparison between the examination performance before Apothekerorganisation. Die beengt werdenden räumlichen Verhältnisse and after the changes in the legal regulations (2nd AAppO-ÄndV, 2000). stellten die Verantwortlichen jedoch vor große Herausforderungen. Es gelang dem BAV aber ‒ trotz zum Teil anders lautender Pläne ‒ die Villa Methods: For this purpose the average performance and the failure rates größtenteils im ursprünglichen Zustand zu bewahren und darüber hinaus of these examination modules have been analyzed separately for the auch modernen Standards anzupassen. Die Apothekerkammer zog in periods before and afterthe changes in the legal regulations (2nd AAppO- den 90er Jahren aus und suchte sich ein neues Domizil ‒ der Verein ÄndV, 2000). Moreover, trend tests were performed to detect significant verblieb in seinem Vereinshaus. Heute gilt es die Carmerstrasse 3 als differences between these 2 time periods in focus. Baudenkmal. 2019 befindet es sich seit nunmehr 87 Jahren (Stand 2019) in Besitz des BAV, der selbst bald wieder sein eigenes Results: The arithmetical means of examination grades calculated since Bestehensjubiläum feiern wird. 1984 for (I) – (IV) amount to (I) 3.23, (II) 3.13, (III) 3.10, (IV) 3.13. The average failure rates were (I) 15.4 %, (II) 18.4 %, (III) 14.9 %, and This research was kindly funded by a stipend from Berliner Apotheker-Verein. Apotheker- (IV) 13.3 %. Verband Berlin (BAV) e.V. The arithmetical means of the grades were fairly stable for each of the analyzed periods. Neither positive nor negative trends were detected Apotheker Semmel am 26. November 1932., zitiert in Pharmazeutische Zeitung 102 (1957), S. (trend test, n.s.). Yet, for each of the submodules average grades indicate 1305. a decay in performance within the new concept (2nd period): (I) 3.14 → ABAV-OG-HAUS-1932-1993; sowie Auskunft des Landesdenkmalamtes Berlin. 3.25, (II) 2.97 → 3.25, (III) 2.91 → 3.30, (IV) 2.96 → 3.28. ABAV-HR-O(RS-TH)-1945-1947, Rundschreiben vom 9. April 1947; B Rep 079 Nr. 113 (Microfilm); sowie J. Hadrich (1947) in Pharmazeutische Zeitung 83 (1947), S. 77f. Conclusion: The introduction of new subjects -apparently- increases the ABAV-HR-O(RS-TH)-1945-1947, Rundschreiben vom 9. April 1947; complexities of each of the respective MCQ submodules (and, hence, ABAV-S14-RS-1968-1971. 22. Mai 1969. Rundschreiben Nr. 2 und Geschäftsbericht 25. Juni 1970. their difficulties) as well as the complexity of the whole 4 independent ABAV-S14-RS-1978-1982. Geschäftsbericht 1981 und VHS-Videos im ABAV. submodules. However, failure rates have remained largely unchanged. It ABAV-OG-HAUS-1932-1993. Abschrift vom 23. Mai 1935 can be concluded that the change process was smooth and successful; Norbert Bartetzko zum 50jähigen Kammerjubil äum. Vgl. 50jahre-akberlin.de/zeitzeugen und yet, the character of the examination modules have changed to some 50jahre-akberlin.de/die-kammer/standorte extent. A. Adlung. Der Berliner Apotheken-Verein in seiner geschichtlichen Entwicklung (1932), S. 11.

POS.90 POS.91 Stammsitz der Berliner Apotheker ‒ Chronik der 128jährigen Geschichte des Hauses in der Carmerstraße 3 Strengthening of the pharmacy-specific focus of the first state examination through the AAppO revision of 2000: More than a decade of MCQ state exams with human Ingmar Allisat1; Christoph Friedrich2 1 Philipps-Universität Marburg, Institut für Geschichte der Pharmazie, Roter Graben 10, 35037 biology and basic pharmaceutics Marburg, Deutschland

2 Philipps-Universität Marburg, Institut für Geschichte der Pharmazie, Roter Graben 10, 35037 Judith Schubert1, Nikola Winter1, Hilde Spahn-Langguth1, Jasmin Marburg, Deutschland Schleißmann1, Hossein Shahla1, Jana Jünger1, Volker Schillings1, Andreas Braun1 Der Berliner Apotheker-Verein (BAV) gilt als die zweitälteste Vereinigung 1IMPP (Institut für medizinische und pharmazeutische Prüfungsfragen), Fachbereiche von Apothekern in Deutschland. Seine Anfänge reichen dabei zurück bis Pharmazie und EDV, Statistik und Dokumentation, Malakoff-Passage, Rheinstr. 4, 55116 Mainz das Jahr 1723. Damals bestand ein lockerer Zusammenschluss von Apothekern, die aus gegebener Veranlassung ihre Treffen abhielten. Auf In other countries than Germany the pharmacy curriculum may determine Befehl Friedrich Wilhelm I. erwarben sie 1732/33 ein Wohnhaus. Ein that pre-pharmacy studies cover the students’ education in basic großer Vorteil erwuchs den Apotheker daraus aber nicht, da der sciences. In Germany, this basic part is integrated into the pharmacy „Soldatenkönig“ derartige Maßnahmen nur im eigenen Interesse curriculum and supposed to be pharmacy relevant. According to the durchführte, weil er beabsichtigte, die Stadt Berlin zur königlichen AAppO, in general, exam objectives (as derived from and agreed within Residenzstadt auszubauen. Den Ansprüchen der Berliner constructive alignment) need to be of pharmaceutical relevance. Apothekerschaft genügte das Haus nicht. Sie verwarfen in der Mitte des The two entirely new subject fields introduced into the exams (via a 18. Jahrhunderts den Plan dort eine gemeinsame Apotheke einzurichten revision of the AAppO in the year 2000) are human biology (clinical und verkauften das Objekt während der Regierungszeit Friedrich II. Ab anatomy and physiology) as well as pharmaceutics with focus on drug 1774 fanden die Versammlungen der „Berliner Apotheker-Conferenz“ formulation (compounding), and both of them are of high intrinsic wieder in angemieteten Räumen statt. 1856 benannte man sich in pharmaceutical relevance. The first part of the state examination consists „Verein der Apotheker Berlins“ um und fasste 1888 den generellen of 4 independent submodules (= 4 separate MCQ exams = 4 subject Entschluss ein neues Haus zu kaufen und es als Vereinshaus groups): (I) pharmaceutical chemistry, (II) pharmaceutical biology, einzurichten. Bis zur machbar scheinenden Umsetzung des Beschlusses biochemistry/biotechnology, human biology, (III) physics, physical teilte man sich solange mit dem Deutschen Apotheker-Verein (DAV) ein chemistry, drug formulation, (IV) pharmaceutical analysis and QC. gemeinsames Haus. 1925 wurde ein Hausbaufond eingerichtet und 1932 The candidates’ performance within these new subject fields is schließlich das ehemalige Professorenhaus „Villa Hirschfeld“ in der characterized via the analyses of their contribution to the overall outcome Carmerstraße 3 erworben. Im Besitz des BAV verblieb dieses allerdings in the respective exam submodules, (II) for “biology”=day 2 and (III) for nur kurze Zeit. Bald begann die Zeit des Nationalsozialismus und der “physics”=day 3. Verein wurde in die eigene Selbstauflösung getrieben. Sein gesamter Besitzstand ging an die nationalsozialistischen Organisationen über. Aim: Retrospective analyses of the 2 out of a total of 4 exam days include Glücklicherweise überstand das Apothekerhaus den Zweiten Weltkrieg a survey of the exam contents, potentially favored topics within the relativ schadlos. Die britische Militärverwaltung setzte eine blueprint, of the test question performance parameters, and of the treuhändische Verwaltung ein, die für den Besitzstand ehemaliger candidate performance, from year 2006 onwards. Reichsorganisationen verantwortlich war. Unter ihrer Ägide wurden die

121 • DPhG Annual Meeting 2019 Conference Book POSTERS

Methods: The test question analyses include the calculation of average Funding by the DFG within FOR1738 and SFB 813 is gratefully acknowledged. performance parameters (r- and p-values) for each exam, i.e., a total of 24 performance parameters resulted, since two exam dates per year References: have been analyzed. The p-value indicates the difficulty of an item, the r- 1. Kühl, T., Imhof, D.: ChemBioChem 2014, 15(14):2024–2035. value the discriminatory power of an exam item. 2. Wißbrock, A. et al.: J. Med. Chem. 2017, 60(1):373–385. 3. Atamna, H., Boyle, K.: Proc. Nat. Acad. Sci. 2006, 103(9): 3381–3386. (II): “Biology” consists of residual pharmaceutical biology (RPB) + human 4. WIßbrock, A. et al.: Biosci. Rep. 2019, 39(1): BSR20181940. biology (HB), 73 vs. 27 test items; 5. Brewitz, H.H. et al.: Biochim. Biophys. Acta 2016, 1860(6): 1343–1353. (III): “Physics” consists of physics and physical chemistry (PPC) + physical pharmacy and formulations (PPF), 60 vs. 20 test items.

Results: With respect to human biology as well as pharmaceutical POS.93 compounding, the selected topics of items are evenly distributed over the respective catalogue with some enhanced focus on the (central) nervous Towards scaffold-diverse DNA-encoded small molecule and the cardiovascular systems. libraries (II): Within-exam average r-values yield arithmetical means 0.27 for HB and 0.28 for RPB (ranges, 0.23 – 0.30 for HB and 0.25 – 0.31 for RPB), average p-values amounts to 60.4 % for HB (range, 48.0 % - 65.7 %) and Andreas Brunschweiger 60.1 % for RPB (range 55.2 % - 64.6 %). Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Str. 6, 44227 (III): Arithmetical means of the respective average r-values for physics to Dortmund, Germany drug formulation amount to 0.22 for PPF and 0.27 for PPC (range, 0,17 – 0,25 for PPF and 0,25 – 0,30 for PPC), p-values were 64.9 % for PPF Compound libraries barcoded with DNA, called DNA-encoded libraries (range, 50.8% - 73.3%) and 58.4 % for PPC (range, 51.4% - 65.1%). (DELs), offer an efficient, high-throughput platform for target-based screening.[1] DELs translate molecular biology techniques for molecular Conclusions: The retrospective analyses indicate that - for candidates - evolution such as DNA barcoding, selection, DNA amplification, and DNA human biology is as difficult as residual pharmaceutical biology and sequencing to combinatorial compound synthesis and identification.[2] physical pharmacy and drug formulation appears less difficult than We developed computer-based statistics tools to design DNA barcodes, physics and physical chemistry. No significant trend of r-/p-values was and to deconvolute sequencing data for screening outcome detected in any of the sequences (trend-test). interpretation. The productivity of any screening library depends not the least on chemical space coverage. Access to diverse compound structures depends on a combination of available starting materials and synthesis methodology. Solution-phase DEL synthesis requires chemical POS.92 methods that show a combination of DNA-compatibility, water-tolerance and fast kinetics to operate on highly diluted DNA-tagged reactants. His-/Tyr-based heme-binding peptides: From heme binding Ideally, they are operationally simple and furnish target compounds from to catalytic function diverse starting materials with high yields. Only a very few chemical reactions meet all these demands, access to encoded heterocyclic chemistry is even rare. In order to expand methodology for encoded Kühl T1; Syllwasschy, B1; Beck, M1; Družeta I; Hopp, M.-T.1; Imhof D1 1Pharmaceutical Biochemistry and Bioanalytics, University of Bonn, An der Immenburg 4, compound synthesis, we are developing DEL coding strategies that are 53121 Bonn initiated with solid phase-bound DNA strands.[3,4] Solid phase synthesis enables the use of a broad scope of organic solvents for target compound synthesis, and harsher reaction conditions because the DNA is fully Transient heme binding to proteins is known to change their structure protected. A solid phase-based DEL strategy allowed for translation of and consequently may alter their function and corresponding several chemistries for encoded library synthesis, among them physiological and/or cellular responses. [1] In addition, in some cases cycloaddition reactions and isocyanide MCR chemistry (Figure 1a).[5] In (e.g. Alzheimer’s disease) it has been shown that interaction with heme a second strategy, we develop bespoke catalysts for encoded library could also lead to the development of a catalytic activity of the complex synthesis based on oil-in-water micelles. Micellar catalysis is an attractive similar to the action of peroxidases. [2,3] principle for DEL synthesis due to both reaction acceleration and DNA In an approach to classify and evaluate motifs for heme binding to short, shielding. Amphiphilic block copolymers formed spherical nanoreactors surface-located peptide stretches in proteins - so-called heme-regulatory and immobilized a catalyst in the DNA-inaccessible hydrophobic core. motifs - a concept was derived to discriminate different binding modes Sulfonic acid-substituted micelles converted DNA-tagged aldehydes by and to predict potential heme-regulated proteins and their heme-binding Povarov and Groebke reactions to diverse substituted sites. [4] This classification was based on the existence of different iron- tetrahydroquinolines and imidazopyridines without noticeable DNA coordinating amino acids as well as distinct surrounding amino acids in degradation, and facilitated tBoc group removal from amines (Figure a short nonapeptide sequence. Extensive studies on more than 150 1b).[6] Furthermore, a Cu(I)-immobilizing micelle mediated the DNA- heme-peptide complexes have been carried out in order to understand compatible oxidation of DNA-tagged alcohols to aldehydes by the their interactions with heme. [4] Peptides containing histidine or tyrosine Cu(I)/bipyridine/TEMPO system. Thus, the concept of micellar catalysis as the iron-coordinating amino acid were studied in more detail earlier. merits investigation of a broad scope of reactions for encoded compound [5] In further attempts to understand heme binding to these peptides we synthesis. then focused on specific subclasses of such histidine- and tyrosine- based motifs. In addition, we examined how these peptides may influence the peroxidase-like activity of heme. In a recent approach we have shown that peptides other than amyloid-beta may possess such a catalytic activity and that these may differ in length and amino acid composition. [2] It turned out that the most promising results were obtained for peptides containing histidine or tyrosine as the coordinating amino acid, however, not all of them showed a peroxidase-like activity Figure 1: Expanding the chemical space of DNA-encoded compound despite a high affinity for heme binding. This motivated us to have a libraries. a) Exploring the scope and limitations of solid phase-based closer look at a selection of app. 50 peptides of different sequence synthesis approaches to encoded libraries. b) A micelle-based reaction compositions to understand requirements for the development of a system for encoded synthesis. peroxidase-like activity in non-covalently linked heme-peptide complexes. Moreover, such complexes would not only be physiologically References 1. Salamon, H. et al.: ACS Chem. Biol. 2016, 19: 296-307. relevant to better understand disorders such as Alzheimer’s disease or 2. Klika Škopić, M. et al.: Med. Chem. Commun. 2016, 7: 1957-1965. lipid oxidation, yet could also represent a starting point for the 3. Klika Škopić, M. et al.: Chem. Sci. 2017, 8: 3356-3361. development of a new class of mimics for proteins such as cytochrome 4. Klika Škopić, M. et al.: Org. Biomol. Chem. 2017, 15: 8648-8654. P450 as diagnostic tools to determine the metabolic pathway of a 5. Potowski, M. et al.: Med. Chem. Commun. 2019, DOI: 10.1039/C9MD00042A drug/prodrug. 6. submitted

DPhG Annual Meeting 2019 Conference Book • 122 ANALYTICS AND TOXICOLOGY

POS.95 4.3 Analytics and toxicology Development of stable bond thiol modified silica with high POS.94 loading density Analytical Toolbox for the Characterization of Coenzyme A- capped Ribonucleic Acids Wolter, Marc1; Lämmerhofer, Michael1 1 University of Tuebingen, Institute of Pharmaceutical Sciences, Auf der Morgenstelle 8, 72076 Tuebingen, Germany Christian Löcherer1, Andres Jäschke1 1 Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, INF 364, 69120 As thiol modified silica is a commonly used material in a wide range of Heidelberg, Germany chromatographic areas, e.g. adsorption and separation of cationic ions, protein separation by thiol-disulfide interchange of sulfhydryl containing proteins in covalent chromatography and in development of stationary NH2 phases as basic material for further modification, this work reports on a N N O O O O study conducted on the synthesis of thiol modified silica gel by thermically N N HS O P O P O induced and photo-induced polymerization reactions. Thereby, poly(3- N N H H O O O mercaptopropyl)methylsiloxane (PMPMS) was coated onto vinyl OH O OH modified silica by radical thiol-ene click polymerization reaction forming O P O a crosslinked layer on its surface. According to literature such bonding O chemistry showed significant better stability in comparison to brush-type analogs with bifunctional siloxane bonding to the silica. Aiming a sulfhydryl group rich, thin PMPMS layer on the surface of the silica several factors such as quantity of PMPMS, radical starter and solvent were investigated. In addition, reaction time and temperature were 3' studied as well. In thermally induced polymerization reactions 2,2'- azobis(isobutyornitrile) (AIBN) was used as radical starter, in photo- Coenzyme A (CoA), synthesized from its precursor pantothenic acid induced reactions 2,2-dimethoxy-2-phenylacetophenone (DMPA) was (Vitamin B5), is a molecule with versatile functions, mainly renowned as used instead. As the thickness of the PMPMS layer correlates with the a metabolic key cofactor throughout all organisms. It essentially total amount of incorporated PMPMS, it was determined indirectly by the participates in the anabolism and catabolism of fatty acids, sugars and sulfur content measured by elemental analysis. For the purpose to amino acids as well as in the aerobic energy production. Furthermore, its assess the amount of unreacted sulfhydryl groups a chemical reaction unique carboxylic acid activation in the form of thioesters is used for the with 2,2'-dipyridyl disulfide (DPDS) was carried out. Here, accessible and acetylation of small molecules, i.e. for the physiological production of the reactive sulfhydryl groups react quantitatively with DPDS to generate neurotransmitter acetylcholine or the phase II biotransformation of drugs equimolar amounts of 2-pyridyl thiol, which was determined by HPLC- like sulfonamides. Biomolecules like histones are acetylated as well, UV/Vis. The estimation of the sulfhydryl group content is necessary, as resulting in a flexible epigenome. Current research suggests not only an an adequate amount is needed for chromatographic or further acetyl transfer to proteins, but additionally a redox-dependent modification issues such as implementation of selective ligand molecules modification of cysteine residues by a disulfide bridge with free CoA itself, on the silica surface. Consequently, suitable reaction conditions were termed CoAlation. figured out in order to generate a thin PMPMS layer on the silica surface All mentioned examples rely on the reactivity of the terminal thiol of CoA, containing a sufficient amount of unreacted sulfhydryl groups. while its nucleoside part seemed to be redundant until recently. In 2009, Ribonucleic acids (RNAs) modified with the cofactors nicotinamide adenine dinucleotide (NAD) and CoA at their 5´-end were identified in bacteria in addition to the canonical triphosphate. Further efforts on the characterization of NAD-RNAs propose that this cap structure also exists in eukaryotes, in parallel to the mRNA cap structure POS.96 7-methylguanylate (m7G). No successful progress was reported for CoA- Determination of the content of albendazole, mebendazole RNAs, though. and praziquantel in pharmaceutical preparations available The development of specific analytical methods for CoA-RNAs will reveal in Northern Tanzania their functional scope in comparison with CoA and other RNA 5´-ends in vitro as well as their relevance in vivo. Biosynthetic strategies to access 3´-dephospho-Coenzyme A (dCoA), Klapper, S.1, Seitzer, M.2, Müller, A. 2, Humphrey, M.3, Holzgrabe, U.1 1 University of Wuerzburg, Institute for Pharmacy and Food Chemistry, Am Hubland, 97074 dCoA thioesters and other analogs were established and resulting Wuerzburg, Germany products are incorporated into model RNAs by in vitro transcription. 2 Klinikum of Wuerzburg Mitte gGmbH, Medical Mission Hospital, Dept. of Tropical Medicine, Functional studies are then performed, i.e. the decapping of CoA-RNAs Wuerzburg, Germany 3 Department of Medical Parasitology and Entomology, Catholic University of Health and Allied by pyrophosphatases to yield monophosphorylated 5´-ends, and Sciences, Mwanza, Tanzania analyzed by thiol-mercury affinity electrophoresis. In vivo experiments address CoA capping rates of E. coli RNAs during different growth conditions on the one hand. Therefore, RNAs are Soil-transmitted helminth infections are one of the most common digested and analyzed by liquid chromatography-tandem mass infections worldwide. Deprived communities in tropical and subtropical spectrometry (LC-MS/MS). On the other hand, CoA-RNA specific areas show an increased prevalence, in particular in sub-Saharan Africa sequencing protocols aim to identify highly CoA-modified RNA [1]. sequences, which are examined in detail by customized Northern blot However, anti-infective drugs like anthelmintics are highly affected by analyses in a next step. pharmaceutical counterfeiting [2, 3]. The outcomes will disclose an entire new perspective on both, the Due to the incomplete data on substandard and falsified medicinal cofactor CoA and RNA cap structures. products, in total 42 batches of anthelmintic drugs, including albendazole (ABZ), mebendazole (MBZ) or praziquantel (PZQ),were collected in the northwest of Tanzania from randomly selected local suppliers to References: determine the content of the active pharmaceutical ingredient (API) by 1. Gout, I.: Biochem Soc Trans. 2018, 46(3): 721–728. HPLC-UV. In addition, 54 batches were collected in West Africa (Côte 2. Kowtoniuk, W.E. et al.: PNAS 2009, 106(19): 7768-7773 3. Chen, Y.G. et al.: Nat Chem Biol. 2009, 5(12): 879-81 d'Ivoire, Ghana and Burkina Faso). The method used for the 4. Cahová, H. et al: Nature 2015, 519: 374–377 determination of the content of ABZ, MBZ and PZQ are related to the 5. Jiao, X. et al: Cell 2017, 168(9): 1015-1027 assay described in the International Pharmacopeia [4] and European Pharmacopeia [5] respectively.

123 • DPhG Annual Meeting 2019 Conference Book POSTERS

115 Introduction: Meaningful size characterization of nanoparticles is a major prerequisite 110 for the successful development and use of nanoparticles in pharmaceutics. Size characterization of nanoparticles involves not only 105 the determination of the physical stability during storage but should also be used to ensure sufficient stability of the particles during in-vivo studies 100 [1]. Today, most frequently techniques include microscopic techniques, dynamic light scattering and laser diffraction. However, these techniques are not always predictive and thus other techniques might be necessary 95 to yield discriminative and meaningful results.

content [%] Methods: 90 In this study the Coulter-Counter principle (Multisizer 4e, Beckmann- Coulter, Germany) was used for the characterization of three different 85 nanocarrier systems (nanocrystals, lipid nanoparticles, solid lipid nanoparticles). Results obtained were compared to the results obtained 80 by the classical methods, i.e. dynamic light scattering, laser diffraction ABZ MBZ PZQ and light microscopy. Measurements were performed in water and in isotonic sodium chloride solution. In addition, for all samples, zeta potentials were analysed in each dispersion medium. Figure 1: Boxplot of all tested samples divided into API-classes Results: Classical size characterization of the nanoparticles yielded mean particle sizes of about 150 - 200 nm for the lipid nanoparticles and of about 400 115 nm for the nanocrystals. In contrast, much larger sizes were observed, when the particles were analysed with the Multisizer. Reasons for this 110 are the need to analyse the particles in isotonic sodium chloride solution, which led to a strong decrease in zeta potentials (decrease > 25 mV) and thus to a partial agglomeration of the formulations. The destabilizing 105 effect of the sodium chloride solution was not reliably detected by dynamic light scattering and laser diffractometry measurements. 100 Reasons for this might be the formation of rather loose and low amounts of large agglomerates which were too large to be detected by dynamic light scattering and/or were destroyed by the stirrer of the laser 95 diffractometer. Conclusion: content [%] Classical methods for particle size characterization, e.g. dynamic light 90 scattering and laser diffraction, yield reproducible results when samples are analysed in water. However, large agglomerates can be overlooked. 85 In this study, destabilization of the samples due to the addition of electrolytes was only reliably detected by utilization of the Coulter- Counter principle. In fact, if size characterization is done to predict the 80 physical stability of samples in isotonic solutions, e.g. cell culture media, A B C D E impedance measurements utilizing the Coulter-Counter principle are advantageous.

Figure 2: Results of the batch-analysis of the tested drug products (♦ ABZ, ● MBZ, ■ PZQ) with at least one sample with API content out of References: 1. Stahr, P. et al.: Mater. Sci. Eng. C 2019, submitted. specification limits (dotted lines)

Depending on the availability, one to six samples per batch were examined. So far, 94 samples out of 33 batches from Tanzania were POS.98 tested. Unravelling seven samples out of five batches did not complied with specifications of the current pharmacopeias. Binding affinity and binding geometry of the NAD(P)H cofactor to alcohol dehydrogenases studied by circular Acknowledgments: University of Wuerzburg: Klaus Schilling and Sebastian Schmidt. dichroism spectroscopy

1. WHO Fact sheet: Soil-transmitted helminth infections, 2019. Available from: Lüdeke, S.1; Marolt, M.1 https://www.who.int/news-room/fact-sheets/detail/soil-transmitted-helminth-infections (accessed 1Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstr. 13.06.2019). 25, 79104 Freiburg, Germany 2. WHO Fact sheet: Substandard and falsified medical products, 2018. Available from: https://www.who.int/en/news-room/fact-sheets/detail/substandard-and-falsified-medical- products (accessed 13.06.2019). The initial step in reactions catalyzed by NAD(P)H-dependent alcohol 3. Delepierre, A., Gayot, A., Carpentier, A.: Med Mal Infect. 2012, 42(6): 247-255. dehydrogenases (ADHs) is the binding of the cofactor to the active site. 4. The International Pharmacopoeia - Seventh Edition (WHO Department of Essential Medicines and Health Products) 2017. We monitored the binding of the cofactor in circular dichroism (CD) 5. European Pharmacopeia – Ninth Edition (EDQM) 2017. spectra recorded at different concentrations of NAD(P)H in presence of purified enzymes (ADH from horse liver, HLADH; ADH-A from Rhodococcus ruber; YGL157w from Saccharomyces cerevisiae) or enzyme-containing whole cell extract (ADH from Lactobacillus brevis, POS.97 LbADH). In previous studies, we have demonstrated that matrix least- squares global fitting of chiroptical data can be used to elucidate Utilization of the Coulter-Counter principle for size analysis thermodynamic parameters and other properties of chiral dynamic of nanoparticles systems.[1-3] In a similar way, we analyzed the NAD(P)H-titration spectra to determine the proportions of binding and non-binding NAD(P)H and the associated dissociation constants (Kd) on the basis of a law of mass action-model (Figure 1). Furthermore, the analysis allowed Knoth, D.1; Keck, C. M.1 the isolation of the CD spectrum associated with the cofactor in its bound 1Philipps-Universität Marburg, Department of Pharmaceutics and Biopharmaceutics, Robert- conformation.[4] Through comparison to CD spectra calculated at the Koch-Str. 4, 35037 Marburg, Germany sTD-DFT level we found that NADH and NADPH exhibit an opposite

DPhG Annual Meeting 2019 Conference Book • 124 ANALYTICS AND TOXICOLOGY

orientation of the cofactor nicotinamide ring in their bound conformations. This coincides with a switch of the sign of the most prominent band in the POS.100 associated CD spectra.[4] The development of a second dimension for the 2D-LC separation of oligonucleotides

Li, F. Y.1; Bäurer, S.1; Lämmerhofer, M.1 1Eberhard-Karls-University Tübingen, Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, Auf der Morgenstelle 8, D-72076, Tübingen, Germany

In the recent years, the use of biopharmaceuticals has become increasingly important. Between January 2014 and July 2018, 47% of genuinely new approved drugs in the US were based on biopharmaceuticals. Additionally, 40 % of the 6,000 or more products in clinical development worldwide are biopharmaceuticals. Besides monoclonal antibodies (mAbs), which remains the dominant branch of biopharmaceuticals, other gene- and nucleic acid-based products like Figure 1. Effect of cofactor binding on the circular dichroism (CD) antisense oligonucleotides (ASOs) have also been established [1]. From spectrum. Fomivirsen as the pioneer ASO treating cytomegalovirus (CMV) retinitis to Zolgensma for a new gene therapy by spinal muscular atrophy (SMA), the list of such nucleic acid-based approvals is very long. Hence, a better References: understanding of these complex synthetic oligonucleotides is essential Rüther, A., et al.: Angew. Chem. Int. Ed. 2017, 56(16): 4603–4607 and demands suitable analysis methods. Conventionally, the Diedrich, D., et al.: Chem. Eur. J. 2016, 22(49): 17600–17611. characterization of oligonucleotide is performed by ion-pair reversed- Rüther, A., et al.: J. Phys. Chem. B 2014, 118(14): 3941–3949. Marolt, M., Lüdeke, S.: Phys. Chem. Chem. Phys. 2019, 21: 1671–1681. phase liquid chromatography (IP-RP-LC) coupled to a mass spectrometry (MS) detection. But the use of ion-pairing reagents like triethylamine (TEA) causes significant ion suppression which decreases the sensitivity of MS [2]. A possibility to avoid this problem is to add a second chromatographic dimension featuring a complementary separation mechanism without any MS-incompatible additives in the mobile phases. Recently, our group has reported a LC-method using POS.99 mixed mode chromatography (MMC) to separate oligonucleotides which New approaches for (Smartphone-) Imaging of Lateral Flow could be considered as the first chromatographic dimension in a 2D-LC setup [3]. In this work, we want to develop an appropriate complementary Immune Assays (LFIAs). Enhancing the capability for point LC-method for the second dimension with a focus on the hydrophilic of care diagnostics (POCTs). interaction liquid chromatography (HILIC-LC) which is expected to be an effective alternative to IP-RP for LC-MS analysis of oligonucleotide. This method should then be combined with the above mentioned HPLC Ruppert, C.1,2,3; Phogat, N.1,2,3; Laufer, S.3 Kohl, M.1,2; Deigner, H-P.1,2,4 1 Furtwangen University, Medical and Life Sciences Faculty, Jakob-Kienzle Str. 17, D-78054 method as 1D in a 2D-LC setup. Villingen-Schwenningen, Germany 2 Furtwangen University, Institute of Precision Medicine, Jakob-Kienzle Str. 17, D-78054 Villingen-Schwenningen, Germany References: 3 University of Tuebingen, Department of Pharmaceutical Chemistry, Auf der Morgenstelle 8, D- 1. Walsh, G.: Nature Biotechnology. 2018, 36: 1136-1145. 72076 Tuebingen, Germany 2. Limbach, P. A. et al.: J. Chromatogr. A. 2019, 1595: 39-48. 4 Fraunhofer Institute IZI, Leipzig, EXIM Department, Schillingallee 68, D-18057 Rostock, Germany 3. Lämmerhofer, M. et al.: J. Chromatogr. A. 2019, 1593: 110-118.

Lateral Flow Immune Assays (LFIAs) are easy-to-use sensing systems for home monitoring of e.g. drugs and biomarkers. Nanoparticle dyes, like gold nanoparticles or fluorescent quantum dots can be used to increase sensitivity, multiplexing potential or allow for imaging and quantification by smartphone. We introduce an imaging system for monitoring of cardiac glycoside digoxin using a smartphone as readout device and includes a customized Shiny App for data processing based on a gold-nanoparticle labelled LFIA [1]. This approach is now extended to a quantum dot labelled duplex LFIA using an improved version of our smartphone readout device in combination with data processing software to develop a portable and fast assay for sepsis biomarkers: C-reactive protein (CRP) & Interleukin 6 (IL-6). We compare imaging hardware and probes composed by different nanoparticles for the design of easy to use-, low cost LFIA point of care devices matching the need for fast diagnostics in precision medicine.

Acknowledgments: Support by Bundesministerium für Bildung und Forschung (MultiFlow project, 03FH046PX4) is gratefully acknowledged

References: 1. Ruppert, C. et al.: Michrochim. Acta (2019) 186:119

125 • DPhG Annual Meeting 2019 Conference Book POSTERS

POS.102

PKM2 Activation Depletes Upstream Glycolytic 4.4 Cancer Intermediates Leading to Reduced TXNIP Expression, POS.101 Glucose Uptake, and Interestingly Depleted the Intracellular Levels of the Onco-metabolite Fumarate Insight into the cisplatin resistance of W1CR ovarian carcinoma cells to decipher novel targets for sensitization Fadi Almouhanna1, Biljana Blagojevic1, Ali Ghanem1, Michael Büttner2, and Stefan Wölfl1 1Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer 1 1 1 2 Wantoch von Rekowski, K. ; Henze, S. ; König, P. ; Januchowski, R. ; Feld 364 Heidelberg, Germany 69120 Bendas, G.1 2 Metabolomics Core Technology Platform, Center for Organismal Studies (COS), Heidelberg 1Pharmaceutical Department, University of Bonn, D-53121 Bonn University, Im Neuenheimer Feld 360, 69120 Heidelberg, Germany. 2Department of Histology and Embryology, University of Poznań, P- 61-781 Poznań

Pyruvate kinase plays a central role in regulating tumor metabolism by Ovarian carcinoma is one of the most lethal gynaecological conferring metabolic and non-metabolic benefits to cancer cells. Its low malignancies. Cisplatin is well established as cytostatic agent in the affinity to phospho-enol pyruvate leads to accumulation of upstream therapy of ovarian carcinoma. However, since there is a high proportion glycolytic metabolites to be shunted onto different anabolic pathways of advanced stage cases at diagnosis, the overall curative rate is less branching from glycolysis. In this study, we investigated the efficacy of than 40% across all stages. Although most ovarian cancer patients pharmacological PKM2 activator (DASA-58 and TEPP-46) on the respond initially to front-line Pt-based chemotherapy, resistance to metabolic phenotypes of different breast cancer (BCa) cells. The energy cisplatin is commonly observed during treatment, which massively crisis caused by DASA-58 treatment leads to AMPK phosphorylation and restricts the therapeutic regime. Circumventing cisplatin resistance, TXNIP reduction in BCa cells. DASA treatment was also associated with therefore, remains a critical goal for cancer treatment and considerable reduced G6PD activity in MCF7 cells leading to ROS accumulation and efforts have been taken to solve this problem throughout the past years. impaired ROS scavenging machinery. The reduction in TXNIP is seemingly pro-survival as DASA-58 treatment increases BCa cells Tumor cell binding to microenvironment components such as collagen vulnerability to other chemotherapeutics. Activating PKM2 was also type 1 (COL1) attenuates the sensitivity to cytotoxic drugs like cisplatin, associated with depletion of fumarate, an oncometabolite supporting the referred to as ‘cell adhesion mediated drug resistance’ (CAM-DR) [1,2]. elevated glycolytic phenotype in many cancers. Despite that, DASA-58 Aiming to better comprehend the functional diversity of ovarian tumor cell only mildly affected BCa cells proliferation. Nevertheless, the energy lines we pursue this strategy characterizing a new ovarian carcinoma cell crisis and reprogrammed glycolysis caused by the PKM2 activator line W1, or the cisplatin resistant subtype W1CR, which presents different rendered BCa cells more vulnerable to pharmacological intervention with resistance features, such as collagen overexpression [3]. The aim of this other chemotherapeutics and metabolic modulators. study was to provide insight into the molecular mechanisms of cisplatin chemoresistance and the elucidation of potential targets for sensitization strategies.

To evaluate CAM-DR as target for sensitization strategies, we analyzed POS.103 cytotoxicity (MTT-assay) of cisplatin in both cell lines in absence or Non-essential amino acid-mediated regulation of Sestrin 2 presence of COL1 and the associated signaling pathways at the protein in hepatocellular carcinoma cells level (SDS-PAGE/WB, proteome profiler array) or by RNA microarray. To further focus on the role of integrin on cellular resistance, a β1 integrin knockdown was performed in W1/W1CR cells by a lentiviral approach. Blagojevic, Biljana1; Wölfl, Stefan1. 1 Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer MTT data confirmed that the cisplatin resistant subline W1CR displayed Feld 364, 69120, Heidelberg, Germany a 3- to 10-fold higher EC50 value of cisplatin cytotoxicity compared to W1 cells. If subjected to integrin activating treatment (COL1), both W1 and W1CR cells showed a significant increase in resistance to cisplatin, Hepatocellular carcinoma (HCC) is representing more than 80% of liver associated with the upregulation of various signaling pathway cancer and is accompanied with low survival rate and high resistance to components, which finally refer to an impact of adhesion mechanisms on existing therapy [1,2]. Understanding regulation of mTORC1, as being resistance. Furthermore, inhibition of integrin-linked kinase (ILK) or focal one of the major controllers of cellular growth and survival, might place adhesion kinase (FAK) led to a higher gain in sensitivity, especially in us a step closer to designing a successful anti-cancer therapy for this cells grown on COL1. In line with these data, COL1 lost its resistance type of liver cancer [3]. Sestrin 2 as a negative regulator of mTORC1 has triggering effect in the W1 β1-integrin knock-down cells, while the a unique function of recognising stress condition (for example: oxidative resistant W1CR ß1-integrin knock-down cells behave differently, stress, nutrient deprivation, ER stress, DNA damage) and forwarding indicating a more rapid rewiring of the resistance signaling in the W1CR signals toward downstream metabolic and apoptotic/survival pathways, cells. giving the cell chance to adapt to the stress [4-7]. Here we investigated the regulation of Sestrin 2 levels in various HCC cell lines representing The data provide evidence for a potential link between integrin activation early (HepG2) and late (SKHep and HLE) stage of liver cancer upon non- and tumor cell resistance against cytotoxic drugs and thus sheds new essential amino acid deprivations. light on CAM-DR as novel therapeutic targets in oncology. Further work We observed amino acid-specific up-regulation of Sestrin 2 protein levels is needed to investigate and simulate the underlying mechanisms of after 48h of deprivation that were in an inverse correlation with cellular CAM-DR, such as transferring into a 3D cell culture model that can better survival under same conditions. In order to further investigate the role of mimic the natural in vivo setting of tumors than conventional monolayers this up-regulation of Sestrin 2 we artificially over-expressed Sestrin 2 in (2D) cell culture. In addition, further signaling pathways and other cellular HCC cells and characterized observed phenotypes. Surprisingly, Sestrin contacts with the microenvironment components should be investigated 2 over-expression had different effects on the survival of investigated cell as potential targets. lines with only SKHep showing characteristic mTORC1 inhibition-induced reduction of proliferation rate. Overall cellular response to this genetic modification showed diverse phenotypical changes, mostly mTORC1- References: dependent indicating rather cell specific and contextual effects of Sestrin 1. Meads, MD. et al.: Nat Rev Cancer, 2009, doi:10.1038/nrc2714/nrc2714. 2 over-expression. Finally, over-expression of Sestrin 2 rescued cell 2. Jakubzig, B. et al.: Cancers, 2018, 10 doi: 10.3390/cancers10120495. survival upon arginine, serine, cysteine, and tyrosine deprivation but only 3. Januchowski, R. et al.: J.Cancer. 2016, 7:1295-310. in case of SKHep. Further investigations on how crucial is the upregulation of Sestrin 2 in response to non-essential amino acid deprivations are still needed.

DPhG Annual Meeting 2019 Conference Book • 126 CANCER

The strong responses to Sestrin 2 over-expression indicate treatment potential of targeting Sestrin 2 functional role. Nevertheless, cell line specificity of this over-expression suggests that targeting Sestrin 2 is highly contextual and depending on the genetic background of cancer as well as on its metabolic phenotype.

1. Allemani C, et al.: Global surveillance of cancer survival 1995-2009: analysis of individual data for 25,676,887 patients from 279 population-based registries in 67 countries (CONCORD- 2). Lancet 2015, 385(9972): 977-1010 2. Nwosu ZC, et al.: Liver cancer cell lines distinctly mimic the metabolic gene expression pattern of the corresponding human tumours. J Exp Clin Cancer Res 2018, 37(1): 211 3.Saxton RA, Sabatini DM.: mTOR signaling in growth, metabolism, and disease. Cell 2017, 169(2): 361-371 4. Budanov AV, et al.: Identification of a novel stress responsive gene Hi95 involved in regulation of cell viability. Oncogene 2002, 21: 6017-6031 5. Lee JH, Cho US, Karin M.: Sestrin regulation of TORC1: Is Sestrin a leucine sensor? Sci Signal. 2016, 9(431): re5 6. Lee JH, Budanov AV, Karin M.: Sestrins orchestrate cellular metabolism to attenuate aging. Cell Metab. 2013, 18: 792–801 7. Kim H, et al.: Janus-faced Sestrin2 controls ROS and mTOR signaling through two separate functional domains. Nat Commun. 2015, 6: 10025

127 • DPhG Annual Meeting 2019 Conference Book POSTERS

4.5 Clinical Pharmacy POS.105 POS.104 A physiologically-based pharmacokinetic model of the GLICEMIA 2.0 – a randomized, controlled trial in secondary CYP1A2 substrate theophylline and its application for the and tertiary prevention in a type 2 diabetic population: prediction of drug-drug interactions with fluvoxamine, preventive care in pharmacies, Germany rifampicin and smoking

Prax, K1; Schmiedel, K2; Hepp, T3; Schlager, H4; Friedland, K5 1Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Professorship for Molecular and Britz, H.; Hanke, N.; Lehr, T. Clinical Pharmacy, Cauerstraße 4, 91058 Erlangen, Germany Clinical Pharmacy, Saarland University, Campus C2 2, 66123 Saarbrücken, Germany 2Kur-Apotheke, Kegetstraße 4, 91438 Bad Windsheim, Germany 3Rheinische Friedrich-Wilhelms-University Bonn, Department of Medical Biometry, Informatics and Epidemiology, Faculty of Medicine, Venusberg-Campus 1, 53127 Bonn, Germany Background and Objectives: CYP1A2 accounts for about 13% of the 4Scientific Institute for Prevention in Health Care (WIPIG), Bavarian Chamber of Pharmacists, cytochrome P450 (CYP) content in the liver and is involved in the Maria-Theresia-Straße 28, 81675 Munich, Germany 5Johannes-Gutenberg-University Mainz, Institute of Pharmacy and Biochemistry, metabolism of 15% of all therapeutic drugs [1,2]. The US Food and Drug Staudingerweg 5, 55128 Mainz, Germany Administration (FDA) recommends theophylline as a sensitive CYP1A2 substrate (fraction metabolized of 0.7 [3,4]), fluvoxamine as a strong CYP1A2 inhibitor, rifampicin as a moderate and smoking as a strong Background: Type 2 diabetes patients have a high risk for micro- and CYP1A2 inducer, for drug-drug interaction (DDI) studies [5]. The macrovascular complications. Hereby, a poor glycaemic control can lead objectives of this study were to establish a whole-body physiologically- to a lower quality of life and reduced life expectancy [1]. Therefore, based pharmacokinetic (PBPK) model of theophylline and to apply this structured programs not only supporting patients in improving physical model for the investigation of theophylline DDIs with fluvoxamine, exercise and nutrition but also providing medication management are of rifampicin and smoking. high medical need and social benefit. Therefore, we developed a Methods: PBPK modeling was performed with PK-Sim® and MoBi® structured program focusing on secondary and tertiary prevention of type (version 7.3.0., www.open-systems-pharmacology.org). Theophylline 2 diabetes patients in pharmacies. This program is based on the concept drug-dependent parameters (e.g. logP, solubility) and theophylline of GLICEMIA – a primary prevention program. GLICEMIA significantly plasma concentration-time profiles (37 clinical studies, dosing range 125 reduced the risk for type 2 diabetes in patients who had a high probability – 426.6 mg as intravenous and 79 – 500 mg as oral dose) were gathered developing this disease [2]. The aim of GLICEMIA 2.0 is now to evaluate from literature and used for model development. Model evaluation was the impact of an intensive pharmaceutical care on the glycaemic control performed by comparison of predicted plasma concentration-time profiles of type 2 diabetes patients. to observed data of theophylline in clinical DDI studies with fluvoxamine Methods: GLICEMIA 2.0 is a randomized, controlled trial in pharmacies (2 studies), rifampicin (3 studies) and smoking (5 studies). located in Bavaria, Germany. Eligible participants were type 2 diabetes Results: The established theophylline model incorporates metabolism patients with among others, a minimum HbA1c of 7 % and at least 18 via CYP1A2 and CYP2E1 as well as glomerular filtration with years of age. The pharmaceutical care in the intervention group reabsorption in the renal tubule. The metabolic pathways were consisted of personal consultations and group meetings with a focus on implemented as saturable Michaelis-Menten kinetics. The theophylline lifestyle intervention and medication management. The control group fraction metabolized via CYP1A2 and the plasma concentration-time received written materials and the usual care. Assessments took place profiles of theophylline are well described by the model, with 34/37 of the at baseline, after six and after twelve months. As primary outcome the predicted area under the plasma concentration-time curve (AUC) values study examines if there is a statistically significant difference in HbA1c- and 36/37 of the predicted peak plasma concentration (Cmax) values values (Point-of-Care) between intervention and control group. As within two-fold of the observed values. To predict the impact of secondary outcome further laboratory data (fasting level of blood fluvoxamine on the theophylline pharmacokinetics, the mixed inhibition glucose, weight, blood pressure), physical activity, life quality, of CYP1A2 by fluvoxamine was modeled using inhibition constants (KIc adherence, number of medications and drug related problems are and KIu) of 10 nmol/l [6]. During co-administration with fluvoxamine, the evaluated. Furthermore, a cost analysis takes place. observed theophylline DDI AUC ratio (AUC DDI / AUC control) and DDI Results: 26 pharmacies took part in GLICEMIA 2.0 and recruited 198 type Cmax ratio (Cmax DDI / Cmax control) are 2.55 (n = 2) and 1.13 (n = 2), 2 diabetes patients in total. Thereof, 183 fulfilled the inclusion criteria. 30 respectively. Fluvoxamine-theophylline DDI modeling predicts a patients quit prematurely. During the study period of one year, the mean theophylline DDI AUC ratio of 2.63 (n = 2) and a DDI Cmax ratio of 1.11 HbA1c value (±SD) in the intervention group (n=96) was reduced from 8.3 (n = 2). To predict the impact of rifampicin on the theophylline % ± 1.3 % to 7.3 % ± 1.0 % [median: from 8.0 % (IQR: 7.5 % - 8.6 %) to pharmacokinetics, the induction of CYP1A2 and CYP2E1 was modeled 7.3 % (6.5 % - 7.9 %)]. The control group (n=86) only showed a reduction using a half-maximal induction concentration (EC50) of 0.34 μmol/l for from 8.1 % ± 1.0 % to 7.8 % ± 1.2% [median: from 7.9 % (IQR: 7.3 % - both CYP enzymes and maximal induction effects (Emax) of 0.65 for 8.6 %) to 7.6 % (IQR: 7.1 % - 8.2 %)]. Including all eligible patients the CYP1A2 and of 0.8 for CYP2E1 [6,7]. During co-administration with difference adjusted via linear mixed model with several co-variates rifampicin, the observed theophylline DDI AUC ratio and DDI Cmax ratio between intervention and control group (n=181) in mean change of HbA1c are 0.84 (n = 3) and 1.05 (n = 2), respectively. Rifampicin-theophylline from baseline was statistically significant (intervention effect: -0.5 %, DDI modeling predicts a theophylline DDI AUC ratio of 0.78 (n = 3) and p=0.0007). No statistically significant difference in fasting plasma glucose a DDI Cmax ratio of 0.94 (n = 2). To predict the impact of smoking on the levels could be proven. 23% of patients in the intervention group could theophylline pharmacokinetics, the induction of CYP1A2 was modeled reduce their weight by 5 % or more. 91 % of the intervention group using a 1.54-fold higher CYP1A2 enzymatic activity. Theophylline plasma evaluated participating in GLICEMIA 2.0 as beneficial. concentration-time profiles of smokers can be successfully predicted with Conclusion: Summarizing the first results of GLICEMIA 2.0, the study 5/5 of the predicted AUC values and 5/5 of the predicted Cmax values resulted in a significant reduction of HbA1c-values compared to the within two-fold of the observed values. control group. Almost one in four participants of the intervention group Conclusion: A whole-body PBPK model of theophylline has been reached the target of reducing his or her weight by 5 % or more. A developed, that accurately describes and predicts the plasma beneficial effect of pharmaceutical care in type 2 diabetes patients has concentration-time profiles of theophylline for the entire range of reported already been identified. The influence on physical activity, life quality, doses and administration protocols. Furthermore, this model was adherence as well as medication management is still under evaluation successfully applied to predict the effects of fluvoxamine, rifampicin and and once completed, will conclude the results of the study. smoking on the pharmacokinetics of theophylline.

Acknowledgments:

GLICEMIA 2.0 was funded by Dr. August und Dr. Anni Lesmüller-Stiftung, Förderinitiative References: Prävention e. V. 1. Shimada, T. et al.: J. Pharmacol. Exp. Ther. 1994, 270(1): 414–23. References: 2. Zhou, S.F. et al.: AAPS J. 2009, 11(3): 481–94. 1. Müller-Wieland, D., Kröger, J.: Deutscher Gesundheitsbericht Diabetes - Die 3. Karjalainen, M.J., Neuvonen, P.J., Backman, J.T.: Drug Metab. Dispos. 2006, 34(12): 2091– Bestandsaufnahme (Deutsche Diabetes Gesellschaft und Deutsche Diabetes-Hilfe) 2018. 6. 2 Schmiedel, K. et al.: Diabetes care 2015, 38(5): 937-939. 4. Lu, P. et al.: Drug Metab. Dispos. 2003, 31(11): 1352–60.

DPhG Annual Meeting 2019 Conference Book • 128 CLINICAL PHARMACY

5. U.S. Food and Drug Administration. Drug development and drug interactions: table of rhythm to describe the chronotropic effect of nicotine. The tolerance substrates, inhibitors and inducers 2017. https://www.fda.gov/drugs/drug-interactions- model proved superior to a classical Emax model and was able to predict labeling/drug-development-and-drug-interactions-table-substrates-inhibitors-and-inducers heart rate peaks occurring rapidly after pulmonary and i.v. nicotine intake. 6. Britz, H. et al.: CPT Pharmacometrics Syst. Pharmacol. 2019, 8(5): 296–307. Conclusion: We successfully developed a whole-body PBPK/PD model 7. Hanke, N. et al.: CPT Pharmacometrics Syst. Pharmacol. 2018, 7(10): 647–59. of nicotine and its main metabolite cotinine which is able to predict nicotine and cotinine plasma profiles in smokers and nonsmokers for different dosing regimens and various application routes in an excellent way. Furthermore, the model successfully predicts the chronotropic effect POS.106 of nicotine. This may help improving strategies of smoking cessation in A physiologically based order to decrease tobacco addiction and providing insights into the pharmacokinetic/pharmacodynamic (PBPK/PD) parent- involvement of nicotine in pathophysiological processes. metabolite model of nicotine including its chronotropic effect and CYP2A6/CYP2B6 metabolism 1. Benowitz N. L.: N. Engl. J. Med. 2010, 362(24) 2295-303. 2. Eissing T. et al.: Front Physiol. 2011, 2(4). Kovar, L.1; Britz, H.1; Selzer, D.1; Benowitz, N. L.2; Kohl, Y. L.3; Bals, 3. Hukkanen J., Jacob P., Benowitz N. L.: Pharmacol Rev. 2005; 57(1): 79–115. 4. Benowitz N. L., Jacob P.: Clin Pharmacol Ther. 1993, 53(3): 316–23. 4 1 R. ; Lehr, T. 1 Clinical Pharmacy, Saarland University, Campus C2 2, 66123 Saarbrücken, Germany 2 Department of Medicine, University of California, San Francisco, 94115, California, USA 3 Fraunhofer Institute for Biomedical Engineering IBMT, Joseph-von-Fraunhofer-Weg 1, 66280 Sulzbach, Germany 4 Department of Internal Medicine V, Saarland University, Kirrberger Straße, 66421 Homburg, POS.107 Germany Dose adjustment of risperidone in Cytochrome P450 2D6 intermediate- and poor metabolizer based on Introduction and objectives: Since nicotine is the pharmacologically physiologically-based pharmacokinetic model active substance in tobacco responsible for addiction it also is a proximate cause of smoking-induced diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer [1]. Hence, a Kneller L.A.1; Abad-Santos F.2; Hempel G.1 detailed understanding of the pharmacokinetics and pharmacodynamics 1 Institute of Pharmaceutical and Medicinal Chemistry, Clinical Pharmacy, Westfälische Wilhelms-Universität Münster, Corrensstr. 48, 48149 Münster, Germany of nicotine is desirable to derive new strategies of smoking cessation and 2 Clinical Pharmacology Department, Hospital Universitario de La Princesa, Universidad to get a better understanding of the role of nicotine in pathophysiological Autónoma de Madrid (UAM), Instituto de Investigación Sanitaria La Princesa (IP), Diego de processes. Our goal was to develop a whole-body physiologically based León 62. 28006 Madrid, Spain. pharmacokinetic/pharmacodynamic (PBPK/PD) model to investigate the fate of nicotine including its main metabolite cotinine after intravenous Background: The genetic polymorphism of Cytochrome P450 (CYP) (i.v.), oral (p.o.), transdermal and pulmonary administration in 2D6 plays an influential role on the appearance of positive and adverse nonsmokers and smokers. drug reactions to different antipsychotics, especially in risperidone (RIS). Methods: A parent-metabolite PBPK model of nicotine and cotinine was Due to non-functional alleles of the CYP2D6 gene, the poor metabolizer built in PK-Sim® and MoBi® (version 7.4.0) [2]. Physicochemical model (PM) phenotype appears to be associated with more adverse drug input parameters as well as plasma profiles and nicotine-induced reactions, going along with frequent discontinuation of the therapy [1]. chronotropic effect data were obtained from published literature. For Based on that large inter-individual variability in RIS’s and 9- PBPK model building, 63 plasma profiles from 27 clinical studies were Hydroxyrisperidone’s (9-OH-RIS) plasma concentrations [2], a split into an internal training (12 plasma profiles) and an external test (51 successful development of a physiologically-based pharmacokinetic plasma profiles) dataset. Initially, a cotinine model was established by (PBPK) model provides a powerful tool to calculate the optimal dose for fitting unknown model parameters to plasma profile training data from i.v. different CYP2D6 phenotype. The objective of the study is to optimise administration in healthy volunteers. Subsequently, the model was phenotype-related dosing of RIS, using a whole-body PBPK approach. complemented by the parent compound nicotine using i.v. plasma Methods: A PBPK model for RIS and 9-OH-RIS was successfully concentration-time profiles. Model evaluation was performed by developed to predict the pharmacokinetics on genotyped healthy comparing observed plasma profiles from the external test dataset with volunteers treated with single-dose RIS, considering relevant CYP2D6 predicted profiles. In addition, nicotine clearance was adjusted for and -3A4 related changes in the different CYP2D6 phenotypes: extensive smokers and the model extrapolated to p.o., transdermal and pulmonal metabolizer (EM), intermediate metabolizer (IM), PM and ultra-rapid application of nicotine. Transdermal and p.o. absorption kinetics were metabolizer (UM) [3]. Based on the developed PBPK models, steady- estimated by fitting relevant model parameters. Conclusively, a PBPK/PD state conditions with the administration of 3 mg RIS twice a day were heart rate-tolerance model was implemented using heart rate data from simulated. This simulated dosage of 6 mg/day corresponds to the most 7 different studies. recommended clinical dose for schizophrenia patients. For those Results: The model includes nicotine metabolism to cotinine via CYP2D6 genotypes in which plasma concentration of the active moiety CYP2A6 and CYP2B6 which accounts for about 75% of nicotine (RIS plus 9-OH-RIS) was outside the therapeutic range (20-60 µg/L), a elimination. The corresponding Michaelis-Menten constants KM were dose adjustment was performed [4]. fitted within the range of literature values to 31 µM and 820 µM, Results: Based on the previously developed PBPK models, steady-state respectively, whereas the catalytic rate constant kcat was fitted to 13 min- conditions were simulated. Here, steady-state trough plasma 1 both for CYP2A6 and CYP2B6. Additionally, nicotine is cleared by an concentration of PM was +106% higher than those of EM (25.3 µg/L in unspecific hepatic clearance (first order kinetics) and renal clearance EM and 52.4 µg/L in PM) at the same dose. Even peak plasma (GFR fraction of 1.00). The elimination routes are consistent with concentration of PM was significantly higher (+53.1%) in PM than in EM published literature [3]. For cotinine, the model contains an unspecific (50.5 µg/L in EM and 77.4 µg/L in PM). This was also evident in the hepatic metabolism (first order kinetics) and a glomerular filtration with CYP2D6 IM type. Here, trough plasma concentration was +62.3% higher tubular reabsorption (GFR fraction of 0.07). The final model was capable than those of EM (25.3 µg/L in EM and 41.1 µg/L in IM), as well as peak to precisely describe and predict all i.v. profiles of the internal and plasma concentration which was +38.5% higher in IM than in EM external dataset. Geometric mean fold errors (GMFEs) of predicted and (50.5 µg/L in EM and 70.0 µg/L in IM). Consequently, RIS’s dose was observed areas under the plasma concentration-time curve (AUC) were reduced by -10% for IM and by -25% for PM. After dose adjustment 1.07, 1.30 and 1.08 for nicotine, cotinine metabolite and administered trough and peak plasma concentrations of the active moiety were within cotinine profiles, respectively. According to published data, nicotine the therapeutic reference range and showed similar plasma clearance in smokers appears to be about 15% lower compared to concentrations like those of EM with 6 mg RIS. nonsmokers [4] resulting in higher AUC values. By adjusting the model Conclusion: PBPK modelling can provide a valuable tool to predict RIS’s to a lower nicotine metabolic capacity, we were also able to predict all and 9-OH-RIS’s pharmacokinetics in subjects genotyped for CYP2D6, plasma profiles including the concomitant increase in AUC after i.v., p.o., taking CYP3A4 into account. These models are able to ultimately support transdermal and pulmonary administration in smokers (GMFEs: 1.07, decision making regarding dose optimization strategies, especially for 1.20, 1.13 and 1.27 for nicotine; cotinine metabolite: 1.30, 1.15 and 1.16 subjects showing lower CYP2D6 activity. Therefore, dose adjustment for i.v., p.o. and transdermal administration). The PBPK model was during steady-state that compensate for genetically caused differences complemented by a heart rate-tolerance model including circadian

129 • DPhG Annual Meeting 2019 Conference Book POSTERS in active moiety’s plasma concentrations amounts to -10% for IM and - outcomes of tigecycline’s effect in in vitro experiments. Using freshly 25% for PM in comparison to EM, respectively. prepared, stabilized ca-MHB for experiments with tigecycline should be implemented in lab routines and guidelines. The stabilized tigecycline solution will be used in upcoming Hollow Fiber infection model References: experiments to evaluate alternative dosing regimens for tigecycline. [1] Leon J de et al.: J Clin Psychiatry 2005;66(1):15–27. [2] Balant-Gorgia AE et al.: Ther Drug Monit 1999;21(1):105–15. [3] Kneller LA, Abad-Santos F, Hempel G. Clin Pharmacokinet 2019 [under review]. [1] Pfizer. TYGACIL(R) FULL PRESCRIBING INFORMATION 2005. [4] Hiemke C. et al.: Pharmacopsychiatry 2018;51(01/02):9-62. [2] Jitkova Y et al. A Novel Formulation of Tigecycline Has Enhanced Stability and Sustained Antibacterial and Antileukemic Activity. PLoS One 2014;9:e95281. [3] Sevillano D et al. Exposure–response analysis of tigecycline in pharmacodynamic simulations using different size inocula of target bacteria. Int J Antimicrob Agents 2010;36:137– POS.108 44. Stability studies with tigecycline in bacterial growth [4] Bradford PA, et al. Tigecycline MIC Testing by Broth Dilution Requires Use of Fresh Medium medium and impact of stabilizing agents: A prerequisite for or Addition of the Biocatalytic Oxygen-Reducing Reagent Oxyrase To Standardize the Test in-vitro PK-PD experiments Method. Antimicrob Agents Chemother 2005;49:3903–9.

Lisa F. Amann1, Emilia Ruda Vicente1, Mareike Rathke1, Astrid Broeker1, Sebastian G. Wicha1 1Institut für Pharmazie, Abt. Klinische Pharmazie, Universität Hamburg POS.109 Physiologically-based pharmacokinetic modeling of trimethoprim drug-drug interactions with the CYP2C8 Tigecycline, as a representative of the class of glycylcyclines, is a last victim drugs repaglinide and pioglitazone resort, broad-spectrum antibiotic. It shows activity against Gram-positive and Gram-negative, an- and aerobic bacteria including strains, that are resistant to many other antibiotics. Currently tigecycline is indicated for Türk, D.; Hanke, N.; Lehr, T. patients with complicated skin and intra-abdominal infections, as well as Clinical Pharmacy, Saarland University, Campus C2 2, 66123 Saarbrücken, Germany community-acquired pneumonia. The manufacture recommends a 100 mg loading dose, followed by 50 mg twice a day as a standard treatment Background and Objectives: Trimethoprim, an inhibitor of bacterial folic protocol [1]. Previously published in-vitro infection models showed, that acid metabolism, is used for treatment of bacterial infections. It is applied a bacterial regrowth occurs in particular at the standard dosing protocol as monotherapy or in combination with sulphonamides. Trimethoprim is [3], indicating the need to optimise and potentially revise the standard one of the most commonly prescribed antibiotics in Germany, ranking dosing regimen. fifth after penicillines, cephalosporines, macrolides and fluorchinolones Tigecycline is known to be sensitive to light and oxygen, requiring its use (108 standard units of trimethoprim were prescribed in Germany in 2010) without delay after reconstitution [2,4]. To test new dosing regimens incl. [1]. The antibiotic is a weak inhibitor of cytochrome P450 (CYP) 2C8 and intensified infusion durations in in-vitro Hollow Fiber infection models, a potent inhibitor of multidrug and toxin extrusion (MATE) 1 and 2-K [2]. tigecycline’s stability must be enhanced. A novel formulation identified Due to the frequent prescription of trimethoprim, investigation of its drug- tigecycline to be stable up to one week in 0.9% saline solution by adding drug interaction (DDI) potential is clinically relevant. Co-administration of 3 mg/mL ascorbic acid and 60 mg/mL sodium-pyruvate as oxygen- trimethoprim increases the area under the concentration-time curve reducing agents [2]. Due to inconsistency in minimal inhibitory (AUC) of repaglinide and pioglitazone [2,3], which are both predominantly concentrations (MIC), it was revealed, that there is a medium-age related metabolized by CYP2C8 and are therefore recommended by the U.S. effect on susceptibility, affected by the amount of dissolved oxygen in Food and Drug Administration as CYP2C8 victim drugs in DDI studies medium [4]. [4,5]. Polymorphism of the gene encoding for CYP2C8 also affects the The objective of these stability studies was (i) to quantify the degradation pharmacokinetics of repaglinide and pioglitazone (drug-gene interaction, process in cation-adjusted Mueller Hinton Broth (ca-MHB) and evaluate DGI): expression of the CYP2C8*3 allele leads to decreased repaglinide the transferability of the previously mentioned antioxidant reagents in and pioglitazone AUCs compared to wild type CYP2C8 [3,6]. fresh and aged broth, and (ii) to evaluate the potential impact of the Physiologically-based pharmacokinetic (PBPK) modeling is a valuable antioxidants on bacterial growth. tool to describe and predict the pharmacokinetics of drugs and to To improve the stability of tigecycline in in-vitro infection models, it’s investigate DDIs, DGIs and even DDGIs (drug-drug-gene interactions). degradation in fresh and aged ca-MHB were compared to stabilized fresh The objectives of this study were to develop a whole-body PBPK model and aged ca-MHB containing 3 mg/mL ascorbic acid and 60 mg/mL of the CYP2C8 perpetrator drug trimethoprim and to predict the DDIs of sodium-pyruvate. The stabilizing components were tested alone and in trimethoprim with repaglinide and pioglitazone, as well as the DDGI of combination. The stability studies were conducted at 37 °C, ambient air, trimethoprim with pioglitazone in carriers of the CYP2C8*3 allele [3]. pH 7, protected from light, for 24 hours relating to typical conditions used Methods: A whole-body PBPK model of trimethoprim was built and in the Hollow-Fiber infection model. High-pressure liquid chromatography evaluated using PK-Sim® and MoBi® (version 7.4.0, www.open- (HPLC) was applied for concentration analysis and time-kill studies were systems-pharmacology.org). Evaluation of the trimethoprim model was used to evaluate the potential impact on bacterial growth. performed by comparison of predicted to observed plasma The measurements revealed a different degradation rate regarding the concentration-time profiles, AUC and peak plasma concentration (Cmax) age of ca-MHB and presence of stabilizing agents in ca-MHB: In aged, values and calculation of geometric mean fold errors (GMFEs). To predict non-stabilized ca-MHB, we observed a rapid variable degradation larger the DDIs with repaglinide and pioglitazone, an inhibitory constant (Ki) than 24 % within 24 h. In contrast to that, in fresh, non-stabilized ca-MHB describing the competitive inhibition of CYP2C8 was added to the more than 80% of tigecycline remained. Addition of both antioxidants to trimethoprim model and the model was subsequently coupled to ca-MHB resulted in tigecycline concentrations of more than 91% after 24 previously developed models of repaglinide and pioglitazone [7]. The h, regardless of the ca-MHB age. The time kill curve experiments with performance of the DDI models was evaluated by comparison of Staphylococcus aureus (ATCC29213) showed no change in bacterial predicted to observed victim drug plasma concentration-time profiles, growth by adding 0,3% ascorbic acid, and 0,09 % pyruvate, adjusted to DDI AUC ratios (AUC DDI / AUC control) and DDI Cmax ratios (Cmax DDI / pH 7. This reduced concentration of antioxidant was calculated as Cmax control). For the DDGI model, comparison of DDGI AUC ratios, pyruvate’s concentration, diluted to the central compartment of later DDGI Cmax ratios and calculation of GMFEs was performed. planned Hollow Fiber experiments. More time kill curves will be Results: The final trimethoprim model applies transport via P- conducted to test if tigecycline’s antibiotic activity and antibiotic-free glycoprotein, an unspecific hepatic clearance process, tubular secretion bacterial growth, is unaffected by adding even higher amounts of via MATE1 and passive glomerular filtration. Comparison of predicted antioxidants. and observed trimethoprim AUC and Cmax values show low GMFEs of To conclude, we confirmed the stabilizing effect of ascorbic acid and 1.21 (range 1.00-1.88, n=27) and of 1.19 (range 1.00-1.82, n=27), sodium-pyruvate on tigecycline in solution. We revealed, that the respectively, demonstrating the good model performance. Application of recommended concentrations of oxygen reducing agents stabilized the newly developed trimethoprim model for DDI prediction results in ca-MHB to a similar extent as described for saline solution. These results plasma concentration-time profiles of the victim drugs (repaglinide and show the importance of using freshly prepared ca-MHB for every pioglitazone) during trimethoprim co-administration that adequately experiment and the need for stabilizing agents to determine exact match the observed profiles. The trimethoprim-repaglinide DDI model

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predicts DDI AUC and Cmax ratios of 2.10 and 1.64, respectively, which is parameters, while only four accounted for interoccasion variability. in good agreement with the observed DDI AUC and Cmax ratios (1.60 and Besides the inclusion of body weight the covariates differed as well as 1.42, respectively [2]). The trimethoprim-pioglitazone DDI model predicts the handling of endogenous factor VIII levels and the underlying drug DDI AUC and Cmax ratios of 1.79 and 1.18, respectively, which also is in product: four accounted for the patients age, three distinguished the good agreement with the observed DDI AUC and Cmax ratios (1.42 and applied drug type and four included an endogenous baseline. 1.18, respectively [3]). Predicted vs. observed DDGI AUC ratios and The accuracy and precision of the prediction based on scenario (i) varied DDGI Cmax ratios show GMFEs of 1.29 (range 1.21-1.38, n=3) and of 1.05 substantially resulting in rBias values between -16 and 102 (rRMSE 46 - (range 1.02-1.08, n=3) for the trimethoprim-pioglitazone DDGI (taking 263). In general most of the models overpredicted trough levels and CYP2C8*3 homozygous-, heterozygous- and non-carriers into account), underestimated peak levels. In scenario (ii) the predictive performance indicating a good predictive performance of the PBPK models. improved in all models (except the model of Nestorov et al., 2015 [1] and Conclusion: A whole-body PBPK model of the CYP2C8 inhibitor the one-compartment model, Karafoulidou et al., 2009 [2]). The rBias was trimethoprim has been successfully established. The model precisely reduced from 37 to 22 on average (rRMSE 143 to 87). describes the plasma concentration-time profiles of trimethoprim The models with the smallest metrics (rBias <15 + rRMSE <65) in observed in clinical trials and accurately predicts the co-administration of scenario (ii) accounted for body weight on CL and V1; a baseline of trimethoprim with repaglinide or pioglitazone. In future studies this PBPK <1 IU/dL or stratified on the severity of the disease; and the inclusion of DDI network can be extended with models of MATE victim drugs. IIV on CL and baseline. The best predictive performance was displayed using the model by Abrantes et al., 2017 [3] and Zhang et al., 2017 [4] in particular regarding 1. Van Boeckel, T.P. et al.: Lancet. Infect. Dis. 2014, 14(8): 742-50. trough level prediction. Despite these levels are most critical in 2. Niemi, M. et al.: Br. J. Clin. Pharmacol. 2004, 57(4): 441-7. 3. Tornio, A. et al.: Drug. Metab. Dispos. 2007, 36(1): 73-80. prophylaxis treatment we emphasize the two models may not be best in 4. U.S. Food and Drug Administration. Drug development and drug interactions: table of every purpose, i.e. reaching target levels in on-demand or perioperative substrates, inhibitors and inducers. 2017, (last access into the TDMx software (www.TDMx.eu, [5]). 21 May 2019). 5. U.S. Food and Drug Administration. Clinical drug interaction Studies - Study Design, Data Analysis, and Clinical Implications. Draft Guidance for Industry 2017. [1] I. Nestorov et al. Clin. Pharmacol. Drug Dev. 2015 4: 163-174 6. Niemi, M. et al.: Clin. Pharmacol. Ther. 2003, 74(4): 380-7. [2] A. Karafoulidou et al. Eur. J. Clin. Pharmacol. 2009 65: 1121–1130. 7. Türk, D. et al.: Clin. Pharmacokinet. 2019, published online. [3] J. A. Abrantes et al. Clin. Pharmacol. Ther. 2015 102: 977-988. [4] Y. Zhang et al. J. Thromb. Haemost. 2017 15: 1106-1114. [5] S. G. Wicha et al. Int. J. Antimicrob. Agents 2015 45: 442-444.

POS.110 Model based dose finding in Hemophilia A: Evaluation of POS.111 the predictive performance of ten published Bioanalytical method validation for the quantification of pharmacometric models for Factor VIII voriconazole in microdialysate: An assay for small sample volumes and low concentrations. 1 2 2 2 2 Uster, D.W. , Diaz Garcia, C. , Aradom, E. , Musarara, M. , Riddel, A. , 1 2 1 2 1 Schulz, J. ; Joseph, J. F. ; Kloft, C. Chowdary, P. , Wicha, S.G. 1Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, 1 Dept. of Clinical Pharmacy, Institute of Pharmacy, University of Hamburg, Hamburg, Germany Kelchstr.31, 12169 Berlin, Germany 2 Katharine Dormandy Haemophilia Centre and Thrombosis Unit, Royal Free London NHS 2Core Facility BioSupraMol, Institute of Pharmacy, Freie Universitaet Berlin, Koenigin-Luise- Foundation Trust, London, UK Straße 2+4, 14195 Berlin, Germany

Hemophilia A, an x-linked inherited disorder with a deficiency of the Objectives: Voriconazole (VRC), a broad-spectrum triazole, is frequently coagulation glycoprotein FVIII, is one of the most common bleeding prescribed as prophylaxis and used as first-line therapy of invasive fungal disorders. To prevent and treat possible life-threatening bleedings the infections. After standard dosing highly variable exposure is observed, substitution of the aforementioned protein is key element in the demanding for a better understanding of the influential factors to guide management of this disease. optimised dosing [1]. For this, knowledge of target-site pharmacokinetics Current issues of hemophilia A are high interpatient variability in the is an essential prerequisite. These can be assessed by microdialysis, a pharmacokinetics (PK) of factor VIII, the variable product response and minimally invasive method to sample unbound drug concentrations high treatment costs. Nonetheless, dosing according to instruction inserts directly at the site of action. As concentrations in microdialysate are low only takes into account the body weight of the patient and the therapeutic and sample volume is limited, developing and validating an assay with a indication of factor VIII treatment (e.g. prophylaxis, surgery, heavy low limit of quantification (LLOQ) requiring as little microdialysate volume bleeding). Hence, therapeutic drug monitoring (TDM) facilitated by as possible was aimed for. population PK models has gained interest to individually tailor factor VIII dosing. Methods: A LC-MS/MS assay was developed using an Agilent LC Regarding the prophylactic treatment the time spent below a plasma system that was connected to a triple quadrupole MS/MS system. All concentration of 1 IU/dL should be as short as possible as this strategy samples were prepared by diluting 5 µL of microdialysate with 195 µL leads to fewer bleeds and hemarthroses. Thus, especially the precise water containing the internal standard (VRC-D3) at a concentration of 0.5 prediction of trough levels is important. The objective of the present study ng/mL. For chromatographic separation, an InfinityLab Poroshell 120 was to investigate the predictive performance of population PK models in Phenyl Hexyl column (RP, 2.1x100 mm, 2.7 µm, Agilent Technologies, this crucial step. Waldbronn, Germany) was used and temperature controlled at 30°C. For the evaluation, a clinical dataset was provided comprising 35 The injection volume was set to 2 µL. As eluents, methanol and ultra- hemophilia A patients receiving five different FVIII-products with a total pure water were utilised both containing 0.1% [V/V] formic acid to of 209 one stage assay observations. improve the ionisation process. Elution and separation of analytes was Ten published population PK (pop PK) models were recreated and achieved by a gradient method with increasing organic solvent over time processed using NONMEM® 7.4. The models were evaluated regarding using a flow rate of 0.350 mL/min. Total run time was 6.4 min. Ionisation their predictive performance, i.e. scenario (i): a priori estimation based on in the MS system was accomplished by an electrospray ionisation source dosing and covariate information; and scenario (ii): Bayesian forecasting, (ESI) and ion acquisition was performed in multiple reaction monitoring i.e. dosing and covariate information incl. a single previously measured (MRM). Thereby, two transitions were monitored for the internal standard trough sample to predict the factor VIII concentrations in a richly sampled VRC-D3 (m/z 353  m/z 284, 127) and three for VRC (m/z 350  m/z occasion (n=5 measurements). The results were graphically examined 281, 224, 127) , respectively. using goodness of fit plots and visual predictive checks. Furthermore, forecasting metrics were compared including relative bias (rBias) and Results: The assay was successfully validated for selectivity, carry-over, relative root mean square error (rRMSE). LLOQ, calibration function, accuracy, precision, matrix effect and stability Nine of the population PK models were two-compartment models, and according to the Europeans Medicines Agency (EMA) guideline on one was a one-compartment model. The statistical sub models included bioanalytical method validation [2]. Assay linearity was observed across interindividual variability (IIV) on the clearance (CL) or on multiple a large concentration range (5 – 5000 ng/mL), indicated by a coefficient

131 • DPhG Annual Meeting 2019 Conference Book POSTERS of determination (R²) ranging from 0.9938 to 0.9998 (n=14, for the evaluation of the genotype implementation, in which the enzyme mean: 0.9982). The signal at the LLOQ was on all occasions ≥5 times activity of genetic variants of DPYD are described through the ratio of U higher than blank samples, indicating the selectivity of the method. All and its first metabolite dihydrouracil (DHU). The final models were used back-calculated concentrations of the calibration functions were within for dose optimization by simulating a 400 mg/m2 5-FU bolus injection the accuracy limits (±15% of the nominal concentration, ±20% at LLOQ) followed by a 600 mg/m2 continuous infusion over 22 hours for wildtype of the EMA guideline (90.6% - 118.8%). The between-day accuracy of patients. The resulting exposure described as the area under the plasma quality control (QC) samples varied between 102% and 108% and concentration-time curve (AUC) was used as reference value. Exposure between-day precision calculated as coefficient of variation was <7.25% for different genotypes were simulated and their corresponding dose was for all five concentration levels (n=16 per level). The mean concentrations adapted stepwise until matching exposures compared to wildtype were of QC samples (n=5-6 per level and day, in total 80 samples) were in all reached. three runs within ±15% of their nominal value and only 6.25% of Results: Whole-body PBPK models for 5-FU and its three metabolites, individual values were outside. Due to the large concentration range, as well as for U and DHU were developed. The compiled data consist of carry-over could not be prevented completely. Detector responses of 36 5-FU studies (as bolus injection, continuous infusion or peroral blank samples injected after high VRC concentrations were occasionally solution in 226 patients) and two peroral U studies (as peroral solutions). above the maximum of 20% of LLOQ response. Therefore, a full The model precisely predicts the PK of 5-FU in wildtype patients and randomisation of sample order was avoided and after high heterozygous patients for the gene variant *2A. Continuous infusions concentrations, blank samples were processed. VRC showed no showed a circadian pattern in plasma concentration which was instability in the investigated conditions (3 freeze-thaw cycles, 24 h in the accurately described by implementing a time-dependent sine function for autosampler, 12 h storage at room temperature) as mean concentrations enzyme activity of DPD. Ratio of predicted to observed AUC values for ranged from 93.9% to 111% of their nominal concentration. wildtype patients was 1.1 for 5-FU. DHU/U ratios for the four gene variants as well as predicted and observed DGI ratios (AUC Conclusion: The validated LC-MS/MS assay for VRC quantification heterozygous/AUC wildtype) for the most prominent gene variant achieved high accuracy and precision in the large concentration range DPYD*2A lied within the two-fold acceptance limits. Dose from 5 to 5000 ng/mL while using only 5 µL of microdialysate. This recommendations were derived for combinations of hetero- and enables the generation of high-resolution concentration-time curves at homozygous variants. Model predicted dose reductions were 25% for the target site since sampling intervals can be considerably shortened. DPYD*2A for the initial bolus injection, and 50% for the following As a next step, bioanalytical method validation for the quantification of continuous infusion. The current CPIC guideline recommends a 50% VRC and its N-oxide metabolite in human plasma shall be performed. reduction of the 5-FU dose altogether [5]. Conclusion: A comprehensive set of PBPK models for 5-FU and its 1. J. Schulz et al.: Drug Metab Rev [In revision] metabolites were successfully developed. The models captured the 2. European Medicines Agency (EMA): Guideline on bioanalytical method validation 2012. important impact of drug-gene interactions and circadian rhythms and could play an important role in decreasing the occurrence of potential life threatening adverse drug effects by deriving alternative dosing regimens for DPD deficient patients. POS.112 Optimizing 5-fluorouracil chemotherapy with regard to DPD Supported by the Robert Bosch Stiftung (Stuttgart, Germany) and the European Commission drug-gene interactions and circadian effects utilizing a Horizon 2020 UPGx grant 668353 physiologically-based pharmacokinetic (PBPK) modelling approach References 1. Casale F. et al. Pharmacol. Res. 2004, 50(2): 173-9. 2. Diasio R.B. et al., Clin. Pharmacokinet. 1989, 16(4): 215-37. Marok F.Z.1, Wojtyniak J.G.1,2, Schwab M.2,3,4, Lehr T.1 3. Sistonen J. et al., Pharmacogenomics 2014, 15(4): 1653-66. 1 Clinical Pharmacy, Saarland, University Saarbrücken, C 2 2, 66123 Saarbrücken Germany 4. Hishinuma E. et al., Biochem. Pharamcol. 2017, 143: 118-128. 2 Dr. Margarete Fischer-Bosch-Institut für Klinische Pharmakologie, Auerbachstraße 112, 70376 5. Johnson J.A. et al., Clin. Pharmacol. Ther. 2017 102(3): 397-404. Stuttgart, Germany 6. Eissing T. et al., Front. Physiol. 2011 2: 4. 3 Department of Clinical Pharmacology, University Hospital Tübingen, Auf der Morgenstelle 8, 72074 Tübingen, Germany 4 Department of Pharmacy and Biochemistry, University Tübingen, Auf der Morgenstelle 8, 72074 Tübingen, Germany POS.113 A survey on continuing education of pharmacists: Status Background and Objectives: The intravenously administered cytotoxic quo, perspectives and outlook drug 5-fluorouracil (5-FU) is used as a first line agent in the treatment of colorectal cancer and further tumours including breast, head and neck cancer [1]. Due to its poor oral bioavailability and high toxicity 5-FU is administered as a continuous infusion over 22 to 46 hours [2]. Müller, M. A.; Louis, C.; Görgen, M.; Lehr, T. Dihydropyrimidine dehydrogenase (DPD) catalyses the rate-limiting Clinical Pharmacy, Saarland University, Campus C2 2, 66123 Saarbrücken, Germany process in the inactivation of fluoropyrimidine based drugs such as 5-FU [3]. However, DPD’s enzyme activity is highly variable depending on for example polymorphisms in DPYD, the encoding gene for DPD or Background and Objectives: Continuing education refers to post- circadian rhythms. Changes in DPD’s enzyme activity can affect 5-FU secondary learning programs directed to professionals to preserve or plasma levels and ultimately increase the risk for severe toxicities. Thus, extend knowledge. In Pharmacy, new active pharmaceutical ingredients it is important to quantify the influence of circadian expression patterns are approved to the markets regularly. About half of all systematic as well as DPYD polymorphisms to optimize 5-FU therapy regiments and reviews are not up to date anymore after five years [1]. Comparably, consequently limit the occurrence of adverse drug effects [4]. Hence, the medical guidelines are updated every third year averagely. Due to that, objective of this work was to build a physiologically-based pharmacists are obliged to educate themselves further. In Germany, this pharmacokinetic (PBPK) model for 5-FU and its metabolites 5,6- requirement is written down in the professional code of conduct of every dihydrofluorouracil, α-fluoro-β-ureidopropionic acid and α-fluoro-β- federal state’s chamber of pharmacists. However, a uniform catalog alanine and furthermore, develop dose recommendations for the regarding specifications as well as an obligatory burden of proof does not clinically relevant DPYD gene variants c.1905+1G>A (DPYD*2A), exist. As evidence of their dedication, pharmacists may apply to a c.1679T>G (DPYD*13), c.2846A>T (DPYD*9B), and c.1129-5923C>G voluntary certificate; an extent of 150 points (one point equals 45 min of (HapB3) [5]. training) has to be completed within three years. A few chambers of Methods: PBPK model development was performed with PK-Sim® and pharmacists recently started to control pharmacists’ commitment to MoBi® (version 7.4.0) as part of the Open Systems Pharmacology Suite continuing education [2]. Therefore, the objectives of this study were 1) [6]. Data for model development were extracted from literature, including to quantify the extent of pharmacists’ continuing education efforts physicochemical parameters and plasma concentration-time profiles for concerning workspace and scope of medication-related services all compounds and for various DPYD genotypes. Data were separated provided, 2) to evaluate current, and future pharmacists’ opinion on into a training and test dataset for model development and evaluation, continuing education, a burden of proof, motivation, and shortcomings respectively. Additionally, an uracil (U) model was developed and used and 3) to illustrate the status quo using an online survey.

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Methods: An extensive literature search was performed to look for studies on the impact of continuing education, its scope and extent for both pharmacists and other professions such as physicians in Germany and abroad. It includes an analysis of the various professional code of conducts for pharmacists. Each federal chamber of pharmacists was asked to provide the number of voluntary certificates existing concerning the overall count of pharmacists. The online survey was conducted with the tool SoSci Survey [3] and was disseminated in specialist pharmaceutical news media as well as to pharmaceutical organizations such as the DPhG to share it with their members and forward them to this survey. It was directed to both pharmacists (community and hospital pharmacists) and future pharmacists (students of Pharmacy, pharmacists in practical internship) to include their expectation as well. Concerning one’s profession, the survey addressed the knowledge about the duty to continuing education, the necessary extent, the benefit, and the motivation or demotivation. Pharmacists also were asked to their extent of continuing education, their workspace, the extent of medication- related services, integration in practice and time of proficiency. In advance, a pretest was completed successfully. Results: The literature search revealed wide variability in the phrasing to continuing education in the professional codes of conduct. A proof of burden, including consequences for failing, only exists in one pharmacist chamber’s district so far. The voluntary certificate of the chambers is not a significant factor yet. Studies on physicians’ performance evidenced a benefit of continuing education on their skills and patient outcomes [4] proving the value, function, and requirement of continuing education. However, opportunities to pharmacists to educate themselves widely vary among districts. Online tutorials are still of minor importance; a nationwide network does not exist. The online survey revealed broad interests among pharmacists. Overall, 1011 datasets were completed. As 128 participants did not meet the inclusion criteria, 883 valid cases were included (698 licensed pharmacists, 136 students, and 49 pharmacists in internship). Participants from every pharmacist chamber’s district took part in the survey. Among licensed pharmacists, 58.6% (n = 698) worked in a community pharmacy, and 41.4% in a hospital pharmacy. First results show that those working in hospital were more engaged in continuing education (median: 45 points vs. 30 points per year). Asked for the minimum yearly extent of continuing education mandatory, licensed (community and hospital) pharmacists deemed 30 points (median) on average appropriate, students and those in practical internship called for less (median: 24 points). A control mechanism of one’s dedication to continuing education was favored by the majority of the students (77.2%, n = 136). Among licensed pharmacists, the majority (51.1%, n=698) rejected that. In support of such a mechanism were 37.1%. Acceptance was slightly higher among hospital pharmacists (42.6% vs. 33.3%). Conclusion: The combination of literature search and an online survey directed to pharmacists allows drawing a holistic image of the status quo of pharmacists’ continuing education in Germany. A strength is that this study also includes the perspectives, motivation, and requests of colleagues, and those still in training, referring to continuing education, which allows identifying pitfalls to overcome. In mind of the political discussion of further pharmacists’ competencies, such as medication management-related services, it will be crucial that pharmacists ensure to maintain their position as the drug expert. Therefore, harmonization of pharmacists’ activities and dedication to continuing education in Germany is necessary.

References: [1] Shojania, K. G. et al.: Ann Intern Med. 2007; 147(4):224–33. [2] Apothekerkammer Sachsen-Anhalt: Hinweise zur Fortbildungspflicht. https://www.ak- sa.de/bildung/fortbildung/vorgaben/beschr-begriff-fobi.html; accessed 17th of June, 2019. [3] soSci der onlineFragebogen (version: 3.2.01-i): soSci Survey GmbH, München. https://www.soscisurvey.de/de/index; accessed 17th of June, 2019 [4] Cervero, R. D. et al.: Journal of Continuing Education in the Health Professions. 2015; 35(2): 131-38.

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as access to probes for functional studies. NCA pertained as the most 4.6 Natural Compounds active member and was not only effective against cell proliferation but also migration, a novel and so far overlooked activity. The potent anti- migratory effects could also be confirmed in an in vivo breast carcinoma mouse model. To decipher the molecular mode of action, we applied chemical proteomics for target discovery and revealed that NCA targets POS.114 cancer cell migration via irreversible binding to the largely Antioxidative Activities of Phylloxanthobilins, Abundant uncharacterized synaptic vesicle membrane protein VAT-1. A Natural Products Derived from Chlorophyll corresponding knockout of the protein confirmed the phenotype and pull- down studies showed the interaction with an intricate network of key migration mediators such as Talin-1. Overall, we introduce VAT-1 as a Karg C., Wang P., Vollmar A.M., Moser S. promising novel target for the development of selective migration Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich inhibitors with the perspective to limit toxicity in absence of anti- proliferative effects.

In the course of the last three decades, the abundant bilin-type products of Chlorophyll breakdown, named phyllobilins, have come to our attention. Considered ‘mere’ left overs of a controlled biological POS.116 Chlorophyll detoxification originally, phyllobilins are now believed a new Vioprolide A reduces pro-inflammatory processes in opportunity for discovering relevant bioactivities contributing to health human endothelial cells – inhibition of protein translation promoting effects1. Phyllobilins are abundant in leaves, fruit and in some as central mechanism? vegetables2,3. Recently, yellow phyllobilins, the phylloxanthobilins, were shown to occur in unprecedented abundance and diversity in Echinacea purpurea, a plant with undoubted high importance in phytomedicine. Burgers, L. D.1; Luong, B.1; Reichel, C. A.2; Müller, R.3; Fürst, R.1 Even in fresh green leaves - without any ‘obvious’ chlorophyll breakdown 1 Institute of Pharmaceutical Biology, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt 4 am Main, Germany - phylloxanthobilins were discovered . 2Department of Otorhinolaryngology, Head and Neck Surgery, Walter Brendel Centre of Many different pharmacological activities have been demonstrated for Experimental Medicine, Clinical Centre at the University of Munich, 81377 Munich, Germany Echinacea purpurea, nevertheless, neither a single compound nor a 3 Helmholtz Institute for Pharmaceutical Research Saarland, Department of Microbial Natural Products and Department of Pharmaceutical Biotechnology, Saarland University, Campus E8.1, compound class has been identified that can account for all of the 66123 Saarbrücken, Germany efficacies. Caffeic acid derivatives are considered a relevant class of constituents with anti-oxidative activity. We are able to demonstrate a high anti-oxidant potential of the phylloxanthobilins isolated from Inflammation is a biological process with the purpose of protecting the Echinacea, superior to caffeic acid in in vitro experiments and body against pathogens and altered cells. Leukocyte recruitment towards comparable to caffeic acid in cellular approaches. the site of inflammation and activation of pro-inflammatory signalling A structurally different type of phylloxanthobilin has recently been pathways are essential for the elimination of the inflammatory stimuli. identified in de-greened leaves of savoy cabbage5. We show that the After successful elimination of the trigger, inflammation must be resolved newly identified phylloxanthobilin closely resembles bilirubin, an for rebuilding homeostasis. However, through defects which prevent the established strong antioxidant that plays a role in the prevention of body from resolution and rebuilding homeostasis, inflammation becomes various diseases. Savoy cabbage, too, is known to have beneficial chronic and causes the emergence of diseases such as chronic effects on human health, attributed to its high content in antioxidants. respiratory diseases or rheumatoid arthritis. Vioprolide A, a cyclic peptide When investigating the in vitro antioxidative potential of the newly derived from the myxobacterium Cystobacter violaceus, was identified to identified phylloxanthobilin using two different approaches, both influence TNF-induced ICAM-1 expression during the screening of a demonstrated an even higher antioxidative activity for the myxobacterial compound library. ICAM-1 is an adhesion molecule that phylloxanthobilin from savoy cabbage compared to bilirubin. promotes the tight adhesion of leukocytes to the endothelium which is a The identification of phylloxanthobilins as powerful anti-oxidative key step in the extravasation of leukocytes. constituents in plants will serve as incentive for further studies on the We aimed to investigate the effects of vioprolide A on inflammatory potential bioactivities of these underexplored natural products. processes in the endothelium and to get insights into the underlying mechanisms of action. Initial data revealed that vioprolide A reduced TNF-induced leukocyte adhesion to primary human umbilical vein 1. Moser S., Kräutler B., Isr. J. Chem., 2019, 59 (5), 420-431 2. Müller T., Ulrich M., Ongania K.H., Kräutler B., Angew. Chem. Int. Ed., 2007, 46, 8699 endothelial cells (HUVECs) after 24 h. Intravital microscopy of 3. Roiser M., Müller T., Kräutler B., J. Agric. Food Chem., 2015, 63 (5), 1385 postcapillary venules of the cremaster muscle in mice confirmed a 4. Karg C. A., Wang P., Vollmar A. M., Moser, S., Phytomedicine 2019, 152969. reduction of firm adhesion and transmigration through the vascular 5. Karg C. A., Schilling C.M., Allmendinger L., Moser, S., J. Porphyr. Phthalocyanines 2019, in endothelium in vivo. Leukocyte adhesion is promoted by adhesion press. molecules like E-selectin, ICAM-1 and VCAM-1. As examined by flow cytometry, vioprolid A concentration-dependently (0.3–10 nM) reduced the TNF-induced cell surface expression of these molecules after short (4 h) as well as long (24 h) treatment periods. The expression of these adhesion molecules is mainly regulated via the NF-κB signalling pathway. In order to test if the reduced adhesion molecule cell surface expression was caused by an effect on gene transcription, mRNA POS.115 expression of these adhesion molecules was investigated. Although Neocarzilin A is a potent inhibitor of cancer cell motility mRNA expression decreased after long (16 h) treatment periods, short targeting VAT-1 controlled pathways (6 h) treatment periods surprisingly revealed elevated mRNA levels. This is in contrast to the reduced cell surface expression after 4 h and indicated that vioprolide A might rather interfere with protein translation Carolin M.-L. Gleissner1‡, Carolin L. Pyka2‡, Wolfgang Heydenreuter1‡, than mRNA expression. Hence, general protein synthesis upon Thomas F. Gronauer1, Carina Atzberger2, Vadim Korotkov1, Weiting vioprolide A treatment was examined showing a concentration Cheng2, Stephan M. Hacker1, Angelika M. Vollmar2, Simone Braig2* and dependent (0.3–10 nM) and time dependent impairment with a maximum Stephan A. Sieber1* † Center for Integrated Protein Science at the Department of Chemistry, Technische Universität effect after 16 h. Since the activation of the pro-inflammatory NF-κB München, Lichtenbergstrasse 4, Garching, D-85747, Germany. signalling pathway relies on the binding of TNF to its cell surface receptor § Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich (TNFR1), its protein expression was analysed. Interestingly, vioprolide A (LMU), Butenandtstrasse 5-13, Munich, D-81377, Germany. reduced the protein expression in a time-dependent manner showing a maximum effect after 16 h corresponding to the findings of general protein synthesis analysis. After 24 h TNFR1 protein expression started The natural product neocarzilin A (NCA) was discovered decades ago to rise again. qPCR experiments verified that the reduced TNFR1 protein and despite its potent cytotoxic effects no mode of action studies were expression upon vioprolide A treatment (10 nM) was due to an impact on performed up to date. Synthesis of neocarzilins A, B, C and a protein level and not mRNA expression as there was no reduction over stereoisomer of NCA provided insights into structural preferences as well time in TNFR1 mRNA levels. Since the effect on protein synthesis was

DPhG Annual Meeting 2019 Conference Book • 134 NATURAL COMPOUNDS stronger after 16 h than after 24 h, leukocyte cell adhesion as well as adhesion molecule cell surface expression were re-analysed after 16 h. In both cases vioprolide A (10 nM) had a stronger effect after 16 h than 24 h, indicating that the effects on leukocyte migration and adhesion molecule expression might be connected to the observed impairment of translation. Taken together, vioprolide A may inhibit pro-inflammatory mechanisms such as leukocyte adhesion by impeding the expression of cardinal proteins such as TNFR1 caused by an impairment of translation. The underlying mechanism of translational regulation is currently studied.

POS.117 Enrichment of bioactive phytochemicals from natural extracts by Centrifugal Partition Chromatography using polarity adjusted solvent regimes

Termer M.1; Fromme A.2; Lopattschenko M.2; Schembecker G.2; von Hagen J.3; Keck C.1 1 Department of Pharmaceutics and Biopharmaceutics, Philipps-Universität Marburg, Marburg, Germany 2 Department of Biochemical and Chemical Engineering, Technische-Universität Dortmund, Dortmund, Germany 3 Merck KGaA, BU Performance Materials, Darmstadt, Germany

Plant extracts contain various types of bioactive compounds and their isolation is a major challenge for the identification and characterization of the biological activity.1 The aim of this work was to develop a polarity adjusted solvent fractionation to predict the required parameters for the isolation of the biological active substances from a complex natural extract using Centrifugal Partition Chromatography. Selected phytochemical standards with different polarity were chosen to obtain information for the required solvent systems and the prediction of retention times by partition coefficients. In the first step suitable solvent mixtures and suitable concentrations were identified, which enabled not only an enlargement of the required design space but also a baseline separation for all reference substances. In the next step the operating conditions were transferred to a natural plant extract containing the phytochemicals used above for defining the corresponding design space. The results show that the retention times of the phytochemicals in the plant extract corresponded with those of pure substances with high purity. In fact, by using Centrifugal Partition Chromatography, we were able to separate the main phytochemicals of a complex natural extract by developing a polarity adjusted fractionation protocol and predicting optimal operation conditions. In the next steps, bio activity tests of the purified phytochemicals will be performed in comparison to the reference substances. Furthermore, the efficiency of this approach will be transferred to further plant extracts and other phytochemicals.

1 Handa, S. et al.: Extraction Technologies for Medical and Aromatic plants (ICS-UNIDO) 2008

135 • DPhG Annual Meeting 2019 Conference Book POSTERS

Results of a thermal shift assay revealed that the carbazole derivative 4.7 Inflammation C81 shows the highest binding affinity to a number of kinases such as the BMP-2-inducible kinase (BMP2K/BIKE), the adaptor-associated kinase 1 (AAK1) as well as to the CDC-like kinase 1 and 4 (CLK1/4) and the proto-oncogene, serine/threonine kinase PIM3. Since the vascular POS.118 endothelium crucially regulates inflammatory processes, we NHC Gold compounds supress immune responses by hypothesized that these kinases might play a pathophysiological role in inducing the AHR-TGF β1 signalling pathway in vitro and in the inflammation-activated endothelium. The actions of these kinases scurfy mice have not been characterized in the vascular endothelium so far. Therefore, we aimed to analyze the pharmacological potential of C81 and to investigate the role of these kinases in inflammatory processes in Rodrigo. A. Gama-Brambila1, Iart Luca Shytaj2, Stefanie Haeberle3, vascular endothelial cells. Ingo Ott4, Marina Lusic2, Eva N. Hadaschik3, Stefan. Wölfl1, Xinlai To analyze the potential involvement of the aforementioned kinases an Cheng1 in vitro cell adhesion assay using human umbilical vein endothelial cells 1Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany (HUVECs) and the monocyte-like THP-1 cell line was performed. Here 2German Center for Infection Research (DZIF), Heidelberg, Germany only endothelial cells were treated with respective kinase inhibitors. The 3Department of Dermatology, University Hospital Heidelberg, Germany 4Institute of Medicinal and Pharmaceutical Chemistry, Technical University of Braunschweig, inhibition of AAK1, CLK1/4 or PIM3 in endothelial cells did not impair the Germany adhesion of THP-1 cells onto a TNF-activated HUVEC monolayer. Of note, RNAi silencing of BMP2K in HUVECs reduced the adhesion of THP-1 cells significantly. The immunosuppressing activity of certain gold complexes has long been Initial experiments using C81 show that only high concentrations of the identified and broadly studied. Nevertheless, the precise mechanisms compound affected the viability of HUVECs after 24 hours of treatment remain unclear. Using a novel liver-on-a-chip system we found that gold (IC50: 171 μM). complexes, containing the planar N-heterocyclic (NHC) carbene moiety In vivo results of intravital microscopy in the murine cremaster muscle as ligand, are potential ligands of the Aryl Hydrocarbon Receptor (AHR). demonstrated that the adhesion of leukocytes was significantly reduced Our leading compound MC3 has been further studied and was found to after C81 treatment. In in vitro cell adhesion assays C81 treatment activate the TGF-β1 signalling pathway, resulting in the suppression of significantly decreased the adhesion of THP-1 cells onto inflammatory- + CD4 T-cell activation in vitro, in human and mouse T-cells. Genetic activated endothelial cells under static and under flow conditions, which knockdown, chemical antagonists of the AHR or inhibition of the of the better mimic the physiological situation. Importantly, also the TGF-β1 signalling pathway decreased the activity of MC3. Treatment transmigration of THP-1 cells through a TNF-activated endothelial with MC3 on Scurfy mice, a mouse model of the human IPEX syndrome, monolayer in the direction of a chemoattractant gradient was significantly reduced the autoimmune response and prolonged the lifespan to up to reduced by C81. As the interaction of leukocytes and the endothelium is 60 days. In conclusion, these results demonstrate that a NCH-gold (I) mainly mediated by cell adhesion molecules (CAMs), the effect of C81 complex has immunosuppressive effects in scurfy mice; suggest that the on their expression was analyzed (western blot, flow cytometry, qPCR) immunosuppressive effect of gold complexes in enhanced using planar in HUVECs. The total and the surface protein expression of ICAM-1, NCH moieties as ligands to activate AHR-related pathway; and open a VCAM-1 and E-selectin as well as their mRNA levels were strongly new clinically potential target and treatment of autoimmune diseases. decreased after C81 treatment. Of note, mRNA levels and the surface expression of CAMS were not attenuated in BMP2K knockdown cells. Acknowledgments: Thank others for any contributions. Funding This work supported by the Moreover, C81 significantly reduced the TNF-induced activation of the DFG grant program (CH 1690/2-1) and the BMBF grant programs Drug-iPS (FKZ 0315398A- MAPK JNK, while the phosphorylation of p38 was not impaired. FKZ 0315398B) and SysToxChip (FKZ 031A303A-FKZ 031A303E). Interestingly, western blot analysis revealed that C81 significantly decreased the total protein expression of the TNF receptor 1, while its References: mRNA level remained unaffected. Initial results of the analysis of effects X. Cheng et al.: Molecular cáncer. 2014, (13): 221 of C81 on the inhibition of the de novo protein synthesis indicate that the X. Cheng et al.: NHC Gold compounds supress immune responses by inducing the AHR-TGF compound might affect the translation of proteins with short half-life β1 signalling pathway in vitro and in scurfy mice (under revision) resulting in a reduced expression of the TNF receptor 1 after C81 treatment. Our study provides first insights into the anti-inflammatory action of the carbazole derivative C81 in vitro and in vivo. Since the inhibition of POS.119 BMP2K seems to be responsible only for some of the pharmacological The role of the carbazole derivative C81 in the actions of C81 and the involvement of kinases predicted by the thermal inflammatory activated vascular endothelium shift assay can be excluded for the anti-inflammatory actions of the compound, we will further investigate the role of other potential targets. The precise role of BMP2K in inflammatory endothelial processes as well Bischoff I.1, Hatemler G. M.1, Strödke B.3, Knapp, S.4,5, Reichel, C. 6, as the involved pathways during BMP2K silencing and C81 treatment will Mittmann, L. 6, Bracher F.3 and Fürst R.1 be further elucidated. 1 Institute of Pharmaceutical Biology, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany 2 Pharmaceutical Biology, Center for Drug Research, University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany 3 Department of Pharmacy - Center for Drug Research, University of Munich, Butenandtstr. 5- 13, 81377 Munich, Germany 4 Institute for Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Str. 9, 60438 POS.120 Frankfurt am Main, Germany Beyond leukotriene formation-Influence of 5-lipoxygenase 5 Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus, Headington, Oxford OX3 7BN, United Kingdom on gene expression. 6 Walter Brendel Centre of Experimental Medicine and Department of Otorhinolaryngology, Head and Neck Surgery, Klinikum der Universität München, Ludwig-Maximilians-Universität München, Marchioninistr 15, D-81377 Munich, Germany Kreiss, M.1; Liu, C. Y.1; Sürün, D.2; Sorg, B. L.1; Steinhilber, D.1; Häfner, A. K.1 1Institute of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Str. 9, 60438 During inflammation leukocytes migrate through the activated endothelial Frankfurt/Main, Germany, [email protected] barrier into the underlying tissue where they differentiate into distinct 2Department of Molecular Hematology, Goethe University Medical School, 60590 Frankfurt am macrophage phenotypes amplifying the inflammatory response. In the Main, Germany healthy organism this process is physiologically terminated, whereas the pathophysiological situation of chronic inflammatory diseases, such as 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of psoriasis or rheumatoid arthritis, is characterized by ongoing leukocyte leukotrienes and specialized proresolving lipid mediators (SPM) [1, 2]. It infiltration resulting in severe tissue damage. Therefore, there is a great is mainly expressed in leukocytes and is part of the innate immune need for the discovery of new drug leads and targets for system. 5-LO can shuttle between the cytosol and the nucleus. Upon cell pharmacotherapeutic treatment of these disorders. activation the protein translocates from soluble cellular compartments to the nuclear membrane. Besides the 5-LO activating protein (FLAP) which

DPhG Annual Meeting 2019 Conference Book • 136 Inflammation is required for cellular leukotriene and SPM formation, 5-LO interacts with other proteins like coactosin-like protein (CLP) [3], Dicer [4], β-catenin [5] and p53 [6]. Here, we give an insight into the role of 5-LO in the regulation of cell proliferation and differentiation and its biological functions apart from leukotriene and SPM formation. We present data, how 5-LO may act as a STRaND (shuttling transcriptional regulator and non-DNA binding) influencing the gene expression of target genes. Therefore, we introduced a 5-LO knockout (KO) in the myeloid cell line MonoMac6 and compared the mRNA expression levels of target genes between KO and wild type cells.

References: 1. Samuelsson B, et al.: Science 1987; 237: 1171-1176. 2. Serhan CN, et al.: Cold Spring Harb. Perspect. Biol. 2015; 7: a016311. 3. Provost P, Samuelsson B and Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881- 1885. 4. Dincbas-Renqvist V, et al.: Biochim. Biophys. Acta - Gene Regul. Mech. 2009; 1789: 99-108. 5. Roos J, et al.: Cancer Res. 2014; 74: 5244-5255. 6. Gilbert B, et al.: Biochim. Biophys. Acta - Gene Regul. Mech. 2015; 1849: 1003-1016.

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4. Dhand, R., et al., Improving usability and maintaining performance: human-factor and aerosol-performance studies evaluating the new reusable Respimat inhaler. Int J Chron 4.8 Pharmaceutical Technology Obstruct Pulmon Dis, 2019. 14: 510-512.

and biomaterials

POS.121 POS.122 Modelling a coextrusion blow molding process with a Relationship between disintegration and dissolution of neural network enteric capsules

Gattig, P.1; Langguth, P.2 1 Boehringer Ingelheim, Binger Str. 173, 55216 Ingelheim am Rhein, Germany 1 1 1 2 2 2 Johannes Gutenberg-Universität Mainz, Staudingerweg 5, 55128 Mainz, Germany Fu,M.Q. ; Blechar,J.A. ; Al-Gousous,J. ; Sauer,A. ; Lesser,I. ; Eggers,T.2, Langguth,P.1 1 Pharmaceutical Technology and Biopharmaceutics, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Introduction: Classically, the goal of pharmaceutical development is to Mainz D-55128, Germany 2 Shin-Etsu Pharma & Food Materials Distribution GmbH, Wiesbaden D-65203, Germany design a quality product and its manufacturing process in such a way that the defined performance of the product (QTPP - quality target product profile) is consistently achieved (ICH Q8). This approach is called QbD - Quality by design. Following the approach, the effects of material and Disintegration testing is an important quality control (QC) test for process parameters on the product CQAs are determined. This functional evaluation of solid dosage forms. However, since complete disintegration connection between input and output can be determined by means of does not necessarily mean complete dissolution, more research has prior knowledge, experimental data, the control strategy or a combination focused on dissolution rather than disintegration testing. Nevertheless, thereof. [1] due to its simplicity, in some cases, disintegration testing seems to be an Objective: Establish a model, which can be found via an analytical attractive alternative to dissolution testing as recognized for example by solution, a simulation or a parameter study. [2] A standard method might the International Conference on Harmonization guideline Q6A. be DOE (Design of Experiments) which would provide a linear or a Nevertheless, based on published studies, it is recommended to analyze quadratic approximation, at best. Usually, a DOE requires a set of for a correlation between disintegration and dissolution for each type of experiments [3], which should be avoided and instead, pre-existing data dosage form and formulation composition before substituting the should be used. dissolution test in favor of the simpler disintegration test [1]. Enteric coated oral dosage forms continue to be popular for formulating Background: Boehringer Ingelheim developed and produces the acid labile drugs, or drugs that are harmful to the stomach wall, or for the Respimat, a propellant-free soft mist inhaler. This consists of a device aim of intestinal targeting of drug release [2]. Furthermore, these types and a drug-filled cartridge. The latter consists of a double-walled plastic of dosage forms have shown unreliable performance in vivo in patients, container inside an aluminum casing. The plastic container consists of suggesting that improvements are needed, both with respect to the outer and inner foils and is produced by means of a coextrusion blow formulation but also regarding the in vitro test procedures [3]. molding process. The production of such a container is dependent on An important quality parameter of enteric oral dosage forms is the release many, partly mutually influencing, parameters. [4] of the drug following gastric emptying. Recent studies have shown that the release in simulated enteric media may be very dependent on the test procedures [4]. The relationship between gastro-resistant capsule disintegration and Input variables Output variables ‐ mechanical settings dissolution has been investigated in this study using enteric coated ‐ in process controls ‐ process parameters Black Box HPMC capsules and enteric hypromellose capsules (DRcaps®, Artificial neural network Capsugel) containing caffeine as model substance. Environment variables ‐ humidity Production efficiency HPMC size 0 hard capsules were filled with a mixture of caffeine and L- ‐ air pressure HPC, lactose, silicon dioxide and magnesium stearate and coated with enteric HPMCP (HP-50) coating solution in ethanol/water containing talc Figure 1: Black box of the functioning of the simulation program as anti-tacking agent. Capsule coating was carried out in a drum coater (Bosch Solidlab 1, Germany). For each trial the pan was loaded with 0.368 kg (700 ml) capsules prefilled with 350 mg of powder. At the end Experimental: A simulation software was programmed to describe the of the coating process the capsules had received 10 mg/cm2 of solid blow molding process of the plastic container. This makes it possible to coating materials. Alternatively, ready-to-use enteric hypromellose estimate the influence of modified input variables on the CQAs. In capsules (DRcaps®, Capsugel) were filled with the same powder mixture addition to the 26 process parameters, the environmental variables and tested for disintegration and dissolution. Tests were carried out in a (absolute air pressure and air humidity) are also taken into consideration PhEur/USP disintegration tester at testing conditions specified in the (see figure 1). The simulation uses a five-layer neural network (multilayer pharmacopoeia and additionally in a 15 mM phosphate buffer pH 6.5 perceptron) and was trained with data from production monitoring. The which previously has demonstrated improved biopredictivity in terms of simulation program is linked to several databases of the company onset of drug release [5]. Dissolution tests were carried out in a Boehringer Ingelheim and enables an easy operation due to its PhEur/USP type 1 tester in 700 ml 0.01N HCl (1 hr) followed by transfer interactive structure. to phosphate buffers at pH 6.8 (50 mM) and alternatively pH 6.5 (15 mM).

The concentration of caffeine in the dissolution fluid was quantified by UV Results: Connections between the machine settings/ process spectrophotometry at 275 nm. parameters/ environment variables on the output variables are thus The time of disintegration was defined as the duration between test onset intuitively visible and quantitatively documented. In addition, the results and visible appearance of cracks in the coating of enteric dosage forms. of the simulation can be used for process control as they solve the multi- The results of both tests share the same trend, demonstrating a linear dimensional optimization problem. relationship between disintegration times and times for 5% (10%) drug dissolution. Therefore, the disintegration test applied to enteric hard capsules can predict the results of the dissolution test in terms of onset Acknowledgments: Boehringer Ingelheim, Johannes Gutenberg Universität Mainz, Dr. Herbert of drug dissolution and could be used in lieu of dissolution testing for in Wachtel vitro lag times in intestinal media.

References: 1.ICH Expert Working Group, ICH guideline Q8 (R2) on pharmaceutical development (Step 5), [Acknowledgments] in Linking material attributes and process parameters to drug product CQAs. 2017, European This work was supported by a scholarship from China government (CSC). medicines Agency: 14. 2. Struckmeier, J., Mathematische Modellierung und Simulation, U. Hamburg, Editor. 2004: Fachbereich Mathematik: 4-5. [References] 3. Institut für Technologie und Arbeit, Design of Experiment (DoE), Statistische 1.Gupta A, Hunt RL, Shah RB, Sayeed VA, Khan MA. Disintegration of highly soluble Versuchsplanung. 2013, Optimus-Spitzencluster: 5. immediate release tablets: a surrogate for dissolution. AAPS PharmSciTech. 2009,10: 495-499

DPhG Annual Meeting 2019 Conference Book • 138 PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS

2. Cole, E. T.et al. Enteric coated HPMC capsules designed to achieve intestinal targeting.Int. J. The optimal required lecithin concentration was identified to enable Pharm., 2002,231:83–95 stabilised particles in a sodium chloride solution imitating physiological 3. Al-Gousous J.et al. Unpredictable Performance of pH-Dependent Coatings Accentuates the condition. Need for Improved Predictive in Vitro Test Systems. Mol Pharm. 2017,14(12):4209-4219 Furthermore, for targeting issues the amphiphilic apolipoprotein E (ApoE) 4. J. Al-Gousous and P. Langguth: European versus United States Pharmacopoeia-specified disintegration testing for enteric-coated soft gelatin capsules. Dissolution Technologies was added after preparation with the aim of an adsorptive binding to the 2015,22(3) amphiphilic lecithin without affecting the protein’s functionality [1]. 5. Al-Gousous J, Amidon GL, Langguth P. Toward biopredictive dissolution for enteric coated The bound amount of ApoE was determined by SDS-PAGE and dosage forms. Mol. Pharm. 2016, 13(6):1927-36 correlates well with the nanoparticle yield. The particle cores were labelled with the fluorescent dye Lumogen F Red 305 enabling tracking in cell culture experiments as POS.123 well as to simulate drug embedment. All characteristics were preserved Influence of PEG spacer selection on active targeting of during this procedure, which proved the capability of this system to trastuzumab-modified nanoparticles possibly deliver an embedded drug. The abilities as drug delivery system were tested by analysing the nanoparticles in cell culture studies at endothelial cells and at an in vitro Barth C., Mulac D., Langer K. blood brain barrier (BBB) model, because ApoE modified lipid Institute of Pharmaceutical Technology and Biopharmacy, University of Münster, Corrensstr. 48, nanoparticles are a promising strategy to overcome the BBB [1, 2]. 48149 Münster, Germany In conclusion, a new, biocompatible drug delivery system based on cholesteryl oleate, which mimics physiological lipoproteins, has been Despite new and innovative cancer therapeutics being approved for successfully established. clinical use in the last years systemical side effects are still a central problem in conventional chemotherapeutics. Therefore, therapy options References: specifically targeting tumor tissue are a desired approach. One promising 1. Rajora, M.A. et al.: Chem Sci 2017, 8: 5371-5384 idea to overcome this obstacle is using nanoparticulate drug vehicles in 2. Nikanjam, M. et al.: Int J Pharm 2007, 328: 86-94 combination with ligands (e.g. antibodies) which address specific target structures on the tumor [1]. The effect of this active targeting has been investigated for many ligands and their targets for cancer treatment in the last years [2]. In this context attractive targeting structures for POS.125 nanoparticulate drug delivery systems are receptors which are Surface modification of PLGA nanoparticles with cysteine overexpressed in tumorous tissue. In the present study nanoparticles (NP) modified with the humanized for oral drug delivery: Analysis of a modified stabilizing monoclonal antibody trastuzumab were used to target the human agent as an example for polyvinyl alcohol modification via epidermal growth factor receptor 2 (HER2) on HER2 overexpressing divinyl sulfone. breast cancer cells. NP based on human serum albumin were prepared using a desolvation method and stabilized with glutaraldehyde. Subsequently the surface of Alberding, G.; Mulac, D.; Langer, K. Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster, the NP was modified with a bifunctional polyethylene glycol (PEG) Corrensstr.48, 48149 Muenster, Germany spacer, using two different molecular weights (5 kD and 10 kD). The antibody trastuzumab was thiolated with 2-Iminothiolane and covalently linked to the PEG spacer. Oral application is the most favorable route of administration respecting Cell culture studies on active targeting were performed using the HER2 patient compliance. However, many active pharmaceutical ingredients overexpressing breast cancer cell line BT-474 [3]. To investigate the cell (API) show limited oral bioavailability due to poor solubility or permeability association of the NP and the influence of the different PEG spacers on at the gastrointestinal barriers. Embedding the API into nanoparticles this process, different methods, including flow cytometry, life cell imaging (NP) is a promising opportunity to overcome these obstacles. A and fluorescence microscopy, were used. functionalized NP with the ability to pass the intestinal membrane can be In conclusion stable NP modified with trastuzumab using two different used as carrier for several different API to increase their oral PEG spacers varying in molecular weight could be prepared. Active bioavailability. Poly(lactic-co-glycolic acid) (PLGA) is a promising starting targeting of HER2 overexpressing cells was achieved, and therefore a material for such NP as its in vivo application is already approved. M- receptor mediated uptake of the NP into the cells with both tested PEG cells, as a discussed transport region for unmodified PLGA NP in vivo, spacers was proven. are less numerous than e.g. enterocytes in the intestine [1]. Therefore, surface modification of NP is necessary to increase the permeability by addressing specific transportation mechanisms. Chemical modification of PLGA is difficult since the functional end group, a carboxylic acid, is not References: available in some versions of PLGA, limiting the applicability of 1. Bertrand N. et al.: Adv. Drug Deliv. Rev. 2014, 66: 2-25 modifications at this position. 2. Bazak R. et al.: J. Cancer Res. Clin. Oncol., 2015, 141(5): 769-784 3. Subik K. et al.: Breast Cancer (Auckl), 2010, 4: 35-41 In this study, a novel method of surface alteration [2] via modification of polyvinyl alcohol (PVA) with divinyl sulfone (DVS) is varied, analyzed and tested for preparation of PLGA NP. PVA represents a common stabilizer for preparation of PLGA NP. Since PVA is located at the surface of the NP after preparation [3] the modification of PVA leads to a functional POS.124 surface of the NP. Lipoprotein Mimicking Nanoparticles as Colloidal Drug PVA 4-88 (MW: 25-30 kDa) was coupled with DVS (MW: 118.15 g/mol), Delivery System leading to a product containing a reactive vinyl group (PVA-VS). Reaction time was controlled by adjusting pH value (Fig.1). After purification via dialysis (membrane: regenerated cellulose, MWCO: 3.5 kDa) analysis Wünsch, A., Mulac, D., Langer, K. via 1H-NMR spectroscopy proved the presence of the vinyl group. Institute of Pharmaceutical Technology and Biopharmacy, University of Münster, Corrensstraße 48, 48149 Münster, Germany O x y CH2 z x y S 0.1N NaOH Lipoproteins form a colloidal transport system for triglycerides, OH O CH3 + O OH O CH3 H2C O pH 13 cholesterol and cholesteryl esters in the human body. Therefore, the idea O O O 90 s S of this work is the imitation of this natural occurring system as a new drug PVA DVS O PVA-VS delivery system. CH2 The main components of physiological lipoproteins are phospholipids Figure 1: Reaction of PVA with DVS. and apolipoproteins surrounding a lipophilic core. The new developed nanoparticle system imitates this structure by using cholesteryl oleate as PVA-VS can be versatilely used for reaction with substances containing a solid lipophilic core, coated by lecithin as emulsifying agent, preventing nucleophilic structure characteristics (e.g. particle agglomeration. -NH2, -SH). L-Cystein (CYS; MW: 121.16 g/mol) was used due to its high

139 • DPhG Annual Meeting 2019 Conference Book POSTERS nucleophilicity of the thiol group. Cystein modified NP are plausible for oral application, since the physiological transport mechanisms for amino Conclusion: acids can be addressed. The product of the reaction of PVA-VS with CYS Results prove that tablets made from paper are not only feasible for an (PVA-VS-CYS) was purified via dialysis, structural analysis via 1H-NMR individualized drug therapy with a manual processing of smartFilms®, spectroscopy showed that no unreacted vinyl groups were left. The e.g. in an official pharmacy, but can also be produced in large-scale by amount of bound CYS was quantified indirectly analyzing the quantity of the production of pellets made from paper, which are subsequently unreacted CYS via reaction with Ellman’s reagent and photometric transferred into tablets. Results demonstrate again, that tablets made analysis. Variation in reaction time of PVA with DVS (30 s, 90 s, 120 s) from paper are a real alternative to classical powder tablets, especially led to different amounts of vinyl groups in the product PVA-VS which was for the formulation of actives with poor aqueous solubility and low oral verified by an increased amount of covalent bound CYS in the product bioavailability. PVA-VS-CYS. PLGA NP were prepared with unmodified PVA as well as with PVA-VS- [1] Lemke, S. et al.: German Patent Application 2016, DE102016000541A1. CYS and analyzed via photon correlation spectroscopy (PCS). NP [2] Stumpf, F. et al.: Int. J. Pharm. 2018, 584(2): 812-819. prepared with PVA or PVA-VS-CYS led to systems with comparable [3] Stumpf, F., PhD-thesis, Philipps-Universität Marburg, 2019. physicochemical characteristics. Modification of PVA before using it as stabilizer for preparation of PLGA NP allowed a quantitative and qualitive analysis of the product and ensured covalent binding of the ligand. The results clearly show that preparation of stable, surface modified PLGA POS.127 NP via PVA-VS modification is possible. Finasteride nanocrystals for dermal application to References overcome the severe side effects of the oral administration 1. Beloqui, A., des Rieux, A., Préat, V.: Adv Drug Deliv Rev 2016 106(B): 242-255. route. 2. Raudszus, B., Mulac, D., Langer, K.: Int J Pharm 2018 536(1): 211-221. 3. Spek, S. et al.: Appl Surf Sci 2015 347: 378-385. Steffen F. Hartmann1, Ralph-Walter Eckert1, Franziska Bär1, Cornelia M. Keck1 1Philipps-Universität Marburg, Department of Pharmaceutics and Biopharmaceutics, Robert- POS.126 Koch-Str. 4, 35037 Marburg, Germany Tablets made from paper – pelleting and industrial scale up

Androgenetic Alopecia is a common disease, which often is Bär, F.1; Stumpf, F.1; Knoth, D.1; Keck, C. M.1 accompanied with a major loss of life quality. One of its main causes lies 1 Philipps-Universität Marburg, Department of Pharmaceutics and Biopharmaceutics, Robert- in a genetic hypersensitivity for dihydrotestosterone of the hair follicles. Koch-Str. 4, 35037 Marburg, Germany An established treatment for this malady are 5-alpha-reductase inhibitors, like finasteride. Unfortunately, the common oral way of application leads to severe side effects, like constant loss of libido, Introduction: continuing sexual malfunction and severe depression [1]. Tablets made from paper are a newly developed carrier system based To still provide the patients with the benefits of finasteride and on smartFilms® [1]. The incorporation of an active into an ordinary paper dramatically reduce the severity of the side effects, it is of major (e.g. copy paper, coffee filter) matrix leads to an amorphous stabilization importance to find alternative ways to the common oral application route. of the active, which then leads to a pronounced increase in solubility and Recent studies have shown, that such a promising alternative might be a bioavailability. Hence, the technology is especially interesting for actives local application of the active and preferentially a targeting to the hair with low solubility, i.e. BCS class II and IV drugs [1]. Furthermore, it has follicles, where it can act as a depot [2]. Due to the poor aqueous been shown that smartFilms® could easily be transferred into tablets just solubility of finasteride, a formulation as nanocrystals seems to be by compressing them, without any further processing or additives. These feasible [3]. Therefore, the aim of this study was to develop a nanocrystal tablets fulfil every requirement of the European Pharmacopeia 8.0 (EP) suspension of finasteride for the development of dermal applicable and are a real alternative to classical tablets being derived from powders formulations. [2]. Nevertheless, smartFilms® don’t fit into the matrix of a tablet press For this study, state-of-the-art NanoWitt LAB-100 milling equipment and possess poor flowability. Therefore, the aim of this study was the (Frewitt fabrique de machines S.A, Fribourg, Switzerland) was used. development of flowable smartFilms® pellets for the automatized Finasteride suspensions were prepared with 1% (w/w) finsateride and production of tablets made from paper. 1% (w/w) Brij L23 as stabilizing non-ionic surfactant. The circulation of Methods: the suspension was set to 500 ml/min for 120 minutes. Samples were Commercially available paper tissues were used as paper basis for the taken after 15 min, 30 min, 45 min, 60 min, 90 min, and 120 min, production of pellets. At first the paper was shredded with a knife mill. respectively. The rotor of the milling chamber was adjusted to 1500 rpm, The resulting cellulose fibres were loaded with rutin in a fluidized-bed with a bead to suspension-ratio of 60 : 40 (V/V). Suspension temperature reactor and subsequently granulated with a 1:1 solution of water and was monitored during the hole milling procedure and was constantly kept sucrose, where sucrose was used to improve the plastic deformation between 15 to 20°C. Three different sizes (small: 100 µm, medium: capacity of the pellets [3]. The pellets were sieved to obtain narrowly 300µm and large: 400 µm) of yttria stabilized zirconia beads (SiLibeads sized pellets and were subsequently compressed into tablets by using an Type ZY-E, Sigmud Lindner GmbH, Switzerland) were used. Size EK0 tablet press in continuous mode. Both, the pellets and the resulting measurements were performed by static light scattering (SLS) using a tablets were tested regarding their pharmaceutical quality according to Mastersizer 3000 (Malvern Panalytical, Germany) and dynamic light the EP. scattering (DLS) using a Nanosizer ZS (Malvern Panalytical, Germany). Results: To determine the optimal production conditions the D(v)0.95 values Optimized pellets possessed a mean particle size of about 782 µm. The obtained by SLS, and the average sizes (hydrodynamic diameter and Hausner ratio was 1.18 ± 0.01 and the Carr index was 15.3 ± 0.9, polydispersity index) obtained by DLS were compared for the different indicating good flowability and good tableting properties of the pellets. production conditions. For the medium sized beads, the average sizes The resulting tablets (Fig. 1) fulfilled all requirements (e.g. mass were 200 nm (90 min) and 200 nm (120 min). With sizes of 195 nm uniformity, content uniformity, friability, resistance to crushing, (90min) and 185 nm (120min) for the small sized beads, there were no disintegration, dissolution) according to the EP. relevant differences in size with respect to bead size or time. For the large beads the average sizes were slightly larger (280 nm (90min), 270 nm (120 min)), but also here no relevant difference in size between the two milling times could be detected. The previously shown DLS data were consistent with the SLS measurements, since no relevant differences in the measured sizes could be detected between 90 min and 120 min of processing time, for the small and medium sized beads. Compared to an initial D(v) 0.95 of Fig. 1: Production steps for tablets made from paper. Left: Paper basis; 10 µm, every condition applied led to a pronounced reduction in size. second from left: shredded paper fibres; third from left: rutin loaded After 90 min of processing time the small sized beads showed a D(v) 0.95 pellets; right: tablets made from paper. of 0.51 µm whereas the medium sized beads showed a D(v) 0.95 of 0.49

DPhG Annual Meeting 2019 Conference Book • 140 PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS

µm. In according to the DLS data, the D(v) 0.95 of the large beads was slightly larger with 1,2 µm. Due to a more effortless handling of the medium sized beads, which also is an economical advantage, it can be Acknowledgments: The authers thank Cornelia Wiegand for providing the Moleculight® i:X. This concluded, that the optimal production parameters are the medium sized work was supported by the German Research Foundation (FI 899/4-1, FI 899/4-2, PL 320/3-1, beads and a milling time of 90 minutes. PL 320/3-2). In conclusion, in this study small sized finasteride nanocrystals were successfully developed. The resulting nanosuspensions can now be References: used for the formulation of dermal drug products to treat androgenetic 1. Bos, A.C. et al.: J. Cyst. Fibros. 2017, 16, 13-23. alopecia without severe side effects of the oral application route. 2. Klinger-Strobel, M. et al.: Expert Opin Drug Deliv. 2015, 12, 1351-1374. 3. Ungaro, F. et al.: J. Contr. Rel. 2012, 157, 149-59.

Acknowledgments The authors kindly acknowledge the support of Frewitt fabrique de machines S.A., Switzerland to provide the NanoWitt LAB-100 milling equipment. The study was partly financed by ZIM POS.129 project ZF4114902SB7. Second-Skin SmartLipids® for advanced Corneotherapy

References: 1. DHCP-letter 05/07/2018, BfArM,Germany Almohsen, N.1; Köpke, D.2; Pyo, S. M.2; Müller, R. H.2; Keck, C. M.1 2. Pelikh, O. et al.: Eur J Pharm Biopharm 2018, 128: 170– 178. 1Philipps-Universität Marburg, Institut für Pharmazeutische Technologie und Biopharmazie, 3. Patzelt, A. et al.: J. Controlled Release 2011, 150: 45-48. 35037 Marburg, Germany 2Freie Universität Berlin, Institut für Pharmazie ,12169 Berlin, Germany

POS.128 Corneotherapy is a dermal treatment principle with the aim to maintain, Ex Ovo Shell-less Hen´s Egg Test on the Chick Area strengthen or repair the skin barrier [1]. The aim of this study was to investigate the skin carrying properties of “second-skin - smartLipids® Vasculosa as a Test Model for Inhalable Formulations (S3L) on healthy and impaired skin. S3L belong to the third generation of the lipid nanoparticles and are composed of a mixture of various liquid Müller-Bötticher, L.1; Ernst, J.1; Warncke, P.1; Klinger-Strobel, M.2; and solid lipids. The special feature of the S3L is their lipid matrix which Makarewicz, O.2; Pletz, M.2; Fischer, D.1 is composed of lipids that mimic the intercellular lipid layer of the stratum 1 Friedrich Schiller University Jena, Department of Pharmaceutical Technology and corneum, e.g. ceramides, free fatty acids and cholesterol [2]. The skin Biopharmacy, Lessingstraße 8, 07743 Jena, Germany carrying properties of the S3L were assessed by determination of the bio- 2 Jena University Hospital, Institute for Infectious Diseases and Infection Control, Am Klinikum 1, 07747 Jena, Germany physical skin properties, e.g. transepidermal water loss (TEWL) and skin hydration (Corneometer values) prior to and after the dermal application of the S3L. After dermal application lipid nanoparticles form an invisible patch, which was described earlier [3] and was again proven in this study Lung infections can be efficiently treated with inhalable formulations like by Frictiometer measurements. The film formation resulted in an antibiotics, e.g. for patients with cystic fibrosis with tobramycin (Tb) [1]. increase in skin hydration but had no influence on the TEWL, indicating Especially nebulized nanoparticles show suitable properties for that the S3L were able to form a protecting but breathable film on the skin, pulmonary applications avoiding exhalation and aggregation but also which results in a pleasant skin feel upon dermal application and skin overcome obstacles of mucus or biofilm [2]. Nevertheless, there is a lack carrying and protecting properties at the same time. Results provide first of test models to eliminate the limitations of animal tests in mice and rats evidence for the skin carrying properties of second-skin - smartLipids® or higher species. Therefore, in the present study the ex ovo shell-less (S3L) and the suitability of their use in advanced corneotherapy. hen’s egg test on the chick area vasculosa (HET-CAV) was investigated as alternative test system to examine antimicrobial activity and Acknowledgement: biocompatibility under nebulization conditions. Noor Almohsen is grateful for the financial support by the Yousef Jameel Scholarship. Tobramycin loaded nanoparticles (NP) as model drug were prepared by a double-emulsion evaporation method using poly(lactic-co-glycolic acid) (PLGA) with and without poly(ethylene glycol) (PEG) [3]. References: 1. Kligman, A. M.: Int. J. Cosmetic Sci. 2011, (33):197-209. Physicochemical characteristics of the particles were determined by 2. Köpke, et al., Annual Meeting of the German Society of Dermopharmacy. 2019, 25-27 March, dynamic light scattering (DLS) confirming a hydrodynamic diameter of Düsseldorf/Germany. 200 nm. Lyophilized NP were resuspended in 0.9% NaCl solution for 3. Müller, R. H., Sinambela, P., Keck, C. M.: EURO COSMETICS. 2013, (6): 20-22. nebulization by vibrating-mesh technology (IH 50, Beurer, Germany) and investigated regarding aerodynamic characteristics for lung delivery with a mass median aerodynamic diameter between 4.6 µm to 5.4 µm and 50% fine particle fraction. In vitro biocompatibility studies showed no POS.130 cytotoxicity in the human lung cell line A-549 up to a concentration of 1 mg/mL after 24 h incubation. Lipid-polymer hybrid nanoparticles for mRNA delivery in The susceptibility towards bacterial spreading was analyzed using Dendritic cells: Impact of lipid composition infected HET with aliquots of diluted bacteria suspensions and antibiotic solutions both pipetted into an O-ring placed on the CAV. Decreased spreading of Pseudomonas aeruginosa on the egg surface with an Kliesch, L. 1; Delandre, S.2; Schulze, K.2; Guzmán, C. A.2; Loretz, B. 1; antibiotic treatment over 24 h incubation was visualized using bacterial Lehr, C-M. 1 1Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Campus E8.1, D-66123 autoflourescence imaging (Moleculight® i:X). Additionally, stability and Saarbrücken efficacy of released Tb after vibration-mesh nebulization were confirmed 2Helmholtz Center for Infection Research (HZI) on the ex ovo hen’s egg model using a tailor-made setup utilized with a 3D printed cone and P. aeruginosa infected eggs. Toxicological profiles of the applied particles confirmed the biocompatibility on a complex Vaccination is considered as an effective way to prevent the outbreak of biological surrounding displaying no toxic effects like haemorrhage, a disease. Vaccines are often injected in the muscles or into the dermis. vascular lysis or thrombosis. To overcome syringe-associated limitations like safety issues, patient In conclusion, we demonstrated that polyester NP displayed excellent compliance and logistic constraints, needle-free vaccination delivering properties as biocompatible drug delivery systems for antibiotics. The antigen to the abundant antigen presenting cells (APCs) of the skin is a nebulization of NP offers a highly suitable approach to deliver NP efficient promising option. Our aim is to reach these immunocompetent cells to the deep lungs. HET-CAV offers an alternative test system with without harming the stratum corneum via the transfollicular route [1] or beneficial conditions especially for bacterial growth in a complex by minimally invasive methods [2]. We are developing a lipid-coated biological surrounding. Ultimately, this ex ovo model is applicable for polymeric nanocarrier loaded with mRNA encoding for the antigen. Such inhalative formulations with a modified setup which confirmed chargos have gained considerable attention in resent research due to antimicrobial efficacy and biocompatibility of tobramycin loaded their good safety profile, the high flexibility in exchanging targeted nebulized NP. diseases and the easy and fast production compared to other vaccine

141 • DPhG Annual Meeting 2019 Conference Book POSTERS types. Another advantage is the amplification of the signal during the All NPs showed a hydrodynamic diameter about 250 nm. The zeta translation in the cells compensating the low bioavailability. The potential indicated a stable nanoparticle dispersion (-15 to -27 mV). TEM particulate carriers should protect the mRNA and deliver it into cutaneous visualisation showed spherically shaped particles. All NPs showed an APCs, in particular dendritic cells, and, in ideal case, co-deliver an excellent biocompatibility after systemic injection in the dynamic blood adjuvant to increase and tailor the immune responses. The translated flow of the hen’s egg test. As a proof of concept, the human whole blood protein antigen is processed and presented in a MHC-restricted manner assay demonstrated a stronger decrease of pro-inflammatory 5-LOX to lymphocytes in the draining lymph node. Such activation pathway products (e.g. t-LTB4, LTB4) for the diflapolin-NPs in comparison to the enables potent immune responses including humoral and cellular free compound. Conclusively, the NPs may protect the diflapolin from immune responses. plasma protein binding resulting in an enhanced efficiency. In conclusion, polyester-based NPs are an excellent and biocompatible To use a non- or minimally-invasive delivery route to the skin in a drug delivery system for safe, efficient and anti-inflammatory treatment sufficient way in vivo the applied nanoparticles must meet certain with the dual FLAP/sEH inhibitor diflapolin. requirements, which are most importantly mRNA protection and high transfection efficiency. As an in vitro model, we use a murine dendritic Acknowledgments: The authors thanks Ramona Brabetz and Angela Herre for technical cell line DC2.4 to select the optimal particle composition. Starting from a assistance. This works was supported by the DFG-funded Collaborative Research Centre recently developed cationic lipid-coated polymer particle (DOTMA- PolyTarget (SFB 1278, projects A04 and C02). PLGA)[3], we investigated the influence of adding specific ratios of a second, pH-dependently charged lipid for coating. First, we assessed References: colloidal properties such as hydrodynamic size, zeta potential, colloidal 1. Garscha, U. et al.: Scientific Reports 2017, 7 (1): 9398. stability and the preparation method. All tested ratios achieved suitable 2. Klinger-Strobel, M.; Int. J. Nanomed. 2016, 11:575-83. size (≤ 240 nm), homogenous size distributions (PDI ≤ 0,14) and sufficient colloidal stability. Then biological parameters like cytotoxicity by live-/dead-staining, cellular uptake into DCs and the transfection efficacy using flow cytometry for detection of either fluorescent-labelled NPs or POS.132 translated fluorescent reporter gene mCherry were determined. While technological aspects of all particles were comparable, the cell-based Development of tailor-made quercetin nanocrystals for assays revealed a non-linear relationship between lipid composition and target-oriented dermal drug delivery cellular effect. Therefore, the consideration of results from cytotoxicity together with transfection efficiency assays leads to the selection of the Ralph-Walter Eckert1, Steffen F. Hartmann1, Daniel Knoth1, Cornelia M. most promising candidates for in vivo testing in the mouse model. Keck1 1Philipps-Universität Marburg, Department of Pharmaceutics and Biopharmaceutics, Robert- Koch-Strasse 4, 35039 Marburg, Germany [1] Mittal et al., Nanomed. Nanotechnol. Biol. Med. 2015, 11(1): 147-154 [2] L. Engelke et al., Poster Galenus Workshop, 2016 [3] Yasar et al., J Nanobiotechnol. 2018, 16: 72 Nanocrystals are a simple and efficient formulation principle for active pharmaceutical ingredients (APIs) to increase the aqueous solubility and thus the bioavailability of poorly water-soluble drugs (BCS class II and IV) [1]. Recent studies suggest that, besides an enhanced bioavailability POS.131 after oral administration, nanocrystals also exhibit many positive effects Development of a polyester-based drug delivery system for after dermal application, i.e. an excellent skin adhesiveness, improved dermal penetration and a depot effect due to uptake of the nanoparticles the anti-inflammatory dual FLAP/sEH inhibitor diflapolin into the hair follicles [2]. Recent research suggests that nanocrystals of approx. 200 nm promote the penetration of an API through the stratum Grune, C.1; Kretzer, C.2; Kattner, S.1; Thamm, J.1; Werz, O. 2,3; Fischer, corneum, whereas sizes from 400 to 600 nm are preferentially located in D.1,3 the hair follicle, where they act as depot [1,3]. Hence, the production of 1 Friedrich Schiller University Jena, Department of Pharmaceutical Technology and nanocrystals with tailor-made size is desirable to pursue a target-oriented Biopharmacy, Lessingstraße 8, 07743 Jena, Germany dermal drug delivery. 2 Friedrich Schiller University Jena, Department of Pharmaceutical and Medicinal Chemistry, The aim of this study was the development of tailor-made quercetin Philosophenweg 14, 07743 Jena, Germany 3 Friedrich Schiller University Jena, Jena Center for Soft Matter (JCSM), Philosophenweg 7, nanocrystals with a state-of-the-art NanoWitt LAB-100 milling equipment 07743 Jena, Germany (Frewitt fabrique de machines S.A, Fribourg, Switzerland) under industrial conditions. Quercetin was chosen as model drug due to its prominent antioxidative activity, which is beneficial for prevention of Diflapolin is the first dual inhibitor of the 5-lipoxygenase-activating protein photoaging of the skin [4,5]. Suspensions were prepared with 5% (w/w) (FLAP) and the soluble epoxide hydrolase (sEH) and demonstrated a quercetin and 1% (w/w) D-ɑ-tocopheryl polyethylene glycol succinate high activity in vivo and in vitro. FLAP supports the conversion of (TPGS) as stabilising surfactant. The milling was carried out with arachidonic acid to pro-inflammatory leukotrienes by 5-lipoxygenase (5- NanoWitt LAB-100 milling equipment in continuous mode configuration. LOX), whereas sEH degrades anti-inflammatory epoxyeicosatrienoic Circulation of the suspension was set to 500 ml/min for 120 minutes. The acids (EETs). Through the inhibition of both enzymes, a synergistic anti- rotor of the milling chamber was adjusted to 1500 rpm, with a inflammatory effect can be achieved. Despite the high potency, bead/suspension-ratio of 60/40 (V/V). Suspension temperature was inappropriate biological and physicochemical properties such as high constantly kept between 15 to 20°C. Three different sizes (small: 100 lipophilicity, low water solubility and high plasma protein binding hamper µm, medium: 300µm and large: 400 µm) of yttria stabilized zirconia the efficiency of diflapolin. To overcome these hurdles, diflapolin was beads (SiLibeads Type ZY-E, Sigmud Lindner GmbH, Switzerland) were encapsulated in poly(lactic-co-glycolic acid)-based nanoparticles (PLGA- used to control the resulting particle size distribution over time. The NP) to develop an efficient and biocompatible drug delivery system. volumetric particle size distribution of the nanosuspensions was Diflapolin-loaded NPs were prepared by an emulsion-diffusion- determined by static light scattering (SLS) using a Mastersizer 3000 evaporation method using PLGA with and without poly(ethylene (Malvern Panalytical, Germany). The span ((D(v)0.9-D(v)0.1)/D(v)0.5) glycol)[2]. The physicochemical properties of the NPs were characterized was calculated as a parameter for the broadness of the particle size by photon correlation spectroscopy, laser Doppler anemometry and distribution. transmission electron microscopy (TEM). The drug load of the NPs was Results showed, that the smallest bead size is suitable for a drastic size determined by high performance liquid chromatography with UV/VIS reduction. After 30 min, the D(v)0.95 value reached 330 nm with a span detection, and in vitro drug release kinetics were investigated. of 3.11. Further 90 min milling reduced the D(v)0.95 to 216 nm with a Biocompatibility of the NP were tested in vitro in cell-based assays and span of 2.64, indicating a relatively narrow particle size distribution. This ex ovo in a shell less hen’s egg test on the chick area vasculosa (HET- would make the manufacturing process suitable for nanosuspensions CAV). The anti-inflammatory effects were assessed on with an enhanced skin penetration of the active through the stratum polymorphonuclear neutrophils (PMNL) and in a human whole blood corneum. The medium and large bead size were able to achieve assay by lipid mediator metabololipidomics using UPLC-MS/MS to nanosuspensions with a more suitable particle size for hair follicle evaluate inhibition of 5-LOX product formation. targeting with D(v)0.95 values of 390 nm (medium, 60 min) and 450 nm (large, 90 min), respectively. The calculated span of the large bead size

DPhG Annual Meeting 2019 Conference Book • 142 PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS was slightly higher with a value of 3.57, compared to 3.42 for medium bead size. Finally, the D(v)0.95 after 120 min were 338 nm (large) and molar ratio 1:1 molar ratio 2:1 molar ratio 3:1 268 nm (medium) with a span(large) = 3.24 and a span(medium) = 2.94. Thus, the largest bead size resulted in the broadest particle size distribution. 0,2 In conclusion, tailor-made quercetin nanocrystals for target-oriented dermal drug delivery can be easily and reproducible manufactured using 0,1 different bead sizes, utilized in the milling process. Nevertheless, future investigations have to clarify the skin penetration properties of the nanosuspensions. 0,0

Intrinsic solubility [mM] solubility Intrinsic Acknowledgments The authors kindly acknowledge the support of Frewitt fabrique de machines S.A., Switzerland to provide the NanoWitt LAB-100 milling equipment. The study was partly supported by ZIM- Project No. ZF4114902SB7. In the present contribution, the analytical results of the created salts will References: be presented. 1. Pelikh, O. et al.: Eur J Pharm Biopharm 2018, 128: 170– 178. 2. Zhai, X. et al.: Int J Pharm 2014, 470(1-2): 141-150. 3. Patzelt, A. et al.: J Controlled Release 2011, 150: 45-48. 4. Eckert, R.-W, PhD-thesis, Philipps-Universität Marburg, in. prep. 1. Domingos, S.; Andre, V.; Quaresma, S.; Martins, I. C.; Minas da Piedade, M. F.; Duarte, M. 5. Kakran, M. et al.: Eur J Pharm Biopharm 2012, 80: 113-121. T. J Pharm Pharmacol 2015, 67, 830-46. 2. Agharkar, S.; Lindenbaum, S.; Higuchi, T. Journal of Pharmaceutical Sciences 1976, 65, 747- 749. 3. Serajuddin, A. T. Adv Drug Deliv Rev 2007, 59, 603-16. 4. Kalant, H. Addiction 1997, 92, 267-277. POS.133 5. Schiff Jr., P. L. American Journal of Pharmaceutical Education 2002. Mimicking nature to solve the problem of poor water solubility of alkaloids by means of salt formation with large organic acids POS.134 Paul Güntzel, Lorenz Meinel and Ulrike Holzgrabe From in-vitro assays to in-vivo application studies – new Institute of Pharmacy, Am Hubland, University of Würzburg, DE-97074 Würzburg, Germany cationic lipoplexes in comparison

Most of the drugs on the market are suffering from low aqueous solubility, 1,* 2,3 1 even though the drug development process tries to take care of this Julia Giselbrecht , Jeroen Bussmann , Andreas Langner , Christian 1 property. To overcome the problem, the formation of an appropriate salt Wölk 1 Martin Luther University Halle-Wittenberg, Institute of Pharmacy, Department of Biochemical may help to improve the poor solubility and, hence, typically salt Pharmacy, Halle (Saale), Germany screenings are performed.1 In the case a drug substance forms a well- 2 Leiden Academic Centre for Drug Research, Leiden, The Netherlands organized lattice characterized by strong lattice forces, it is difficult to 3 Leiden Institute of Chemistry, Leiden, The Netherlands * [email protected]] achieve sufficient water solubility. Using large counterions, which are able to interfere with the lattice, may be an alternative to the classical salt screening with small ions, such as chlorides, bromides, mesylates, and sodium and potassium, respectively. Interestingly, nature provides a large number of sterically “demanding” anions, perhaps because in the plant vacuoles a lot of basic secondary metabolites have to be dissolved for emergency reasons.2,3 However, to the best of our knowledge it is not well understood, whether the large counterions prevent the crystallization of the metabolites and what is the physical background. In order to study the influence of natural organic acids on the solubility of alkaloids we have chosen opium as a model system. It consists of easily available papaverine and morphine on the one hand and citric acid, malic acid, tartaric acid and meconic acid on the other hand.4,5 In order to collect information about aggregation, solubility, zeta potential, and dissolution rates the formed salts were investigated by means of XRPD, DSC, and IR in solid state as well as NMR spectroscopic methods, photometrical Cationic lipids in combination with a helper lipid complex therapeutic DNA and potentiometric solubility measurements, DLS and zeta potential and are an efficient system to transport nucleic acids into cells. In the last measurements in solution. In the series studied here the papaverine as decade cationic lipids are an often used transfection agent because of a citrate salt showed an enhanced water solubility by factor of 3 in their high loading capacity, low cytotoxicity, low immunogenicity and comparison to papaverine hydrochloride. An explanation for the absence of oncogenic risk. After an extensive physico-chemical analysis, increased water solubility of the citrate salt is the amorphous nature of the biocompatibility of newly designed lipoplex formulations gets into the salt as well as the colloidal dispersion of the citrate solution in focus of our research activity. What will happen after systemic application comparison to the crystalline papaverine hydrochloride. This is an of the lipoplexes? Therefore various in-vitro and in-vivo assays regarding important step forward in our efforts to make use of the principles of their hemocompatibility, biodistribution and pharmacodynamic activity nature for solubility enhancement. are necessary. For evaluating the interactions of the cationic lipoplexes with blood components we performed hemolysis assay, particle size measurement and erythrocyte aggregation assay. For in-vivo studies an animal model gives the opportunity to estimate the behaviour and toxicity of the lipoplexes in the organism. The zebrafishembryo as an in-vivo model has many advantages because of their small body size and optical transparency for studying the delivery of nanomedicines [1]. Especially the optical transparency allows observing the biodistribution direct in the living animal by microscopic observation. Besides that, the zebrafish has a high genomic and molecular similarity to humans and the discoveries in zebrafish experiments can give hints on biocompatibility in humans [2]. In addition, the experiments on

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Zebrafishemryos do not count as animal testing as long as they are Figure 1: Schematic representation of the polyelectrolyte multilayers completed before day 5 after fertilization. (PEMs) with embedded lipoplexes using layer-by-layer technique. The The presented experiments show the systemic administration of lipoplex PEMs consist of alternating layers of hyaluronic acid (HA) and chitosan formulations, which show excellent in-vitro results. The in-vivo (CHI). Polyethylenimine (PEI) is used as starting layer. biodistribution and transfection efficiency of this formulations were evaluated in the zebrafishembryo. The in-vitro and in-vivo analysis of the Acknowledgments: The project is funded by the Deutsche Forschungsgemeinschaft (DFG, lipoplexes showed no hemolysis, low cytotoxicity and an efficient German Research Foundation) – project grand number 396823779 transfection.

References: [1] Hacobian, A.R., et al.: Eur Cell Mater, 2016. 31: p. 191-204. 1. Campbell, F., et al., Directing Nanoparticle Biodistribution through Evasion and Exploitation of [2] De Laporte, L.; L.D. Shea: Advanced drug delivery reviews, 2007. 59(4-5): p. 292-307. Stab2-Dependent Nanoparticle Uptake. ACS Nano 2018, 12: p. 2138-2150.

2. Bhattacharya, M., et al., Therapeutic applications of zebrafish (Danio rerio) miRNAs linked with human diseases: A prospective review. Gene, 2018. 679: p. 202-211. POS.136 PVM/MA-MCT nanocapsules – overcoming hydrolysis of

nanoparticles polyanhydride nanoparticles for oral POS.135 delivery. DNA-loaded polyelectrolyte multilayer scaffolds for local transfection in regenerative medicine De Souza, L.1a, Mäder, K.1 1 Institute of Pharmacy, Faculty of Biosciences, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120, Halle/Saale, Germany Husteden, C.1; Repanas, A.2; Groth, T.2; Wölk, C.1 a CAPES Foundation, Ministry of Education of Brazil, Caixa Postal 250, Brasília – DF, 70040- 1Martin-Luther-University Halle-Wittenberg, Institute of Pharmacy, Department of Biochemical 020, Brazil Pharmacy, Wolfgang-Langenbeck-Str. 4, 06120 Halle, Germany 2Martin-Luther-University Halle-Wittenberg, Institute of Pharmacy, Department of Biomedical Materials, Heinrich-Damerow-Str. 4, 06120 Halle, Germany Poly(methyl vinyl ether-alt-maleic anhydride) - PVM/MA is a commercially available polymer that has been described as a functional excipient for drug delivery during the last years. Due to the ability to Therapeutic treatment of insufficient bone regeneration is a challenging develop nonspecific adhesions to mucous membranes, PVM/MA in its problem and a topic of on-going search for novel treatment strategies. A native or cross-linked form, is considered to be suitable as a polymeric promising approach to treatment and improvement utilizes non-viral gene matrix for nanoparticles for oral applications [1,2]. PVM/MA has a delivery using the bone morphogenetic protein-2 gene (BMP-2) to moderate chemical reactivity due to the presence of the anhydride bonds, achieve local and sustained expression of this growth factor [1]. The allowing grafting of the polymer backbone with primary amines and introduction of DNA vectors encoding for therapeutic genes makes DNA- alcohols without the use of complex chemical reactions [3]. In an aqueous loaded material films suitable for stimulating bone formation. Particularly environment, the maleic anhydride group is hydrolyzed into two useful for application for implementation in regenerative medicine is a carboxylic groups, yielding the water-soluble free acid. The hydrolysis will nucleic acid delivery-system with controlled release of DNA from change the microenvironment and leads to the dissolution of biocompatible systems.[2] nanoparticles or the formation of nanohydrogels (for cross-linked In this study, we have developed a multi-layered polyelectrolyte film, that nanoparticles). The hydrolysis of plain and cross-linked PVM/MA permit both, the immobilization and controlled release of DNA from the nanoparticles (NP and NP-CL, respectively), was investigated over time surface of glass cover slips. Our approach makes use of the layer-by- at relevant physiological pH values (1.2, 5.0, and 7.4). The dense layer method for the assembly of nanostructured thin films consisting of polymeric nanoparticles are solubilized at different rates that are alternating layers of hyaluronic acid (HA) as polyanion and chitosan (CHI) influenced not only by time, but also by the pH of the medium. Dynamic as polycation. Here, lipid/DNA complexes (lipoplexes), consisting of light scattering (DLS), nanoparticle tracking analysis (NTA), auto-titration, novel cationic lipids in combination with a helper lipid are embedded and ATR-FTIR results showed that at pH 7.4, the nanostructures were within polyelectrolyte multilayers (PEMs) (Figure1). Thus, a multilayer completely solubilized in less than 20 minutes, while the cross-linked system is to be produced, which enables localized, surface-based structure formed a mesh with hydrogel character (Fig. 1). Hence, the transfection. We focused on the development of methods to achieve instability of PVM/MA nanoparticles in neutral pH hinders its application effective loading of the PEMs with DNA and on the intensive surface as a useful drug delivery system. To circumvent PVM/MA-nanoparticle characterization using confocal fluorescence microscopy, ellipsometry, drawbacks, enhancing the drug loading, encapsulation efficiency, and atomic force microscopy and zeta potential measurements. Interactions keeping the ability of PVM/MA to develop adhesiveness with the mucosa between C2C12 myoblasts with the functionalized PEMs were along the GIT, nanocapsules composed of PVM/MA and medium-chain investigated using confocal microscopy. triglycerides (PVM/MA-MCT NC) were developed and characterized. We have successfully established a system that allows a loading of DNA Plain and cross-linked PVM/MA-MCT NC were produced without the in the PEM-film, and which is also capable of transfecting C2C12 cells. need for any type of stabilizers. The stability of plain and cross-linked Ellipsometric measurements were used to monitor the thickness growth PVM/MA-MCT NC was also evaluated at different physiological pH of the PEMs after treatment with cationic lipoplexes. In addition, further conditions. Particle size distribution was determined by laser diffraction cell studies on C2C12 cells showed focal adhesion to extracellular matrix. (LD), and dynamic light scattering (DLS). Particle morphology was First in-vivo experiments were carried out, in which a good transfection revealed by cryo- and FF-TEM (Fig. 2). The results showed the pH of the could also be achieved with our established system. medium did not change the particle size over time, although an effect on the PdI of samples at pH 1.2 was observed. Thus, for the first time PVM/MA-MCT nanocapsules prepared employing only acetone as an organic solvent and without stabilizers were described and characterized, being a promising drug delivery system for oral administration.

DPhG Annual Meeting 2019 Conference Book • 144 PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS

pH values and forms physically gelling hydrogels under physiological conditions. Furthermore, the density of free amino functionalities in chitosan (4.7 µmol/g with 80% degree of deacetylation (DD)) is a lot higher than in the collagenous peptides (0.4 µmol/g). With the oligomer oPNMA-10 (oligo(pentaerythritol diacrylate monostearate (PEDAS)-co- N-isopropylacrylamide-co-maleic anhydride) with 10 MAeq per PEDAS, 7% in dimethylformamide (DMF)), stable hydrogels could be generated with different volumes of chitosan solutions containing between 1% and 2% chitosan (DD 80%) dissolved in acetate buffer with pH 3.0 and 4.76. No gelation was observed on addition of DMF without oligomer. Depending on the ratio between oligomer and chitosan, as well as pH, component incorporation between 75% and 100% could be achieved.

Rheological characterization gave storage moduli between 11.8 kPa and 32.5 kPa. Water content of the generated 2K-hydrogels ranged from 81.3% to 94.7%, with the highest storage moduli being found for the hydrogels with the lowest water contents. Fig. 1 - Cryo-TEM Fig. 2 - Size distribution (DLS) and With these results, the general suitability of chitosan as the amine- micrographs of NP and NP- FF-TEM of PVM/MA-MCT NC containing component in 2K-hydrogels could be shown. The low pH CL freshly prepared (left) Sacale bar 100 nm. values of the employed buffer system means that the hydrogels need to and 30 min after dilution in be prefabricated and washed to exchange the acidic reaction medium for PBS pH 7.4 (1:1). Scale bar a more physiological buffer, which makes them not suitable for direct cell 200 nm. incorporation. Nevertheless, the high adaptability of this system and the attractive components for tissue engineering/regenerative medicine Acknowledgments: CAPES Foundation, Ministry of Education of Brazil, Caixa Postal 250, makes this material platform highly attractive for 3D-printing applications. Brasília – DF, 70040-020, Brazil - CAPES scholarship Proc. nº 99999.010146/2013-00”.

References: 1. Arbós, P., et al.: J. Control. release 2002, 83: 321–330. Acknowledgements: Funding by the DFG (SFB TRR 67/A1) is gratefully acknowledged. 2. Irache, J. M. et al.: Molecules 2005, 10: 126–45. 3. Schmidt, U., et al. J. Appl. Polym. Sci. 2002, 87: 1255–1266. References: 1. Loth T. et al. React Funct Polym 2013, 73: 1480-1492 2. Loth T. et al. Biomacromolecules 2014, 15: 2014-2118 3. Kohn, C. et al. Biomaterials science 2016, 4 (11): 1605–1621 4. Kohn-Polster. C. et al. Int J Mol Sci 2017 18:1104

POS.137 POS.138 Formulation of 2-component-hydrogels from anhydride- containing oligomers and chitosan Anhydride-containing amphiphilic oligomers for nanoparticle stabilization and functionalization Krieghoff, J.1; Maqsood, I.1; Schulz-Siegmund, M.1; Hacker, M. C.1 1 Institute of Pharmacy, Pharmaceutical Technology, Faculty of Medicine, Leipzig University,

Germany Schmid M, Nawaz HA, Mitrach F, Schulz-Siegmund M, Hacker MC Pharmaceutical Technology, Institute of Pharmacy, Faculty of Medicine, University Leipzig, Eilenburger Straße 15A, 04317 Leipzig, Germany

Hydrogel-forming materials of both natural and synthetic origin are The need for improved ways of drug delivery is growing along with the materials of great interest for applications in drug delivery, tissue identification and development of highly selective or DNA editing API’s, engineering and regenerative medicine. Our group developed an especially for the treatment of cancer, autoimmune or neurological anhydride-containing oligomer platform for cross-linking of amine- diseases.[1] Polymeric or polymer coated nanostructures are of special containing hydrogel forming materials like collagenous peptides and interest as their physico-chemical properties can be tuned precisely gelatin, which are derived from naturally occurring collagen and during synthesis.[2] inherently biocompatible, –degradable and cell adhesive [1, 2]. The We hypothesize that amphiphilic anhydride group (red boxes) containing resulting 2-component-hydrogels (2K-hydrogels) feature adjustability of oligomers represent a versatile group of polymeric material to stabilize their biochemical and biophysical properties by variation of the metal, metal oxide or mineral NP’s via colloidal association and ionic component ratio, oligomer composition and cross-linking conditions. interactions. The incorporation of anhydride groups in oligomers has Reacting part of the anhydrides in the oligomer with other amine- several advantages in this context. Anhydride groups are neutral which containing molecules allows for covalent modification of the hydrogels [3, supports oligomer polymerization in anhydrous organic solvents without 4]. The 2K-hydrogels can be employed for different applications, for solubility problems. The oligomers can be purified and isolated by instance for the generation of nerve regeneration structures by manual repeated precipitation and vacuum dying. Prior to application of the fabrication or 3D-printing. material the anhydride groups can by hydro- or aminolyzed in order to

obtain polyanionic structures for interaction with minerals or metals. Via

HO O NH partial derivatization of the anhydride groups the oligomers can be CH2OH R: H decorated with additional (bio)functional groups to impart specific O O O biological functionality. y x OH O O O O O O O O Based on our expertise with maleic anhydride (MA) containing O HN oligomers,[3] we began to synthesize derivatives with enhanced 8 x + y = 20 B R amphiphilicity by incorporating oligo(ethylene glycol) (PEG) and fatty Fig.1: (A) Cross-linking oligomer oPNMA-x; (B) Monomers of chitosan: alcohols (14) to the get terpolymers of the o14PEGMA type. Expansion glucosamine (R = -H) and N-acetylglucosamine (R = -Acetyl). with a functionally inert filler monomer like 4-acryloylmorpholine (MO) is optional. The pristine anhydride-containing oligomers were characterized In this work, the use of chitosan as the amine-containing component in for chemical composition and comonomer incorporation as well as the 2K-hydrogels is investigated. Chitosan is an aminopolysaccharide molecular weight distribution. The chemically characterized oligomers and is derived from naturally occurring chitin by deacetylation. The were converted into water-soluble salts that already proved suitable for polymer is used in research and development of medical products in a calcium phosphate NP stabilization. We strive to develop functionalized variety of ways, for instance with chitosan-based hollow tubes that are NP’s for siRNA delivery particularly for application to the brain. available for peripheral nerve regeneration. The production of chitosan-containing 2K-hydrogels requires changes of the reaction conditions, as chitosan is insoluble at neutral and alkaline

145 • DPhG Annual Meeting 2019 Conference Book POSTERS

In this study hCMEC/D3, a well-characterised cell line with good blood- brain barrier properties [4], was used as an in vitro BBB model. PEGylated liposomes, mainly consisting of 1,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), with the addition of cholesterol and a rhodamine-conjugated lipid for fluorescent detection, were produced using dual asymmetric centrifugation [5] and loaded with a hydrophilic small drug molecule. Peptide-linked DSPE-PEG(2000)-maleimide was incorporated into the liposomes for either cell-penetrating and/or targeting purposes. Acknowledgments: This project has been funded by the European Regional Development To study cellular uptake, a modified version of a previously established Found Saxony (EFRE). Furthermore we gratefully appreciate and acknowledge the funding by the Sächsische Aufbaubank (SAB). liposome uptake assay was used [6]. Cells were incubated with fluorescent liposomes in different concentrations for 2 hours. Concentration of liposomal components in lysed cells after incubation References: and washing and the corresponding uptake efficiency were determined 1. Ben-Akiva, E.; Est Witte, S.; Meyer, R. A. et al. Biomater. Sci., 2019, 7, 14–30. using fluorescent spectroscopy. Uptake was also visualised using 2. Du, J.-Z.; Li, H.-J.; Wang, J. Acc. Chem. Res., 2018, 51, 2848–2856. confocal laser scanning microscopy. 3. Loth, T.; Hennig, R.; Kascholke, C. et al. React. Funct. Polym., 2013, 73, 1480–1492. First results show a significantly higher uptake of the tested surface-

modified PEGylated liposomes than of non-modified PEGylated

liposomes. The highest uptake efficiency generally occurred at a

liposome concentration of 0.125 mM total lipids. Confocal microscope POS.139 images confirm abovementioned results. SensorTransBBB – Establishment of a Microfluidic Chip In further studies, modified liposomes are to be tested in transwell models to elucidate not only uptake into but also permeation across brain Model of the Blood-Brain-Barrier capillary endothelial cell monolayers. Studies with isolated and functional intact rodent brain capillaries as well as in vivo pharmacokinetic studies Sebastian Trennheuser1, Felix Schmitt-Hoffner2, Julia Botta2, Martin with drug-loaded liposomes will complete this study. Stelzle3, Gert Fricker1 1Department of Pharmaceutical Technology, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 329, 69120 Heidelberg, Germany Acknowledgments: Lipoid GmbH (Ludwigshafen, Germany) for phospholipid samples; Prof. Dr. 2Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Jörg Huwyler, Department of Pharmaceutical Sciences, University of Basel (Basel, Switzerland) Feld 364, 69120 Heidelberg, Germany for hCMEC/D3 cells. 3NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstrasse 55, 72770 Reutlingen, Germany References: 1. Feigin, Valery L. et al.: Lancet Neurol. 2017, 16 (11): 877–97 2. Rubin, Lee L.; Staddon, James M.: Annu. Rev. Neurosci. 1999 22. (1): 11–28 The human blood-brain-barrier (BBB) is a highly selective cellular 3. Sercombe, Lisa et al.: Front. Pharmacol. 2015, 6 (286) structure which separates central nervous system (CNS) and circulating 4. Poller, Birk et al.: J. Neurochem. 2008, 107 (5): 1358–68 blood stream. It plays a very important role for the protection of the CNS 5. Massing, Ulrich; Cicko, Sanja; Ziroli, Vittorio: J. Control. Release 2008, 125 (1): 16–24 and therefore prevents various pharmaceutical substances from reaching 6. Thöle, Marc et al.: J. Drug Target. 2002, 10, (4): 337–44 the brain. Furthermore, a variety of diseases of the CNS are associated with dysfunctions of the BBB. In order to investigate barrier functions of the BBB and drug transport under physiological and pathophysiological conditions, the SensorTransBBB system was developed. The microphysiological chip in POS.141 microwell format, produced by micro-injection molding, consists of ten Gelatin/pectin blend for soft capsule manufacture: A parallel culture channels which can be perfused with cell culture medium. rheological study Porcine brain capillary endothelial cells (PBCECs) were cultivated on the surface of a synthetic hydrogel matrix thus forming a barrier between a perfusion and a hydrogel matrix channel. Important cell culture conditions Claudia Klein, Gabriele Reich like seeding density, attachment time, medium volume etc. were University of Heidelberg, IPMB, Department of Pharmaceutical Technology and Biopharmaceutics, INF 329, 69120 Heidelberg, Germany determined in order to obtain a tight cell layer under perfusion. The viability of PBCECs was assessed by calcein stainings and cell layer integrity was determined by a permeability assay using FITC dextran. Purpose Furthermore, tight junction protein ZO-1 was present at the cell boarders Soft capsules are a versatile single-unit oral dosage form used in both as visualized by immunostainings, indicating a functional BBB. the pharmaceutical and the food and nutrition market. Due to its characteristic thermoreversible sol/gel transition gelatin is the most The project was funded in part by the BMBF and the Baden-Württemberg Stiftung. frequently used polymer for the manufacture of capsules. Enteric properties of the capsule shell can be achieved by an additional coating

process or directly by blending gelatin with a gastroresistant polymer, e.g.

gelatin/pectin (G/P) blends. Interestingly, the temperature dependent

rheological characteristics of highly concentrated G/P blends used as soft POS.140 capsule shell material and their machinability on the Rotary Die process Uptake of Peptide-modified Liposomes into Human Blood- has not been studied in detail. The purpose of this work was to investigate Brain Barrier Cell Line hCMEC/D3 the viscoelastic properties of G/P blends containing glycerol as a plasticizer by oscillating rheology over the temperature range between 70 and 25 °C followed by an isothermic ageing process at 25 °C. Laura Dehm1, Gert Fricker1 1Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy and Methods Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 329, 69120 The gelatin used in this study was a 160 Bloom Limed Bone gelatin from Heidelberg, Germany GELITA AG, Eberbach, Germany. The pectin was a low-methoxyl grade from CP Kelco, Atlanta GA. Two different polymer concentrations were Neurodegenerative and neurological disorders are becoming more and prepared, with either 35 % (w/w) and 40 % (w/w) solid content and a 2:1 more prevalent in our society, partly due to a generally longer life-span ratio of glycerol relative to the solid content. For G/P blends two different of the population [1]. The central nervous system (CNS) uptake of a ratios ([6/1] and [8/1]) were applied. Pure gelatin samples were used as majority of drugs, especially hydrophilic compounds that could potentially references. be used for the treatment of these diseases, is hindered by the presence Gelatin was swollen in the water-glycerol mixture for two hours at room of the blood-brain barrier [2]. temperature and then kept at 70 °C for three hours (gelatin formulation) The use of liposomes as drug vehicles can be an option to overcome or one hour (G/P blends) in a water bath. For the preparation of the G/P such barriers and has been topic of numerous investigations [3]. blends, the pectin powder was suspended in the water-glycerol mixture

DPhG Annual Meeting 2019 Conference Book • 146 PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS by ultrasonic treatment, then heated up to 70 °C for four hours in a water POS.143 bath, mixed with the gelatin solution and then treated again with ultrasound at 70 °C for two hours. Characterization of Cell Penetrating Peptides as Enhancers Freshly prepared samples were measured with an oscillatory HAAKE of Oral Liposome Delivery MARS III rheometer (Thermo Scientific, Germany). A linear cooling ramp from 70 to 25 °C with a temperature gradient of 0.5 °C/min was applied, followed by a 90 min ageing period at 25 °C. The storage modulus (G’) Fidelj, V1, Mühlberg, E.1,2, Öhlenschläger K.1, Mier, W.2, Fricker, G.1 1Department of Pharmaceutical Technology and Biopharmacy, Institute for Pharmacy and and the loss modulus (G’’) during cooling and isothermic ageing at 25 °C Pharmaceutical Technology (IPMB), University of Heidelberg, Germany were measured in controlled shear deformation mode with 1 % 2Department of Nuclear Medicine, Heidelberg University Hospital, Germany deformation and a frequency of 1 Hz.

Results Liposomes still present a promising nano-sized drug delivery system due Rheological measurements in the temperature range between 70 and to the biocompatibility of their components and their variability in 25 °C revealed significantly higher values of the loss modulus (G’’) for functionality and targeting. While the internalisation mechanism of the G/P blends than for the gelatin formulation at the same solid content. liposomes via the gastrointestinal route is still not completely clear, the In the gel state (T = 25 °C), the storage modulus (G’) was not significantly use of uptake enhancers seems to be inevitable in order to gain different for the G/P blends and the gelatin formulation with a 35 % solid reasonable permeability through the gastrointestinal epithelial barrier. content. In contrast G/P formulations with 40 % solid content showed a significantly higher G’ compared to pure gelatin samples. This clearly The aim of this study was to generate arginine rich liposomes, implies that the impact of pectin on G’ strongly depends on the solid characterize them and test them for possible toxicity in cell tissue culture content in the formulation. (CaCo-2 cells).

Conclusion Four different poli-L-arginine structures were synthesized and coupled to Oscillating rheology is a promising approach to gain a better a linker lipid which was then incorporated into the liposomal formulation. understanding of the temperature dependent viscoelastic properties of The well-known cell penetrating peptide (CPP) Penetratin was used to G/P blends. The study revealed that the viscosity increase associated compare with the novel CPP-decorated vesicles. Liposomes were with the addition of pectin to the gelatin in the sol state is a great generated using dual centrifugation. The standard lipid compositions challenge for the Rotary Die machinability and narrows the formulation contained a ratio of 40 : 60 cholesterol : egg phosphatidlycholine (EPC). window necessary to achieve gastroresistant capsule shell properties. When CPP-linked lipid was added to the formulation in concentrations of 0,1 mole-%, 0,5 mole-% and 1 mole-%, the adequate amount of EPC was substituted. The obtained liposomal formulations were then characterized regarding size, size distribution and zeta-potential. The effect of the CPPs on the liposomal membrane fluidity was assessed, POS.142 using fluorescence anisotropy of diphenylhexatriene (DPH). In addition, the toxicity of the novel formulations at the highest CPP concentration Influence of Biorelevant Media on Membrane Fluidity of was tested. CaCo-2 cell monolayers were exposed to the formulation for Tetraether Liposomes 4 hours at different concentrations, after which an AlamarBlue® assay was preformed, testing the viability of the treated cells.

Stefan Martin, Stephanie Prosek, Gert Fricker As expected an increasing CPP concentration results in larger liposomes Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 329, 69120 with a higher positive charge. Two out of the four formulations have an Heidelberg, Germany effect on the viability of the cells, which is why they have been ruled out as potential candidates for our formulation. The stability of liposomes in gastric and intestinal fluids is of central interest for oral application of liposomal preparations. Encapsulated In the following steps the effect of the formulation on the membrane substances can display various instabilities towards the surrounding fluidity of a Caco-2 cell monolayer will be evaluated. The most promising media as well as insufficient permeability through the gut wall when formulation will be tested in an animal experiment, using a model cargo liposomes become leaky. Only through maintaining API. liposomal integrity, the payload can be successfully delivered to possible regions of interest along the gastrointestinal tract (GIT). It has been demonstrated that membrane fluidity is directly correlated to Acknowledgements: Lipoid GmbH (Ludwigshafen Germany) for the Lipids the permeability of low molecular solutes and protons [1]. Therefore, the objective of this study was to assess the influence of simulated gastric

(FaSSGF) and intestinal fluid (FaSSIFv2) on liposomal membrane fluidity. Fluidity was measured using anisotropy of diphenylhexatriene POS.144 (DPH), a fluorescent probe that shows different polarization depending EPR spectroscopy as a tool to evaluate poly-anhydride on its microenvironment. Liposomes with different compositions were nanostructures – how polymer hydrolysis changes the tested: All liposomes consisted of either egg phosphatidyl choline (EPC) or distearoylphosphatidylcholine (DSPC) as main lipid with varying added nanoenvironment? concentrations of cholesterol. 10 mole-% of main lipid was then substituted for glyceryl caldityl tetraether (GCTE) to elucidate the De Souza, L.1a, Metz, H.1, Mäder, K.1 influence of this membrane spanning, archaeal tetraether lipid on fluidity. 1 Institute of Pharmacy, Faculty of Biosciences, Martin Luther University Halle-Wittenberg, DSPC membranes showed the overall lowest fluidity and highest Wolfgang-Langenbeck-Str. 4, D-06120, Halle/Saale, Germany resistance against gastric and intestinal fluids. A high cholesterol content a CAPES Foundation, Ministry of Education of Brazil, Caixa Postal 250, Brasília – DF, 70040- 020, Brazil seems beneficial for stability in the GIT while no relevant effect of GCTE could be observed on membrane fluidity as measured by DPH anisotropy. Poly(methyl vinyl ether-alt-maleic anhydride) – PVM/MA was proposed Further studies will investigate a possible correlation between observed to be the polymeric matrix of nanoparticles for oral and topical membrane fluidity changes and drug permeability through the membrane administration considering its ability to develop adherence with mucosa upon exposure to FaSSGF and FaSSIF. Also, the influence of different and moderate chemical reactivity due to the presence of the anhydride concentrations of bile salts on the fluidity of GCTE containing liposomes bonds[1,2]. In an aqueous environment, the maleic anhydride group is will be investigated. hydrolyzed into two carboxylic groups, yielding the water-soluble free acid. The hydrolysis will change the microenvironment and leads to (i) the dissolution of nanoparticles or (ii) the formation of nanohydrogels (for Acknowledgments: Lipoid GmbH (Ludwigshafen, Germany) for phospholipid samples cross-linked nanoparticles). At pH 7.4, the nanostructures were completely solubilized in less than 20 minutes (Fig. 1), while the cross- References: linked structure formed a mesh with hydrogel character. It was described 1. Lande M.B., Donovan J.M., Zeidel M.L.: J. Gen. Physiol. 1995, 106(1): 67–84 that a nanocapsules system made with PVM/MA and medium-chain

147 • DPhG Annual Meeting 2019 Conference Book POSTERS triglycerides (MCT) can circumvent PVM/MA-nanoparticle drawbacks, smartLipids, the further development of solid lipid nanoparticles (SLN) enhancing the polymer stability in aqueous medium, the drug loading and nanostructured lipid carriers (NLC), were made with a lipid phase capacity and encapsulation efficiency [3]. Electronic paramagnetic composed of ceramide IIIB and ceramide VI (70%), cholesterol (15%), spectroscopy (EPR) is a non-invasive technique that permits the and 6 of the fatty acids (15%) being most present in natural skin lipids. quantitative measurement of micropolarity, microviscosity and, using Comparable to SLN and NLC smartLipids forms a film on skin allowing special probes, it is also possible to quantify microacidity and oxygen the restoration and reinforcement of skin’s natural lipid film [3] – thus content inside a sample. Stable free radicals, such as nitroxides, are where named smartLipids 2nd skin. widely employed as model drug reporting the microenvironment of smartLipids 2nd skin suspensions were prepared by hot high pressure pharmaceutical formulations [4]. In this study EPR was used to evaluate homogenization at 500 bar for 1-5 cycles. Surfactant screening was the microenvironment of the nanostructures and their ability to retain the performed to determine the type and concentration of appropriate spin probe TEMPO-Benzoate (Fig.1). The presence of immobilized spin surfactant for stabilizing 10% of the lipid phase. Particle size was probe at the first-time point shows that the nanoparticles are dense characterized by photon correlation spectroscopy (PCS), laser polymeric structures. The signal of this immobilized spin probe diffractometry (LD), and light microscopy. Melting behavior of the lipid decreased over 10 h, at intrinsic pH, and after 1h at pH 7.4, suggesting mixture was investigated by differential scanning calorimetry (DSC). the dissolution of the nanoparticle protective environment. The results According to DSC measurements the lipid mixture melts at over 50°C also demonstrated the presence of two distinct milieus in the and therefore stays solid on skin. Due to the complexity of the matrix nanocapsules formulation (TB I and TB II). First one with polarity and composed of 9 lipids, its physical stabilization was challenging. Among viscosity equal to that observed for pure MCT, and the second with the 19 screened surfactants at 1.5% only 2 (Lanette E and TegoCare intermadiate viscosity and polarity, suggesting the presence of an 450) led to liquid suspensions with PCS mean particle sizes of 187 nm interfacial area. This interfacial area was affected by the environmental (5 cycles) and 248 nm (3 cycles), respectively. All other formulations pH presenting reduced mobility of the spin-probe at pH 1.2. Although solidified or formed visibly large aggregates already one day after partially hydrolyzed, the amphiphilic polymer stabilizes the interface of production. Most stable physical formulation was obtained with the skin the nanocapsules giving a stable structure, which is able to retain the friendly and ECOCERT Lanette E. No gelation or formation of visible model drug over time. Hence, comparing both PVM/MA colloidal aggregation occurred during 2 months of storage at room temperature. systems, nanoparticles and nanocapsules, the nanocapsules offer a Increasing or decreasing the Lanette E concentration led to poorer more protective environment for a lipophilic drug model than the storage stabilities (non redispersible aggregates, viscosity increase). nanoparticles, being a promising drug delivery system for oral smartLipids 2nd skin was successfully developed being promising in administration. restoration and reinforcement of the skin lipid barrier in a natural way. As the formulation consists exclusively of natural ingredients, it follows the latest trend of organic cosmetics and can therefore be incorporated in a wide variety of dermal products following an anti-pollution strategy. Also, smartLipids 2nd skin can be loaded with natural antioxidants, e.g. coenzyme Q10, in order to neutralize ROS already formed in the skin, enabling to create a system with dual anti-pollution effect.

Fig. 1 –EPR spectra of (A) TB in water, MCT and dry PVM/MA. (B) NP- [References] TB at 5 min and 10 h after preparation. NP-TB diluted (1:1) in PBS pH 1. https://www.who.int/airpollution/en/ [accessed 20th May 2019] 7.4 at 1 hour after preparation. (C) NC-TB at 1 h and 1 day, and of NC- 2. Weerheim A, Ponec M, Arch. Dermato.l Res. 2001, 293(4): 191–199. TB after 24 h of dialysis against buffered solutions at pH 7.4 or at pH 1.2. 3. Müller, R H, Sinambela, P, Keck, C M, Euro Cosmetics. 2013, (6): 20-22.

Acknowledgments: CAPES Foundation, Ministry of Education of Brazil, Caixa Postal 250, Brasília – DF, 70040-020, Brazil - CAPES scholarship Proc. nº 99999.010146/2013-00”.

References: 1. Irache, J. M. et al.: Molecules 2005, 10: 126–45. POS.146 2. Schmidt, U., et al. J. Appl. Polym. Sci. 2002, 87: 1255–1266. Development of orodispersible tablets for delivery of 3. De Souza, L.E., Poly(vinyl methyl ether-alt-maleic anhydride) based nanoparticles and nanocapsules: formulation and characterization, PhD diss., Martin-Luther Universität, 2017, probiotic bacteria to the oral cavity urn:nbn:de:gbv:3:4-21991 4. Kempe, S, Metz, H, Mäder, K, Eur. J. Pharm. Biopharm 2010, 74: 55-66. Hoffmann, A.; Daniels, R. Department of Pharmaceutical Technology, Auf der Morgenstelle 8, 72076 Tübingen, Germany

Lactobacillus species can be classified as probiotics and are part of the POS.145 physiological microbiome of the human oral cavity and intestinal tract. nd Several positive effects are described, like anti-inflammation [1], positive smartLipids 2 skin – restoration and reinforcement of the influence on the immune system [2], and antimicrobial activity against skin's natural lipid film as anti-pollution strategy P. gingivalis [3]. Thus, probiotics might be an option to treat gingivitis associated inflammation of periodontal tissue. A probiotic formulation Köpke D1 and Pyo, S M1 concept based on an orodispersible tablet (ODT) was developed. This 1Freie Universität Berlin, Kelchstr. 31, 12169 Berlin, Germany convenient dosage form has outstanding benefits, because water is not needed for intake and it is appropriate for children and the elderly. The fast tablet disintegration is usually accompanied by rapid removal of the According to WHO reports, air pollution continues to increase and is probiotic bacteria from the mouth due to saliva flow and swallowing reaching a health alarming level in over 91% of urban population [1]. The leading to a too short residence time in the oral cavity to show the skin as the largest organ of the body thereby offers a great contact and expected positive effects. The retention time of the bacteria can be attack surface. Molecules such as polycyclic aromatic hydrocarbons extended adding mucoadhesive polymers [4]. Nevertheless, the (PAH), localized on the surface of particulate matter, can penetrate and disintegration time of the tablets should be below 30 s to still meet the activate the aryl hydrocarbon receptors in skin. Decreased skin moisture, acceptance level from the FDA. To support fast disintegration on the one hyperpigmentation, increased inflammation tendency and ROS (reactive hand and to attain intimate contact of bacteria (Lp299v) and the oxygen species) formation are some consequences. Both adhesion and mucoadhesive polymer on the other hand, granulation of Lp299v and the penetration are efficiently prevented by the protecting properties of the mucoadhesive with a methacrylic acid copolymer was performed first. natural lipid film of the epidermis. Inspired by this natural anti-pollution Three mucoadhesive polymers, carbomer, HPMC and chitosan, were effect a dermally applicable formulation was developed mimicking the assessed. The comparison with the ungranulated formulations showed natural skin lipid’s physicochemical properties [2] and thus its anti- clear differences between the polymers. Granulation of carbomer and pollution effect. Lp299v resulted in a distinct acceleration of tablet disintegration. This

DPhG Annual Meeting 2019 Conference Book • 148 PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS was due to electrostatic repulsion of the negatively charged polymer and screened. Suitable for preservation are the classical preservative the bacteria themselves. Moreover, the very fine carbomer spheres were Microcare PEHG (2%) and pentylene glycol. They did not affect the bound to the probiotics leading to a dramatically reduced amount of free physical stability during storage, size increase was similar or even below carbomer. Granulation of Lp299v and non-ionic HPMC also led to the non-preserved reference formulation. accelerated tablet disintegration. Surprisingly, an opposite effect was found for the cationic chitosan. Here the interaction of the polycation and In conclusion, rutin smartCrystals concentrates are now available, being the polyanion during granulation prolonged the disintegration time of the industrially producible on large scale. They are PEG-free, and – if desired tablet in comparison to the formulation that was not granulated with the – also available preservative-free. This meets the present consumer methacrylic acid copolymer. The mucoadhesion of the formulation was expectations and market needs. assessed with a novel mucoadhesion test, which is related to physiological conditions in the oral cavity [5]. In the presence of the mucoadhesive polymers 20 – 30 % of the applied bacteria adhered to the References: mucosa, which represented a two- or threefold increase compared to 10 1. Romero, G. B., et al.: Int. J. Pharm. 2015, 482(1-2): 54-60. % when a formulation without any mucoadhesive additive was applied. 2. Petersen, R.: Patent PCT/EP2007/009943. 2007. Tablets consisting of a granulated mixture of bacteria and mucoadhesive polymer were proven to have good storage stability in tablet containers with a desiccant bag for 30 months (2 - 8 °C). For all three polymers a reduction of the mucoadhesive effect was detected after storage, but the formulation including carbomer still proved superior mucoadhesion POS.148 compared to HPMC and chitosan. Determination of antioxidant capacity in skin

Acknowledgments: We want to gratefully acknowledge financial support from Symrise AG. Alnemari R.1, Carrillo-Hormaza L 2., Pinnapireddy. S. R.1, Keck C.M.1 1 Department of Pharmaceutics and Biopharmaceutics, University of Marburg, Robert-Koch-Str. 4, 35037 Marburg, Germany 2 Bioactive substances research group, Universidad de Antioquia, Cra 56 No 61-30, 050010 [1] Schmitter, T. et al.: J. Oral Microbiol. 2018, 10(1): 1-10. Medellin, Colombia. [2] Cheng, P.-C.; Tsai, Y.-T. and Pan, T.-M.: Appl. Microbiol. Biotechnol. 2012, 96(4): 853-862. [3] Koll-Klais, P. et al.: Oral Microbiol. Immunol. 2005, 20(6): 354-361. [4] Hoffmann, A; Daniels, R.: Eur. J. Pharm. Biopharm. 2019, 13: 240-245. Oxygen Radical Absorbance Capacity assay (ORAC) is a sensitive [5] Hoffmann, A; Daniels, R.: Int. J. Pharm. 2018, 551(1): 141-147. antioxidant assay used to evaluate the antioxidant potential capacity (AOC) in food and cosmetic industries. Due to the impact of reactive oxygen/nitrogen species (ROS/RON) on aging and pathogens, ORAC is of great value in cosmeceuticals research [1, 2]. In skin aging, applying antioxidants to treat skin conditions increases the resistance towards POS.147 oxidative stress and improves the signs of aging [3]. In this study, ORAC assay was validated and used to measure the AOC Antioxidative rutin smartCrystals – commercial of two different antiaging cosmetic products, and to measure the concentrates with skin friendly stabilization penetration efficacy of these products into the stratum corneum layers of fresh porcine ears. Antiaging products were applied on porcine ears and incubated for 2 h at 30 °C. Tape stripping was then performed to remove Hespeler, D.; Pyo, S. M.; Müller, R. H. stratum corneum and the tapes were washed using ethanol/water. Freie Universität Berlin, Kelchstr. 31, 12169 Berlin, Germany Validated ORAC assay as described by Ou et. al. was performed using fluorescein as a fluorescent probe, AAPH (2,2'-Azobis(2- Dermal nanocrystals (smartCrystals) are a formulation to increase the amidinopropane) dihydrochloride) as a peroxyl radical generator, and penetration of poorly soluble cosmetic actives and drugs into the skin, Trolox as an internal standard [4]. The AOC results of cosmetical especially molecules being poorly soluble simultaneously in water and in products and stratum corneum extracts were expressed as µM Trolox lipophilic media/oils. They are commercially available as concentrates [1] equivalents/ 1 mL extract. which are simply admixed to the water phase of dermal formulations. Trolox calibration curve showed good linear relationship between net Originating from pharmaceutical development, the stabilizers used were area under the curve and Trolox concentrations with a correlation elected under pharma aspects, i.e. being well tolerated and ideally even coefficient (r2) ≥ 0.98. The method precision, which is expressed as intravenously injectable. Thus, smartCrystals concentrates are stabilized %RSD was ≤ 10%, and the accuracy varied from 97% to 107%. In the with Tween 80 or Poloxamer 188. However, the trend in dermal delivery results obtained, the first cosmetic serum (product A) showed much – cosmetics and dermotherapy – is the use of ECOCERT classified higher AOC (6.5-fold) than the second one (product B). This could be due ingredients. Ideally the products should be PEG-free and preservative- the nature of the products, i.e. product A being classified as a natural free. cosmetic while the other is not. Furthermore, also the AOC that was determined from the tape strips, yielded higher AOC values for product Nanocrystals are of increasing interest for anti-pollution products, A than for product B and non-treated skin, respectively and indicated a because anti-oxidants are an essential part in the molecular barrier deeper penetration of product A into the skin. strategy. Many interesting anti-oxidants are of plant origin and poorly ORAC assay was successfully used to evaluate the efficacy of anti-aging soluble, e.g. rutin, hesperetin, quercetin etc. Using them as suspension serums by measuring the AOC of the products and the AOC of stratum formulation with µm-sized crystals provides insufficient bioavailability in corneum tape stripping extracts. This convenient, economical and the skin. versatile ex-vivo model can be a valuable tool to predict the potency and the penetration efficacy of cosmeceuticals. Thus, in this study, a smartCrystals formulation was developed using dermofeel® G10L as ECOCERT stabilizer. Rutin was elected as active References because of its proven activity [2]. Formulation was designed that large- [1] Wink, D. A., et al. AAAS, 1991, 254(5034) p.1001–1003. scale production as concentrate is feasible, i.e. rutin content of 11%. [2] Nguyen, T., et al. Proc Natl Acad Sci U S A, 1992, 89(7): p.3030–3034. Production was performed using a bead mill PML-2 with a 1.2 L [3] Masaki, H.J, Dermatol Sci, 2010, 58(2): p.85–90. continuous flow chamber (Bühler, Switzerland). Optimized production [4] Ou, B., et al, J. Agric. Food Chem., 2001, 49(10): p.4619–4626. parameters were used (e.g. rotation speed, no. of passages).

The crystal size was about 330 nm, i.e. above the threshold of 100 nm for being legally classified as nanomaterials. They are thus classified as submicron crystals (>100 nm to < 1 µm), considering the increasing POS.149 concern of the public about “nano”. Storage stability of the concentrate Stability Studies of Proteins during Biofabrication was monitored over 6 months at storage temperature of 4-6°C. There was a small size increase by 80 nm, negligible for a dermal product and crystal performance. Various preservatives/preserving agents were Eiber, J. F. 1, Ruopp, M. 1, Lorson, T. 1, Lühmann, T.1

149 • DPhG Annual Meeting 2019 Conference Book POSTERS

1Institute of Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, 97074 and physical degradation. Protein aggregation is known to be the most Wuerzburg, Germany common physical instability and may occur at various points throughout the lifetime (e.g., formulation development, production, processing, storage, and packaging) and even during administration of protein-based Background and objective: therapeutics [1]. The formation of these larger particles depends on Within the field of tissue engineering, biofabrication is a promising intermolecular interactions and may be of different quantity and quality technology in order to obtain functional tissue equivalents not only such as size and morphology. Aggregates can considerably range in size containing living cells, biomaterials and bioactive factors but also from dimers to multimers and up to sub-visible and visible particles, mimicking the hierarchical structure of natural tissues [1]. One approach endangering the safety and efficacy of the drug product due to an to produce such constructs is bioprinting, commonly using printable increased immunogenicity [2]. It is therefore important to understand the formulations based on hydrogel-forming biopolymers [2]. Little is known interactions and characterization of aggregates in order to control protein about the impact of the printing process on the biological integrity of the aggregation to enable stable and successful drug products [1]. The main components and possible interactions between them, crititical aspects for goal of this study was to investigate whether analytical measures can a future medical application. provide useful screening tools to estimate the aggregation propensity of Our aim is to investigate the stability of murine fibroblast growth factor 2 a model protein in liquid formulations. The analytical techniques used (mFGF-2) and enhanced green fluorescent protein (eGFP) before, during were based on the assessment of conformational and colloidal stability and after the printing process correlated to their bioactivity and functional [3], including the determination of the melting temperature (Tm) as properties. suitable indicator of protein conformational stability [4], the determination Methods: of the diffusion self-interaction parameter (kD) and the osmotic second mFGF-2 was recombinantly produced and purified via heparin- virial coefficient (B22) as measures of colloidal stability [3,5]. sepharose affinity chromatography on a HiTrap Heparin HP column (GE Material and Methods: In this study three batches of lysozyme in liquid Healthcare) as previously described [3]. His-tagged eGFP was formulations were investigated in terms of their biophysical recombinantly produced and purified via immobilized metal ion affinity characteristics, and conformational and colloidal stability. Lysozyme was chromatography on a HisTrap FF column (GE Healthcare) as previously formulated in 20 mM acetate buffer pH 4.5 and in the absence or described [4]. Successful expression and purification of the proteins was presence of sodium chloride. Protein concentration series ranged from 2 confirmed by HPLC, MALDI-MS and ESI-MS analysis after trypsin to 14 mg/mL. Dynamic (DLS) and static light scattering (SLS) were used digestion. to determine the diffusion self-interaction parameter (kD) and the second The proteins were formulated into several commercially available bioinks virial coefficient (B22), respectively. The denaturation temperature (Tm) of based on alginate and gelatin (Bioink by Cellink, GelMa by Cellink, lysozyme in solution was investigated by intrinsic tryptophan Alginat by Allevi). HPLC profiles of the formulations were compared to fluorescence spectroscopy. The samples were also stored for 3 months those of the pure protein solutions. Therefore, measurements of the at 5 °C and were then analyzed for visual appearance and sub-visible formulations were performed on a BioSep-SEC-s2000 column particles by DLS. (Phenomenex), using 0.1 M PBS, 0.3 M NaCl, pH 6.6 as mobile phase Results and Conclusion: The diffusion self-interaction parameter (kD) at a flow rate of 0.5 mL/min and 15 min run time. Proliferation of NIH 3T3 and the second virial coefficient (B22) indicated that repulsive interactions fibroblast cells in response to mFGF-2 was measured by WST-1 between lysozyme and solvent molecules are present at pH 4.5 in the colorimetric assay. Analysis of the structural conformation of eGFP was absence of sodium chloride. In the presence of 400 mM sodium chloride, performed fluorimetrically. the DLS and SLS measurements showed attractive interactions between Results: the molecules, indicated by negative kD and B22 values. At pH 4.5, HPLC measurements revealed that the stability of both proteins was lysozyme carries a positive net charge accompanied with a maximal significantly decreased in pure alginate hydrogels. In contrast, if alginate number of cationic sites. The change from net repulsion (positive values) was combined with other components such as cellulose nanofibers, the to net attraction (negative values) in presence of salt, results from the stability of both proteins was less reduced. The stability data are currently transition of charge-mediated protein-protein repulsion at low salt correlated with bioactivity studies in order to investigate the influence of concentration to attraction due to short-range protein interactions (van the composition of the bioink on protein performance. der Waals forces, hydrophobic effects, etc.). The B22 values of lysozyme Conclusion: formulations in the presence of sodium chloride are within the In conclusion, first destabilizing effects on proteins in bioink formulations crystallization slot, indicating a higher risk of forming precipitate, because were demonstrated. of stronger protein-protein attractions. Lysozyme in acetate buffer pH 4.5 was therefore expected to be the most stable formulation compared to Acknowledgments: Federal Ministry of Education and Research (BMBF, FKZ 13XP5071D) formulations in presence of sodium chloride. These findings were confirmed by the study of melting point determination. Tm of lysozyme was reduced in the presence of sodium chloride. This indicates that the addition of salt has a very unfavorable effect on the protein thermal References: stability at pH 4.5. The formulations were also stored at 5 °C for 3 months [1] Groll, J. et al: Biofabrication 2016 1–5 and were visually inspected at the end of the storage time. Formulations [2] Ferris, C. J., et al: Appl. Microbiol. Biotechnol. 2013, 97(10) 4243–4258 in the absence of salt and showing positive B22 values outside the [3] Hongshi, Z. et al: J. Struct. Biol. 2014, 186(3) 420–430 [4] Wandrey, G. et al.: J. Biol. Eng. 2016, 10(11) 1–13 crystallization slot were visibly clear, whereas formulations in the presence of salt and presenting negative B22 values within the crystallization slot showed precipitation. Based on the observed findings, kD, B22 and Tm values can be used to choose optimal formulation conditions to influence the protein critical solution behavior and thus to POS.150 estimate colloidal and conformational stability of liquid formulations. Such Biophysical characterization methods to assess the studies could be performed within a few days and within a miniaturized stability of formulated protein-based drugs setting, instead of real time formulation screenings.

Dauer, K.1; Wagner, K. G.1; Pfeiffer-Marek, S.2; Kamm, W.2 References: 1 Institute of Pharmaceutical Technology and Biopharmacy, Rheinische Friedrich-Wilhelms- 1. Mahler H.-C. et al.: J. Pharm Sci. 2009, 98(9): 2909–2934. Universität, 53121 Bonn, Germany 2. Sorret, L. L. et al.: Biophys. J. 2016, 111(9): 1831–1842. 2 Sanofi-Aventis Deutschland GmbH, Industriepark Hoechst, 65926 Frankfurt am Main, 3. Hofmann, M. and Gieseler, H.: J. Pharm Sci. 2018, 107(3): 772–777. Germany 4. Manning, M.C. et al.: Adv. Protein Chem. Struct. Biol. 2018, 112: 1–59. 5. Pindrus, M.A. et al.: Mol Pharm. 2018, 15(8): 3133–3142 Introduction: The characterization of protein-based drugs within drug product development poses several challenges. There is increasing interest in early characterization and formulation optimization to POS.151 accelerate drug development and thus to avoid costly late-stage failures. Early-stage development is often accompanied by tight timelines, limited Apparent Effective pKa of Bicarbonate in the Boundary API availability, limited knowledge of drug properties and the impact of Diffusion Layer and its Implications for pH-dependent Oral formulation conditions. Due to the complex structure of proteins, they Drug Delivery show limited stability in solution and are thus susceptible for chemical

DPhG Annual Meeting 2019 Conference Book • 150 PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS

wet oscillated granulates was much lower than the bulk density of the roller compacted ones. Al-Gousous J1,2, Salehi N3,Ruan H1,4, Blechar JA1, Langguth P1,Ziff The different amounts of the fillers compared to the hydrogen generating RM3, Amidon GE2, Amidon GL2 ingredient did not affect the results to a great extent. Granulates from 1Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany each group of filler showed nearly the same results in every prepared 2College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, MI 48109, USA concentration in the starting powder mixture. However, the different types 3Department of Chemical Engineering, University of Michigan, 2300 Hayward St, Ann Arbor, MI of fillers showed differences in the results compared to each other. 48109, USA 4Department of Chemical Drug Research, Zhejiang Institute for Food and Drug Control, Overall the method of preparation had the biggest impact on the Hangzhou, Zhejiang 310052, China properties of these granulates. The type of filler that is used is also very important. The different amounts of the fillers compared to the hydrogen generating ingredients did not change their characteristics noteworthy. The disintegration and dissolution of enteric-coated (EC) dosage forms has been observed to be considerably delayed in vivo compared to in 1. Ohsawa, I. et al.: Nature Medicine volume 2007, 13: 688–694 vitro, even with instances of associated clinical failure.1 The reason 2. Li, H. et al.: Medical gas research 2018, 7(4): 265-272. behind this seems to be the lack of responsivity of enteric polymer 3. Tomofuji, T. et al: Sci. Rep. 2014, 4, 5534; dissolution to bicarbonate which is the main buffering species in human intestinal fluid, with the bicarbonate molarities present in vivo not providing sufficiently prompt enteric polymer dissolution. This is caused by the anomalous buffering action of bicarbonate in the boundary layer POS.153 surrounding a dissolving ionisable solute. For, owing to the relatively slow Sustainable Nano Products: Production and equilibration of the CO2-H2CO3 interconversion, the effective pKa of bicarbonate in the boundary layer would be far lower than in the bulk. For characterization of PlantCrystals from the waste of black instance, in a 30 micron boundary layer, the apparent effective pKa of tea as a source of antioxidant phenolic compounds bicarbonate would be around 4.6. This impairs the ability of bicarbonate to buffer the surface of a dissolving enteric polymer at pH values that 1 1 1 facilitate prompt dissolution. This leads to unexpected failures of EC Abraham, A. ; Alnemari, R. ; Keck, C.M. 1Philipps-Universität Marburg, Department of Pharmaceutics and Biopharmaceutics, Robert- dosage forms to disintegrate properly in vivo and showcases the need Koch-Str. 4, 35037 Marburg, Germany for more in vivo-predictive dissolution testing methodologies in order to minimize the risk of such occurences. Introduction: Acknowledgment: Black tea is one of the most common beverages consumed worldwide. It This work was supported by Grant HHSF223201510157C and Grant HHSF223201310144C by is derived by fermentation of the dried green leaves of Camellia sinensis the U.S. Food and Drug Administration (FDA). This report represents the scientific views of the (Theaceae). Tea is usually used due to its taste and high polyphenols’ authors and not necessarily those of the FDA. content [1]. Its residue is usually thrown away. But this wasted residue might still contain a fortune, like polyphenols. Such active constituents References: Grosser, T. et al.: Circulation 2013, 127(3): 377-385. can therefore help to treat several oxidative stress disorders like Alzheimer's disease. However, its poor solubility and low bioavailability lead to reduced biological activity. Recent studies showed that nanosizing of medicinal plants and their wastes is a promising, smart and easy method to release their active constituents [2]. Therefore, the waste POS.152 of black tea was chosen in this study to investigate the effects of the Formulation and evaluation of effervescent hydrogen nanosizing process on its antioxidant capacity. The characteristics of the generating granulates using wet (oscillating granulation) resulted PlantCrystals from black tea waste were compared with the bulk and dry (roller compacting) granulation material, including the antioxidant activity in-vitro.

Methods: Moritz Rosch1,2, Peter Langguth2, Ulrich Pöschl1, Kurt Lucas1 One percent of black tea was boiled in hot water for about one minute. 1Multiphase Chemistry Department, Max Planck Institute for Chemistry, Hahn-Meitner-Weg 1, The residue obtained was dried and grinded. The resulted residue was 55128 Mainz, Germany suspended in 1% (w/w) Plantacare2000® surfactant solution. A Small- 2Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudingerweg 5, 55128 Mainz, Germany scale bead milling method was performed on the obtained bulk- suspension [3]. The produced nanocrystals were analysed using photon correlation spectroscopy (PCS), laser diffraction (LD) and light The antioxidant and selectively reducing effects of hydrogen on cytotoxic microscopy. radicals were first described in 2007 [1]. Since then the interest in Total polyphenols, flavonoids and carotenoids contents were determined. hydrogen as a therapeutic gas is growing quickly [2]. Most of the The antioxidant capacity (AOC) was detected using DPPH (2,2-diphenyl- experiments are performed with hydrogen which is applied to animals via 1-picrylhydrazyl) assay [4]. The characteristics were conducted on the inhalation, injection or as hydrogen enriched drinking water (HRW). HRW obtained PlantCrystals and compared with those of the bulk material and can for example be prepared by electrolysis [3]. tea. In order to transfer hydrogen therapeutic potential to human medical use we aim to develop an effervescent hydrogen releasing tablet as a Results: pharmaceutical dosage form to freshly prepare HRW. To achieve this Nanosizng process applied to the waste of the black tea bulk material led hydrogen releasing effervescent granulates were manufactured by to sizes of about 280 nm. The antioxidant capacity increased with different granulation techniques (wet oscillating granulation and roller decreasing size, i.e. the antioxidative potential increased about nine fold compacting). Moreover the type and amount of possible fillers were upon nanosization, leading to the highest AOC value from the evaluated in order to explore a suitable manufacturing technology, type suspensions analysed. The different contents’ tests used in this study and amount of filler for the formulation. demonstrated that the produced PlantCrystals possess the highest Granulates were characterized by measuring different parameters (angle polyphenols, flavonoids, and carotenoids contents, hence, the available of repose, flowing time, bulk density). content was higher due to the release occurred upon the nanosizing The results demonstrate that the roller compacted granulates are more process. In contrast, the produced nanocrystals showed higher AOC than favourable regarding the flowing time and bulk density when compared the bulk material. However, according to DPPH assay the prepared tea to the wet oscillated granulates, while their angle of repose was in boiled water had the best AOC followed by the obtained PlantCrystals satisfying. Concerning the angle of repose, the granulates that were with IC50 values of 0.013 and 0.127 mg/ml, respectively. prepared by oscillating granulation performed slightly better than the roller compacted ones, while the flowing time was not as good but still Conclusion: sufficient for a later tabletting process. However, the bulk density of the Black tea wastes were successfully nanosized. The bulk-suspension possesses weak antioxidant activity, which was improved by the

151 • DPhG Annual Meeting 2019 Conference Book POSTERS

nanosization process, i.e. the obtained Nano-Wastes demonstrated the References: best antioxidant activity. Therefore, we can conclude that plants’ wastes 1. Lipner, S. R., Scher, R. K, J. Am. Acad. Dermatol. 2019, 80(4): 835-851. are a rich and sustainable source of phytochemicals and nanonization 2. Lipner, S. R., Scher, R. K, J. Am. Acad. Dermatol. 2019, 80(4): 853-867. can further increase the health beneficial properties. The PlantCrystal- 3. Pötzinger, Y. et al.: Ther. Deliv. 2017, 8(9): 753-761. technology represents a natural, environmentally friendly and cost- effective alternative source of antioxidant phenolic compounds.

POS.155 References: Sterile 3D-bioprinting and mechanical characterization of 1. Lin, Y. S. et al.: J. Agric. Food Chem. 2003, 51: 1864-1873 2. Griffin, S. et al.: J. Environ. Manage. 2018, 210: 114-121. hydrogels 3. Kulkarni SA. and Myerson AS.: Engineering crystallography: from molecule to crystal to functional form 2017, 275-287. 1 1 1 1 4. Sharma, PO. and Bhat, TL.: Food Chem. 2009, 113(4): 1202 – 1205. Ruopp, M. ; Eiber, J. ; Lorson, T. ; Lühmann, T. 1Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074

Würzburg, Germany

POS.154 Biofabrication aims for biologically active constructs consisting of cells, Nail Patches from Bacterial Nanocellulose for the Laser (bio-) materials and biological molecules in hierarchical order using 3D Therapy of Onychomycosis printing technologies [1]. Fabrication yielding consistent, tailor made, and above all microbiologically safe constructs is an important task to translate from research into application [2]. Alginates are widely used Bellmann, T.1; Kischio, L.1; Karl, B.1; Beekmann, U.1; Kralisch, D.1,2; hydrogels because of their good biocompatibility, gelation in contact with Fischer D.1,2 cations such as Ca2+ and their tunability of mechanical properties to the 1 Friedrich Schiller University Jena, Pharmaceutical Technology and Biopharmacy, requirements of different cell types by variation of crosslinker, gelling Lessingstraße 8, 07743 Jena, Germany conditions and molecular weight. These properties make alginate a 2 Jena Center for Soft Matter (JCSM), Friedrich Schiller University, Philosophenweg 7, 07743 Jena, Germany suitable base material for bioinks [3]. Here, we compared compendial sterilization monographs with aseptic manufacturing of 3D-bioprinted alginates with a particular focus on microbiological outcome and the impact on supramolecular structures as assessed rheologically. Onychomycosis is the most common nail infection and is caused by different types of fungi, like dermatophytes, nondermatophytes and Compared to the untreated alginate after autoclaving for 15 min at 121 yeasts [1]. As the established treatment options, local and systematic, °C, alginate hydrogels showed a reduction of the number average require a long treatment time and have a high recurrence rate, the molecular weight by 70 % and a significantly lower viscosity. In addition, patient’s compliance is poor [2]. Laser therapy is an improved treatment after autoclaving, the alginate has transformed into a hydrogel with for onychomycosis with a shorter treatment time, few side effects and reduced gel characteristics. This can be seen by a significant change in promises a higher patient acceptance. In the present study, the suitability the storage and loss modulus. Due to the reduced viscosity and the of the biopolymer bacterial nanocellulose (BNC) as a softening patch for changed flow behaviour, the autoclaved alginate material lost its infected nails prior to laser therapy was investigated to develop an easily printability and therefore its suitability for 3D-bioprinting. Sterile filtration, applicable system that increases the efficacy of laser treatment. on the other hand, had only a minor effect on the rheological behaviour BNC is a biotechnologically synthesized three-dimensional network of and the molecular composition of the alginate hydrogel. nano-sized fibers with many desirable properties, like excellent biocompatibility, high purity, mechanical strength and a high loading We conclude that aseptic production of alginate bioinks is made possible capacity for hydrophilic drugs [3]. BNC was loaded with different amounts by sterile filtration without significantly influencing the rheological of the moisturizing glycerol and urea as a keratolytic agent. For the properties required for 3D-bioprinting. development of a pharmaceutical product, characteristics like form stability, adhesion, prevention of dehydration, transparency, wetting of the nail and a high loading efficacy for urea are demanded. Acknowledgments: Federal Ministry of Education and Research (BMBF, FKZ 13XP5071D) BNC was synthesized by the bacterial strain Komagataeibacter xylinus at 28 °C in 24-well plates for 14 days, alkaline purified and loaded by absorption with glycerol solutions at different concentrations with or References: without the addition of urea. Form stability was characterized by 1. Grolll, J. et al.: Biofabrication 2016, 8(1): 1-5. determining mass loss and compressive strain after pressurizing the 2. Moroni, M. et al.: Trends Biotechnol. 2018, 36(4): 384-402. loaded BNC fleeces. By comparing the absorption of the loaded fleeces 3. Freeman, F. E. et al.: Sci Rep. 2017, 7(1): 1-12. relatively to the maximum opacity transparency was investigated. Horse hoof plates were used as a model to examine the wetting-capacity of the pharmaceutical formulations. To determine the loading capacity of the BNC, urea was quantified in the loading solution before and after the loading procedure. The mechanical stability of the BNC was enhanced by the addition of > 75% glycerol, while the addition of urea did not change the compression stability. The absorption measurements displayed that an increase of the glycerol concentration correlated with an increased transparency of BNC. The addition of glycerol and urea provided a sufficient wetting-capacity over more than 24 hours, which facilitates an overnight application. Investigations of the loading capacity revealed that urea solutions with concentrations up to 40%, which is comparable to commercial pharmaceutical products, could be loaded into the BNC. The combination of BNC with urea and glycerol offers many desirable characteristics for a tailor-made pretreatment of onychomycotic nails prior to laser therapy. The easy to use application as nail patch without additional dressing material and the transparency of the BNC to evaluate the progress of softening without the need to remove the patch are beneficial for the pharmaceutical application.

Acknowledgments: We would like to thank JeNaCell GmbH for providing the K. xylinus culture.

DPhG Annual Meeting 2019 Conference Book • 152 PHARMACOLOGY

References: 1. Marquard, J. et al. Nature medicine, 2015, 21(4): 363-372. 4.9 Pharmacology 2. Carpenter, C. et al.: Brain Research, 1988, 439: 372–375. 3. Netzer, R. et al. European Journal of Pharmacology, 1993, 238: 209-216. POS.156 2+ Dextromethorphan inhibits KATP and Ca currents in pancreatic β-cells POS.157 A. Gresch1; M. Düfer 1 Exotoxins from Staphylococcus aureus regulate 1University of Münster, Pharmaceutical and Medicinal Chemistry, Dept. of Pharmacology, specialized pro-resolving mediators that promote Corrensstraße 48, 48149 Münster, Germany resolution of inflammation

Question: The NMDAR antagonist dextromethorphan (DXM) and its Jordan, P.M.1; Gerstmeier, J.1; Pace, S. 1; Bilancia, R. 1,2; Rao, Z.1; metabolite dextrorphan (DXO) have been recommended for treatment of Arakandy, V.3; Gutierrez-Gutierrez, O.4; Tuchscherr, L.3; González- type 2 diabetes mellitus because of their beneficial effects on insulin Estévez, C.4; Serhan, C.N.5; Werz, O.1 secretion [1]. It is also known that DXM and DXO, although the latter less 1Institute of Pharmacy, Friedrich Schiller University Jena, Philosophenweg 14, D-7743 Jena, 2+ Germany; potent, not only inhibit NMDA receptors but also voltage-gated Ca 2Department of Pharmacy, School of Medicine, University of Naples Federico II, Via Domenico channels [2, 3]. This study investigates the effects of DXM and DXO in Montesano 49, 80131-Naples, Italy; 3 islets and single β-cells on [Ca2+]c, Ca2+ current and KATP current, which Institute of Medical Microbiology, Jena University Hospital, Am Klinikum 1, D-07747 Jena, Germany; regulate essential steps of the stimulus-secretion coupling. 4Leibnitz Institute on Aging (FLI), Beutenbergstraße 11, D-7745 Jena, Germany; Methods: Islets or β-cells were isolated from wildtype (WT) and SUR1- 5Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America deficient mice (SUR1-KO) of a C57Bl/6N background. [Ca2+]c was determined by fluorescence microscopy. Electrophysiology was performed with the patch-clamp-technique. Insulin release from islets The knowledge about the mechanisms by the host to actively resolve was determined by radioimmunoassay. bacterial infections is incomplete. A major characteristic of inflammation Results: WT islets stimulated with 8 mM glucose showed a typical resolution is the biosynthesis of specialized pro-resolving mediators 2+ oscillatory pattern in [Ca ]c measurements. DXO (100 µM) was added (SPM) by macrophages. These antiphlogistic endogenous lipid 2+ for 10 minutes to the bath solution and slightly elevated mean Ca of mediators (LM) lead to reprogramming of the immune response to whole islets (control 0.94±0.03 a.u. vs. DXO 1.05±0.04 a.u., n=17, accelerate the termination of inflammation, enhance bacterial clearance 2+ p≤0.01). Acute application of 100 µM DXM first stopped the Ca and promote tissue regeneration. Pathogenic bacteria stimulate oscillations, then after approximately 5 minutes the islets switched into a cyclooxygenase and lipoxygenase (LOX) pathways in human plateau pattern. Regarding the last 5 minutes of substance addition, DXM macrophages to produce differential LM profiles in a phenotype- 2+ significantly increased mean [Ca ]c (control 0.97±0.03 a.u. vs. DXM dependent manner [1]. However, the underlying factors and mechanisms 1.27±0.04 a.u., n=14, p≤0.001). In comparison, DXM increased mean that originate from bacteria to induce LM generation in these host cells 2+ [Ca ]c significantly more than DXO (DXO 1.05±0.04 a.u., n=17 vs. DXM remain vague. Here, we identify the pore-forming toxin α-hemolysin (Hla) 1.27±0.04 a.u., n=14, p≤0.05). The sudden transient inhibition of Ca2+ from Staphylococcus aureus as selective and potent activator of 15-LOX- oscillations in WT islets by DXM was further elucidated by measuring 1 which is a key enzyme in SPM biosynthesis in human monocytes- Ca2+ currents in the whole-cell configuration. DXM (100 µM) inhibited derived M2 macrophages and in vivo in murine peritoneal infections. Hla- 2+ partially but significantly the Ca current in single β-cells (control -22±2 deficient Staphylococcus aureus mutants or depletion of Hla using a pA vs. DXM -17±3 pA, n=5, p≤0.05). capturing Hla-antibody failed to activate 15-LOX-1 and thus related SPM The KATP channel current of WT β-cells was measured in the whole-cell biosynthesis but were still efficient to induce proinflammatory eicosanoids configuration. In the presence of 0.5 mM glucose application of 100 µM like arachidonic acid-derived prostaglandins (PGs) and leukotrienes DXO acutely inhibited the KATP current (control 25±3 pA/pF vs. DXO 10±2 (LTs). Genetic manipulation by si-RNA and pharmacological inhibition by pA/pF, n=5, p≤0.001). DXM reduced the KATP current dose-dependently. BLX-3887 [2] of human 15-LOX-1 in M2 macrophages exclusively The effect was significant at 50 and 100 µM DXM (control 20±3 pA/pF reduced the Hla-dependent SPM biosynthesis upon challenge with vs. DXM 50 µM 16±2pA/pF, n=6, p≤0.001; control 24±2 pA/pF vs. DXM bacterial secreted toxins. Moreover, LM isolates from HLA-stimulated M2 100 µM 7±2pA/pF, n=5, p≤0.001). that contain abundant SPM levels but lack proinflammatory PGs and LTs, Insulin secretion in WT islets was elevated by 100 µM DXO, when islets promoted tissue regeneration in Planaria. Conclusively, our data suggest were pre-incubated for 1.5 h with DXO before 1-h stimulation with 15 mM that bacterial pore-forming toxins, besides initially harming the host, may glucose (15 mM glucose 6±1 ng/(islet*h) vs. DXO 9±1 ng/(islet*h), n=6, also exert beneficial functions by stimulating SPM production to resolve p≤0.05). In SUR1-KO islets DXO only tended to rise insulin secretion inflammation. when stimulated with 15 mM glucose (15 mM glucose 2.5±0.5 ng/(islet*h) vs. DXO 3.6±0.7 ng/(islet*h), n=6, n.s.). In agreement with insulin 1. Werz, O.; Gerstmeier, J. et al.: Nature communications 2018, 9(1), 59. 2. Werner, M.; Jordan, P.M. et al.: FASEB J. 2019, 33(5):6140-6153. secretion, [Ca2+]c experiments with SUR1-KO islets showed just a marginal increase in response to a 10-minute application of 100 µM DXO (control 1.08±0.02 a.u. vs. DXO 1.15±0.04 a.u., n= 13, p≤0.05). Insulin release of WT islets stimulated with 100 µM DXM along with 15 mM glucose was significantly elevated (15 mM glucose 4.6±0.4 ng/(islet*h) POS.158 vs. DXM 8±1 ng/(islet*h), n=10, p≤0.05). Astonishingly, in SUR1-KO A Ruthenium(II) N-Heterocyclic Carbene (NHC) Complex islets insulin secretion stimulated by 15 mM glucose was not elevated by with Naphthalimide Ligand Triggers Apoptosis in 100 µM DXM (15 mM glucose 2.5±0.5 ng/(islet*h) vs. DXM 2.6±0.4 Colorectal Cancer Cells via Activating the ROS-p38 MAPK ng/(islet*h), n=6, n.s.). [Ca2+]c measurements with whole SUR1-KO islets even showed a significant decrease when DXM was added for 10 Pathway. minutes to the bath solution (control 1.15±0.05 a.u. vs. DXM 0.99±0.03 1 1 2 1 1 a.u., n= 12, p≤0.001). Schmid, A. ; Dabiri, Y. ; Ott, I. ; Wölfl, S. ; Cheng, X. 1 Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer 2+ Conclusion: DXM directly inhibits KATP and Ca currents, while its Feld 364, 69120 Heidelberg, Germany metabolite DXO only reduces KATP current. Our investigations reveal that 2 Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, the stimulatory effects of DXM and DXO on insulin secretion rather Beethovenstraße 55, 38106 Braunschweig, Germany depend on inhibition of the KATP channel than on an interaction with NMDAR. The direct effect of DXM on the L-type Ca2+ channel can cause unreliable alterations of insulin secretion. We conclude from our data that The p38 MAPK pathway is known to influence the anti-tumor effects of DXM and DXO are no suitable candidates for treatment of type 2 diabetes several chemotherapeutics, including that of organometallic drugs. mellitus. Previous studies have demonstrated the important role of p38 both as a regulator and a sensor of cellular reactive oxygen species (ROS) levels. Acknowledgment: We thank Prof. Dr. Gisela Drews (Institute of Pharmacy, Department of Investigating the anti-cancer properties of novel 1,8-naphthalimide Pharmacology, University of Tübingen, Germany) for providing SUR1-KO islets. derivatives containing Rh(I) and Ru(II) N-heterocyclic carbene (NHC)

153 • DPhG Annual Meeting 2019 Conference Book POSTERS ligands, we observed a profound induction of ROS by the complexes, C57BL/6 with the unspecific cyclooxygenase (COX) inhibitor diclofenac which is most likely generated from mitochondria (mtROS). Further (5mg/kg BW) reduced the extravasation to 2.26±0.28 (n=6 each, “P < analyses revealed a rapid and consistent activation of p38 signaling by 0.0001”) and ibuprofen (12 mg/kg BW) reduced the effect to 2.72±0.15 the naphthalimide-NHC conjugates, with the Ru(II) analogue-termed (n=5, “P < 0.0001”). Identical effects were seen using LD. In comparison, MC6-showing the strongest effect. In view of this, genetic as well as a lesser reduction was observed using the selective COX-1-inhibitor SC- pharmacological inhibition of p38α, attenuated the anti-proliferative and 560 (n=5 each, BK to 3.44±0.19, “P < 0.01” and LD to 3.04±0.22, “P < pro-apoptotic effects of MC6 in HCT116 colon cancer cells, highlighting 0.05”), while the more COX-2 specific celecoxib achieved a highly the involvement of this signaling molecule in the compound's toxicity. significant reduction in BK (to 3.08±0.27, n=6, “P < 0.0001”) but also in Furthermore, the influence of MC6 on p38 signaling appeared to be LD treatment (to 3.05±0.09, n=6, “P < 0.05”). In an additional approach dependent on ROS levels, as treatment with general- and mitochondria- this effect was reproduced on already induced extravasation. Treatment targeted anti-oxidants abrogated p38 activation in response to MC6 as of C57BL/6J with diclofenac 10 min after i.d. injections also reduced well as the molecule's cytotoxic- and apoptogenic response in HCT116 extravasation significantly (n=6 each, BK to 3.22±0.18, “P < 0.0001” and cells. Altogether, our results provide new insight into the molecular LD to 3.13±0.11, “P < 0.05”). No effect was observed on His-induced mechanisms of naphthalimide-metal NHC analogues via the ROS- extravasations by any COX-inhibitor. Our results suggest that the induced activation of p38 MAPK, which may have therapeutic interest for generation of prostaglandins in response to BK formed by both COX-1 the treatment of various cancer types. and COX-2 contributes to edema formation in small dermal blood vessels of mice.

POS.160 Activation of AT2 potentiates bradykinin-induced extravasation – role of ACE

Gholamreza-Fahimi, E.1; Krybus, M.1; Bisha, M.1; Kurz, T.2; Hansen, F. K.3; Kojda G.1 1Institute of Pharmacology and Clinical Pharmacology, Heinrich Heine University, Düsseldorf, Germany. 2Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Germany. 3Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Leipzig University, Medical Faculty, Leipzig, Germany.

Non-allergic angioedema arises from swelling of mucosal and sub- mucosal tissue and can be potentially life-threatening, e.g., if the swelling occurs in the larynx. Non-allergic angioedema falls into different subtypes Acknowledgements: depending on the underlying molecular mechanism. In any case, 1. Deutsche Forschungsgemeinschaft (DFG) grant program (CH 1690/2-1), Cheng, X. bradykinin-induced activation of bradykinin receptor type 2 (B2) has been 2. Landesgraduiertenförderung (LGF) fellowship program for individual doctoral training from shown to be crucially involved in animal studies [1] and in clinical trials, Universität Heidelberg, Dabiri, Y. e.g. in ACE inhibitor induced angioedema [2]. While non-allergic 3. Bundesministerium für Bildung und Forschung (BMBF) grant programs; Drug-iPS (FKZ angioedema induced by ACE inhibitors is most likely caused by inhibition 0315398A/B) and SysToxChip (FKZ 031A303A/E), Wölfl, S.; Cheng, X. of bradykinin degradation, the molecular mechanism of non-allergic angioedema induced by angiotensin II receptor type 1 (AT1) inhibitors References: (sartans) such as telmisartan is still unclear. Blockade of AT1 disrupts the 1. Dabiri, Y. et al. A Ruthenium(II) N-Heterocyclic Carbene (NHC) Complex with Naphthalimide negative feed-back on renin secretion and increases plasma levels of Ligand Triggers Apoptosis in Colorectal Cancer Cells via Activating the ROS-p38 MAPK Pathway: Int J Mol Sci. 2018, 19(12). pii: E3964. angiotensin II (AT2) likely resulting in a more pronounced activation of 2. Reczek, C. R.; Chandel, N. S. The Two Faces of Reactive Oxygen Species in Cancer: Annu angiotensin II receptor type 2 (AT2). Therefore, we sought to investigate Rev Cancer Biol. 2017. 1: 79-98. whether AT2 might be involved in the molecular mechanism of sartan 3. Streciwilk, W. et al. Fluorescent organometallic rhodium(I) and ruthenium(II) metallodrugs induced angioedema. To accomplish this, we synthesized the selective with 4-ethylthio-1,8-naphthalimide ligands: Antiproliferative effects, cellular uptake and DNA- non-peptide AT2 agonist compound 21 (C21, Fig 1). We verified its interaction: Eur J Med Chem. 2018, 156: 148-161. pharmacologic specificity in organ bath studies using aortic rings of AT2 knockout mice. Furthermore, in two different human primary endothelial cell cultures we were able to show that C21 reduces ACE activity in cell homogenates [3]. As for an in-vivo approach, we quantified dermal POS.159 extravasation induced by intradermal injection of bradykinin in mice Role of prostaglandins in bradykinin-induced skin edema (Miles Assay). As compared to vehicle (0.1 % dimethyl sulfoxide), C21 in mice significantly increased dermal extravasation from 3.9±0.2-fold to 4.7±0.2-fold (n=11, P<0.05). This effect was abolished by co-treatment with the AT2 antagonist PD123,319 (PD) and dependent on activation of Krybus, M.1; Gholamreza-Fahimi, E.1; Khosravani, F.1; Bisha, M.1; B2. The selective B2 antagonist icatibant strongly reduced extravasation Kojda, G.1 and abolished the difference between vehicle and C21. In striking 1Institut für Pharmakologie und klinische Pharmakologie, Universitätsklinikum der Heinrich- contrast, extravasation induced by the synthetic B2 agonist labradimil, Heine-Universität Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany which cannot be hydrolyzed by ACE, showed no dependence on pretreatment with C21 (P>0.05). In another approach we compared The inflammatory mediator bradykinin (BK) is involved in non-allergic tissue and supernatant ACE protein content by Western blot in lungs of angioedema. Prostaglandins presumably contribute to BK-induced C57BL/6J incubated in oxygenated buffer solution at 37°C for 30 min in edema. We used the Miles assay to quantify dermal extravasation of the the presence or absence of C21. C21 incubation caused a numerical albumin-bound dye Evans blue in mice’s dorsal skin. Anaesthetized mice decrease of ACE in tissue lysates to 0.81±0.05-fold, while ACE protein received the dye intravenously (i.v.). After shaving the dorsal skin, 30 µl in supernatants was significantly increased to 1.33±0.17-fold (n=6, of physiologic buffer solution (PBS), 2 nmoles of BK, the proteolytic stable P<0.05, Tukey's multiple comparisons test following One-Way ANOVA). BK-analogue labradimil (LD) and histamine (His) were injected The difference of ACE protein content between tissue and supernatant intradermally (i.d.). The resulting extravasates were excised after was significant as well (P<0.01). Likewise, lung ACE protein content as sacrificing those mice. Then the dye was extracted and quantified determined following in-vivo treatment of mice with C21 significantly spectrometrically. In C57BL/6, BK induced a dose-dependent increase of decreased to 0.5±0.1-fold (n=6, P<0.05) as compared to treatment with extravasation expressed as fold increase (±SEM) in comparison to PBS vehicle. A similar reduction of ACE protein to 0.42±0.1-fold (n=6, P<0.05) reaching at 2 nmoles 4.41±0.11 (n=6, “P < 0.0001”). Pretreatment of was observed after combined treatment with C21 and telmisartan, while

DPhG Annual Meeting 2019 Conference Book • 154 PHARMACOLOGY telmisartan alone showed no effect. These data suggest that activation specific drug etoricoxib using the same approach in another 9 healthy of AT2 reduces ACE activity and that this effect is likely caused by volunteers show a similar tendency (n=9). shedding of membrane bound ACE. Circulating ACE loses its second These data suggest that cyclooxygenase activity contributes to catalytic domain and provides less catalytic capacity. This process might bradykinin induced extravasation in humans. These findings and further contribute to non-allergic angioedema occurring in patients treated with identification of the COX subtypes and the corresponding prostaglandins sartans. involved as well as the corresponding receptors may rise new treatment options for non-allergic angioedema. In addition, our findings may provide a rationale for the routine use of glucocorticoids in the current standard of care for this condition [2].

1. Bisha M, Dao, VT; Gholamreza-Fahimi E, Vogt M, van Zandvoort M, Weber S, Bas M, Khosravani F, Kojda G, Suvorava T. The role of bradykinin receptor type 2 for spontaneous extravasation in mice skin: Implications for non-allergic angio-oedema. Br. J. Pharmacol. 2018, 175(10):1607-20. 2. Bas M, Greve J, Stelter K, Havel M, Strassen U, Rotter N, Veit J, Schossow B, Hapfelmeier A, Kehl V, Kojda G, Hoffmann TK. A randomized trial of icatibant in ACE-inhibitor-induced Fig. 1. Selective AT2 receptor agonist C21 angioedema. N Engl J Med 2015; 372:418-425.

1. Bisha M, Dao, VT; Gholamreza-Fahimi E, Vogt M, van Zandvoort M, Weber S, Bas M, Khosravani F, Kojda G, Suvorava T. The role of bradykinin receptor type 2 for spontaneous extravasation in mice skin: Implications for non-allergic angio-oedema. Br. J. Pharmacol. 2018; 175(10):1607-20. POS.162 2. Bas M, Greve J, Stelter K, Havel M, Strassen U, Rotter N, Veit J, Schossow B, Hapfelmeier Differences in meropenem pharmacokinetics between A, Kehl V, Kojda G, Hoffmann TK. A randomized trial of icatibant in ACE-inhibitor-induced obese and non-obese surgery patients are largely angioedema. N Engl J Med 2015;372:418-425. explained by body size and estimated creatinine clearance 3. Gholamreza-Fahimi E, Bisha M, Hansen FK, Kurz T, Kojda G. Inhibition of angiotensin- converting enzyme by selective stimulation of angiotensin II type 2 receptor. Naunyn- Schmiedeberg's Arch Pharmacol 2017;390(Suppl 1):S38, abstract (P64) David Busse1,2, Philipp Simon3, Lisa Ehmann1,2, David Petroff4, Christoph Dorn5, Wilhelm Huisinga6, Robin Michelet1, Hermann Wrigge3, Charlotte Kloft1 1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Germany, POS.161 2 and Graduate Research Training program PharMetrX, Germany, 3 Dept. of Anaesthesiology and Intensive Care Medicine and Integrated Research and A bradykinin in skin edema trial (ABRASE) – implications Treatment Center (IFB), Adiposity Diseases, University of Leipzig, Germany, for non-allergic angioedema 4 Clinical Trial Centre Leipzig, University of Leipzig, Germany, 5 Dept. of Clinical Pharmacy, University of Regensburg, Germany, 6 Institute of Mathematics, University of Potsdam, Germany Gholamreza-Fahimi, E.1; Bisha, M 1; Hahn, J.2; Straßen, U. 3; Krybus, M. 1; Khosravani, F.1; Hoffmann, T.2; Hohlfeld, T.1; Greve, J.2; Bas, M.3; Background and objectives: Meropenem (MER), a broad-spectrum β- Twarock, S.1; Kojda, G.1 lactam antibiotic, is used for the treatment of (multi-)resistant soft tissue 1Institute of Pharmacology and Clinical Pharmacology, Heinrich Heine University, Düsseldorf, Germany infections and in calculated empiric antimicrobial therapy. Although 2Department of Oto-Rhino-Laryngology, Head and Neck Surgery, Ulm University Medical obesity has been identified as a risk factor for surgical site infections [1], Center, Germany quantitative evaluations of pharmacokinetics (PK) of anti-infectives such 3Otorhinolaryngology Department, University Hospital Rechts der Isar, Munich Technical University, Munich, Germany as MER in obese patients are rare and often lack crucial information on exposure at the target-site, i.e. the interstitial space fluid (ISF) of subcutaneous (s.c.) adipose tissue. The aim of this analysis was (i) to Non-allergic angioedema induced by drugs such as ACE inhibitors, quantify MER tissue distribution and elimination by developing a sartans, sacubitril, or tissue plasminogen activators, develop as a nonlinear mixed-effects (NLME) PK model based on concentrations both consequence of increased bradykinin plasma concentration and the in plasma and ISF and (ii) to identify individual patient characteristics subsequent stimulation of bradykinin-receptor-type-II (B2), respectively. which explain the variability of PK parameter estimates. Experiments in transgenic mice with an endothelium-specific Methods: Data originating from a clinical study including 15 obese overexpression of B2 revealed the involvement of prostaglandins in (BMI=38.1-81.5 kg/m2) and 15 non-obese patients (BMI=20.5-27.1 bradykinin induced effects. Unspecific inhibition of cyclooxygenase by kg/m2) receiving uniform singe-dose 1000 mg MER (30-min i.v.) for diclofenac abolished the marked contractile response of aortic rings of infection prophylaxis prior to abdominal surgery performed at the Leipzig mice in organ bath studies and significantly diminished extravasation University Hospital (EudraCT 2012-004383-22) were considered in this induced by bradykinin in the skin of these mice and the control strain analysis. Rich PK sampling data were available over 8 h in plasma C57BL/6 [1]. (n=239) and via microdialysis in the ISF of s.c. adipose tissue in both To study whether cyclooxygenase activity is involved in bradykinin arms of each patient (n=585) [2]. Microdialysis catheter calibration was induced extravasation in humans, we conducted the multicentre study “A performed using retrodialysis. In addition, information about potentially bradykinin in skin edema trial” (ABRASE). Healthy volunteers of either influential covariates (body size, serum creatinine, liver enzyme sex received an intradermal injection of 20 µl (18.9 nmoles) bradykinin, concentrations, etc.) was collected. NONMEM 7.4.3, PsN 4.8.1, Pirana while 0.9% saline served as control injection. The size of the resulting 2.9.6 were employed in model development using an integrated plasma wheals at the injection sites were measured for 120 min at predefined and micro-/retrodialysis modelling approach [3] and R 3.4.4 was used for time points. After further 60 min, the unspecific cyclooxygenase inhibitor pre- and post-processing. Model adequacy was assessed by plausibility ibuprofen was orally administered as a single dose of 600 mg. Following and precision of parameter estimates, goodness-of-fit plots and visual another 60 minutes, which is the usual time to achieve maximal or near predictive checks. maximal plasma concentrations, injections were performed as described Results: A catenary three-compartment model with a chain of two above and the resulting wheal sizes were measured again over 120 min. peripheral compartments (ISF attributed to the first peripheral The primary endpoint of ABRASE was the mean time to complete compartment) adequately described MER PK in obese and non-obese resolution of wheals (TTCR) as compared to no treatment. Ibuprofen patients and yielded plausible and precise parameter estimates. To more treatment significantly lowered the TTCR (n=39, 82.6 min, 95% mechanistically describe elimination processes, MERclearance (CL) was confidence interval [CI], 74.3 to 90.9, P<0.0001, Wilcoxon matched-pairs divided into renally filtrated and non-renal CL. Based on the substantial signed rank test) as compared to no treatment (99.5 min, 95% CI, 92.2 renal excretion of unchanged MER, renal CL was determined by linear to 106.8). The secondary endpoint of the study was the change of the scaling using estimated creatinine clearance via the Cockcroft-Gault mean maximal wheal size induced by ibuprofen. Again, ibuprofen formula (CLCRCG) [4,5]. The remaining non-renal CL was scaled based treatment significantly reduced this endpoint from 468.9 mm2 (95% CI, on theory-based allometry using adjusted body weight [6]. Renal CL 378.8 to 559.0) to 385.5 mm2, (95% CI, 295.8 to 475.2, P=0,0044). (5.75 L/h) accounted for 54% of total CL (10.9 L/h). Theory-based Furthermore, preliminary data obtained with the more cyclooxygenase-2 allometric scaling for volume of distribution parameters of the central and

155 • DPhG Annual Meeting 2019 Conference Book POSTERS

peripheral compartment substantially reduced unexplained References: interindividual variability (27 and 31% relative reduction, respectively) 1. Cheng, X. et al. Indirubin derivatives modulate TGFβ/BMP signaling at different levels and and largely explained differences in individual PK parameter values trigger ubiquitin-mediated depletion of nonactivated R-Smads: Chem Biol. 2012, 19(11):1423- between the two patient populations. 36. 2. Matsuzaki, K. Smad phospho-isoforms direct context-dependent TGF-b signaling: Cytokine Conclusion: An NLME PK model was successfully developed to Growth Factor Rev. 2013, 24(4):385-99. describe concentration-time profiles of MER in the obese and non-obese 3. Fouassier, L. et al. Signaling networks in cholangiocarcinoma: Molecular pathogenesis, population. The model structure suggested further distribution of MER targeted therapies and drug resistance: Liver Int. 2019, 1:43-62. from ISF into a deeper compartment, e.g. the cellular space. Differences in MER PK parameter values between the two patient populations were largely explained by body size and CLCRCG. A covariate analysis using haemodynamic, hepatic and inflammation biomarkers will be performed POS.164 to further explain interindividual variability in PK parameters. Vacuolar (H+)-ATPase critically regulates specialized pro- resolving mediator pathways in human M2 macrophages [1] Winfield R et al.: Am Surg. 2016, 82(4): 331-336. and resolution of inflammation [2] Simon P et al.: Contemp Clin Trials Commun. 2019, 16. [3] Minichmayr I et al.: Clin Pharmacokinet. 2017, 56(6): 617-633. [4] Harrison M et al.: Xenobiotica. 1993, 23(11): 1311-1323. Rao, Z. 1; Gerstmeier, J. 1; Jordan, P.M.1; Pace, S. 1; Börner, F. 1; [5] Cockcoft DW et al.: Nephron. 1976, 16(1): 31-41. Troisi, F1; Bilancia, R. 1,2; Andreas, N. 3; Kamradt, T. 3; Menche, D. 4; [6] Kleiber M et al.: Physiol Rev. 1947, 27(4): 511-541. Rossi, A. 2; Serhan, C.N.5; Werz, O.1 1Institute of Pharmacy, Friedrich Schiller University Jena, Philosophenweg 14, D-7743 Jena, Germany; 2Department of Pharmacy, School of Medicine, University of Naples Federico II, Via Domenico Montesano 49, 80131-Naples, Italy; 3Institute of Immunology, Jena University Hospital, 07743 Jena, Germany; POS.163 4Kekulé-Institut für Organische Chemie und Biochemie der Rheinischen Friedrich-Wilhelms- Targeting TGFβ/BMP Signaling Through Modulating R- Universität Bonn, Bonn, Germany; 5Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, 02115, Smad Activity: Implications in Cholangiocarcinoma (CCA) United States of America

Dabiri, Y.1; Cheng, X.1; Dropmann, A.2, 3; Dooley, S. 2, 3; Wölfl, S.1 1 Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Vacuolar (H+)-ATPases (vATPases) are ATP-dependent proton pumps Feld 364, 69120, Heidelberg, Germany that are present within the membrane of various organelles in numerous 2 Department of Medicine II, Molecular Hepatology Section, Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3 (H42, Floor 4), 68167, Mannheim, Germany cells and are essential to maintain pH homeostasis. Although vATPase 3 Molecular Hepatology Section, Medical Faculty Mannheim, Heidelberg University, Theodor- is fundamental in cytokine trafficking and secretion in human monocytes Kutzer-Ufer 1-3 (H42, Floor 4), 68167, Mannheim, Germany [1] and was implicated in the M2 polarization of murine macrophages [2], its functional roles in lipid mediator (LM) biosynthesis remain elusive. Alternative (M2)-polarized macrophages possess high capacities to The canonical TGF/BMP signaling pathway involves the activation of produce specialized pro-resolving lipid mediators (SPM) that regulate key regulatory Smads (R-Smads)–Smad2/3 and Smad1/5/8–by means of functions in resolution of inflammation and tissue regeneration. Here, we receptor-mediated C-terminal phosphorylation. Activated R-Smads are exploited the role of vATPase using the well-established vATPase then subjected to further modification at several serine/threonine inhibitor Archazolide (ArchA) in LM biosynthetic pathways in human M2 residues in the linker region in response to several kinases including macrophages. Monocyte-derived human macrophages were pretreated cyclin-dependent kinases (CDKs), glycogen synthase kinase3 with ArchA (30 nM, 15 min), polarized towards M1 and M2 for another 48 (GSK3), and mitogen-activated protein kinases (MAPKs), creating hrs, and stimulated with pathogenic E. coli for 180 min to produce LM different phosphorylated isoforms with both anti- and pro-tumorigenic profiles that were analyzed by UPLC-MS/MS [3]. Blockade of vATPase functions. The differentially phosphorylated forms determine the outcome during human M2 polarization abrogated 15-lipoxygenase-1 (15-LOX-1) of the signaling as well as the stability of R-Smads, as they are marked expression and prevented the related biosynthesis of SPM. Targeting for ubiquitination, which poses the molecules for proteasomal vATPase neither influenced the IL-4-triggered JAK-STAT6 pathway that degradation. is known to regulate 15-LOX-1 expression [4], nor the mTORC1 signaling Although the ubiquitin-proteasome pathway (UPP) plays an important cascade that promotes the M2 phenotype [2], but strongly suppressed role in terminating TGF/BMP signaling through degradation of activated MEK and ERK phosphorylation in M2, indicating an essential role of the R-Smads, its role in the regulation of non-activated R-Smad levels is less MEK-ERK cascade in the regulation of 15-LOX-1 and SPM formation in known. Here, we present an indirubin derivative, designated as E738–a M2. Targeting vATPase in vivo delayed resolution of zymosan-induced known kinase inhibitor–as a modulator of TGF/BMP signaling. E738 murine peritonitis accompanied by decreased SPM biosynthesis without showed a significant depletion of total R-Smad pools in cell lines from affecting pro-inflammatory leukotrienes or prostaglandins during the both cancerous and non-cancerous tissue origin, involving components resolution phase after 24 h. Together, our data propose that vATPase is of the UPP (e.g., USP34). Further investigation suggested that required for ERK-1/2-mediated 15-lipoxygenase-1 expression and modulation of specific linker phosphorylation sites by E738 may be an consequent SPM biosynthesis during M2 polarization, implying a crucial important player in the small molecule-mediated R-Smad degradation. role for vATPase in the resolution of inflammation. Importantly, E738's inhibitory activity on certain R-Smad phospho- isoforms appeared to be cell type-specific and was found to antagonize the oncogenic R-Smad signaling in a panel of patient-derived 1. Thomas, L. et al.: Biochem Pharmacol 2017,130, 71-82 cholangiocarcinoma (CCA) cells. This could lead to new therapeutic 2. Kimura, T. et al.: Nature communications 2016, 7, 13130. strategies, exploiting the requirement of R-Smads in cellular survival and 3. Werz, O. et al.: Nature communications 2018, 9(1), 59. differentiation. 4. Heydeck, D. et al.: Blood 1998, 92(7), 2503-2510.

POS.165 The microtubule-targeting agent pretubulysin impairs inflammatory key features in endothelial cells in vitro and in vivo

Acknowledgments: 1. Landesgraduiertenförderung (LGF) fellowship program for individual doctoral training from Primke T.F.1; Schwenk R.1, Schäfer R.1 Fabritius M.P.2, Reichel C. A.2, Universität Heidelberg, Dabiri, Y. Ullrich A.3, Kazmaier U.3 and Fürst R.11 2. Bundesministerium für Bildung und Forschung (BMBF) grant programs; Drug-iPS (FKZ 1Institute of Pharmaceutical Biology, Biocenter, Goethe University, 60438 Frankfurt, Germany 0315398A/B) and SysToxChip (FKZ 031A303A/E), Wölfl, S.; Cheng, X. 2Department of Otorhinolaryngology, Head and Neck Surgery, Walter Brendel Centre of Experimental Medicine, Clinical Centre at the University of Munich, 81377 Munich, Germany

3Institute of Organic Chemistry, Saarland University, 66123 Saarbrücken, Germany

DPhG Annual Meeting 2019 Conference Book • 156 PHARMACOLOGY

The process of inflammation is a physiological mechanism inevitable for providing protection against noxious stimuli. An inflammatory response is usually comprised of the recruitment of leukocytes to the site of inflammation and the elimination of the noxious stimuli, which is followed by resolution of the inflammatory state and repair of damaged tissue. However, if failed to be resolved, the mechanisms underlying inflammation can contribute to a broad range of pathological conditions, like chronic inflammatory diseases, which are characterized by constant infiltration of leukocytes, ultimately leading to tissue damage and even cancer. Microtubule-targeting agents (MTAs), which are primarily known for their use as chemotherapeutic drugs, have also been shown to have anti-inflammatory properties, with colchicine being one of the lead compounds. In this context, the aim of this study is to investigate the impact of the novel MTA pretubulysin (PT) and already established MTAs on leukocyte-endothelial cell interactions. The endothelium plays a pivotal role in the migration of leukocytes to the site of inflammation and has long been neglected in anti-inflammatory drug research. By using intravital microscopy of postcapillary venules of the cremaster muscle in mice, we found that the treatment with PT decreases the TNF- induced firm adhesion of leukocytes onto and their transmigration through the vascular endothelium in vivo. In addition, in vitro cell adhesion assays showed that the adhesion of monocytic cells (THP-1) onto TNF-activated endothelial cells is reduced when endothelial cells were pre-treated with the microtubule-destabilizing drugs PT, vincristine (VIN) or colchicine (COL). In contrast, the TNF-induced adhesion of THP- 1 cells onto an endothelial cell monolayer was not impaired when endothelial cells were pre-treated with the microtubule-stabilizing agent paclitaxel (PAC). Based on this data, the influence of PT and other MTAs on the surface expression of the cell adhesion molecules (CAMs) ICAM- 1, VCAM-1 and E-selectin (on endothelial cells) was tested by flow cytometry. While the TNF-induced surface expression of ICAM-1 and VCAM-1 is decreased by the pre-treatment of endothelial cells with PT, VIN and COL, the surface expression of ICAM-1 was not and that of VCAM-1 only slightly affected when TNFα-activated endothelial cells were pre-treated with PAC. In addition, pre-treatment with PT had no effect on the surface expression of E-selectin. It cannot be excluded that a reduction of the surface expression is not caused by effects of the used MTAs on the microtubule-mediated transport of the examined CAMs. However, western blot and RT-qPCR analysis showed that total protein content as well as mRNA levels for ICAM-1 and VCAM-1 were significantly down-regulated after treatment with PT, VIN and COL in TNF-activated endothelial cells. Surprisingly, the total protein as well as mRNA levels of E-selectin were also down-regulated upon treatment with these compounds. Based on these findings, the influence of PT, Vin, Col and Pac on the translocation of NFκB into the nucleus of TNF-activated endothelial cells was examined microscopically (immunocytochemistry) but was shown to be unimpaired by any of the tested compounds. In consequence, the influence of PT and other MTAs on the NFκB and AP- 1 promoter activity was determined by a dual luciferase reporter assay. It could be shown that the pre-treatment of TNF-activated endothelial cells with PT, VIN and COL causes only a slight reduction of the promoter activity for both transcription factors, whereas this process was not affected when endothelial cells were treated with PAC. Taken together, this study shows that the microtubule-destabilizing agents PT, VIN and COL interfere with leukocyte-endothelial cell interactions. At least in part, this influence is caused by an effect of these MTAs on the NFκB or AP-1 signaling cascades in endothelial cells. However, since these results are insufficient to explain the striking effects of these compounds on the expression of CAMs involved in leukocyte- endothelial cell interactions, further investigations into potential effects downstream of any transcription factor-induced gene regulation are required.

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Methods: CaP-NP were fabricated and loaded with siRNA using 4.10 Poster Short Talks nanoprecipitation. Stabilisation of CaP-NP was analysed for different concentrations of terpolymer (2 µM, 5 µM, 10 µM) and siRNA (1 µg, 10 µg). For determination of particle sizes, we compared Nanoparticle tracking analysis (NTA) and Laser diffraction analysis (Mastersizer 3000: Hydro SV unit with a volume of 7 ml). In vitro transfection efficiency of PST.01 stabilized siRNA-loaded CaP-NP was investigated for the rat glioblastoma cell line F98 with the WST-8 cell viability assay. Intracochlear PLGA based implants for dexamethasone Results & Discussion: Particle size determination by NTA revealed the release: Challenges and solutions presence of NP whereas a microscopic analysis still showed the presence of aggregates. In comparison to NTA, Laser diffraction analysis enables particle size analysis in a wide size range from nano- to 1 2 3 3 1 Lehner E , Gündel D , Liebau A , Plontke S , Mäder K micrometer allowing for simultaneous illustration of NP and aggregates 1Institute of Pharmacy, Martin Luther University Halle-Wittenberg, D-06120 Halle (Saale), Germany) during measurement. Furthermore, Laser diffraction analysis enables for 2 Department of Nuclear Medicine, Martin Luther University Halle-Wittenberg, D-06120 Halle a time-dependent measurement. The small volume dispersion chamber (Saale), Germany allowed for affordable determination of particles size of siRNA loaded 3 Department of Otorhinolaryngology-Head and Neck Surgery, Martin Luther University Halle- Wittenberg, D-06120 Halle (Saale), Germany particles. Based on these findings, we chosed Laser diffraction analysis as the method of choice for further particle size determinations. We analyzed the ability of the terpolymer for CaP-NP stabilization using The effective treatment of diseases of the inner ear is still an unmet concentrations of 2 µM, 5 µM and 10 µM. For polymer concentrations of medical need [1]. Local controlled drug delivery to the cochlea is 2 µM and 5 µM we observed the formation of aggregates, whereas 10 challenging due to the hidden location, small volume and high sensitivity µM terpolymer were shown to successfully stabilize the precipitated NP of this organ. A local intracochlear delivery of drugs would avoid the at a size <100 nm. We further analysed the behavior of stabilized CaP- problems of intratympanic (extracochlear) drug application, but is more NP diluted in cell culture medium and in the presence of serum. Here, invasive. The requirements for such a delivery system include a small CaP-NP prepared with 10 µM terpolymer remained stable (particle sizes size and appropriate flexibility. The delivery device must be rigid enough <100 nm) for at least 30 min. This stabilization remained if CaP-NP were for surgical handling but also flexible to avoid traumatizing cochlear loaded with 1 µg siRNA. Furthermore, stabilized CaP-NP showed structures. We developed biodegradable dexamethasone loaded PLGA satisfying in vitro transfection efficiencies independent of terpolymer extrudates for the controlled intracochlear release. concentration as well as low cytotoxicities. In order to achieve the desired flexibility, Polyethylene glycol (PEG) was Conclusion: Here, we present the successful use of polymer-stabilized used as a plasticizer. In addition to the drug release, the extrudates were siRNA loaded CaP-NP (<100 nm) for in vitro transfection of brain tumour characterized in vitro by differential scanning calorimetry (DSC) and cells. Using a small volume dispersion unit, Laser diffraction analysis texture analysis. Simulation of the pharmacokinetics of the inner ear allowed to simultaneously observe NP and aggregates formation in support the expectation that a constant perilymph drug level is obtained different media. after few hours and retained over several weeks. Ex vivo implantation of the extrudates into a guinea pig cochlea indicate that PEG containing We like to thank the European Regional Development Found Saxony (EFRE) and the SAB extrudates have the desired balance between mechanical strength and (Sächsische Aufbaubank (Saxony, Germany)) for funding this project. We further like to thank flexibility for direct implantation into the cochlea. The location of the Prof. Dr. Achim Aigner & Dr. Alexander Ewe, Rudolf -Boehm-Institute of Pharmacology and implant was visualized by computer tomography. Toxicology, University Leipzig for their support with the NTA. In summary, intracochlear administration of drug releasing biodegradable References: implants is a new and promising approach to achieve local drug delivery 1. Mehta, A.M. et al.: Neurotherapeutics 2017, 18: 358-371. to the cochlea for an extended time [2]. 2. Young, J.S. et al.: World Neurosurg. 2018, 117: e698-e704. 3. Saucier-Sawyer, J.K.: J Control Release 2016: 232: 103-112.

References 1. Mäder, K. et al, Hearing Research 2018, 368: 49–66. 2. Lehner, E et al, Int. J. Pharm. X; 2019, https://doi.org/10.1016/j.ijpx.2019.100015 PST.03

Optimization of thermogelling emulsions with enhanced PST.02 substantivity for use in treatment of chronic skin diseases

Stabilization of Calcium Phosphate nanoparticles (CaP-NP) Markus Schmidberger, Dominique Jasmin Lunter as carrier system for siRNA 1Department of Pharmaceutical Technology, Eberhard Karls University, Auf der Morgenstelle 8, 72076 Tuebingen, Germany

Mitrach, F.1; Schmid, M.1; Dukic-Stefanovic, S.2, Brust, P.2, Hacker, M.C.1; Schulz-Siegmund, M.1 Topical formulations are an important pillar in the therapy of skin 1Pharmaceutical Technology, Institute of Pharmacy, University Leipzig, Eilenburger Straße 15A, 04317 Leipzig, Germany diseases. Nevertheless, after application the formulation will be exposed 2Department of Neuroradiopharmaceuticals, Helmholtz-Zentrum Dresden-Rossendorf, to environmental effects. Contact with other surfaces will reduce the Permoserstraße 15, 04318 Leipzig, Germany available amount of formulation and drug substance. The consequences for therapy range from reduced effects up to therapeutic failure. The Introduction: Convection-enhanced delivery (CED) represents a removed active ingredient also contaminates patients’ environment. promising technique to deliver therapeutics through the narrow interstitial The aim of this work was to develop preparations which remain at the spaces of the brain via a mild pressure gradient [1]. Application of NP by application site. These will enhance safety and efficiency und thus this technique allows for high local drug concentrations as well as a improve therapy of skin diseases. Therefore, we developed polymer homogenous distribution in brain tumours [2]. One prerequisite for a stabilised emulsions that show thermogelling properties. Emulsions with successful CED application are particle sizes <100 nm [3]. different methyl cellulose concentrations and macrogol of different Aim & Objectives: In this study we investigated the ability of a newly molecular weight were investigated. The dispersed phase consisted of synthesized terpolymer to stabilize siRNA loaded CaP-NP intended for nonivamide as active pharmaceutical ingredient dissolved in medium- transfection of brain cancer cells. Consequently, time dependent chain triglycerides. Rheological properties, droplet size, substantivity [1, changes in particle size distributions as well as transfection ability of the 2] and ex vivo penetration experiments on porcine skin were performed NP were investigated. The terpolymer consisted of a lipophilic anchor, to characterize the developed formulations. polyethylene glycol (PEG) and maleic anhydride that had been Droplet size and rheological parameters were affected by the hydrolyzed in order to obtain an amphiphilic anionic oligomer that composition of the preparations. The tested formulations showed interacts with Ca(II) ions and CaP. benefits in their substantivity compared to a conventional semi solid cream. We found a residual amount of up to 100% at application site. As shown in figure 1 the drug levels in viable epidermis were comparable to

DPhG Annual Meeting 2019 Conference Book • 158 POSTER SHORT TALKS those obtained with a conventional formulation and were found to be in a enhanced binding of the CPP-liposomes in contrast to standard therapeutic range. liposomes was obtained. Furthermore, a high increase in the transport of The developed emulsions are a promising vehicle to improve therapy of vancomycin over rat mucosal tissue in Ussing chamber studies in chronic skin diseases. contrast to the free peptide was shown. Oral application in a rat model revealed that the novel liposomal formulation led to a fivefold increase in the bioavailability of vancomycin. The functional efficiency of this uptake enhancement was demonstrated by a strong increase of the antibiotic efficiency in a (MRSA) systemic infection model.

Conclusion: The promising results clearly raise the hope that this novel liposomal formulation can be used as platform-technology for oral application of a variety of peptide drugs. Bioavailability studies in dogs are ongoing.

Figure 1: Penetrated amount of nonivamide after 4 h from conventional formulation (HNC 0.05%) and a developed thermogelling emulsion containing 0.3% nonivamide mean ± SD, n = 3 References: [1] Hertlein, T., et al. "Bioluminescence and 19F magnetic resonance imaging visualize the efficacy of lysostaphin alone and in combination with oxacillin against Staphylococcus aureus in murine thigh and catheter-associated infection models." Antimicrobial agents and chemotherapy The authors would like to thank PD Dr. Martin Schenk from the Department of Experimental 58.3 (2014): 1630-1638. Medicine at the University Hospital of Tübingen for providing pig ears and Shin-Etsu Chemical [2] Uhl, P., et al. "A liposomal formulation for the oral application of the investigational hepatitis Co. for the generous donation of methyl cellulose. Also thanks to Symrise AG for kindly B drug Myrcludex B." European Journal of Pharmaceutics and Biopharmaceutics 103 (2016): providing avobenzone. Dominique Lunter is supported by the European Social Fund and the 159-166. Ministry of Science, Research and Arts Baden-Württemberg. [3] Uhl, P., et al. "Oral delivery of vancomycin by tetraether lipid liposomes." European Journal of Pharmaceutical Sciences 108 (2017): 111-118.

[1] Herrmann S, Daniels R, Lunter DJ. 2016. Methods for the determination of the substantivity of topical formulations. Pharmaceutical Development and Technology.1-5. [2] Schmidberger M., Daniels R, and Lunter DJ, Method to determine the impact of substantivity PST.05 on skin permeation. EJPB, 2018 Identification and quantification of microdialysis variability using a dynamic in vitro microdialysis system and PST.04 nonlinear mixed-effects modelling

Cell penetrating liposomes enable the oral delivery of Ilia, L.1*; Michelet, R.1*; Busse, D.1,2; Ehmann, L.1,2; Kloft, C.1 peptide drugs 1 Freie Universitaet Berlin, Institute of Pharmacy, Dept. of Clinical Pharmacy & Biochemistry, Kelchstr. 31, 12169 Berlin, Germany 2 Graduate Research Training Program PharMetrX, Germany Philipp Uhl1, Max Sauter1, Tobias Hertlein2, Dominik Witzigmann3, Knut * Contributed equally Ohlsen2, Gert Fricker4 and Walter Mier1 1Department of Nuclear Medicine, Heidelberg University Hospital, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany 2Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Straße 2/D15, 97080 Würzburg, Germany 3Nanomedicines Research Group, Life Sciences Institute, University of British Columbia, Objectives: In the guidelines on the use of pharmacokinetics (PK) and Vancouver BC, V6T 1Z3, Canada pharmacodynamics (PD) in the development of antiinfectives, the 4Ruprecht-Karls-University, Institute of Pharmacy and Molecular Biotechnology, Im European Medicines Agency (EMA) Committee for Medicinal Products Neuenheimer Feld 329, 69120 Heidelberg, Germany for Human Use (CHMP) emphasises the importance of the determination of unbound drug concentrations at target sites [1] such as interstitial Purpose: space fluid (ISF) to foster predictions for probability of PK/PD target Oral delivery of peptide drugs is limited due to their instability in the attainment. This knowledge can be gathered using the minimally invasive gastrointestinal tract and low mucosa penetration. A novel approach to microdialysis (µD) sampling technique [2], which is based on passive facilitate oral bioavailability of peptide drugs such as vancomycin is the diffusion of molecules from ISF across a semipermeable membrane at use of liposomes containing tetraether lipids (TELs) and cell penetrating the tip of a µD catheter. peptides (CPPs). Unbound drug in the ISF can be determined by measuring the drug Methods: concentration in the collected dialysate and retrodialysis needs to be Tetraether lipid isolation of S. acidocaldarius was performed by Soxhlet performed to calibrate the catheter to account for the relative recovery, extraction. The cyclic cell penetrating peptide R9C was synthesized by which is often less than 100%. However, in a clinical study [3] high solid-phase synthesis and coupled to maleimide-functionalized variability of in vivo retrodialysis was observed, which prompts in vitro phospholipids. Purification of the novel conjugates was achieved by investigation to identify and quantify its sources. Previous in vitro preparative HPLC and confirmed by HPLC/MS analysis. Preparation of investigations in Ringer’s solution showed that the method of the µD the liposomal formulations was performed by dual asymmetric sampling technique as such can be excluded as a relevant source for the centrifugation (DAC). For in vivo studies, the oral uptake of the peptide observed in vivo variabilities [4]. In this work, the antibiotic linezolid (LIN) antibiotic vancomycin was determined by radiolabelling. Therapeutic was studied in an in vitro dynamic µD system (dIVMS) [5] in different trials in a (MRSA) systemic infection model were performed according to types of artificial ISF with a focus on relative recovery and their impact on Hertlein et al [1]. µD concentrations, compared to in vivo µD. The objective was to Results: investigate the impact of ISF composition on µD variability. All liposomes were characterized by Zetasizer measurements. The increase in the zeta potential indicated the successful incorporation of the Methods: An in vivo ISF LIN concentration-time profile (C(t) profile) CPP-phospholipid-conjugates into the liposomes. TEM and cryo-TEM obtained from the typical patient profile of a clinical µD study [3, 6] was micrographs confirmed the appropriate liposomal size and morphology. mimicked in the dIVMS. Samples were taken from the flask, which The preparation of the liposomes by DAC enabled high encapsulation represents the ISF and collected from three different µD catheters (CMA efficiencies (up to 60%) as verified by HPLC and FCS measurements [1, 60, 20kDa cut-off) simultaneously. Retrodialysis was consecutively 2]. Microscopy studies using Caco-2 cells showed that a strongly performed as a calibration method and this was repeated on four different

159 • DPhG Annual Meeting 2019 Conference Book POSTERS occasions using the same catheters. Nonlinear mixed-effects (NLME) subject, i.e. pharmacokinetics, within a clinical setting. After completing modelling was performed using NONMEM® (7.4.3) to characterise the the e-learning case, students can additionally attend a tutorial with the LIN C(t) profile in the dIVMS and to quantify variability between catheters same subject, led by a medical student, in order to discuss particular and occasions. An integrated ISF and micro-/retrodialysis modelling problems even further. Any unresolved questions from the tutorials are approach was chosen to evaluate data from all three available sources fed back to the main lecturer, who can discuss them within the lecture if (ISF, micro- and retrodialysis) simultaneously [7]. Afterwards, variabilities needed. The whole course is organised via the learning platform associated with relative recovery were compared to variability from in Moodle®. vivo clinical data. Model adequacy was assessed by goodness-of-fit Preliminary Results plots, parameter precision and visual predictive checks. We have started the third term of P3, attendance is increasing over time, for the e-learning cases as well as the tutorials. To date we trained three Results: The LIN C(t) profile in the dIVMS was successfully described instructors (all medical students), designed 14 e-learning cases which using a one compartmental model with linear clearance describing LIN were retrieved 300 times; and held 18 tutorials (some themes were elimination from the flask and three µD measurement compartments. offered twice due to high demand) with 144 participants altogether. The predictive performance of the PK model was adequate and typical Evaluation shows that students appreciate the peer-to-peer situation and parameter estimates (95% CI) were in line with the experimental settings. relaxed atmosphere during the tutorials as well as the overall structure of The model-predicted and experimentally derived relative recovery were the e-learning cases with different questions types and detailed comparable. Recovery variability was split up in intra- and intercatheter explanations. Students also choose particular subjects rather than variability which were lower in vitro compared to 27.2%CV (21.8 – attempting all subjects. 32.0%CV) and 26.1%CV (16.7 – 33.8%CV) in vivo. Discussion Evaluation of participants shows that students appreciate to have Conclusion: The established dIVMS and model-based analysis provide protected opportunities to apply theoretical knowledge in a more clinical quantitative and qualitative insights into the µD sampling technique. and practice-based setting. Participation rates in particular for the e- Changing the target site fluid from Ringer’s solution to artificial ISF learning cases however are extremely low compared to the number of increased intra- and intercatheter variability which were still lower than in students enrolled in the pharmacology lecture. Further qualitative vivo. This suggests that the current in vivo assessment of sources of µD evaluation will be needed to explore reasons for non-participating in e- variability might be confounded by further factors such as variability in the learning as well as the tutorials. We hope to provide P3 in an sampled target site or location of the µD catheter. An integrated interprofessional context in near future by inviting medical students to approach considering additional in vitro and in vivo data might identify take part in both, e-learning and tutorials. these confounding factors and thus aid in further elucidating the high variability in clinical µD studies. We like to thank Lehre@LMU for the financial support of the project.

References 1. EMA/CHMP (594085/2015): Use CfMPfH. 2016. 1. Bertsche T, Schwalbe O, Högger P. Klinische Pharmazie: Eine Bestandsaufnahme an 2. Plock, Buerger, Kloft: Biomed. Chromatogr. 2005, 19: 237–244. deutschen Universitäten. Pharmazeutische Zeitung Online. 2009. Online erhältlich: 3. Simon et al.: Anästh. Intensivmed. 2018, 59: 399. https://www.pharmazeutische-zeitung.de/ausgabe-482009/eine-bestandsaufnahme-an- 4. Ilia, Michelet, Kloft: 29th ECCMID, Amsterdam, Netherlands, 2019. deutschen-universitaeten/, zuletzt aufgerufen: 27.05.2019. 5. Simmel et al.: Int. J. Clin.Pharmacol. Ther. 2010, 48: 695–704. 2. Bundesapothekerkammer. Kompetenzorientierter Lernzielkatalog Pharmazie - 6. Ehmann: 26th PAGE, Budapest, Hungary, 2017. Perspektivpapier "Apotheke 2030". Berlin: Bundesapothekerkammer; 2017:52. 7. Minichmayr et al.: Clin. Pharmacokinet. 2017, 56: 617-633. 3. Erpenbeck J, Sauter S, Sauter W. E-Learning und Blended Learning. Wiesbaden: Springer Fachmedien, 2015 4. Kononowicz AA, et al. Virtual patients--what are we talking about? A framework to classify the meanings of the term in healthcare education. BMC Med Educ. 2015;15:11. PST.06

Pharmacology meets (clinical) pharmacy – virtual patients PST.07 for pharmacy students Addressing targets relevant in neurodegenerative disorders: synthesis and biological activity of Pudritz YM1,2, Fischer MR3, Wahl-Schott C2,4, Biel M2 1Apotheke des Klinikums der Ludwig-Maximilian-Universität München, Campus Innenstadt, multifunctional heterobivalent carboline derivatives Pettenkoferstr. 8a, 80336 Munich, Germany 2 Department Pharmazie – Zentrum für Pharmaforschung, LMU Munich, Butenandtstr. 7-15, 81375 Munich, Germany Schwarthoff, S.1; Sager, H.1; Schätz, B.1, Robaa, D.2; Arndt, H.-D.3; 3Institut für Didaktik und Ausbildungsforschung in der Medizin, LMU Munich, Pettenkoferstr. 8a, Gaube, F.1; Winckler, T.1 80336 Munich, Germany 1 Pharmaceutical Biology, Institute of Pharmacy, Friedrich-Schiller University of Jena, 4Institut für Neurologie, Medizinische Hochschule Hannover (MHH), Carl-Neuberg-Str.1, 30625 Semmelweisstrasse 10, 07743 Jena, Germany Hanover, Germany 2 Pharmaceutical Chemistry and Clinical Pharmacy, Institute of Pharmacy, Martin Luther University of Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle (Saale), Germany 3 Organic Chemistry I, Institute for Organic Chemistry and Macromolecular Chemistry, Friedrich- Background Schiller University of Jena, Humboldtstrasse 10, 07743 Jena, Germany Clinical pharmacy is the ‘youngest’ of the five main subjects taught in the German pharmacy curriculum being introduced in the year 20011. It is the In an ageing society, therapy of neurodegenerative disorders such as only clinical subject amongst subjects related to natural sciences like Alzheimer`s disease or Parkinson`s disease is becoming increasingly chemistry. One particular challenge of clinical sciences is that they need important. Due to the complex nature of these diseases, therapeutics that to be taught in a practical and clinical context, i.e. with patients present. combine several pharmacophores into one molecule and act as In order to close the gap between theory -the lecture-, and practice -the multitarget drugs may improve the effectiveness of therapy. Recently, it patient, a blended learning project was introduced to the pharmacology was reported that compounds consisting in a tetrahydro-gamma- curriculum in 2018. carboline (2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole) scaffold linked to Aim phenothiazine block N-methyl-D-aspartate receptors (NMDAR) and also Closing the gap2 between theory and clinical practice in pharmacology butyrylcholinesterase (BChE), but are inactive on acetylcholinesterase and clinical pharmacy through the introduction of a blended learning (AChE) [1]. In our own work, we previously found that homobivalent concept3 using e-learning cases4 and tutorials in addition to the traditional carbolinium derivatives consisting of two linked gamma-carboline (5H- lecture. pyrido[4,3-b]indole) scaffolds are potent inhibitors of NMDAR in cell- Methods based assays with IC50 values similar to the marketed drug memantine, The blended learning concepts consist of the lecture in pharmacology, yet they similarly inhibit both cholinesterases in the nanomolar range [2]. which is being held over the course of four semesters, e-learning cases, In the present study we produced novel heterobivalent compounds based and tutorials. With each introduction of a new subject in the on a lead structure presented by Makhaeva et al. [1] and performed pharmacology lecture, a correspondent e-learning case is accessible for structure-activity relationship to evaluate whether the unique combination students, providing an opportunity to review/rehash the pharmacological

DPhG Annual Meeting 2019 Conference Book • 160 POSTER SHORT TALKS of a carbolinium scaffold with phenothiazine produces improved NMDAR- inhibitory activity while retaining strong selectivity for inhibition of BChE. We found that, in contrast to homobivalent compounds, N-methylation of Background: Lately, the crosstalk between tumour cells and blood the pyridine to obtain permanently charged carbolinium moieties was platelets has been the objective of multiple investigations in oncological dispensable in heterobivalent carboline-phenothiazine compounds to act research. It was shown that direct contact with certain types of tumour as NMDAR blockers. The new compounds retained their high selectivity cells activates platelets leading to a release of soluble factors, which in turn can bind to surface receptors on the cancer cell. While most of the as cholinesterase inhibitors, as they blocked BChE with IC50 in the low nanomolar range while they were inactive at AChE. Selected compounds work focused on the metastatic behaviour of these circulating cancer were furthermore characterized in vitro with regard to their physico- cells, the impact of platelets on the mechanisms of chemoresistance chemical properties and potential mutagenic potentials. remains unclear. An increased resistance to chemotherapeutic drugs is often associated with the upregulation of ATP-binding cassette transporters (ABC-transporters) and the concomitant drug efflux. ABCB1, ABCC1 and ABCG2 are particularly well studied because of their responsibility for multi-drug-resistance in cancer therapy. Nevertheless, a functional axis of platelet activation and transporter upregulation of tumour cells has not been described before. Aim: The project aims on elucidating the influence of platelets on the chemoresistance of different tumor cells in vitro and the underlying molecular mechanisms, e.g. a probable involvement of ABC- transporters. The elucidation of a platelet / tumour cell resistance activation axis should provide novel targets for a pharmacological sensitization strategy. Methods: AsPC1 human pancreatic cancer cells and MDA-MB231 human breast cancer cells were co-incubated with human platelets. The Lead structures survival in presence of the chemotherapeutic drugs doxorubicin and mitoxantrone was detected by MTT assays, ABC-transporter activities were followed by the conduction of fluorescence assays using transporter References: substrates and inhibitors. The expression of ABCB1, ABCC1 and ABCG2 1. Makhaeva, G. F. et al: Sci. Rep. 2015, 5: 13164. 2. Otto, R. et al.: J. Med. Chem. 2015, 58(16): 6710–6715. was visualized via Western blot. In order to verify the role of EGFR / MAPK signalling for cell activation and resistance, the cancer cells were co-incubated with inhibitors of key molecules of the pathway in addition to the treatment with platelets. PST.08 Results: MDA-MB231 and AsPC1 tumour cells displayed a higher resistance against doxorubicin and mitoxantrone induced cytotoxicity “Gender Medicine” in the 18th century?! Pharmacotherapy when cells have been treated with platelets. The chemoresistance could in the hessian hospital Merxhausen. be associated with the involvement of the transporters ABCB1 and ABCG2, confirmed by higher activity of these transporters in the fluorescence transporter assays. Western blot data confirm the Borchers, M.-K.1, Friedrich, C.2 connection between platelets and transporters by showing increased 1Institut für Geschichte der Pharmazie der Philipps-Universität Marburg. Roter Graben 10. expression of aforementioned transporter proteins. When treated with an 35032 Marburg. Germany 2Institut für Geschichte der Pharmazie der Philipps-Universität Marburg. Roter Graben 10. inhibitor of CREB – a transcription factor and important signal transductor 35032 Marburg. Germany in both EGF- and PI3K-signaling – the effects of the platelet / tumour cell interaction is partly reversed referring to the critical role of the EGF- pathway in these terms. Since the founding of the „Hohen Hospitäler“ in the middle of the 16th Conclusion: It can be assumed that platelets influence circulating century, a few charity institutions for the rural population in Hesse existed. cancer cells, besides the known effects on attenuating the attacks of the The hospital in Merxhausen was a sanctuary for old, infirm or disabled immune cells, also with respect to induce a higher resistance against a women as well as for those with mental-health diseases. Once a week, chemotherapeutic treatment, which is closely related to the clinical a surgeon visited the hospital to take care of the women in cases of acute prognosis. Therefore, the interference with platelet / tumour cell sickness or injury. Basically, the surgeon prepared the medicine himself communication and especially the EGF-pathway appears as a promising and was requested to issue a calculation of the expended drugs at the therapeutic perspective for sensitization strategies. end of the year.

We analyzed these medical calculations from the 18th century as an origin for practical pharmacotherapy of the past. Therefore, these hand-written manuscripts including various alchemical signs had to be transcribed; we identified the drugs, arranged them accordingly to the dosage form and PST.10 described the commonness. We exemplary present the analyze based on the year 1755. The surgeon Glycoloytic Flux and p53 status influence Growth Inhibition treated 523 patients during that year and included 660 drugs in his calculation. It’s promising to compare the outcome with the analyze for in response to the G6PD Inhibitor DHEA in Colorectal another hessian hospital in Haina, which only treated men.1 In fact, there Cancer Cells are differences in the most commonly used dosage forms and in the medical range. In summary we considered some indications for gender 1 1 1 specific pharmacotherapy in the past. Fadi Almouhanna , Biljana Blagojevic , and Stefan Wölfl 1Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364 Heidelberg, Germany 69120 1. Mendel, A. M.: Zur Alltagsgeschichte der Arzneimitteltherapie im 18. Jahrhundert. Die Arzneimitteltherapie im Hohen Hospital Haina zwischen 1732 und 1800. Stuttgart 2013. (Quellen und Studien zur Geschichte der Pharmazie; 100). Pyruvate kinase and glucose-6-phosphate dehydrogenase contribute to cancer aberrant metabolism. The low pyruvate kinase activity of PKM2 leads to accumulation of upstream glycolytic metabolites to be used in PST.09 different anabolic pathways supporting the cancerous elevated proliferation rate. In this study, we investigated the effects of DHEA, a Platelets induce a chemoresistance of tumor cells by G6PD inhibitor, on colon cancer (CRC) cells with different p53 activity upregulating drug efflux transporters (p53 status). DHEA triggered increased G6PD expression in a ROS dependent mechanism. DHEA also reduced PKM2 activity in the presence and absence of wild-type p53 causing a “Warburg-like” Sebastian Heuter, Katharina Schmutzler, Alessandra Pierantoni, Martin phenotype that restored glucose dependency and enhanced lactate Schlesinger, Gerd Bendas production. PKM2 activation synergizes with DHEA treatment further University of Bonn, Pharmaceutical Institute, An der Immenburg 4, 53121 Bonn

161 • DPhG Annual Meeting 2019 Conference Book POSTERS increasing ROS levels and impairing proliferation in CRC cells. DHEA Comprehensive understanding of the complex interplay between treatment showed efficacy in targeting colorectal cancer cells regardless antibiotic, patient and pathogen and applying this by optimising dosing is of their p53 status and enhanced the anti-cancer effects of other crucial to prevent further emergence and spread of antimicrobial chemotherapeutics when combined together. resistance. Escherichia coli (E. coli) is an important cause of life- threatening diseases, such as sepsis and pneumonia, and fluoroquinolones are critically important antibiotics with a challenging PST.11 resistance situation [1]. The aim of these investigations was to elucidate bacterial adaptation and resistance mechanisms and Proteomic Exploration of IDH-Mutant Brain Tumors pharmacokinetic/pharmacodynamic (PK/PD) relationships exposing bacteria to human target-site concentration-time profiles (C(t) profiles) in a dynamic in vitro infection model (dIVIM) [2]. In addition to sequencing Felix M1,2; Warnken U3; Wick W4,5; von Deimling A1,2; Reuss D1,2 1Clinical Cooperation Unit Neuropathology, German Consortium for Translational Cancer of fluoroquinolone resistance determining regions (QRDR) of the Research (DKTK), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 bacterial genome, PK/PD modelling of time-kill curve data was applied to Heidelberg, Germany unveil phenotypic adaptation. 2Department of Neuropathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany Methods: 3Functional Proteome Analysis, German Cancer Research Center (DKFZ), Im Neuenheimer Two fluoroquinolone resistant E. coli isolates were investigated. Firstly, Feld 580, 69120, Heidelberg, Germany mutations in the QRDR of gyrA and parC and the presence of the 4Clinical Cooperation Unit Neurooncology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 plasmids qnrA, qnrB and qnrS were assessed by polymerase chain Heidelberg, Germany reaction (PCR) and electrophoresis. PCR products of gyrA and parC 5Department of Neurology, Heidelberg University Hospital, Im Neuenheimer Feld 400, 69120 were analysed by Sanger sequencing. Secondly, the isolates were Heidelberg, Germany exposed to C(t) profiles of a 750 mg, 90 min i.v. levofloxacin (LEV) infusion in septic patients (n=4 per isolate), which were mimicked in a Over the past years major improvements have been made in the dIVIM based on a previously published population PK model [3]. Bacterial diagnostics of brain tumors. Thanks to next generation sequencing and and LEV concentrations over time were quantified using a plate counting DNA-methylation analysis, neuropathologists have acquired two assay and a fluorescence assay, respectively. PK/PD analysis was powerful tools to increase their precision and accuracy [1]. Recently, performed for replicates with clinically relevant LEV exposure, which was proteomics using liquid chromatography coupled mass spectrometry determined over time as cumulative area under the LEV C(t) profile (LC-MS) has risen to the next potential tool to unravel existing mysteries (cumAUC(t)). The PD effect was assessed as the cumulative area in life sciences and diagnostics [2]. It is now possible to identify and between the growth control (GC) and the bacterial killing and regrowth quantify thousands of proteins in a single sample. Additionally the curve over time (cumABBC(t)) [4] and normalised to the growth of introduction of powerful and robust workflows using pressure cycling unexposed bacteria, determined as cumulative area under the GC curve. technology (PCT) has created possibilities to prepare samples within a Nonlinear regression was performed in R Studio™ to estimate in a few hours, making it feasible to measure proteomic samples the same sigmoidal maximum effect (Emax) model the cumulative exposure causing day they have been obtained from surgical treatment of the patient [3]. 50% of Emax (cumAUC50) and the steepness of the exposure-effect curves This makes proteomic analysis of brain tissue a viable option for day to for the two different strains. day screening. Currently, a proteomic pipeline including sample Results: preparation, mass spectrometry analysis and data analysis is being Both investigated isolates harboured the gyrA mutation S83L, but no established in the Clinical Cooperation Unit Neuropathology of the DKFZ. mutations in the QRDR of parC were present. Qnr plasmids were only Initial experiments are revealing the vast proteomic landscape of IDH- detectable in strain 2. In the dIVIM, initial reduction of bacterial load and mutant brain tumors and discovered biomarker candidates are being regrowth within 24 h was observed for both isolates. The extent of tested and evaluated under standardized diagnostic procedures. reduction and regrowth was different for the two strains, with a ≤ 2 log10- So far a cohort of fresh frozen IDH-mutant astrocytomas and IDH-mutant fold reduction for strain 1 and a reduction to the lower limit of oligodendrogliomas were analyzed using a data dependent acquisition quantification of the applied assay for strain 2. While strain 1 showed (DDA) method on a Q-Exactive HF-X Hybrid Quadrupole-Orbitrap mass more pronounced regrowth up to the GC level, bacterial concentration of spectrometer. We were able to identify around 9000 proteins over all runs strain 2 did not reach the inoculum concentration again within 24 h. Using with approximately 6000 proteins quantified per run. In total around 300 cumulative areas as dynamic exposure and effect metrics enabled proteins were significantly deregulated between astrocytoma and describing the PK/PD relationship with a sigmoidal Emax model. The oligodendroglioma. Specific proteins with a high fold change and exposure-effect curve was steeper for strain 2 (Hill factor: 0.994) significance were then further evaluated by immunohistochemistry. compared to strain 1 (Hill factor: 0.886). The higher cumAUC50 value for Additionally a data independent acquisition (DIA) pipeline was strain 1 compared to strain 2 (326 (mg·h)/L and 39.6 (mg·h)/L, implemented to further analyze the proteome in a more comprehensive respectively) was in accordance with the observed growth and kill and reproducible fashion. behaviour and the minimum inhibitory concentrations (MIC), indicating a In the future not only could proteomics support neuropathologists in the higher susceptibility of strain 2 (MIC: 2 µg/mL) compared to strain 1 (MIC: diagnostics of brain tumors, but may also help clinicians and researchers 8 µg/mL). For strain 1, cumAUC50 was not reached with the investigated in finding new potential drug targets and predictive biomarkers for tumor dosing regimen. therapy. Conclusion: Detected target-site mutations alone were not capable to fully explain the observed differences in the time-dependent growth-kill behaviour of the investigated clinical isolates. PK/PD modelling strongly indicated 1. Louis DN et al.: Acta Neuropathol. 2016; 131(6):803-20 2. Doll S et al. Proteomics Clin. Appl. 2019; 13(2):e1800113 additional adaptation and LEV resistance mechanisms underlying the 3. Lucas N et al. J. Proteome Res. 2019; 18(1):399-405 different PD parameter values of the strains. The mimicked LEV dosing regimen was not capable to eradicate bacteria in vitro, but might have triggered expression of adaptation mechanisms, such as SOS response. Further investigations will focus on phenotypic adaptation, applying PST.12 nonlinear mixed-effects PK/PD modelling.

Determinants of growth-kill behaviour of fluoroquinolone [1] European Centre for Disease Prevention and Control: Surveillance of antimicrobial resistant Escherichia coli under levofloxacin exposure in a resistance in Europe (EARS-Net) 2018. [2] Michael-Gloede, J. PhD thesis 2011. dynamic in vitro infection model [3] Schaeftlein, A. PhD thesis 2013. [4] Firsov, A. et al.: Antimicrob. Agents Chemother. 1990, 34(7):1312-1317.

1 1 2 1 Seeger, J. , Michelet, R. , Günther, S. , Kloft, C. 1 Freie Universitaet Berlin, Institute of Pharmacy, Dept. of Clinical Pharmacy & Biochemistry, Kelchstr. 31, 12169 Berlin, Germany

2 Universitaet Greifswald, Institute of Pharmacy, Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany

Objectives:

DPhG Annual Meeting 2019 Conference Book • 162 POSTER SHORT TALKS

model supported patient-tailored dosing to ensure treatment success. PST.13 Efforts to increase population-specific genotype data along with ethnicity- Computational treatment simulations to assess the risk for specific imputation of missing CYP2D6 diplotype information could non-efficacy in tamoxifen treatment for breast cancer further improve the predictive power of treatment simulations. patients of different ethnicities

Mueller-Schoell, A1,2., Bielesch, S1., Klopp-Schulze, L.1, Michelet, R.1, [1] Madlensky, L. et al.: Clin. Pharmacol. Ther. 2011, 89: 718-725. Huisinga W.3, Joerger, M.4, Kloft, C.1 [2] Goetz, M. et al.: Clin. Pharmacol. Ther. 2018, 103: 770-777. 1Freie Universität Berlin, Institute of Pharmacy, Dept. of Clinical Pharmacy & Biochemistry, [3] Del Tredici, A.L. et al: Front. Pharmacol. 2018 9: 305 (1-13). Kelchstr. 31, 12169 Berlin, Germany, 2Graduate Research Training program PharMetrX, [4] Klopp-Schulze, L. et al: PAGE Meeting. 2017, Abstr 7314 Germany, 3Institute of Mathematics, University of Potsdam, Karl-Liebknecht-Str. 24/25, 14476 Potsdam, Germany, 4Medical Oncology and Clinical Pharmacology, Dept. of Internal Medicine, Cantonal Hospital St. Gallen, Rorschacher Str. 95/Haus 10, 9007 St. Gallen, Switzerland

Background: The antihormonal drug tamoxifen (TAM) is used worldwide PST.14 for the treatment of breast cancer (BC). TAM is extensively metabolised Triple-color reporter system to follow up directed to its most active metabolite endoxifen (ENDX) via two metabolic differentiation of iPSC towards the three germ layers pathways, both including the cytochrome P450 (CYP) isoenzyme 2D6. CYP2D6 is a highly polymorphic enzyme and isoforms with reduced or 1 1 1 2 impaired function have been shown to result in ENDX minimum Gama-Brambila, R.A .; Wieland, J.E .; Hansen, P .; Mrowka , R.; Wölfl, S1.; Cheng, X1. concentrations at steady-state (Cmin,ss ENDX) below the proposed 1Institute of Pharmacy and Molecular Biotechnology. University of Heidelberg therapeutic threshold of 5.97 ng/mL1. As the frequency of CYP2D6 2 Experimental Nephrology, KIM III, University Clinique, Jena, Germany functional variants and thus the extent of ENDX formation differs between ethnicities, this study, by applying pharmacometric simulation, aimed to assess which populations might be at highest risk for subtherapeutic Ectopic expression of transcription factors, such as OCT4, SOX2, KLF4 Cmin,ss ENDX under TAM standard dosing and therefore benefit the most from therapeutic target concentration interventions. and MYC converts terminally differentiated somatic cells into induced Methods: CYP2D6 diplotype frequencies in 8 ethnic populations pluripotent stem cells (iPSCs), which are comparable to embryonic stem (Africans, African-Americans, Americans (Latin-Americans and cells in terms of self-renewal and differentiation potential to all three germ Canadians), Middle-Easterns, Caucasians (Europeans and North layers. iPSC technology provides a promising approach for tissue Americans), Oceanians, Central Asians and East Asians) were extracted regeneration, autologous transplantation and disease modelling in the from tables of the Clinical Pharmacogenetics Implementation consortium field of regenerative medicine. The clinical application of iPSCs is (CPIC)2. Reported CYP2D6 diplotypes were transformed into CYP2D6 currently limited by the lack of sufficient differentiation protocols leading activity scores (AS), which were translated into genotype-predicted to the desired functional cells. Directed differentiation of human iPSCs is phenotypes (gXM; AS=0: poor metaboliser (gPM), AS=0.5-1: a field widely studied and developed in recent years, which, however, is intermediate metaboliser (gIM) and AS≥1.5: normal metaboliser (gNM)) often a complicated, time-consuming, and cell-line dependent process. according to the most recent CPIC guideline2. As previously proposed, To better understand cellular events occurring during the differentiation, the CYP2D6 wildtype (WT; AS=2) was assigned to patients for who no we generated a reporter hiPSC line, which contains a triple-color reporter allele-defining sequence variations could be identified3. Based on the system, namely GFP, mCherry and iRFP regulated by endoderm-related extracted ethnic-specific AS frequencies, 8 large virtual populations genes (Sox17, Gata4, and, HNF4), mesoderm-related genes (brachuary, (n=10.000), each mirroring one ethnic-specific CYP2D6 variant Hand1, and Eomes) and ectoderm-related genes Pax6 and Sox1, composition, were generated. Using an earlier developed nonlinear respectively. Applying this system, we successfully recorded landscapes mixed effects parent-metabolite PK model of TAM and ENDX4, each of cellular differentiation stimulated by distinct differentiation protocols. population was simulated to receive 20 mg TAM once daily (q.d.). Upon Recently, we performed cell-based high throughput screening and reaching steady-state, the respective number of patients not reaching identified a number of small molecules able to modulate pluri- and multi- potency associated transcription factors. Our next aim is to optimize therapeutic Cmin,ss ENDX was assessed. Results for all ethnicities were compared and ranked according to their respective risk for therapeutic current differentiation protocols by screening our chemical library for the target-non attainment. Simulations were performed in NONMEM (v. 7.4). generation of fully functional differentiated cells. Results: Even though gNM was found to be the most prominent genotype-predicted phenotype in all ethnicities, the frequency of gNM Acknowledgments: Thank others for any contributions. Funding This work supported by the differed up to 2-fold between ethnicities (highest in Oceanians (87.1%) DFG grant program (CH 1690/2-1) and the BMBF grant programs Drug-iPS (FKZ 0315398A- and lowest in Africans (43.3%)). While highest proportions of gIM were FKZ 0315398B) and SysToxChip (FKZ 031A303A-FKZ 031A303E). observed in Africans (37.1%) and African-Americans (36.5%), Caucasians and African-Americans showed the highest proportion of gPM (4.83% and 2.16%, respectively). These findings had direct References: implications on the proportion of patients at risk for subtherapeutic Cmin,ss Dabiri, Y, et al: iScience. 2019, 12: 168-181 ENDX in our simulations (African-Americans: 22%, Africans and Caucasians: 20.9%, East Asians: 19.5%, Central Asians: 18.4%, Americans: 16.5%, Middle Easterns: 16.7% Oceanians: 11%). Genotype data was missing for 18.5% of Africans and 21% of Americans and PST.15 imputed as described above. While the WT was the most common AS in Ketamine promotes early changes in dendritic morphology Americans, it was only the third most frequent AS in Africans (AS 2: 11.2% vs. AS 1.5: 29.0% and AS 1.0: 26.5%). Therefore, we propose to in the hippocampus of a genetic rat model displaying consider ethnic-specific diplotype frequencies prior to AS imputation and depressive-like behaviour to impute the respective most common AS instead of the CYP2D6 WT. Otherwise, the proportion of patients at risk for subtherapeutic Cmin,ss ENDX Treccani G.1,2; Ardalan M.2; Chen F.2; Musazzi L.3; Popoli M.3, might be underestimated, especially in populations (1) for which the most Wegener G.1, Nyengaard J.4, Müller M. B.2, Müller H.K.2 frequent diplotype is not the CYP2D6 WT (e.g. Africans, African- 1 Deutsches Resilienz Zentrum (DRZ) gGmbH, Untere Zahlbacher Straße 8, 55131 Mainz, Americans, East Asians) and (2) for which a high fraction of genotype Germany data is missing. 2 Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Skovagervej 2, 8240 Risskov, Denmark Conclusions: By collecting rich CYP2D6 genotype information and 3 Department of Pharmacological and Biomolecular Sciences, University of Milano, via subsequently applying this information in treatment simulations for 8 Balzaretti 9, 20133 Milano, Italy different ethnicities, we determined Africans, African-Americans and 4 Core Center for Molecular Morphology, Section for Stereology and Microscopy, Department of Clinical Medicine, Palle Juul Jensens Boulevard 99, Aarhus University, 8200 Aarhus N, Caucasians as ethnicities with the highest number of patients at risk for Denmark TAM treatment failure. Within these populations, more than 1 out of 5 patients will not reach the therapeutic ENDX threshold under TAM standard dosing. Thus, we strongly recommend CYP2D6-guided and Background

163 • DPhG Annual Meeting 2019 Conference Book POSTERS

Psychiatric disorders constitute a major burden for society in terms of lipoxygenase (12-LOX) and cyclooxygenase (COX)-1. While a more productivity and years lost to disability. Recently it was shown that pronounced expression of 12-LOX was found in platelets from male rats ketamine (KET), a non-competitive NMDA receptor antagonist, induces than in females, no sex differences were evident in the expression of a rapid and sustained antidepressant effect in treatment-resistant COX-1. Of interest, when platelet aggregation was induced by the patients (1). However, the mechanism by which KET ameliorates addition of AA (20 µM), platelets from female animals aggregated more depressive symptoms is still unclear. efficiently than those from males. Here we used the Flinders Sensitive Line (FSL) rat, and its control strain Together, here we show for the first time a sex-difference in the basal the Flinders Resistant Line (FRL) rat to investigate morphological and expression of key enzymes in the regulation of platelet activation in rats molecular changes in the hippocampus that may be involved in the rapid together with a sex-biased aggregation dependent on the stimulus antidepressant-like effect of KET. utilized. Methods Further experiments will be carried out to understand the molecular To validate the antidepressant-like effects of KET at 1 h post injection, mechanisms underlying these sex differences. we exposed the rats to the forced swim test. For morphological analysis, one hemisphere per animal was processed for the Golgi-cox staining. Molecular studies were performed on the controlater hemisphere (2). Results We found that FSL rats exhibited higher immobility times (p<0.001) while PST.17 KET treatment reduced immobility times (p<0.0001). Moreover, the swimming behaviour was lower in FSL rats compared to FRL (p<0.01) Thiocyanates as a new class of selective Sirt1 inhibitors and it was higher in FSL rats treated with KET compared to FSL vehicle (p<0.05). These data demonstrate an antidepressant-like effect of KET Wössner, N.1, Alhalab, Z.i2, Swyter, S.1, González Nieto, J.3, only 1 h after injection. Schmidtkunz, K.1, Vaquero, A.3, Sippl, W.2, Jung, M.1 Regarding the morphological study, we found a significant increase in the 1 Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstraße 23, number and density of spines in the apical dendrites (p<0.01) in FSL rats 79104 Freiburg, Germany 2 Institute of Pharmacy, Martin-Luther University of Halle−Wittenberg, 06120 Halle/Saale, treated with KET. We also found an overall decrease in the basal Germany dendritic length in the FSL rats (p<0.05) and an effect of KET treatment 3 Josep Carreras Leukaemia Reseach Institute, Muntaner 383, 3r2a, 08021 Barcelona, Spain on spine number in FSL rats treated with KET (p<0.05). At synaptic level, KET decreased the phosphorylation of cofilin and the NMDAR2A subunit level while it increased the HOMER 3 level. Sirtuins are class III histone deacetylases (HDACs)[1] that, unlike other Conclusion HDACs, are not Zinc-dependent but use NAD+ as a cofactor to cleave These data suggest that morphological and synaptic reorganization of off various different acyl groups from the ε-amino residues of lysines.[2] both apical and basal dendrites may be involved in the fast Through their great range of substrates they influence various cellular antidepressant-like effect of KET (2). rocesses like metabolism, stress response, DNA repair, cell survival or apoptosis.[3] Therefore sirtuins are associated with the pathogenesis of various diseases, like cancer, metabolic or neurodegenerative diseases.[4] Of the seven human sirtuin isotypes Sirt1 is the most Acknowledgment extensively studied one. It is linked to aging in general and specifically to Funding was provided for G.T. by a NARSAD Young Investigator Grant from the Brain & age related diseases, like for example Alzheimer`s or Huntington`s Behavior Research Foundation and a postdoctoral research grant from the Danish Council for Independent Research (DFF–5053-00103) Disease (HD). The first Sirt1 inhibitor to pass phase I and II clinical studies, Selisistat is currently examined in a phase III trial for treatment of HD.[5] More modulators of irt1 are needed to exploit and further References characterize its therapeutic use. 1. Zanos P and Gould TD: Mol Psychiatry 2018, 23(4): 801–811. 2. Treccani G et al.: Mol Neurobiol. 2019 [Epub ahead of print] We tested a small library of commercially available compounds proposed by docking studies against Sirt1, 2 and 3. OSSK 221646 was found to selectively inhibit Sirt1 with an IC50 of 13.0 ± 0.6 μM. Analogues showed PST.16 that the thiocyanate structure of the compound was key to the selectivity and high affinity towards Sirt1. To enable cellular studies new analogues of the thiocyanates with higher solubility were identified through docking Sex dimorphism in rat platelet aggregation studies and tested. Compounds that showed micromolar IC50 values in vitro were further studied in cells. Therefore levels of γH2AX, which are Bilancia R.1, Pace S.2, Caiazzo E.1, Troisi F. 2, Rossi A.1, Cicala C.1, lower in Sirt1 KO cells than in WT, were examined.[6] We obtained similar Werz O.2, Ialenti A1. protein levels of γH2AX for Sirt1 KO cells as well as cells treated with 1 Department of Pharmacy, School of Medicine, University of Naples Federico II, 80131 Naples, Selisistat or the thiocyanates. These results show that thiocyanates are Italy. 2 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller- a promising new class of selective Sirt1 inhibitors. University Jena, 07743 Jena, Germany We thank the DFG for funding (Ju295/14-1). Platelets are key players in maintaining the homeostasis controlling the blood loss after vessel injury. However, a not-controlled platelet activation can lead to the formation of pathologic blood clots connected References to several diseases (i.e. myocardial infarction or stroke). Clinical 1) E. Fiorino, M. Giudici, A. Ferrari, N. Mitro, D. Caruso, E. D. Fabiani, M. Crestani, IUBMB Life observation show a sex-biased prevalence and severity of vascular 2014, 66, 89-99. 2) J. L. Feldman, K. E. Dittenhafer-Reed, N. Kudo, J. N. Thelen, A. Ito, M. Yoshida, J. M. Denu, thrombosis and/or mortality, with men having a worse prognosis. Biochemistry 2015, 54, Nonetheless, in the last decades there are conflicting reports on the 3037-3050. effects of sex on platelet aggregation, prompting for a deeper 3) P. Martínez-Redondo, A. Vaquero, Genes Cancer 2013, 4, 148-163. investigation on the mechanisms behind. The purinergic system is 4) a) H. Jęśko, P. Wencel, R. P. Strosznajder, J. B. Strosznajder, Neurochem. Res. 2017, 42, involved in platelet aggregation, in fact, endothelial cell ecto-nucleoside 876-890; b) S. triphosphate diphosphohydrolase 1, E-NTPDase1 (CD39) promotes Voelter-Mahlknecht, U. Mahlknecht, Clin. Epigenetics 2010, 1, 71-83. vascular homeostasis, but how this is modulated by sex and sex 5) a) S. D. Süssmuth, S. Haider, G. B. Landwehrmeyer, R. Farmer, C. Frost, G. Tripepi, C. A. Andersen, M. D. Bacco, C. hormones is still not elucidated. Here we show that the basal expression Lamanna, E. Diodato, L. Massai, D. Diamanti, E. Mori, L. Magnoni, J. Dreyhaupt, K. Schiefele, of CD39 in rat platelets is higher in female rats than in males. Notably, D. Craufurd, C. Saft, M. adenosine-diphosphate (ADP, 1 µM)-induced aggregation was stronger Rudzinska, D. Ryglewicz, M. Orth, S. Brzozy, A. Baran, G. Pollio, R. Andre, S. J. Tabrizi, B. in platelets from male rats as compared with females. Important to note Darpo, G. Westerberg, British is that the efficiency of the CD39 inhibitor ARL was stronger in platelets Journal of Clinical Pharmacology 2015, 79, 465-476; b) AOP Orphan Pharmaceuticals AG, derived from females than males. 2017, Available at: http://www.aoporphan.com/news-media/startpage-detail/artikel/aoporphan- Another metabolic pathway with pivotal roles in platelet activation is the pharmaceuticals-ag-to-acquire-selisistat-a-clinical-stage-drug-candidate-for-the-treatment-of- arachidonic acid (AA) cascade mediated by the activity of 12- huntingtons-disease-hd.html

DPhG Annual Meeting 2019 Conference Book • 164 POSTER SHORT TALKS

[Accessed 12Mar. 2019]; c) V. D. Longo, B. K. Kennedy, Cell 2006, 126, 257-268. 1, we developed different series of novel 7-chloro-9H- 6) R. H. Wang, K. Sengupta, C. Li, H. S. Kim, L. Cao, C. Xiao, S. Kim, X. Xu, Y. Zheng, B. pyrimido[4,5-b]indole-based compounds in order to explore structure- Chilton, R. Jia, Z. M. Zheng, E. activity relationships and optimized this compound class towards GSK-3β Appella, X. W. Wang, T. Ried, C. X. Deng, Cancer Cell 2008, 14, 312-323. inhibition. During the presented optimization study, we kept the 7-chloro- 9H-pyrimido[4,5-b]indole core intact and varied the substituent on the PST.18 piperidine nitrogen as well as the aliphatic heterocycle.

Small molecules as MKK4 inhibitors for the regeneration of hepatocytes

Praefke, B. 1; Pantsar, T.2; Poso A.2,3; Klotz S.3; Zender L.3; Laufer S. 1 1University of Tuebingen, Faculty of Science, Pharmaceutical and Medicinal Chemistry, Auf der Morgenstelle 8, 72076 Tuebingen, Germany 2 School of Pharmacy, University of Eastern Finland, Kuopio, Finland. 3 Division of Translational Gastrointestinal Oncology, Department of Internal Medicine I, Scheme 1. Development of GSK-3β inhibitors derived from tofacitinib. University of Tuebingen, Tuebingen, Germany

About 1 million deaths per year are affiliated with chronic or acute liver The final compounds were evaluated for their inhibitory activity on GSK- failure, having viral infections (hepatitis B/C), non-alcoholic fatty liver 3β in an ADPGlo kinase assay. The biological data of this compound disease (NAFLD) or metabolic syndrome as main causes. As no medical series revealed a crucial role of the nitrile group and highlighted its treatment for liver failure is available, the finding of a molecular target is importance for the compound activity. While the original piperidine moiety pursued. Using RNAi experiments, the mitogen activated protein kinase proved optimal in terms of the ring size of the aliphatic ring, a successful kinase 4 (MKK4) was found by Wuestefeld et al. to play a key role in liver rigidization approach resulted in an increased compound activity in the regeneration. Silencing MKK4 expression with shRNA resulted in an low triple-digit nanomolar range. The most potent compounds were increase in robustness and regenerative potential of the liver via further characterized regarding their metabolic stability in human liver amplified hepatocyte proliferation. MKK4 silencing is suspected to lead microsomes and inhibitory potency on the likely off-target JAK3. to a higher activation of mitogen activated protein kinase kinase 7 (MKK7) Furthermore, we conducted 1 µs molecular dynamics simulations to and thus to a higher phosphorylation and activation of the downstream c- examine the putative binding modes of these compounds within the ATP Jun-N-terminal protein kinase (JNK1) [1,2] binding site of the kinase.

For the finding of a small molecule inhibitor of MMK4 a virtual screening with MKK4 crystal structure (PDB: 3ALO) using Glide and SurflexDock Reference: methods on the Taito: HP Apollo 6000 XL230a/SL230s Supercluster 1. Beurel, E. et al. Pharmacol. Ther. 2015, 148: 114-131.

(taito.csc.fi) Finland was pursued. Docking was carried out using an incremental approach. In the first screening, fast approximated scoring/pose scanning was used and later with more robust/time PST.20 consuming settings. The combined docking lead to 180 compounds, Bioisosteres of the Natural Product Taxifolin and their which were purchased for in vitro testing with DiscoverX KINOMEScan Impact on Amyloid-β 42 Aggregation and Intracellular assay. A POC (percent of control) of 5 @ 10µM for MKK4 and a good Oxidative Stress selectivity profile for a compound was determined, which lacked hydrolytic stability due to an imino group. For the chemically stable J. Hofmann1; S. Gunesch1; C. Stigloher2; M. Decker1 analogue, a POC of 23 @ 10µM was determined for MKK4. Further 1 Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, Julius derivatisation and optimization resulted in a compound with a POC of 5 Maximilian University Wuerzburg, Am Hubland, 97074 Würzburg, Germany 2 Imaging Core Facility, Biocenter, Julius Maximilian University Wuerzburg, Am Hubland, 97074 @ 10µM and 51 @ 1µM. Würzburg, Germany

For the replacement of the imino group to a hydrolytically stable E configured C-C double bond, HWE-reactions were carried out for the first compounds. In the synthesis of the further derivatives, HWE-reaction Several polyphenolic compounds have shown neuroprotective properties was replaced by Heck-reaction. and anti-aggregative effects towards fibrilization of amyloid-β 42 (Aβ42), a key protein in the pathogenesis of Alzheimer disease.[1] Especially the flavanonol (+)-taxifolin is well known for its inhibitory effect on Aβ42 1. Wuestefeld, T et al.: Cell 2013, 153(2): 389-401. aggregation.[2] The chemical structure shows a catechol moiety on the 2. Willenbring, H. and M. Grompe: Cell 2013, 153(2): 283-284 B-ring, three hydroxy groups at position 2, 5 and 7 and a ketone at position 3. (Fig. 1)

PST.19 From JAK to GSK-3β: Synthesis and Biological Evaluation of 7-Chloro-9H-Pyrimido[4,5-b]indoles as Inhibitors of Glycogen Synthase Kinase-3β

Stanislav Andreev 1, Tatu Pantsar 2,3, Michael Forster 1, Mark Kudolo 1, Stefan A. Laufer 1 and Pierre Koch 1,4 1Institute of Pharmaceutical Sciences, Department of Medicinal and Pharmaceutical Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany 2Department of Internal Medicine VIII, University Hospital Tübingen, Otfried-Müller-Str. 14, 72076 Tübingen, Germany 3School of Pharmacy, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland 4Department of Pharmaceutical / Medicinal Chemistry II, Institute of Pharmacy, University of Figure 1 Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany

The glycogen synthase kinase-3β (GSK-3β) represents a relevant therapeutic target for the treatment of human pathologies such as CNS Structure-activity relationship studies could show the importance of the disorders and neurodegenerative diseases [1]. Herein, we present a hydroxy groups of the catechol unit for the inhibition of Aβ42 fibril novel class of GSK-3β inhibitors derived from the pan-Janus kinase formation.[3] Also computational studies suggest that the catechol plays (JAK) inhibitor tofacitinib. A kinome-wide screening campaign identified a crucial role for aggregation inhibition of β-amyloid aggregation via compound 1 as a GSK-3β inhibitor with moderate potency displaying an oxidative formation of an o-quinone. Ginex et al. postulated the aza- IC50 value in the single-digit micromolar range (Scheme 1). Starting from Michael addition as binding mechanism of taxifolin to β-amyloid fibrils.[4]

165 • DPhG Annual Meeting 2019 Conference Book POSTERS

Based on the structure of (+)-taxifolin and supported by computational studies we designed six new bioisosteric compounds with an azobenzene scaffold carrying the catechol moiety.(Fig.1) All compounds show high aggregation inhibition in transmission electron microscopy (TEM) experiments. Additionally, the compounds show protection against glutamate-induced intracellular oxidative stress in HT22 mouse hippocampal neuronal cells at low concentrations. Taken together, we synthesized six bioisosteric compounds which have shown higher activity then the parent compound (+)-taxifolin for inhibition of Aβ42 fibril formation and exhibit neuroprotection on HT22 cells.

[1] Ramassamy C.: Eur. J. Pharmacol. 2006, 545(1): 51-64. [2] Sato, M. et al.: J. Biol. Chem. 2013, 288(32): 23212-23224. [3] Sato, M. et al.: Biosci. Biotechnol. Biochem. 2013, 77(5): 1100-1103. [4] Ginex, T., Trius, M., Luque, F. J.: Chem. Eur. J. 2018, 22: 5813-5824.

DPhG Annual Meeting 2019 Conference Book • 166 AUTHOR INDEX

AUTHOR INDEX

Bauder-Wüst, U...... POS.61 A Bauer, S...... POS.20 Bauerschlag, D. O...... POS.59 Abad-Santos, F...... POS.107 Bäurer, S...... POS.100 Abdelmalek, C. M...... POS.36 Beck, M. S...... POS.92 Abdelsamie, A...... POS.25 Beckford Vera, D. R...... POS.66 Abraham, A...... POS.153 Bednarski, P...... POS.54 Alberding, G...... POS.125 Bednarski, P. J...... POS.42, POS.55, POS.62 Albrecht, W...... POS.7 Beekmann, U...... POS.154 Alexandrov, T...... SL.59 Behnam, M...... POS.35 Al-Gousous, J...... POS.122, POS.151 Behnisch-Cornwell, S...... POS.62 Alhalabi, Z...... PST.17 Behring, L...... POS.10 Allisat, I...... POS.90 Beijer, B...... POS.46 Almohsen, N...... POS.129 Beirow, K...... POS.42 Almouhanna, F...... POS.102, PST.10 Bellmann, T...... POS.154 Alnemari, R...... POS.148, POS.153 Belter, B...... POS.10 Amann, L. F...... POS.108 Bendas, G...... POS.101, PST.09 Amidon, G...... POS.151 Bengel, F. M...... SL.37, POS.63 Andreas, N...... POS.164 Benowitz, N...... POS.106 Andreev, S...... PST.19 Berger, A...... SL.53 Arakandy, V...... POS.157 Berger, B.-T...... POS.20, POS.24 Ardalan, M...... PST.15 Berlin, M...... SL.08 Ardelt, M...... SL.44 Bermudez, M...... SL.36, POS.80 Arimitsu, K...... POS.64 Bernhardt, G...... POS.49, POS.75 Arndt, H.-D...... PST.07 Bertazzo, S...... SL.32 Arnold, G. J...... SL.44 Beziere, N...... SL.43 Assmann, M...... POS.71 Bhatia, S...... POS.57 Atzberger, C...... SL.44 Biel, M...... SL.07, PST.06 Bielesch, S...... PST.13

Bilancia, R...... SL.49, SL.61, POS.157, POS.164, PST.16 Birk, B...... SL.10 B Bischoff, I...... SL.48, POS.119 Bisha, M...... POS.159, POS.160, POS.161 Blagojevic, B...... POS.102, POS.103, PST.10 Baalmann, M...... SL.02 Blechar, J...... POS.151 Bach, A...... SL.07 Blechar, J. A...... POS.122 Bacher, J...... POS.51 Bluck, J. P...... POS.29 Bacmeister, L...... SL.08 Bodem, J...... POS.48 Baecker, D...... POS.82 Boehlich, G. J...... POS.68 Balke, W.-T...... POS.87 Boerner, F...... POS.164 Bals, R...... POS.106 Borchard, G...... PL.1 Bandaru, S...... POS.54 Borchers, M...... PST.08 Bankstahl, J. P...... SL.37, POS.63 Borkhardt, A...... POS.57 Banoğlu, E...... POS.78 Borrelli, F...... SL.61 Bantscheff, M...... SL.41 Boschetti, F...... SL.43 Bär, F...... POS.126, POS.127 Botta, J...... POS.139 Bartenschlager, R...... PL.2 Bracher, F...... SL.06, POS.119 Barth, C...... POS.123 Braig, S...... POS.115 Barth, M...... POS.1 Brandstetter, H...... POS.44 Barthes, N...... POS.28, POS.51 Braun, A...... POS.91 Barthes, N. P. F...... POS.28 Braun, A. M...... POS.89 Bas, M...... POS.161

167 • DPhG Annual Meeting 2019 Conference Book AUTHOR INDEX

Breitinger, H.-G...... POS.36, POS.67 Damaraju, V. L...... SL.38 Breitinger, U...... POS.36, POS.67 Daniels, R...... POS.146 Bresinsky, M...... POS.15 Dauch, D...... POS.26 Britz, H...... POS.105, POS.106 Dauer, K...... POS.150 Brockmann, A...... POS.72 Davies, G...... SL.43 Bröker, A...... POS.108 de Souza, L...... POS.136, POS.144 Bruno, P. B...... POS.29 de VIilliers, E.-M...... PL.5 Brunschweiger, A...... POS.93 de Vries, J...... POS.71 Brunst, S...... POS.4, POS.9 Decker, M...... SL.35, POS.64, PST.20 Brust, P...... PST.02 Dehm, L...... POS.140 Budzinski, J...... SL.34 Deigner, H.-P...... POS.99 Bund, T...... PL.5 Delandre, S...... POS.130 Bünemann, M...... SL.33 Diana, P...... POS.22 Burgers, L. D...... POS.116 Diaz Garcia, C...... POS.110 Buschauer, A...... POS.41, POS.49, POS.75 Dieckmann, S.-M...... POS.77 Buschmann, J...... POS.75 Dieterich, C...... SL.07, SL.08 Busse, D...... POS.162, PST.05 Dimitrov, T...... POS.23 Büssing, R...... POS.12 Dobelmann, C...... POS.74 Bussmann, J...... POS.134 Domhan, C...... POS.46 Büttner, M...... POS.102 Dooley, S...... POS.163 Döring, E...... POS.27 Dorn, C...... POS.162 C Dräger, G...... POS.63 Draheim, C...... POS.87 Caiazzo, E...... PST.16 Dreischulte, T...... SL.26 Camacho Londoño, J. E...... SL.08 Dropmann, A...... POS.163 Camacho-Londono, J...... SL.07 Družeta, I...... POS.92 Cantonati, P...... SL.44 Ducho, C...... POS.44, POS.45, POS.69, POS.70, POS.79 Carina, A...... POS.115 Düfer, M...... POS.156 Carrillo-Hormaza, L...... POS.148 Dukic-Stefanovic, S...... PST.02 Cass, C. E...... SL.38 Chaikuad, A...... SL.48, POS.3, POS.20, POS.24 Chandralingam, S...... POS.56 Chandrawati, R...... SL.32 E Chao, Y.-K...... SL.06 Chen, C.-C...... SL.06 Ebert, R...... SL.03 Chen, F...... PST.15 Eck, M...... POS.27 Chen, X...... POS.64 Eckert, R...... POS.127 Cheng, W...... POS.115 Eckert, R.-W...... POS.132 Cheng, X. SL.47, POS.88, POS.118, POS.158, POS.163, Efferth, T...... SL.56 PST.14 Eggers, T...... POS.122 Chowdary, P...... POS.110 Ehmann, L...... POS.162, PST.05 Christianson, D...... POS.84 Ehrlichmann, W...... SL.38 Cicala, C...... PST.16 Eiber, J...... POS.149, POS.155 Cichutek, K...... SL.53 Eigner Henke, K...... POS.66 Ciciliani, A.-M...... SL.31 Eigner, S...... POS.66 Clark, T...... POS.41 Elsa, A...... POS.110 Clement, B...... POS.59 Emery, F. d. S...... POS.5 Conway, S. J...... POS.29 Engel, J...... POS.27 Cumbana, R...... SL.03 Ermert, J...... POS.76 Czodrowski, P...... SL.13 Ernst, J...... POS.128

D F

Dabiri, Y...... POS.88, POS.158, POS.163 Fabritius, M...... POS.165

DPhG Annual Meeting 2019 Conference Book • 168 AUTHOR INDEX

Fassnacht, M...... SL.62 Gmeiner, P...... SL.34, SL.40, POS.32 Faudone, G...... POS.21, POS.37 Göbel, T...... SL.48 Felix, M...... PST.11 Gohlke, H...... POS.41 Ferreiros, N...... SL.03 González Nieto, J...... PST.17 Fidelj, V...... POS.143 González-Estévez , C...... POS.157 Fiebig, H.-H...... POS.27 Görgen, M...... POS.113 Fiedler, J...... POS.63 Grathwol, C...... POS.62 Filippakopoulos, P...... POS.29 Grätz, L...... POS.13, POS.14, POS.75 Fischer, D...... POS.128, POS.131, POS.154 Gresch, A...... POS.156 Fischer, M. R...... PST.06 Greve, J...... POS.161 Fischer, S...... POS.30 Grimm, C...... SL.06, SL.07 Flockerzi, V...... SL.07 Grimm, M...... SL.04 Flörkemeier, I...... POS.59 Gronauer, T...... POS.115 Flötgen, D...... POS.52 Gronbach, L...... SL.12 Fluck, G...... SL.51, POS.81 Gros, P...... SL.52 Forster, L...... POS.49 Groth, T...... SL.22, POS.135 Forster, M. SL.15, POS.20, POS.23, POS.24, POS.30, Grune, C...... POS.131 PST.19 Grüning, B. A...... SL.17 Franco, R...... POS.13, POS.14 Gündel, D...... PST.01 Frangioni, J...... POS.56 Gunesch, S...... PST.20 Freichel, M...... SL.07, SL.08 Gunkel, N...... SL.25 Fricker, G. POS.139, POS.140, POS.142, POS.143, Günther, M...... POS.27 PST.04 Günther, S...... PST.12 Friedel, D...... POS.47 Güntzel, P...... POS.133 Friedland, K...... POS.104 Gunzer, M...... SL.38, SL.43 Friedrich, C...... POS.90, PST.08 Guse, A. H...... SL.05 Fröhlich, T...... SL.44 Gust, R...... POS.82, POS.86 Fromme, A...... POS.117 Gutierrez-Gutierrez, O...... POS.157 Frotscher , M...... POS.25 Gütschow, M...... SL.14 Fu, M...... POS.122 Guzman, C. A...... POS.130 Fuchs, N...... POS.31 Fuhrmann, G...... SL.32 Fürst, R...... SL.48, POS.116, POS.119, POS.165 H

Haarer, L...... POS.19 G Haase, G.-S...... POS.26 Haberkorn, U...... POS.46, POS.47 Gama-Brambila, R. A...... POS.88, POS.118, PST.14 Hacker, M...... POS.137, POS.138 Gao, D...... POS.85 Hacker, M. C...... PST.02 Garscha, U...... POS.65, POS.78 Hacker, S...... POS.115 Gattig, P...... POS.121 Hadaschik, E. N...... POS.118 Gaube, F...... PST.07 Haeberle, S...... POS.118 Gehringer, M...... SL.15, POS.19, POS.20, POS.23 Haefeli, W. E...... PL.3 Geistlich, S...... SL.38, SL.43 Häfner, A. K...... POS.120 Gellrich, L...... SL.48 Hahn, J...... POS.161 George, S...... POS.9 Haller, V...... POS.30 Gerbes, A...... SL.44 Hamacher, A...... POS.11 Gerndt, S...... SL.06 Hammerschmidt, F...... SL.38 Gerstmeier, J...... SL.49, SL.61, POS.157, POS.164 Hammerschmidt, S...... POS.50 Ghanem, A...... POS.102 Hammond, E...... POS.29 Gholamreza Fahimi, E...... POS.160 Hanke, N...... POS.105, POS.109 Gholamreza-Fahimi, E...... POS.159, POS.161 Hansen, F...... POS.11 Gierse, R. M...... POS.83 Hansen, F. K...... POS.57, POS.58, POS.160 Gilsbach, R...... SL.17, SL.18 Hansen, P...... PST.14 Giselbrecht, J...... POS.134 Harm, M...... POS.65 Gleissner, C...... POS.115 Hartmann, R. W...... POS.4, POS.44

169 • DPhG Annual Meeting 2019 Conference Book AUTHOR INDEX

Hartmann, S...... POS.127 Imberg, L...... SL.51, POS.81 Hartmann, S. F...... POS.132 Imhof, D...... POS.92 Hashmi, A. S. K...... SL.23 Immerheiser, M...... POS.48 Hatemler, G. M...... POS.119 Isaak, A...... POS.74 Hau, M...... POS.17 Isbemer, N...... SL.62 Hauer, J...... POS.57 Haupenthal, J...... POS.4, POS.44, POS.69, POS.83 Heering, J...... SL.48, POS.3, POS.9, POS.21 J Hegenbarth, U...... SL.57 Heib, A...... POS.45 Jaehde, U...... SL.28 Hein, L...... SL.17, POS.28 Janssen, E. H...... POS.63 Heinz, C...... POS.60 Januchowski, R...... POS.101 Heitel, P...... SL.48, POS.37 Jäschke, A...... POS.94 Hellmich, U. A...... POS.31, POS.48, POS.50 Jedamzik, J...... POS.42 Hempel, G...... POS.107 Jedamzik, P...... SL.04 Hendrik, M...... POS.144 Jennings, L. E...... POS.29 Henning, M...... POS.52 Jensen, A...... POS.67 Henze, S...... POS.101 Jensen, A. A...... POS.36 Hepp, T...... POS.104 Jessen-Trefzer, C...... SL.58 Heppner, D. E...... POS.27 Jha, A...... SL.07 Herp, D...... POS.84 Joerger, M...... SL.63, PST.13 Herrlinger, E.-M...... POS.17 Johannsen, S...... POS.83 Hertlein, T...... POS.46, PST.04 Johe, P...... POS.31 Hespeler, D...... POS.147 Jonas, H...... POS.22 Heß, A...... SL.37 Jordan, P. M...... SL.49, POS.157, POS.164 Heussel, C. P...... SL.31 Joseph, J. F...... POS.111 Heuter, S...... PST.09 Juchum, M...... POS.27 Hewings, D. S...... POS.29 Jung, M...... POS.17, POS.28, POS.51, POS.84, PST.17 Heydenreuter, W...... POS.115 Jünger, J. S...... POS.89, POS.91 Higuchi, T...... POS.64 Junker, A...... POS.5, POS.74 Hinz, S...... POS.72 Hirano, M...... POS.64 Hirsch, A. K. H...... POS.4, POS.44, POS.69, POS.83 K Hoffmann, A...... POS.146 Hoffmann, M...... POS.64 Kahnt, A. S...... SL.03 Hoffmann, T...... POS.161 Kaindl, J...... SL.34, POS.41 Hoffmeister, H...... POS.43 Kalinin, D...... SL.51, POS.81 Hofmann, C...... SL.08 Kamm, W...... POS.150 Hofmann, J...... PST.20 kamradt, t...... POS.164 Hohlfeld, T...... POS.161 Kanitz, V...... SL.44 Holzgrabe, U. POS.36, POS.60, POS.67, POS.96, POS.133 Kany, A. M...... POS.4, POS.44 Hopf, C...... SL.60 Karg, C...... POS.114 Hopp, M.-T...... POS.92 Karl, B...... POS.154 Hoppe-Tichy, T...... SL.27 Kassack, M...... POS.11 Hörmann, N...... POS.65 Katoh, Y...... SL.01 Hübner, H...... SL.34 Kattner, S...... POS.131 Huisinga, W...... POS.162, PST.13 Kaya, C...... POS.4 Husteden, C...... SL.22, POS.135 Kazmaier, U...... POS.165 Keane, T...... SL.32 Keck, C...... POS.126 I Keck, C. M. POS.97, POS.117, POS.127, POS.129, POS.132, POS.148, POS.153 Ialenti, A...... SL.61, PST.16 Kehl, T...... SL.17 Ibrahim, P...... POS.41 Keilholz, U...... SL.12 Idris, R. M...... POS.33 Keller, M...... SL.39, POS.75 Ilia, L...... PST.05 Kelter, G...... POS.27

DPhG Annual Meeting 2019 Conference Book • 170 AUTHOR INDEX

Kersten, C...... POS.48 Kroll, H...... POS.87 Kersten, C. F...... POS.31, POS.50 Kronenberger, T...... POS.26 Keßler, K...... POS.87 Krybus, M...... POS.159, POS.160, POS.161 Keul, M...... POS.27 Kudolo, M...... POS.77, PST.19 Khosravani, F...... POS.159, POS.161 Kühl, T...... POS.92 Kilu, W...... SL.48, POS.3, POS.9 Kuhne, K...... POS.10 Kimura, H...... POS.64 Kurlbaum, M...... SL.62 Kircher, B...... POS.82 Kuryshev, V...... SL.08 Kircher, T...... POS.6 Kurz, T...... POS.160 Kirchner, E.-A...... POS.82 Kuswandi ...... POS.60 Kischio, L...... POS.154 Kutzner, L...... SL.03 Klapper, S...... POS.96 Kuwert, T...... SL.40 Klapschinski, T...... POS.72 Klein, C...... POS.34, POS.35, POS.141 Kleist, C...... POS.46 L Klemm, P...... POS.78 Kliesch, L...... POS.130 Lächelt, U...... SL.21 Klika, K. D...... POS.46 Lambris, J...... SL.52 Kling, R. C...... POS.41 Lamers, C...... SL.52 Klinger-Strobel, M...... POS.128 Lämmerhofer, M...... POS.95, POS.100 Klinghammer, K...... SL.12 Landsiedel, R...... SL.10 Klinker, H...... SL.62 Langer, K...... POS.123, POS.124, POS.125 Kloevekorn, P...... POS.7 Langer, L. B. N...... SL.37, POS.63 Kloft, C. SL.63, POS.111, POS.162, PST.05, PST.12, Langguth , P...... POS.152 PST.13 Langguth, P...... SL.31, POS.121, POS.122, POS.151 Klopp-Schulze, L...... SL.63, PST.13 Langner, A...... POS.134 Klösel, I...... POS.32 Lategahn, J...... POS.27 Klotz, S...... PST.18 Laufer, S. SL.15, POS.6, POS.7, POS.19, POS.20, POS.24, Knapp, S...... PL.4, SL.48, POS.20, POS.24, POS.119 POS.27, POS.30, POS.52, POS.77, POS.99, Kneilling, M...... SL.38 PST.18, PST.19 Kneller, L. A...... POS.107 Laufer, S. A...... POS.23, POS.26 Knoth, D...... POS.97, POS.126, POS.132 Laurin, C. M. C...... POS.29 Koch, P...... POS.40, PST.19 Lauterbach, S...... POS.69 Kohl, M...... POS.99 Lehner, E...... PST.01 Kohl, Y. L...... POS.106 Lehr, C.-M...... POS.130 Kojda, G...... POS.159, POS.160, POS.161 Lehr, M...... POS.1 Kolle, S...... SL.10 Lehr, T. SL.44, POS.105, POS.106, POS.109, POS.112, König, L...... SL.44 POS.113 König, P...... POS.101 Lemcke, T...... POS.18 Kopka, K...... POS.61 Leotta, K...... POS.46 Köpke, D...... POS.129, POS.145 Lesser, I...... POS.122 Korff, M...... SL.51, POS.81 Leuschner, F...... SL.08 Korkmaz, Z...... SL.37 Leyerer, K...... POS.45 Korotkov, V...... POS.115 Li, F...... POS.100 Kovar, L...... POS.106 Liebau, A...... PST.01 Koziolek, M...... SL.04 Link, A...... POS.62 Kralisch, D...... POS.154 Lipp, P...... SL.07 Kramer, J. S...... POS.4, POS.9 Littmann, T...... POS.75 Kratz, J. M...... POS.5 Liu, C. Y...... POS.120 Krause, A...... POS.47 Löcherer, C...... POS.94 Kreiss, M...... POS.120 Lopattschenko, M...... POS.117 Kretzer, C...... SL.49, POS.65, POS.78, POS.131 Lorenz, K...... SL.16 Kriebs, U...... SL.07, SL.08 Loretz, B...... POS.130 Krieghoff, J...... POS.137 Lorson, T...... POS.149, POS.155 Krogsaeter, E...... SL.06 Löser, R...... POS.10 Kroiss, M...... SL.62 Louis, C...... POS.113

171 • DPhG Annual Meeting 2019 Conference Book AUTHOR INDEX

Lucas, K...... POS.152 Muallem, S...... SL.07 Lüdeke, S...... POS.98 Mueller-Schoell, A...... PST.13 Lühmann, T...... POS.149, POS.155 Mühlber, E...... POS.143 Lunter, D. J...... PST.03 Mühlberg, E...... POS.46 Luong, B...... POS.116 Mulac, D...... POS.123, POS.124, POS.125 Lusic, M...... POS.118 Müller, A...... POS.96 Müller, C...... POS.72, POS.75 Müller, C. E...... POS.33 M Müller, H. K...... PST.15 Müller, M...... SL.44 Mäder, K...... POS.136, POS.144, PST.01 Müller, M. A...... POS.113 Maes, L...... POS.5 Müller, M. B...... PST.15 Maier, F. C...... SL.38 Müller, R...... POS.116 Maison, W...... POS.2, POS.8, POS.56, POS.73 Müller, R. H...... POS.129, POS.147 Makarewicz, O...... POS.128 Müller-Bötticher, L...... POS.128 Mandour, Y...... POS.67 Müller-Knapp, S...... POS.24 Mapp, A. K...... POS.29 Musarara, M...... POS.110 Maqsood, I...... POS.137 Musazzi, L...... PST.15 Marcus, K...... SL.42 Marok, F. Z...... POS.112 Marolt, M...... POS.98 N Martens, M...... POS.18 Martin, S...... POS.142 Nagl, M...... POS.13, POS.14 Martini, E...... SL.44 Nahidino, P...... POS.23 Marzouk, M...... POS.67 Nakayama, K...... SL.01 Maschauer, S...... SL.40 Napierkowski, M...... POS.54 Matuszczak, B...... POS.65 Naundorf, T...... POS.2 Maurer, A...... SL.43 Nawaz, H. A...... POS.138 Mayr, D...... SL.44 Neumaier, B...... POS.76 Mazigo, H...... POS.96 Ngo, V...... POS.28 Mazurek, A...... POS.18 Nguyen, M. A...... SL.30 Mehling, A...... SL.10 Niro, G...... POS.45, POS.79 Meiers, J...... POS.79 Nothjunge, S...... SL.17 Meinel, L...... POS.133 Nyengaard, J...... PST.15 Menche, D...... POS.164 Merk, D...... SL.48, POS.3, POS.9, POS.21, POS.37 Merten, C. A...... SL.45 O Meßner, M...... SL.44 Metzger, E...... POS.51 Obermoser, V...... POS.82 Meyer, S...... SL.08 Obexer, P...... POS.86 Michelet, R...... POS.162, PST.05, PST.12, PST.13 Öhlenschläger, K...... POS.143 Mier, W...... POS.46, POS.47, POS.143, PST.04 Ohlsen, K...... POS.46, PST.04 Miething, C...... POS.17 Ott, I. SL.24, POS.12, POS.43, POS.53, POS.118, Mihajlović, U...... POS.65 POS.158 Miller, A. K...... SL.25 Millies, B...... POS.48, POS.50 Mistry, I. N...... POS.29 P Mitrach, F...... POS.138, PST.02 Mittmann, L...... POS.119 Pace, S...... SL.49, SL.61, POS.157, POS.164, PST.16 Mohamed, A...... POS.25 Pachmayr, J...... SL.44 Mohsen, A...... POS.67 Painer, C...... POS.18 Morales, M...... POS.10 Pamar, P...... SL.32 Moritz, S...... POS.28 Pantsar, T...... POS.30, POS.52, PST.18, PST.19 Moschopoulou, A...... POS.26 Parazzoli, D...... SL.44 Moser, S...... POS.114 Patberg, M...... POS.74 Mrowka, R...... PST.14 Patel, S...... SL.06

DPhG Annual Meeting 2019 Conference Book • 172 AUTHOR INDEX

Paulke, A...... SL.48 Repanas, A...... POS.135 Peric, N...... POS.56 Reßing, N...... POS.57 Petroff, D...... POS.162 Reuss, D...... PST.11 Pfaffenrot, E...... POS.20, POS.23 Ricklin, D...... SL.52 Pfeifer, A...... SL.07 Riddell, A...... POS.110 Pfeiffer-Marek, S...... POS.150 Ritmaleni ...... POS.60 Pfleger, C...... POS.41 Roatsch, M...... POS.11 Phogat, N...... POS.99 Robaa, D...... POS.28, PST.07 Pica, A...... POS.74 Röder, R...... SL.21 Picaud, S...... POS.29 Rohrbacher, C...... POS.70 Pichler, B. J...... SL.38, SL.43 Romp, E...... POS.65 Pierantoni, A...... PST.09 Rosch, M...... POS.152 Pietzsch, J...... POS.10 Roscher, M...... POS.61 Pinnapireddy, S. R...... POS.148 Rosier, N...... POS.13, POS.14 Pletz, M...... POS.128 Ross, T. L...... SL.37, POS.63 Plontke, S...... PST.01 Rossi, A...... SL.49, SL.61, POS.164, PST.16 Pockes, S...... POS.13, POS.14, POS.15, POS.38 Rothenfußer, S...... SL.44 Pollinger, J...... SL.48, POS.9 Ruan, H...... POS.151 Popoli, M...... PST.15 Ruda Vicente, E...... POS.108 Pöschl , U...... POS.152 Ruf, E...... POS.73 Poso, A...... POS.26, PST.18 Ruopp , M...... POS.149 Poso, A. A...... POS.52 Ruopp, M...... POS.155 Praefke, B...... PST.18 Ruppert, C...... POS.99 Prante, O...... SL.40 Prause, A...... POS.53 Prax, K...... POS.104 S Primke, T...... POS.165 Proschak, E...... SL.48, POS.4, POS.9 Sadek, B...... POS.15 Prosek, S...... POS.142 Sager, H...... PST.07 Pudritz, Y. M...... PST.06 Sager, M...... SL.04 Purnomo, H...... POS.60 Saifudin, A...... POS.60 Pyka, C...... POS.115 Salah, M...... POS.25 Pyo, S. M...... POS.129, POS.145, POS.147 Salcher, S...... POS.86 Salehi, N...... POS.151 Samstag, Y...... SL.07 Q Sauer, A...... POS.122 Sauer-Gürth, H...... SL.57 Sauter, M...... PST.04 Schädel, P...... SL.61 Schaeker-Huebner, L...... POS.58 R Schäfer, M...... POS.61 Schäfer, R...... POS.165 Rajan, R...... POS.16 Schäfer-Korting, M...... SL.09, SL.12 Rao, Z...... POS.157, POS.164 Schahla, H...... POS.89, POS.91 Rathke, M...... POS.108 Schätz, B...... PST.07 Rauh, D...... POS.27 Schebb, N.-H...... SL.03 Redhaber, D...... POS.17 Schembecker, G...... POS.117 Reffert, L. M...... SL.37 Scherf-Clavel, O...... SL.62 Reich, G...... POS.141 Schibli, R...... SL.38, SL.43 Reichel, C...... POS.119, POS.165 Schiedel, M...... POS.29 Reichel, C. A...... POS.116 Schihada, H...... POS.13, POS.14 Reigl, U...... SL.40 Schillings, V...... POS.89, POS.91 Reiner, J...... POS.52 Schirmeister, T...... POS.31, POS.48, POS.50 Reischl, G...... SL.38, SL.43 Schlager, H...... POS.104 Renn, C...... POS.33 Schleißmann, J...... POS.89, POS.91 Renner , M...... SL.53 Schlesinger, M...... PST.09

173 • DPhG Annual Meeting 2019 Conference Book AUTHOR INDEX

Schlund, F...... POS.47 Spahn-Langguth, H...... POS.89, POS.91 Schmid, A...... POS.158 Spohner, K...... SL.03 Schmid, M...... POS.138, PST.02 Spycher, P. R...... SL.43 Schmidberger, G...... POS.26 Stadlbauer, S...... POS.61 Schmidberger, M...... PST.03 Steinborn, B...... SL.21 Schmidt, J...... POS.37 Steinhauer, T. N...... POS.59 Schmidt, S...... POS.8 Steinhilber, D...... SL.03, SL.48, POS.9, POS.120 Schmidtkunz, K...... POS.28, PST.17 Stelzle, M...... POS.139 Schmiedeknecht, G...... SL.55 Stevens, M...... SL.32 Schmiedel, K...... POS.104 Stigloher, C...... PST.20 Schmies, C...... POS.33 Stitz, J...... SL.54 Schmitt-Hoffner, F...... POS.139 Straßen, U...... POS.161 Schmutzler, K...... PST.09 Strobel, O...... SL.07 Schöler, A...... POS.11, POS.57 Strödke, B...... POS.119 Schönauer, E...... POS.44 Stump, K...... POS.87 Schöneweis, K...... POS.47 Stumpf, F...... POS.126 Schönland, S. O...... SL.57 Sürün, D...... POS.120 Schönthaler, M...... POS.65 Swyter, S...... PST.17 Schöpf, A. M...... POS.86 Syllwasschy, B...... POS.92 Schubert, J...... POS.89, POS.91 Schubert, S...... POS.78 Schubert, U. S...... POS.78 T Schüle, R...... POS.17, POS.28, POS.51 Schulz, J...... POS.111 Tahoun, M...... POS.25 Schulze, K...... POS.130 Takakura, Y...... SL.20 Schulzke, C...... POS.54 Temml, V...... POS.65 Schulz-Siegmund, M...... POS.137, POS.138, PST.02 Termer, M...... POS.117 Schumacher, D...... SL.07 Thackeray, J...... POS.63 Schuster, D...... POS.65 Thackeray, J. T...... SL.37 Schütz, S...... POS.65 Thamm, J...... POS.131 Schützenmeister, N...... SL.50, POS.68, POS.71 Thornton, C...... SL.38, SL.43 Schwab, M...... POS.112 Thum, T...... POS.63 Schwaderer, M...... SL.17 Tinhofer-Keilholz, I...... SL.12 Schwarthoff, S...... PST.07 Trapp, C...... POS.10 Schweifer, A...... SL.38 Treccani, G...... PST.15 Schwenck, J...... SL.43 Trennheuser, S...... POS.139 Schwenk, R...... POS.165 Troisi, F...... SL.61, POS.164, PST.16 Scita, G...... SL.44 Tsvilovskyy, V...... SL.07 Seeger, J...... PST.12 Tuchscherr, L...... POS.157 Seitzer, M...... POS.96 Tumbrink, H...... POS.27 Selig, R...... POS.7 Türk, D...... POS.109 Selzer, D...... POS.106 Twarock, S...... POS.161 Serhan, C. N...... SL.49, SL.61, POS.157, POS.164 Seyfried, D...... SL.43 Shinsky, S...... POS.84 U Shkodra-Pula, B...... POS.78 Shytaj, I. L...... POS.118 Uhl, P...... POS.46, PST.04 Sieber, S...... POS.115 Ullrich, A...... POS.165 Silva, D. G...... POS.5 Umstätter, F...... POS.46 Simon, P...... POS.162 Urban, S...... POS.47 Sinatra, L...... POS.11 Uster, D. W...... POS.110 Sippl, W...... POS.28, PST.17 Sobeh, M...... SL.57 Sönnichsen, M...... POS.57 V Sorg, B. L...... POS.120 Sotriffer, C...... POS.67 van Oosten, L...... POS.34

DPhG Annual Meeting 2019 Conference Book • 174 AUTHOR INDEX

Vaquero, A...... PST.17 Wirth, A...... SL.07 Vieider, L...... POS.65 Wissenbach, U...... SL.07 Vielmuth, C...... POS.72 Wittlinger, F...... POS.27 Villmann, C...... POS.67 Witzigmann, D...... PST.04 Vogt, D...... SL.07 Wodtke, R...... POS.10 Völkers, M...... SL.08 Wojtyniak, J.G...... SL.44 Vollmar, A...... SL.44, POS.114, POS.115 Wojtyniak, J.-G...... POS.112 Vollrath, A...... POS.78 Wolber, G...... SL.36, POS.80 von Deimling, A...... PST.11 Wolff, C...... SL.12 von Hagen, J...... POS.117 Wolff, J...... SL.17 Voos, K...... POS.44 Wolff, L...... POS.54 Wolfl, S...... PST.14 Wölfl, S. SL.46, POS.102, POS.103, POS.118, POS.158, W POS.163, PST.10 Wölk, C...... SL.22, POS.134, POS.135 Wabitz, G...... SL.07 Wolter, M...... POS.95 Wachtel, H...... SL.29, SL.31 Wombacher, R...... SL.02 Wagner, B...... SL.52 Wössner, N...... PST.17 Wagner, E...... SL.19, SL.21 Wrigge, H...... POS.162 Wagner, K. G...... POS.150 Wulf, J...... POS.55 Wahl-Schott, C...... SL.07, PST.06 Wulle, S...... POS.87 Wang, P...... POS.114 Wünsch, A...... POS.124 Wang, S...... POS.51 Wünsch, B...... POS.16, POS.22, POS.85 Wantoch von Rekowski, A. K...... POS.101 Wurglics, M...... SL.48 Warncke, P...... POS.128 Wuttke, S...... SL.21 Warnken, U...... PST.11 Wawrzinek, J...... POS.87 Weck, S. C...... POS.79 X Wegener, G...... PST.15 Wehrmüller, J. E...... SL.43 Weikert, D...... SL.34 Weindl, G...... SL.11 Weissgerber, P...... SL.07 Y Weißhaupt, P...... POS.20 Weitschies, W...... SL.04 Yahiaoui, S...... POS.4, POS.44 Wentsch, H. K...... POS.30 Yang, Q...... SL.57 Wenzler, S...... POS.28 Yuan, Y...... SL.06 Werner, M...... SL.49 Werner, R. A...... POS.64 Werth, P...... POS.55 Z Werther, P...... SL.02 Werz, O. SL.49, SL.61, POS.65, POS.78, POS.131, Zahler, S...... SL.44 POS.157, POS.164, PST.16 Zender, L...... POS.26, POS.52, PST.18 Wicha, S. G...... SL.64, POS.108, POS.110 Ziegler, M...... SL.02 Wichelhaus, T. A...... POS.4 Ziff, R...... POS.151 Wick, W...... PST.11 Zimmer, C...... POS.48 Wiehr, S...... SL.38, SL.43 Zimmermann, K...... SL.07 Wieland, J. E...... PST.14 Zimmermann, S...... POS.46 Wifling, D...... POS.41 Zimpel, A...... SL.21 Wild, A.-M...... SL.43 Zlotos, D...... POS.67 Willems, S...... POS.3 Zlotos, D. P...... POS.36 Willmann, M...... POS.76 Zöller, E...... POS.65 Winckler, T...... PST.07 Zoschke, C...... SL.12 Wink, M...... SL.57, POS.46 zur Hausen, H...... PL.5 Winter, N...... POS.89, POS.91 Zwirner, S...... POS.26

175 • DPhG Annual Meeting 2019 Conference Book

Geschäftsführer und Leiter der Geschäftsstelle Apotheker Dr. Michael Stein DPhG Geschäftsstelle Varrentrappstr. 40 - 42 60486 Frankfurt Tel.: 069-7191596-0 Fax: 069-7191596-29 Email: [email protected] http://www.dphg.de

Univ.-Prof. Dr. Michael Wink Universität Heidelberg Institut für Pharmazie und Molekulare Biotechnologie Im Neuenheimer Feld 364 69120 Heidelberg Tel.: + 49 62 21 - 54 48 80 Fax: + 49 62 21 - 54 48 84 E-Mail: [email protected]

20.08.2019

DPhG Annual Meeting 2019 Conference Book • 176

NOTES

177 • DPhG Annual Meeting 2019 Conference Book

NOTES

DPhG Annual Meeting 2019 Conference Book • 178 Annual Meeting of the German Pharmaceutical Society – DPhG

Heidelberg, Germany September 01 – 04, 2019 at Heidelberg University www.2019.dphg.de

ISBN 978-3-9816225-6-0