Polish-German Symposia on Pharmaceutical Sciences

1985, May 13-15 1st Symposium Poznan – Halle, held at Poznań University of Medical Sciences

1998, October 5 2nd Polish-German Symposium in Pharmaceutical Sciences – held at Martin- Luther University Halle-Wittenberg

1999, June 24-25 3rd Polish-German Symposium “Pharmacy Before the Third Century” – held at Poznań University of Medical Sciences

2007, June 6 4th Polish-German Symposium “The Pharmacy in the New Century” - held at Martin-Luther University Halle-Wittenberg

2009, May 15-16 5th Polish-German Symposium “New Challenges for Pharmaceutical Sciences” - held at Poznań University of Medical Sciences

2011, May 20-21 6th Polish-German Symposium on Pharmaceutical Sciences “Perspectives for a new decade” – held at Heinrich-Heine University Düsseldorf

2013, May 24-25 7th Polish-German Symposium on Pharmaceutical Sciences "Interdisciplinary research for pharmacy" – held at Medical University of Gdańsk

Scientific Committee

Chair: Prof. Małgorzata SZNITOWSKA

Members:

Prof. Tomasz BĄCZEK – Gdańsk Prof. Halina EKIERT – Kraków Prof. Edmund GRZEŚKOWIAK – Poznań Prof. Renata JACHOWICZ – Kraków Prof. Roman KALISZAN – Gdańsk Prof. Cornelia KECK – Berlin Prof. Peter KLEINEBUDDE – Duesseldorf Prof. Zenon KOKOT – Poznań Prof. Rainer H. MÜLLER – Berlin Prof. Reinhard NEUBERT – Halle Prof. Christian PEIFER – Kiel Prof. Peter PROKSCH – Duesseldorf Prof. Wiesław SAWICKI – Gdańsk PREFACE

Dear Colleagues, Participants of the 7th Polish-German Symposium on Pharmaceutical Sciences

Polish-German meetings devoted to presentation of the achievements in pharmaceutical sciences and bilateral cooperation have been organized successfully as long as since 1985. The meetings on the Polish side were initiated by the Pharmaceutical Faculty of Poznań, joined in by the Pharmaceutical Faculties from Kraków and Gdańsk in 2007. It is an honour for us, scientists in the Faculty of Pharmacy of the Medical University of Gdańsk to welcome you in Gdansk at the 7th Polish-German Symposium on Pharmaceutical Sciences.

Traditionally scientists from pharmaceutical faculties/institutes in Kiel, Düsseldorf, Halle, Berlin, Poznań and Kraków have been invited to the Symposium. Our invitation to the conference have been accepted by the large group of researchers. We can expect that a special energy will be brought by young scientists, since among 170 participants 61 are Ph.D. students. We do hope that some of them will make an effort in the future to keep going on these important neighborhood meetings for many years.

The Symposium is aimed at providing a forum for high level scientific relations between German and Polish researchers working in the area of pharmaceutical sciences. This is the best chance to learn what are the current scientific achievements of the contributing schools of pharmacy and to develop better collaboration among Polish and German researches. Exceptionally friendly atmosphere is a tradition of our meetings and this must help in our future contacts.

The leading topic of the symposium is „Interdisciplinary research for pharmacy”. We are sure that not only our honorary speakers, prof. Reinhard Neubert and prof. Roman Kaliszan, but also seven lecturers invited from all participating universities and authors of 164 poster presentations will show how much can be achieved in pharmaceutical sciences if broader interdisciplinary cooperation and new research tools as well as new fields of exploration are considered.

The event is taking place in the history-breathing and picturesque Gdańsk. We hope that like for every Polish citizen, also for our German friends this will become a city where one wants to return many times. We wish all participants a joyful but also fruitful time here, with intensive scientific discussions and new contacts.

Gdańsk, May 24-25, 2013

Prof. Tomasz Bączek Prof. Wiesław Sawicki Prof. Małgorzata Szmitowska

Vice-Rector for Research Dean Chair of the Symposium Program

Friday 24th May, 2013 14.00- Registration 16.00 Hanging posters at the poster boards 16.00- Opening Remarks 16.45 Plenary Lecture 16.45- Prof. Reinhard Neubert: “Interdisciplinary research in 17.15 Biopharmaceutics: Investigation of the nano-structure of the stratum corneum lipids” 17.15- Plenary Lecture 17.45 Prof. Roman Kaliszan: “Current trends in search for better drugs” 17.45- Coffee at the poster area 18.45 Saturday 25th May, 2013

Jörg Breitkreutz (Duesseldorf): “Recent advances in orodispersible 8.30- drug formulations” 9.00 9.00- Tomasz Gośliński (Poznań): “Porphyrinoid tattoo pigments with 9.30 various missions” 9.30- Coffee break 10.00 10.00- Thomas Groth (Halle): “Polysaccharide-based materials and surface 10.30 modifications for control of cell behavior” 10.30- Christian Peifer (Kiel): “Design, synthesis and biological evaluation 11.00 of kinase inhibitors” Ewa Skrzypczak-Pietraszek: “Production of therapeutically 11.00- important compounds in in vitro cultures of medicinal plants and 11.30 higher fungi 11.30- Coffee break 12.15 Visits in the Departments 12.15- Cornelia Keck (Berlin): “New developments in dermal delivery with 12.45 lipid nanoparticles and drug nanocrystals” 12.45- Krzysztof Cal (Gdansk) “Targeting to the hair follicles by antibiotic- 13.15 loaded lipospheres” 13.15- Luncheon and posters 14.15 14.15- Selected poster presentation – 3 parallel sessions * 15.15 (10 min x 5) 18.00 Departure of buses to Gdynia Gala dinner at “Dar Pomorza” 19.00 Prizes for the best posters Organizing Committee

Chair: Prof. Małgorzata SZNITOWSKA

Members:

Department of Biofarmaceutics and Pharmacodynamics - Assoc. prof. Paweł WICZLING, Ph.D. - Damian SZCZESNY, M.Sc.

Department of Pharmaceutical Technology - Prof. Krzysztof CAL - Magdalena CZAJKOWSKA, M.Sc.

Department of Physical Chemistry – Piotr PIKUL, M.Sc. - Kamil WŁODARSKI, M.Sc.

Department of Pharmaceutical Chemistry – Piotr KAWCZAK, Ph.D. - Ilona OLĘDZKA, Ph.D.

International Relations Office - Ewa KISZKA, M.Sc.

Table of Content

Lectures ...... 1-11 I - Pharmaceutical Technology ...... 12-71 II - Pharmacodynamics and Biopharmacy ...... 72-84 III - Analysis ...... 85-129 IV – Pharmacognosy ...... 130-151 V - Synthesis and Biological Activity ...... 152-179 VI - Pharmaceutical Practice ...... 180-182 Index ...... 183-186 Lectures

1 7th Polish-German Symposium on Pharmaceutical Sciences Lectures

Interdisciplinary research in Biopharmaceutics: Investigation of the nano-structure of the Stratum corneum lipids

Prof. Dr. Dr. h.c. Reinhard H.H. Neubert Institute of Pharmacy, Martin-Luther-University, Halle (Saale), Germany

The outermost layer of the skin, the Stratum corneum (SC) is the main penetration barrier for drugs active ingredients. The SC lipids are playing an essential role concerning drug penetration. Therefore, interdisciplinary research activities are highly necessary for the investigation of the nanostructure of the SC lipids, particularly of the ceramides, is one key research direction in order (1) to understand the barrier function of the human skin and (2) to generate new concepts for influencing dermal and transdermal drug delivery [1 - 3].

In the focus of the lecture are the influence of the ceramides on the nanostructure of the SC lipids and the search for the substantial ceramide subclasses. In the past, results were published concerning key role of the long chain ceramides concerning the barrier function of ther skin. Therefore, influence of the long chain ceramides on the nanostructure of the SC lipids is shown using the CER EOS [4, 5]. Then the impact of short chain ceramides such as CER AP und CER NP is presented. There is a change paradigm because it is shown that the short chain most polar ceramides appear to be essential for the barrier function of the SC. Based on these studies the armature reinforcement model of the SC lipid matrix was proposed: Neutron diffraction results confirm the armature reinforcement model which can be used to illustrate the nanostructure of the SC lipids [4 – 8, 11].

New results were presented in the literature to create the asymmetry model because the SC lipids form asymmetric bilayers in the SC. The short chain ceramides (e.g. the CER NP) have different long alkyl chains (C18 and C24). The asymmetric bilayers consist of one 45 nm bilayer with the C18 alkyl chain and mainly cholesterol molecules and one 65 nm bilayer with the C24 alky chain and mainly free fatty acids. CER NP appears to play a crucial role in the fully extended conformation for the nanostructure of the SC lipids. These results could be confirmed using neutron scattering experiments and a SC lipid model system as well as the Molecular Dynamic Simulation as theoretical method.

Furthermore, the influence of lipophilic penetration enhancing molecules (penetration enhancer), e.g. oleic acid and isopropylmyristate on the nanostructure of the SC lipids is shown [9 – 10, 12].

A new interdisciplinary approach is presented concerning the use of the NMR spectroscopy and the lipid monolayer technique [13]. Dynamics of the ceramides inside of the SC bilayers can be studied using different NMR techniques. On the other hand, the hydrophilic pathway across the SC is most important for modern highly hydrophilic drugs such as peptides. Therefore, the monolayer technique such as the Langmiur Blodgett method is to be applied in order to study the interaction of the ceramide head groups with hydrophilic penetration enhancing molecules such as urea and taurine.

At the end of the lecture first results concerning the penetration of CER [NP] into the human skin are presented.

[1] Kiselev MA, Ryabova NY, Balagurov AM, Dante S, Hauss T, Zbytovská J, Wartewig S, Neubert RHH. Eur Biophys J, 2005; 34:1030-1040.

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[2] Wartewig W, Neubert RHH. Properties of ceramides and their impact on the stratum corneum structure: A review. Part 1: ceramides, Skin Pharmacol Physiol, 2007; 20: 220-229. [3] Kiselev MA, Ryabova NY, Balagurov AM, Otto D, Dante S, Hauß T, Wartewig S, Neubert RHH. Ceramide 6 Influence on the Structure and Hydration of Multilamellar Dipalmitoylphosphatidylcholine Membrane, Poverchnost. X-ray, synchrotron and neutron investigations, 2006; 6: 30-37. [4] Kessner D, Kiselev, M, Dante S, Hauss T, Lersch P, Wartewig S; Neubert RHH. Arrangement of ceramide [EOS] in a stratum corneum lipid model matrix –new aspects revealed by neutron diffraction studies, European Biophy J Biophys Lett 2008; 37: 989-999. [5] Kessner D, Kiselev MA, Hauß T, Dante S, Wartewig S, Neubert RHH. Localisation of partially deuterated cholesterol in quaternary SC lipid model membranes: a neutron diffraction study, Eur Biophys J Biophys Lett, 2008; 37: 1051-1057. [6] Ruettinger A, Kiselev MA, Hauß T, Dante S, Balagurov AM, Neubert RHH. Fatty acid interdigitation in stratum corneum model membranes: a neutron diffraction study, Eur Biophys J Biophys Lett , 2008; 37: 759- 771. [7] Schröter A, Kessner D, Kiselev MA, Hauß T, Dante S, Neubert RHH. Basic Nanostructure of Stratum Corneum Lipid Matrices Based on Ceramides [EOS] and [AP]: A Neutron Diffraction Study, Biophys J, 97, 4, 2009, 1104-1114. [8] Schroeter A, Kiselev MA, Hauß T, Dante S, Neubert RHH. Evidence of free fatty acid interdigitation in stratum corneum model membranes based on ceramide [AP] by deuterium labelling, Biochim Biophys Acta Biomembr, 2009; 1788:, 2203. [9] Engelbrecht T, Schroeter A, Hauß T, Neubert RHH.Lipophilic penetration enhancers and their impact to the bilayer structure of stratum corneum lipid model membranes: Neutron diffraction studies based on the example oleic acid,Biochim Biophys Acta Biomembr, 2011; 1808: 2798-2808. [10] Engelbrecht T, Hauß T, Süß K, Vogel A, Roark M, Feller SE, Neubert RHH, Dobner B. Characterisation of a new ceramide EOS species: Synthesis, investigation of the thermotropicphase behaviour and influence on the bilayer architecture of stratum corneum lipid model membranes. Soft Mater 2011 (7): 8998-9010 [11] Kessner D, Brezesinski G, Funari SS, Dobner B, Neubert RHH. Impact of the long chain omega-acyl ceramides on the Stratum corneum lipid nanostructure. Part 1: Thermotropic phase behaviour of CER[EOS] and CER[EOP] studied using X-ray powder diffraction and FT-Raman spectroscopy. Chem Phys Lipids 2010; 163: 42-50. [12] Engelbrecht TN, Demé B, Dobner B, Neubert RHH. Study of the Influence of the Penetration Enhancer Isopropyl Myristate on the Nanostructure of Stratum Corneum Lipid Model Membranes Using Neutron Diffraction and Deuterium-Labeling. Skin Pharmacol Physiol, 2012; 25: 281-287. [13] Engelbrecht T, Schroeter A, Hauß T, Deme B, Scheidt H, Huster D, Neubert RHH. The impact of ceramides NP and AP to nanostructure of stratum corneum lipid model membranes. Part I: Neutron diffraction and 2H NMR studies on multilamellar models based on ceramides with symmetric alkyl chain length distribution. Soft Matter 2012 (8): 2599-2607.

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Current trends in search for better drugs

Prof. Dr. Dr. h.c. Roman Kaliszan

Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdask, Gdask, Poland

Pharmaceutics emerged as a scientific disciplines complex, dominated by chemistry. Inputs of great pharmacists to chemistry cannot be overestimated. However, more recently, outstanding pharmacists got recognition mostly for their biological/biomedical research. Still, the strength and attractiveness of Pharmaceutics consists in its interdisciplinary nature. Chemistry is mostly behind the search for new chemical entities as “drug candidates”. Innovative drug companies continue to carry on such a search on a large scale, although success rate seems to decrease with time or, at best, there is a stagnation observed worldwide. Nonetheless, the possibilities of chemical synthesis are practically unlimited. Modern methods of effective preselection of the promising structures involve automated synthesis, along with high throughput screening (HTS) and virtual screening (VS) of individual agents. Molecular biology in Pharmaceutics provides not only valuable biopharmaceuticals. It also offers means to diagnose the patients and to control the course of medical treatment. Due to it, a rational guidance of the search for better drugs is possible. Pharmacogenetics and molecular biology tests (Theranostics = Therapeutics + Companion Diagnostics) form concrete basis for personalized pharmacotherapy. Due to the measures supporting the organism weakened by an aggressive drug therapy, the previously impossible, intensive drug therapy became feasible. Pharmacokinetics of drugs can now be improved by overcoming some physicochemical limitations, e.g., insolubility of an active agent in the blood. Another means are due to binding of drugs to physiological proteins. Modern pharmacy offers the drugs and therapeutics delivery systems, which are cost-effective and which make patient’s compliance to the prescribed pharmacotherapy regime’s easier. Still, pharmacist is by no means exempted from duties to give proper advice and to monitor the risks associated with drug therapy.

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Recent Advances in Orodispersible Drug Formulations

Jörg Breitkreutz

Institute of Pharmaceutics and Biopharmaceutics, Heinrich Heine University, Universitätsstraße 1, 40225 Düsseldorf, Germany [email protected]

Orodispersible drug formulations present dosage forms which rapidly disintegrate into multiple particles in the oral cavity. Whereas oral lyophilisates [1] and orodispersible tablets, ODTs [2], have been used for years in various indications throughout the world, orodispersible films, ODFs [3], and orodispersible minitablets, ODMTs [4], have been licensed recently for the European market. Since last year, ODFs are included in the Ph.Eur.. Some further production methodologies such as electrospinning are currently under investigation, but have not reached industrial production scale yet. The gained popularity of orodispersible drug formulations is due to the fact that these rapidly disintegrating dosage forms combine beneficial properties of both liquid and solid dosage forms. They enable improved drug stability over the shelf-life and usually do not contain any potential harmful ingredients like preservatives or antioxidants, but offer precise dosing, ease of administration and swallowing. Hence, they are ideal dosage forms for patients with disease- related swallowing issues [5], in particular for the paediatric and geriatric population [6]. Some recent advances in material science and engineering processes seem to overcome the previously used energy and time consuming freeze-drying technology for preparing orodispersible formulations. Ready-to-use tabletting excipients promise easy and safe production by simple mixing and direct compression. However, the resulting disintegration times are usually much higher as for the oral lyophilisates. More recently orodispersible minitablets of only 2 mm in diameter or even less were introduced which exhibit short disintegration times below 5 s which is very similar to the marketed oral lyophilisates. Complying with “uniformity of dosage units” specifications is a major issue for these small minitablets. Orodispersible films can be produced by various methods, such as solvent-casting, melt extrusion or drug printing. Disintegration times are higher than for lyophilisates and ODMTs, but often the film is not sensed at the application site and therefore, the disintegration times might be of minor importance for patients’ acceptance and therapy adherence. Although the recent developments are quite promising and offer new therapeutic options in various conditions, pharmacopoeial specifications and the requirements of the regulatory bodies are still not harmonized and mostly even useless. Especially mechanical properties of the orodispersible dosage forms, disintegration and dissolution testing need new validated equipment and analytical methods to ensure the quality of the advanced drug dosage forms.

[1] H. Seager, J. Pharm. Pharmacol. 50 (1998) 375-382 [2] D. Brown, Drug Deliv. Technol. 3 (2003) 58-61 [3] E.M. Hoffmann, A. Breitenbach, J. Breitkreutz, Exp. Opin. Drug Deliv. 8 (2011) 299-316 [4] I. Stoltenberg, Breitkreutz J., Eur. J. Pharm. Biopharm. 78 (2011) 462-469 [5] S. Stegemann, M. Gogol, J. Breitkreutz, Int. J. Pharm. 430 (2012) 197-206 [6] J. Breitkreutz, J. Boos, Exp. Opin. Drug Deliv. 4 (2007) 37-45

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Porphyrinoid Tattoo Pigments with Various Missions

Tomasz Goliski

Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, Poznan;e-mail: [email protected]

Tattoos which are ancient forms of body ornamentation are not definitely going to be the real heroes of this talk. The main subject are porphyrinoids. They have found in fact large scale commercial applications as organic pigments and some of them have been used in tattooing. For the last thirty years many organic pigments, like azo dyes, polycyclic amines, dioxazine, phthalocyanine (green, blue, violet), quinacridone, and arylide have been applied as tattoo inks [1,2]. Nevertheless, the porphyrinoids revealed many other applications in research and industry. They are especially interesting compounds for medicine [3] and of growing popularity in engineering and nanotechnology [4]. Many porphyrinoid photosensitizers have been currently applied in photodynamic therapy (PDT), which is a relatively novel treatment method of various diseases, including cancer. PDT requires photosensitizer, which after irradiation with light of an appropriate wavelength generates reactive oxygen species, particularly singlet oxygen that leads to necrotic and/or apoptotic cell death.

In addition, many publications and patents indicate other than medical applications of porphyrinoids, which are constantly becoming more and more popular as sensors, catalysts, and molecules of increasing significance in photonics [5]. Some studies aim to make use of porphyrinoids in nanotechnology. Due to the self-assembly and self-organization, conductivity and optical properties, porphyrinoids seem to be the key to nanomaterials, the properties of which have not been achieved so far [6,7].

Data embracing many aspects of research on porphyrinoids obtained at the Faculty of Pharmacy, Poznan University of Medical Sciences will be presented.

The author acknowledges financial support for the projects granted from the National Sciences Centre - N N404 069440 and 2012/05/E/NZ7/01204.

[1] De Cuyper C., D’hollander D., Materials used in body art. In de Cuyper C., Pérez-Cotapos S. M.L. (eds.) Dermatologic complications with body art, Springer-Verlag, Berlin Heidelberg (2010) 13-28. [2] Goldstein N., Clin. Dermatol., 25 (2007) 417-420. [3] Allison R.R, Sibata C.H. Photodiagn. Photodyn. Ther. 7 (2010) 61-75. [4] Claessens C.G., Blau W.J., Cook M., Hanack M., Nolte R.J.M., Torres T., Wöhrle D., Monatsh. Chem. 132 (2001) 3-11. [5] Wöhrle D., Schnurpfeil G., Makarov S.G., Kazarin A., Suvorova O.N., Macroheterocycles 5 (2012) 191-202. [6] Rodriguez-Morgrade M.S., Stuzhin P.A., J. Porphyr. Phthalocya., 8 (2004) 1129-1165. [7] Walter M.G., Rudine A.B., Wamser C.C., J. Porphyr. Phthalocya., 14 (2010) 759-792.

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Polysaccharide-based materials and surface modifications for control of cell behavior

Thomas Groth Biomedical Materials Group, Institute of Pharmacy, Martin Luther University Halle Wittenberg, Heinrich-Damerow-Strasse 4, 06120 Halle (Saale), Germany

Here glycosaminoglycans (GAG) were chemically activated for direct covalent immobilisation on model substrata while cellulose was regioselectively sulfated and later immobilised with Layer-by-Layer technique to study their interplay with proteins and cells. Glycosaminoglycans like chondroitin sulfate, hyaluronan (HA), heparin (HEP) were thiolated or oxidized to allow covalent immobilisation on either gold or amino-terminated surfaces. Regioselectively sulfated celluloses (CS) with heparinoid activity were used to generate multilayer surfaces by application of layer-by-layer methods. Surface modification was monitored by water contact angle, zeta potential, ellipsometry and atomic force microscopy. Bioactivity of immobilized polysaccharides was studied measuring adsorption of proteins like aggrecan and fibronectin by surface plasmon resonance and investigation of cell adhesion using fibroblasts and C2C12 mouse myoblast cells. Activation of GAGs by thiol or aldehyde groups enabled their covalent immobilisation on gold or self-assembled monolayers (SAM) having either terminal vinyl or amino groups. Modified GAG were also able to bind their natural protein ligands as shown for aggrecan (HA) or fibronectin (HEP). CS could be immobilized in multilayers, when combined with chitosan as polycation and possesses the ability to bind growth factors FGF-2 and BMP-2. Mass and surface properties of multilayers was dependent on sulfation degree. Studies with cells showed that modification of surfaces with polysaccharides has a strong impact on adhesion and spreading of cells. Comparison with native GAG either adsorbed or covalently bound by standard procedures showed that chemical modified polysaccharides possessed a comparative activity. Immobilised activated GAG and chemical modified polysaccharides like sulfated cellulose may be useful to generate bioactive coatings on implant materials and tissue engineering scaffolds that may control adhesion, growth and differentiation of cells. Acknowledgements: This work was funded partly by the European Union Seventh Framework programme under grant agreement No. NMP-SL-2009-229292 (Find&Bind) and Deutsche Forschungsgemeinschaft under grants Gr 1290/7-1 & Gr 1290/7-2.

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Design, Synthesis, and Biological Evaluation of Protein Kinase Inhibitors

Prof. Dr. Christian Peifer

Pharmaceutical Chemistry, Christian Albrechts University, Kiel

Among human protein kinases (PK) as validated drug targets in oncology, receptor tyrosine kinases (RTK) including VEGFR, PDGFR and c-kit are considered to be key for the development of clinically effective inhibitors. Since most PK´s of the kinome use ATP as cofactor for the phosphorylation of proteins in signal transduction pathways, they share a highly conserved ATP binding pocket that is the molecular binding site of most PK inhibitors. Herein, small molecular differences in amino acid identity adjacent to the central ATP pocket provide selectivity filters for specific inhibitor design. In order to enhance potency and selectivity of inhibitors, structure-based design studies on human RTK were performed, suggesting straight forward lead optimization strategies. In our Medicinal Chemistry projects we identified highly potent RTK inhibitors with IC50 values in the nM range which have interesting properties for further development towards anti-cancer drugs.

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Production of some therapeutically important compounds in in vitro cultures of medicinal plants and higher fungi

Ewa Skrzypczak-Pietraszek, Inga Kwiecie, Boena Muszyska, Katarzyna Sukowska-Ziaja, Agnieszka Szewczyk, Agnieszka Szopa, Halina Ekiert.

Chair and Department of Pharmaceutical Botany Jagiellonian University Collegium Medicum Medyczna 9,30-688 Kraków, Poland e-mail: [email protected]

A search for therapeutically important plant and fungal metabolites from in vitro cultures is the leading research topic of the Chair and Department of Pharmaceutical Botany. In the last two years we have investigated the influence of some media components (Aronia melanocarpa callus cultures, Exacum affine shoot cultures, Ginkgo biloba callus cultures) and the impact of light quality (Ginkgo biloba callus cultures) on endogenous accumulation of phenolic acids. The contents of two selected dibenzocyclooctadien lignans in Schisandra chinensis shoot-differentiating and undifferentiating callus cultures cultivated on different variants of media were also analysed. The influence of sulfate concentration on thiols accumulation in Brassica cretica ssp. botrytis shoot cultures was investigated. Enzymatic potential of Aronia melanocarpa and Schisandra chinensis shoot cultures was used for biotransformation of hydroquinone, added exogenously, into its B-D-glucoside, arbutin. The influence of L-tryptophan addition to the medium on accumulation of indole compounds in mycelial cultures of Agaricus bisporus, Boletus badius, Cantharellus cibarius was investigated. Methanol extracts from the biomasses obtained from in vitro cultures were analysed by means of HPLC methods. The concentrations of two tested plant growth regulators, BAP and NAA, in the LS [1] and MS [2] medium variants have a vital influence on the accumulation of phenolic acids in Aronia melanocarpa cultures. The highest total content was obtained on LS medium with BAP-3 mg/l, NAA-1 mg/l and MS medium with BAP-2 mg/l, NAA-2 mg/l (102,31 mg% and 96,55 mg%, respectively). Enhanced accumulation of phenolic acids in Exacum affine cultures was observed after increasing sucrose concentration, reducing the amount of phosphate ions in the medium, and adding the precursor L-phenylalanine (e.g. increase of free compounds from 56 mg% to 182 mg%). The highest total phenolic acid content (78 mg%) in Ginkgo biloba cultures was obtained on MS medium with picloram - 4 mg/l and BAP - 2 mg/l under UV-a irradiation. The highest deoxyschrizandrin and - schizandrin contents (308,51 mg% and 22.09 mg%, respectively) were obtained in Schisandra chinensis shoot differentiating callus cultures on MS medium with BAP – 3 mg/l, NAA – 1 mg/l and BAP – 2 mg/l, NAA – 2 mg/l, respectively. Medium supplementation with 5 mM of sulfate caused the highest increase of cysteine (from 0,51 to 3,59 umol/g d.w.) in Brassica cretica ssp. botrytis cultures. The maximum content of the biotransformation product – arbutin was obtained in Aronia melanocarpa cultures (6,07%). L-tryptophan addition to Agaricus bisporus culture medium caused the highest increase of 5-hydroxytryptophan (from 14,22 mg% to 17,21 mg%) and 5-methyl-tryptamine (from 0,31 mg% to 3,93 mg%). Our results show that plant and fungal in vitro cultures may constitute a potential source of therapeutically important compounds.  1. E.M. Linsmaier, F. Skoog: Physiol. Plant, 18 (1965) 100-127. 2. T. Murashige, F. Skoog: Physiol. Plant., 15 (1962) 473-496.

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New developments in dermal delivery with lipid nanoparticles & drug nanocrystals

Cornelia M. Keck1,2, Rainer H. Müller2

1 University of Applied Sciences Kaiserslautern, Applied Pharmacy Division, Pirmasens, Germany; 2Freie Universität Berlin, Institut für Pharmazie, Pharmazeutische Technologie, Biopharmazie und Kosmetik, Berlin, Germany, e-mail: [email protected]

Drug nanocrystals possess an increased solubility and dissolution velocity when compared to larger micro or macrocrystals. Thus, they are exploited in case of poorly soluble actives, i.e. BCS class II drug actives. Nanocrystals for dermal application possess an increased concentration gradient at the skin and lead therefore to an improved penetration of poorly soluble actives [1]. Lipid nanoparticles are composed of a solid lipid matrix. Lipophilic actives can be encapsulated into this matrix. Upon dermal application lipid nanoparticles form an invisible film on the skin. This leads to a re-enforcement of the natural lipid film of the skin, to an increase in skin hydration and to improved penetration of actives [2]. Both carrier systems are subject to intensive research work in many work groups. Meanwhile they are exploited in many dermal products. Examples are the products platinum rare (company la prairie) or age line one (company Audorasan). New developments with lipid nanoparticles include the use of especially skin-friendly stabilizers, i.e. polyhydroxy surfactants and the production of ultra-small nano lipid carriers with a size below 100nm. Further aspects are the development of improved characterization methods and a method to produce lipid nanoparticles in ultra-small scale, i.e. with a batch size of 0.5ml. New developments with drug nanocrystals include the formulation of poorly soluble antioxidants, e.g. flavonoids. Due to nanonization these particles possess improved penetration properties into the skin and thus improved in vivo properties, e.g. increased antioxidant capacity. Recently methods to produce tailor-sized nanocrystals and for a more efficient large scale production were also developed [3]. Lipid nanoparticles and drug nanocrystals are innovative carrier systems with many positive properties for improved delivery of actives. Recent developments focus on improved production and characterization methods to enable the production of tailor-made particles in the future

[1] Müller, R.H., S. Gohla, C.M. Keck, (2011) State of the art of nanocrystals - Special features, production, nanotoxicology aspects and intracellular delivery. Eur J Pharm Biopharm, (2011)78: 1-9 [2] Müller, R.H., R. Shegokar, C.M. Keck, 20 Years of Lipid Nanoparticles (SLN and NLC): Present State of Development and Industrial Applications. Curr Drug Discov Technol, (2011) 8 (3): 207-27 [3] Keck, C.M., Nanocrystals and amorphous nanoparticles and method for production of the same by a low energy process, in EP11185527.6. (2011)

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Targeting to the hair follicles by antibiotic-loaded lipospheres

Krzysztof Cal

Department of Pharmaceutical Technology, Medical University of Gdansk, Hallera 107, 80-416 Gdansk, Poland e-mail: [email protected]

The pilosebaceous unit is a complex structure that includes the hair follicle, hair shaft, adjoining arrector pili muscle and associated sebaceous gland(s). The sebaceous glands represent an important therapeutic target site because they are involved in the etiology of acne and androgenic alopecia, the latter by expressing 5-reductase (especially in scalp region), which converts testosterone to the more potent 5-dihydrotestosterone. Various approaches aimed at increasing the distribution of a corresponding drug in that region have already been performed for different active pharmaceutical ingredients. To treat effectively such diseases as hair loss and acne, targeted follicular delivery of active agents is desirable, but not all hair follicles are accessible for penetration. It was found that substances penetrate only into the ‘‘active’’ (open) hair follicles, which are characterised by hair growth and/or sebum production. ‘‘Inactive’’ (closed) hair follicles exhibit neither growth nor sebum production. Roxithromycin is a semi-synthetic macrolide antibiotic with proved anti-Propionibacterium acne (anti-acne) and anti-hair loss activities, predisposing it for topical application. Because lipophilic rather than hydrophilic vehicles are able to improve follicular penetration, roxithromycin was incorporated into glyceryl behenate-based lipospheres – lipid, round in shape particles with diameter about 300 nm. Immediately after ex vivo skin application of the lipospheres loaded with roxithromycin and with the fluorescent dye, the superficial aggregations in the follicular openings were visible. No fluorescence was observed deeper in the skin tissue. After 1 h penetration time, skin sections revealed fluorescence only in the areas of the hair follicles, even up to 1.5 mm in depth. Differential stripping performed in vivo demonstrated the presence of roxithromycin in the hair follicles.

Acknowledgements This work was supported by grant No N N405 674740 from the National Science Centre (Poland).

K. Cal, H. Wosicka: Targeting to the hair follicles: Current status and potential, J. Dermatol. Sci., 57 (2010) 83-89. K. Cal, H. Wosicka: WIPO 10/C PL401625.

11 I – Pharmaceutical Technology

12 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 1

Preliminary studies on the formation of the inclusion complexes between porphyrazines and -cyclodextrin

ukasz Sobotta1, Maciej Wrotyski1, Wojciech Szczoko2, Marcin Wierzchowski2, Tomasz Goliski2, Jadwiga Mielcarek1

1Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland; 2Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland; [email protected]

Introduction: Porphyrazines (Pzs) are analogues of phthalocyanines, which have been intensively researched due to their interesting physicochemical properties like self-assembly, fluorescence, coordination of ions and small molecules. Many Pzs generate singlet oxygen after irradiation with light. This property has been applied practically to treat cancer and is called Photodynamic Therapy (PDT). For the last 50 years many groups of photosensitizers have been applied in PDT, including various porphyrins, phthalocyanines, and chlorins. As Pzs are nearly insoluble in water, which obviously generates application problems, many attempts have been employed to design their pharmaceutical formulation with liposomes or complexes with cyclodextrins (CDs) [1].

Methods: The aim of this work was to determine the stoichiometry of the complexes formed between novel Pzs and -cyclodextrine (-CD) using a Job’s plot approach. The formulae of substances investigated are presented in Fig.1. Mixtures of -CD and Pzs were stirred at 80 ºC for 24 h in DMSO and then their UV-Vis and fluorescence spectra were measured [2].

Results: As shown in Fig.1. complex of Pz-1 and –CD was formed at the ratio of 1 to 4. Nevertheless, the formation of other complexes cannot be excluded at this stage. Analysis of the interaction between the Pzs tested and -CD may suggest incorporation of one of four 2,5- dimethylpyrrolyl moieties present in the macrocyclic periphery into CD’s hole as shown in Fig.2 (inset). [1] Allison RR, Downie GH, Cuenca R, Hu X, Childs CJH, Sibata CH. Photodiagn Photodyn 2004;1:27-42; [2] Tau P, Ogunsipe AO, Maree S, Maree MD, Nyokong T. J. Porphyr. Phthalocya. 2003;7:439-446.

This study was supported by the National Science Centre under Grant No. N N404 069440.

13 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 2

The ready-to-use excipients for preparation of orally disintegrating minitablets with carbamazepine

Hanna Kotowska1, Anna Madanecka2, Magorzata Sznitowska1 1Department of Pharmaceutical Technology, Medical University of Gdask, Gdansk, Poland 1Pharmaceutical Works Polpharma SA, Starogard Gdaski, Poland [email protected], [email protected]

Introduction: Orally disintegrating minitablets (ODMTs, diameter 1-3 mm) are promising peadiatric dosage form combining advantages of minitablets and liquid form. As ODMT disintegrates in oral cavity for maximum 60 seconds is more versatile dosage form comparing to standard minitablets. It can be suitable even for infants and toddlers while minitablets are preferred for children at the age 2-5 years. In the presented study ODMTs containing carbamazepine (1.5 - 2.5 - 3.2 - and 5 mg strenghts) were produced. Investigation of tabletting process and physical parameters of minitablets was performed.

Methods: The carbamazepine ODMTs (2 mm, 2.5 mm and 3 mm size) and orally disintegrating tablets (10 mm) were produced by direct compression method (Korsch XL 100, spherical punches). Compression forces of 5kN, 10kN, 15kN and 20kN were applied. Suitability for tabletting process of the ready-to-use commercial excipients (Prosolv ODT, Parteck ODT, Pearlitol Flash and Ludiflash) dedicated for manufacturing of orodispersible tablets was verified. Tablet masses were characterized with particle size (microscopy and sieve analysis), bulk and tap density, flowability and angle of repose. Impact of the main compression pressure on: hardness, friability and disintegration time of tablets/minitablets was studied.

Results: Flowability and angle of repose results for all tablet masses were satisfactory for minitableting process. The mass uniformity of most minitablet formulations was confirmed. Maximum hardness was obtained for minitablets/standard tablets composed of Parteck ODT, lower for Ludiflash and Prosolv ODT and the minimum - for Pearlitol Flash. Satisfactory friability (below 0.5%) was obtained for minitablets/tablets with Parteck ODT, Ludiflash and Prosolv ODT. Disintegration time below 60 seconds in pharmacopoeial apparatus was measured for 3 mm minitablets consisting of Parteck ODT and Ludiflash (samples obtained for all compression forces). Longer disintegration times were obtained for 3 mm minitablets with Prosolv ODT and Pearlitol Flash, despite their smaller hardness.

Conclusion: Two ready-to-use excipients - Parteck ODT and Ludiflash - appeared to be the most suitable for manufacturing of carbamazepine minitablets with satisfactory physical parameters. The correlation between main compression pressure and hardness, disintegration time or friability was similar for standard (10 mm) tablets and for minitablets. During friability test it was observed that minitablets had lower tendency for capping process comparing with conventional tablets. In addition, application of ready-to-use excipients, can accelerate the development process.

14 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 3

Using a multilayer tablets form for increasing stability of two active pharmaceutical ingredients combined in one tablet Maciej Poom1, Wiesaw Sawicki2

1Formulation Department, Polpharma S.A., Pelpliska 19, 83-200 Starogard Gdaski, Poland, e-mail: [email protected] 2Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland

Introduction: Increasing amount and level of impurities in tablets containing two active pharmaceutical ingredients (API) makes difficulties with establishing a shelf live. Especially when both API`s give interactions with each other. Such interactions are observed in tablets containing atorvastatin calcium and amlodipine besylate. Possible solution is to separate API`s in two different layers and additionally introduce neutral third barrier layer between them. The main purpose of this study was to assess the impact of API separation in comparison with standard, one layer tablets.

Methods: The three layer tablets were produced with granulate of atorvastatin calcium prepared in wet granulation process [pregelatinized starch 1500, microcrystalline cellulose, magnesium carbonate, hydroxypropyl-cellulose, polysorbate 80, croscarmellose sodium, colloidal silica, magnesium stearate] and dry mixed amlodipine besylate [mannitol, microcrystalline cellulose, colloidal silica, croscarmellose sodium, magnesium stearate] and also dry mixed barrier layer containing hydroxypropyl cellulose, lactose monohydrate and colloidal silica. Tableting process was performed on Korsch XL-400 tablet press in tree layer configuration. Initial studies involved the identification of the degradants confirming its chemical structure and degradation pathway. This was done by using HPLC technics. Stability study was performed in accelerated ICH conditions: 40°C and 75% RH, samples were stored for 6 months.

Results: Some difference in purity of trilayer tablets comparing to standard ones were observed. Impurities coming from interaction between two API`s, are not observed and other degradation products remain on comparative level to standard tablets. It was found that, thickness of barrier layer and its type has no influence on final results. Also physico-chemical parameters of multilayer and standard tablets were checked.

Conclusions: On the basis of results it was found that active substances separation in different layers protect formation of impurities coming from interactions. This techniques seems to be useful to obtain combined products of two incompatible active substances in one dosage form.

15 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 4

Synthesis and water-dispersion of ZnO-based quantum dots

Marika Wojewódzka1,2 , Janina Lulek1, Raphaël Schneider2

1Department of Pharmaceutical Technology, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Pozna, Poland, [email protected] 2Université de Lorraine, LRGP - Laboratoire Réactions et Génie des Procédés, UPR 3349 1 rue Grandville, BP 20451, 54001 Nancy Cdex, France

Introduction: Quantum dots are among the most promising tools in the nanomedicine area. The potential uses of these colloidal semiconductor nanocrystals include bioengineering, bioanalytical detection, bioimaging, as well as targeted drug delivery, in vivo monitoring, and photodynamic therapy [1,2]. The possibility of such wide applications is attributed to their unique size-dependent photophysical properties, like narrow and symmetrical emission spectra, broad absorption spectra, high quantum yield, and simultaneous excitation by a single light source. However, toxicity of the QDs is a serious limitation in biomedical applications. Synthesizing biocompatible nanocrystals, composed of weakly toxic ZnO became a leading route in quantum dots nanofabrication. However, uncapped ZnO QDs tend to aggregate and are not stable due to high surface reactivity. In this work various approaches were presented which can be useful to eliminate these drawbacks.

Methods: ZnO QDs capped with oleic acid as well as ZnO/MgO and ZnO/ZrO2 core-shell QDs were synthesized in a sol-gel process and characterized by UV-Vis and fluorescence spectroscopies, transmission electron microscopy (TEM), and powder X-ray diffraction (XRD). Additionally silanization with 3- aminopropyltrimethoxysilane and 3-[2-(2- aminoethylamino)ethylamino]propyltrimethoxysilane were performed and characterized using the same methods. Results: Conjugating with oleic acid as well as introducing an inorganic shell made of higher band-gap semiconductor were the routes to obtain better stability and protection of the ZnO core. As water-stability is the fundamental criterion to apply these nanoparticles in biological applications, silanization was performed, which resulted in exchange of hydrophobic ligands into hydrophilic ones. All the QDs showed good stability and preserved luminescent properties. Conclusions: Obtained results have demonstrated that synthesized materials are very promising and due to good dispersibility, preserved optical properties, flexible surface and non-toxicity should be more developed to be successfully used in medical applications, such as e.g. cancer therapy. Acknowledgement: This study was partially supported by PUMS grant N0 502-01- 03314429-03439

[1] J. Drbohlavova, V. Adam, R. Kizek, J. Hubalek, Quantum dots- Characterization, Preparation and Usage in Biological Systems, Int. J. Mol. Sci, 10 (2009) 656-673. [2] R.-O. Moussodia, L. Balan, R. Schneider, Synthesis and characterization of water-soluble ZnO quantum dots prepared through PEG-siloxane coating, New J Chem, 32 (2008) 1388-1393.

16 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 5

Bioactive silica-based nanomaterials for doxorubicin delivery: evaluation of structural properties associated with release rate

Magdalena Prokopowicz

Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, e-mail: [email protected]

Introduction: The sol-gel process is a very common method for preparing mesoporous sol- gel derived silica xerogels, which find applications in pharmaceutical fields as carrier of drugs. This study aimed at the development of a novel granule-type formulation, composed of a sol-gel derived bioactive xerogel based on a silica-polydimethylsiloxane composite containing calcium and phosphate ions, as a sustained release delivery for an anticancer drug (doxorubicin, DOX).

Methods: The preparation of the DOX loaded granule-type formulation was performed using the sol-gel casting method. Liquid-form of DOX was added to the sol before moulding. The DOX-loaded formulation was immersed in a simulated body fluid (SBF) at pH 7.4 and 37.0#C to study the effect of different drug loads on the release profiles. The bioactivity of these composites, associated with the nucleation of an apatite layer on its surface, was examined in SBF under static mineralization conditions during 50-days. FTIR spectroscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, powder X-ray diffraction, N2- porosimtery were used to monitor changes in bulk and surface of the drug carrier.

Results: It has been observed that all DOX loaded granule-type formulations are characterized by mesoporosity with the uniform pore-size-distribution (~40 Å) and the surface area 340 m2 g-1. The slow drug release from all formulations followed zero order kinetics for 1-50 days. After 50 days of mineralization, a semicrystalline carbonated hydroxyapatite with a highly developed porous structure was formed, indicates their bioactive character (Fig. 1).

(A) (B)

Fig.1 SEM micrograph of DOX loaded mesoporous granule-type formulation before (A) and after 50 days of mineralization in SBF solution (B).

Conclusions: The sustained release of DOX molecules loaded in the granule-type formulations was confirmed, indicating that granule is a candidate for use as a drug carrier in drug delivery systems. Besides its potential carrier properties, this material was highly bioactive (surface-reactive) during in vitro mineralization. Acknowledgments: The author would like to thank the National Centre for Science of Polish State (project no. N N405 024440 for providing financial support to this project.

17 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 6

Amorphization of the indapamide as a method to improve solubility

Wojciech Gostomczyk, Wiesaw Sawicki, Krystyna Mojsiewicz-Piekowska, Damian wieczkowski, Katarzyna Homernik

Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, e-mail: [email protected]

Introduction: The poor oral bioavailability arising from poor aqueous solubility should make drug research and development more difficult. Therefore the various approaches have been developed with a focus on enhancement of the solubility of poorly water-soluble active pharmaceutical ingredients (APIs). A special place is taken by methods of amorphization of a crystalline API [1-3]. The main purpose of this study was to assess the impact of amorphization of indapamide (IND) on the physical properties, particularly solubility. In addition, the aim of the study was a comparative assessment of influence of form of indapamide, crystalline and amorphous obtained by various techniques (quenching, cryogenic grinding, ball milling).

Methods: Initial studies involved the identification of the form of the drug and confirming its chemical structure. Microscopic images, showing the morphology of the crystalline and amorphous forms, were performed and compared. In order to confirm the identity of the IND, 1H- NMR and FTIR spectroscopy were used. As to verify receipt of the amorphous form, the X-ray diffraction XRD and DSC analysis were applied. To determine the saturated solubility of the crystalline and amorphous forms IND in various medium the RP-HPLC method was used.

Results: There was no significant difference in improving the solubility of crystalline IND using different media. It was found that, irrespective of the technique amorphization in each medium model, IND in amorphous form shows an increase in solubility compared to the crystalline form (13 - 42%). In each case there was an increase in the solubility depending on the temperature. Indapamide greatest solubility (151 mg/l) was obtained for cryomilled sample at 37 °C using as a medium 0.1 M hydrochloric acid. The increase in solubility compared to crystalline IND was 42%. It was observed that for each medium, regardless of the temperature, between solubilities for the cryomilled and ball milled sample slight difference exist. The values are higher than for the vitrified sample.

Conclusions: On the basis of results it was found that regardless of methods of amorphization, in each medium, the amorphous forms IND had increase solubility compared to crystalline IND.

[1] P. epek, W. Sawicki, K. Wodarski, Z. Wojnarowska, M. Paluch, L. Guzik: Effect of amorphization method on telmisartan solubility and tableting process, Eur. J. Pharm. Biopharm. 83 (2013) 114-121. [2] P. Ghugare,V. Dongre, P. Karmuse, R. Rana, D. Singh, A. Kumar, Z. Filmwala: Solid state investigation and characterization of the polymorphic and pseudopolymorphic forms of indapamide, J. Pharm. Biomed. Anal. 51 (2010) 532–540. [3] S. B. Murdande, M. J. Pikal, R. M. Shanker, R. H. Bogner: Solubility Advantage of Amorphous Pharmaceuticals: I. A Thermodynamic Analysis, J Pharm Sci., 99 (2010) 1254–1264.

18 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 7

The influence of energy input on producing Nanostructured Lipid Carriers

C.M. Keck1,2, R.H. Müller2, T. Junmahasathien2,

1 Applied Pharmacy Division, University of Applied Sciences Kaiserslautern, Pirmasens, 66953, Germany, [email protected]; 2 Institute of Pharmacy, Freie Universität Berlin, Berlin, 12169, Germany

Introduction: Nanostructured Lipid carriers (NLC) are obtained from blending different lipids to form a matrix which provides a lot of space to accommodate active ingredients. The advantages of NLC are of interest especially for cosmetic field. Cosmetic products are obtained by adding a NLC pre-concentrate, i.e. containing high lipid concentrations to a cream or gel. Up to date little is known about the influence of different lipid contents on optimal production parameters. Therefore, the target of this study was to investigate the influence of the energy input during the production, on size and physical stability of NLC containing different lipid concentrations. Methods: The mixed lipid (10%-25% in the formulation) was melted apart from water phase. Plantacare® 2000 UP 2% was used as a surfactant. The hot water phase was mixed into the melted lipid phase at 80°C and premixed by Ultra-Turrax at 8,000 rpm for 30 minutes. Then the pre-mixed emulsion was homogenized by high pressure homogenizer (HPH) at 500 or 800 bar for 3-10 cycles. The particle size, polydispersity index (PI) and zeta potential were investigated by Photon Correlation Spectroscopy (PCS) and Laser Diffractometer (LD). Results: The optimum energy input depended on the content of lipid in the product. Furthermore, the energy input affected not only the particle size and zeta potential but also the stability of the product. For the HPH at 500 bar, more cycle applied, led to the reduction of particle size which can be seen in 20% and 25% of lipid. While, more HPH cycle did not affect the particle size of 10% (145 nm) and 15% (157 nm) lipid content. On the other hand, HPH at 800 bar could further decrease the particle size for the formulations containing 10% and 15% lipid (123 nm and 142 nm, respectively). However, the products containing 10% and 15% lipid, which were produced at 500 bar, were stable for at least 6 months. Whereas, all formulations, produced at 800 bar were stable for only 60 days. The reason for reduced stability is not only due to the higher energy input. It is also due to the smaller particle size. Smaller sizes possess a larger surface, thus more surfactant is required to coat each particle and prevent particles from aggregation. 2% of surfactant was inadequate for stabilizing particle size smaller than around 150 nm. Interestingly, the energy input, i.e. the number of homogenization cycles, also affected the zeta potential. All formulations showed a minimum of ZP after 5 homogenization cycles. Larger ZP was measured after 3 and 10 HPH cycles, respectively. Changes in ZP indicate changes in the interaction of the stabilizer and the lipid matrix during homogenization. Moreover, ZP decreased with increasing lipid content of the formulations, which might indicate a thinner surfactant layer in formulations with higher lipid content. The reason is a larger surface area, i.e. more particles, which needs to be covered in formulations with higher lipid content by the surfactant. Conclusion: The number of homogenization cycles required to obtain physically stable lipid nanoparticles was found to depend on the lipid content. Higher lipid contents require more energy input and probably higher contents of stabilizer.

19 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 8

Lecithin semisolid dispersion as a matrix for parenteral implants

Marcin Paczek, Anna Bogucewicz, Magorzata Sznitowska

Department of Pharmaceutical Technology, Medical University of Gdask, Hallera 107, e-mail: [email protected]

Introduction: Lecithin and separate phospholipids are used for years as pharmaceutical excipients in different types of parenteral formulations: submicron emulsions, mixed micelles and liposome dispersions. New parenteral drug formulations containing lecithin are also proposed, among them forms (e.g. Vesicular Phospholipid Gels), which release an active substance in a sustained manner [1]. The aim of the study was to prepare and characterize semisolid implants containing egg or soy lecithin (40-60%) dispersed in different aqueous media. To some formulations (containing 50% of egg lecithin) ondansetron hydrochloride (OND) was incorporated as a model drug. Methods: In order to prepare semisolid implants, appropriate amounts of lecithin were mixed (using Unguator mixer) with aqueous phase: water (W) phosphate buffer (PB), 2.4% glycerol (GL), 10% macrogol (PEG) or 10% poloxamer (PL). OND (16 mg/g - final concentration) was added to the aqueous phase before homogenization step. Implants were examined visually and microscopically (also in polarized-light microscope). Physicochemical tests comprised of rheological analysis (using texture analyzer and rheometer), determination of drug content and dissolution test (dialysis-tube method). Results: All prepared implants were semisolid dispersions, which consistency depended on lecithin concentration (40% dispersions were dense and cream-colored, 60% were rigid and dark yellow). Microscopic analysis revealed, that in all formulations small, multivesicular structures are present, which are able to reflection of polarized light (similarly to liposomes). The size of the vesicles decreased with the increase of lecithin concentration and depended on the type of aqueous media (the smallest particles - <1 μm - were found in PL formulations). Rheological test indicated, that the hardest consistency possessed gels contained egg lecithin (60%) dispersed in PB or PL. Incorporation of OND to the gel structure in form of aqueous solution (W, GL) or suspension (the other media) resulted in reduction of lecithin particles size. For all formulations prolonged release of OND was demonstrated (after 24 h, 47,9% – 82,0% of the dose was released), while the kinetics of the process depended on the type of aqueous media. Conclusion: Phospholipid gels contained 50% of egg lecithin can be used as stable matrices for implantation. Into these matrices active substance can be introduced in form of suspension or aqueous solution, and it was shown, that the drug may be released from the formulation in prolonged manner (even over a period of few days). [1] M. Brandl:Vesicular Phospholipid Gels: a technology platform, J. Liposome Res., 17 (2007) 15–26.

20 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 9

Glycyrrhetinic acid smartCrystals®: Production, characterization & stability study Moumita Mishra1, Biswadip Sinha1, Ranjita Shegokar1, Rainer H. Müller1, Sven Gohla2 1Freie Universität Berlin, Institute of Pharmacy, Dept.of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Kelchstr. 31, 12169 Berlin, Germany 2Research & Development of the la prairie group - Juvena, Volketswil, CH-8604, Switzerland Email: [email protected]

Glycyrrhetinic acid (GHA) is obtained from licorice root (Glycyrryha glabra). GHA has excellent antioxidant activity [1], but poor skin penetration property [2]. This is because of the fact that GHA has poor aqueous solubility and this leads to poor skin penetration. The purpose of this study was to produce GHA smartCrystals® to increase its dissolution velocity and skin adhesion property leading to enhanced skin penetration. The smartCrystal® technology is defined as a “tool box” of different combination processes for the production of smaller size nanocrystals [3]. GHA [5% (w/w)] was dispersed in 1% (w/w) aqueous solution of Plantacare 2000 UP to prepare the coarse suspension. smartCrystals® were prepared by using pearl milling (PM, Bühler PML-2, Bühler AG, Switzerland) followed by high pressure homogenization (HPH, Micron LAB 40, APV Deutschland GmbH). Particle size of the smartCrystals® was measured using photon correlation spectroscopy (PCS, Zetasizer Nano ZS, Malvern Instruments, UK) and laser diffractometry (LD, Mastersizer 2000, Malvern Instruments, UK). The zeta potential of the GHA nanosuspension was measured in conductivity adjusted water (50μS/cm) and in the original dispersion media. Light microscopy was performed to detect the presence of aggregates or larger particles. The samples were stored at 4°C, 25°C and 40°C for stability assessment. The crystallinity of the particles was analyzed by powder X-ray diffractometry (PXRD, Philips X-ray Generator PW 1830, Amedo, The Netherlands). Saturation solubility of the drug from the coarse suspension and the smartCrystals® were determined at 25°C in water, after equilibration for 7 days. The smartCrystals® had a mean hydrodynamic diameter (z-average diameter) of 155 nm and polydispersity index of 0.177, when measured by PCS. The data were well correlated with the d (0.5) value obtained from LD, i.e. 121 nm. The zeta potential of -39.8 mV in water and -31.5 mV in original dispersion medium (1% Plantacare 2000 UP solution) indicated a very good physical stability of the particle in suspended state. The formulation was stable for 1.5 years in terms of its particle size and crystallinity. The equilibrium solubility (7 days) of the drug from the coarse suspension and the smartCrystals® were 4 μg/ml and 80 μg/ml, respectively. Second generation smartCrystal® technology has been proved to be successful to generate smaller size GHA nanocrystal, which is stable for almost 1.5 years. The smaller sized GHA nanocrystal has improved saturation solubility in water. GHA nanocrystal formulations can be further admixed to cosmetic or pharmaceutical bases for dermal applications.

[1] X. L. Li, A. G. Zhou, L. Zhang, W. J. Chen: Antioxidant Status and Immune Activity of Glycyrrhizin in Allergic Rhinitis Mic. Int. J. Mol. Sci., 12 (2011) 905-916. [2] K. Sasaki, S. Yonebayashi, M. Yoshida, K. Shimizu, T. Aotsuka, K. Takayama: Improvement in the bioavailability of poorly absorbed glycyrrhizin via various non-vascular administration routes in rats, Int. J. Pharm., 265 (2003) 95-102. [3] C.M. Keck, S. Kobierski, R. Mauludin, R. H. Müller: Second generation of drug nanocrystals for delivery of poorly soluble drugs: smartCrystals technology, Dosis, 24 (2008) 124-128.

21 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 10

The potential antioxidant capacity of coenzyme Q10-loaded ultrasmall lipid nanocarriers (usNLC)

Cornelia M. Keck1,2, Nuttakorn Baisaeng1, Daniel Peters1, Rainer H. Müller1 1Freie Universität Berlin, Institut für Pharmazie, Pharmazeutische Technologie, Biopharmazie und Kosmetik, Berlin, Germany, e-mail: [email protected] ; 2University of Applied Sciences Kaiserslautern, Applied Pharmacy Division, Pirmasens, Germany

Introduction: Coenzyme Q10 (CoQ10) is a vitamin like, poorly water soluble compound with strong antioxidant capacity, making it an interesting drug for treating oxidative stress. However, the bioavailability upon dermal application is very limited. Lipid nanoparticles, e.g. solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) can be exploited to increase the bioavailability of lipophilic, poorly water soluble drugs [1]. These particles are typically produced by hot high pressure homogenization, leading to particle sizes in the range from about 150 to 300 nm. For both SLN and NLC, it is known that the particle size is directly related to their bioactivity, i.e. the smaller size of the nanoparticles, the bioavailability is higher. Preferentially particles should be < 100 nm [2]. Therefore, the aim of this study was to evaluate the physicochemical properties, the in vitro release profile and antioxidant capacity of CoQ10- loaded ultrasmall lipid nanocarriers (usNLC) with particle size below 100 nm compared to classical nanostructured lipid carriers (NLC) and a nanoemulsion (NE).

Methods: CoQ10-loaded NLC, NE and usNLC were produced by hot high pressure homogenization. Size, zeta potential, physical state, physical and chemical stability were analyzed for all systems. The in vitro release profile and the antioxidant capacity of the usNLC was determined and compared to the AOC of the NLC and NE (DPPH test).

Results: NLC, NE and usNLC with a CoQ10 content of 5% w/w were produced. The size of the NLC was approximately 210 nm, the size for the NE and the usNLC was similar (about 85 nm). All samples possessed a similar good physical and chemical stability over a period of 3 months. Differential scanning calorimetry (DSC) confirmed a solid matrix for usNLC to be a super cooled melt. In vitro release studies using static Franz diffusion cells revealed the highest accumulated amount of CoQ10 for the usNLC, followed by the NE and NLC, respectively. The AOC was highest for the usNLC, followed by the NE and NLC. Conclusions: Ultrasmall NLC with a size below 100 nm possess good physical and chemical stability. They proved to enhance the antioxidant capacity and in vitro release profile of CoQ10, when compared to larger NLC and a nanoemulsion. Therefore, usNLC might be a promising carrier system for the improved delivery of CoQ10 into the skin.

[1] Müller, R.H., Lipid nanoparticles: recent advances (2007), Adv Drug Deliv Rev 59(6): p. 375-76. [2] Keck, C. M., Muchow, M. (2009). Nanonized testosterone formulations for improved bioavailability, European Patent Application No. 09 003 370.5, 8 March 2009.

22 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 11

Influence of film forming materials on preparation and properties of orodispersible strips

Katarzyna Centkowska1, Malgorzata Sznitowska1

1 Department of Pharmaceutical Technology, Medical University of Gdansk, Hallera 107, Gdansk 80-416, e-mail: [email protected]

Children’s ability to accept swallowing of solid forms is usually approximately defined as the age above 6 years [1]. Polymer films rapidly dissolving in oral cavity (ODFs) can serve as an alternative for solid dosage forms dedicated for preschool children [2]. The aim of this study was to compare different polymer types and plasticizers in orodispersible films. Casting and solvent evaporation method was applied. Water-soluble polymers such as: cellulose deriviatives (hydroxypropylmethylcellulose, methylcellulose, hydroxyprophyl- cellulose), synthetic polymers (polyvinyl alcohol, polyvinylpyrrolidone) and natural materials (pullulan, sodium alginate) were used. Influence of plasticizers concentration and type, on physical properties of films, were examined. Liquid Plasticizers (glycerol, propylene glycol and macrogol 400) and solid plasticizer (citric acid and sorbitol plasticizers). Films with sartans as model drug substance were made from hypromelose as a matrix polymer. Physical examinations were undertaken: weight, thickness, tensile strength, microscopic observation and disintegration/dissolution behaviour. Drop shape analysis was used to determine wetting behavior of films [3]. All drug-free films dissolved very quickly within 30 s and had proper texture. The lowest contact angle was achieved for pullulan (50°), hypromellose and hydroxypropylcellulose (55°) while the highest one for methylcellulose (80°). The percent elongation was equal to 200% in case of polyvinyl alcohol while for the rest of membranes was in range between 5 to 10%. The elasticity above 10% results in film deformation during packaging and application. Polymer films of satisfying physical properties and short disintegration time (<1 min) with hypromelose have been prepared for valsartan solution. In case of candesartan cilexetil it was only possible to prepare suspension type films due to low solubility of active substance both in solvent (ethanol and water) and plasticizer. Incorporation of both substances resulted in significantly longer disintegration time and reduced wettabillity in comparison to drug-free film.

[1] J. Krause, J. Breitkreutz: Improving drug delivery in pediatric medicine, Pharm Med, 22 (2008) 41–50. [2] R. Dixit, S. Puthali: Oral strip technology: Overview and future potential., J Control Release, 139 (2009) 94-107. [3] V. Garsuch, J. Breitkreutz: Novel analytical methods for the characterization of oral wafers, Eur J Pharm Biopharm, 73 (2009) 195-201.

23 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 12

In vitro dissolution behavior of lipid matrices in ethanolic media

Klaus Knop, Peter Kleinebudde

Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University Duesseldorf, Germany

In vitro dissolution tests of oral solid dosage forms are performed to ensure a specified product quality and/or to predict the in vivo dissolution behavior. FDA guidelines [1, 2] contain several recommendations for the composition of the dissolution medium to mimic the physiological conditions of the gastro intestinal tract, differing in e.g. buffer, pH, ionic strength, enzymes, and surfactants. If a solid dosage form with modified release characteristic is taken up with an alcoholic beverage, the ethanol may change the dissolution behavior and faster drug release - even dose dumping can occur [3, 4]. The aim of the present study was therefore to evaluate the influence of ethanol on the drug release from lipid matrices with prolonged drug release. Small lipid matrices (1 mm diameter, several mm length) containing 50 % anhydrous theophylline as model drug were prepared by solid lipid extrusion (twin screw extruder, extrusion temperature 25°C/33°C). Three different lipids were used as matrix substances: glyceryl trimyristate (Dynasan 114), glyceryl tristearate (Dynasan 118) and glyceryl distearate (Precirol 5 ATO). Dissolution tests were performed using the basket apparatus (Ph.Eur. 2.9.3) with lipid extrudates containing 150 mg theophylline. The dissolution medium consisted of pure water or 20 % rsp. 40 % ethanol, imitating a worst case scenario. All extrudates showed the typical release behavior of insoluble matrix systems. The release profiles could be linearized by plotting the released amount against the square root of time (up to an amount of 70 %). All extrudates released the drug faster in the ethanolic media than in water. The differences could not be explained solely by the higher solubility of theophylline in the ethanolic media. The release from the triglycerides showed large differences between 0 and 20 % ethanol but only small differences between 20 and 40 % ethanol, whereas the release from the diacylglycerol was also strongly influenced by the ethanol content between 20 and 40 %. The results of the in vitro drug release indicate that the in vivo release and in the end the bioavailability of orally administered lipid solid dosage forms can be strongly affected by the simultaneous intake of alcohol in higher concentrations. Acknowledgement: The authors would like to thank Gustavo Petrovick for the preparation of the extrudates and Fulya Yilmaz for the realization of the dissolution experiments. [1] FDA/CDER: Guidance for industry – Dissolution testing of immediate release solid oral dosage forms, http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/default.htm (1997). [2] FDA/CDER: Guidance for Industry – Extended release oral dosage forms: Development, evaluation and application of in vitro/in vivo correlations. http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/default.htm (1997). [3] A.P. Smith, T.W. Moore, B.J. Westenberger, W.H. Doub: In vitro dissolution of oral modified-release tablets and capsules in ethanolic media, Int. J. Pharm. 398 (2010) 93-96. [4] M. Walden, F.A. Nicholls, K.J. Smith: The effect of ethanol on the release of opioids from oral prolonged-release preparations, Drug Dev. Ind. Pharm. 33 (2007) 1101-1111.

24 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 13

Infrared spectroscopic study of drug-loaded bioactive mesoporous silica-polydimethylsiloxane systems

Magdalena Prokopowicz1, Adrian Szewczyk2, Wiesaw Sawicki1

1Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, e-mail: [email protected] 2Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, Scientific Circle of Students, e-mail: [email protected]

Introduction: Nowadays, mesoporous silica-polydimethylsiloxane (M-silica-PDMS) xerogels are one of the most progressive biomaterials. Studies about internal conformation of M-silica-PDMS can be useful in bioactive delivery drug system researches. In this paper, the bioactivity of M-silica-PDMS under in vitro conditions was investigated.

Methods: M-silica-PDMS were prepared by sol-gel method including drug loading [1]. Next, loaded M-silica-PDMS samples were soaked in simulated body fluid (SBF) for 28 days in stirred conditions at 37.4oC. The formation of phosphate network on the samples surface was carried out after 7, 14, 28 days of soaking, respectively by using Fourier transform infrared spectroscopy (FT-IR). Samples were dried, prepared as a KBr tablet and analyzed.

Results: Each spectrum presents broads associated with Si-O-Si and C-H groups (Fig.1) The Si–O–Si band, recorded at 1081 cm1 before soaking, was replaced by band at 1091 cm-1 after 7 day of soaking. Two bands of absorption 603-564 cm-1 and 1030 cm-1 can be observed progressively. This indicated that hydroxyapatite network began formation on silica surface. Moreover broad bands at 1422 cm-1 and 872 cm-1 have also appeared. Both of them are 2- connected with CO3 groups which come from carbonate hydroxyapatite forms.

Fig.1 FTIR spectra of M-silica-PDMS drug loaded samples.

Conclusions: According to IR results a bioactive character of mesoporous silica- PDMS structures has been proved. The progressive formation of carbonate hydroxyapatite during soaking time was 2- observed. Specific bands from CO3 and 3- PO4 functional groups were observed which means that mesoporous silica-PDMS can be a modern bioactive system of drug delivery.

Acknowledgments: This work was supported by the National Centre for Science of the Polish State project no. N N405 024440 awarded to M. Prokopowicz.

[1] M. Prokopowicz: Correlation between physicochemical properties of doxorubicin-loaded silica/polydimethylsiloxane xerogel and in vitro release of drug, Acta Biomater. 5 (2009) 193-207.

25 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 14

Application of magnetic resonance imaging (MRI) in quality assessment of orodispersible tablets

Witold Brniak1, Renata Jachowicz2, Anna Krupa3, Tomasz Skórka4

1Department of Pharmaceutical Technology and Biopharmaceutics Jagiellonian University Medical College, ul. Medyczna 9, 30-688 Kraków, Poland e-mail: [email protected] 2Department of Magnetic Resonance Imaging, Institute of Nuclear Physics, Polish Academy of Sciences, ul. Radzikowskiego 152, 31-342 Kraków, Poland

Introduction: According to the European Pharmacopoeia, orodispersible tablets (ODT) are uncoated tablets intended to be placed in the mouth where they disperse rapidly before being swallowed. Since their properties differ from conventional tablets, there are alternative methods of quality assessment needed for their evaluation. In recent years, the magnetic resonance imaging (MRI) became well established method used for in vitro characterization of many pharmaceutical dosage forms [1]. However, its application in research on fast disintegrating formulations is limited, due to the problems with quick registration of high resolution images, caused by long acquisition time. The aim of this work was to develop a MRI method useful in quality assessment of ODT and to compare it with pharmacopoeial tests, as well as the alternative method introduced by the authors [2]. Methods: There were prepared different kinds of ODT with one of the co-processed ready to use excipients: Pharmaburst, Ludiflash, or F-MELT. They were either placebo or contained prednisolone, ketoprofen, or ibuprofen. Properties of the prepared tablets such as tensile strength, friability, wetting time and water absorption ratio were evaluated. Disintegration time was measured using the pharmacopoeial method and the novel apparatus constructed by the authors. The MRI experiments were performed on the system consisted of a 9.7T/210mm Bruker magnet and the Maran DRX console. A custom designed T1 weighted fast gradient echo sequence (FLASH) was used for data acquisition. Results: The results of MRI, as well as disintegration time measurements with novel method showed influence of excipients on disintegration properties of ODT. Tablets with Pharmaburst disintegrated in the shortest time, and the process was very dynamic. Magnetic resonance images showed extensive swelling of tablet and immediate water penetration into its matrix leading to the disappearance of dry core after the couple seconds. Tablets with F-MELT and Ludiflash absorbed water without significant changes in their dimensions. MRI analysis showed the dry core inside, which slowly disappeared. Conclusions: The research has shown that magnetic resonance imaging (MRI) is a valuable tool in evaluation of orodispersible tablets. It allowed the analysis of different mechanisms involved in disintegration of tablets.

[1] M.D. Mantle: Quantitative magnetic resonance micro-imaging methods for pharmaceutical research, Int. J. Pharm. 417 (2011) 173-195. [2] W. Brniak et al.: Evaluation of co-processed excipients used for direct compression of orally disintegrating tablets (ODT) using novel disintegration apparatus, Pharm. Dev. Technol. 18 (2013) 464-474.

26 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 15

Application of short time exposure (STE) tests to assess an irritation property of new eye drops with choline salicylate

Katarzyna Wróblewska1, Magorzata Kuciska2, Marek Murias2, Janina Lulek1 1 Department of Pharmaceutical Technology, Poznan University of Medical Sciences, 6 Grunwaldzka Str., 60-780 Poznan,Poland [email protected] 2Department of Toxicology, Poznan University of Medical Sciences), 30 Dojazd Str., 60-631 Poznan, Poland Introduction: The evaluation of irritation potential of chemicals is essential to secure the safety of their use. This applies to drugs, cosmetics and other objects that may accidentally get into the eye. Until recently, the standard method for assessing the potential of irritating substances has been the Draize rabbit eye test developed in 1944 [1]. The European Centre for Validation of Alternative Methods and the Coordinating Committee for Validation of Alternative Methods recommend validated short time exposure (STE) in vitro tests as an alternative method for assessing eye irritation [2,3,4]. The aim of our study was to examine the irritation potential of new eyes drops containing choline salicynate (CS) as an active pharmaceutical ingredient (API) and various polymers improving drops viscosity.

Methods: The eye drops containing 2% of CS and different thickening agents (0,5% and 0,25% HEC, 0,5% HPMC, 0,25% MC, 2% PVA and 5% PVP) as well as eye drops with 1%, 2 % or 3% of CS were prepared and sterilized in the autoclave at the temperature of 122 ± 2OC for 20 min. The eye irritation potential of all formulations was determined using rabbit corneal cell line (SIRC) and two in vitro tests: MTT and Neutral Red Uptake (NRU)[5]. Both STE tests use the cell viability (CV) as the endpoint for irritation potential. Data were compared for statistical significance by the Tukey’s Multiple Comparison Test, a probability value (P) of less than 0,05 was considered significantly.

Results: The cell viability of SIRC cell line was greater than 93% (MTT) and 76% (NRU) for all examined drops. Statistically significant differences between the control and the tested formulation were observed in the MTT assay only for drops including 5% PVP and in NRU assay only for formulation containing 2% PVA.

Conclusions: According to the irritation rankings for the STE test all tested eye drops are classified as minimally irritant (Class 1, viability rabbit corneal cells >70%) [3,4]. All examined eye drops regardless of the CS concentration as well as of thickening agent did not show of irritation potential.

Acknowledgement: This study was partially supported by PUMS grant N0 502-14-03314429-08338

[1] Wilhelmus, K.R., The Draize Eye Test. Survey of Ophthalmology 45 (2001), 493-515. [2] Takahashi Y., Koike M., Honda H., Ito Y., Sakaguchi H., Suzuki H., Nishiyama N. Development of the short time exposure (STE) test: An in vitro eye irritation test using SIRC cells. Toxicol. In Vitro 22 (2008) 760–770 [3] Sakaguchi H. , Ota N., Omori T. et al Validation study of the Short Time Exposure (STE) test to assess the eye irritation potential of chemicals. Toxicol. In Vitro 25 (2011) 796–809 [4] Takahashi Y., Hayashi T., Watanabe S., Hayashi K., Koike M., Aisawa N., Ebata S., Sakaguchi H., Nakamura T., Inter-laboratory study of short time exposure (STE) test for predicting eye irritation potential of chemicals and correspondence to globally harmonized system (GHS) classification. J. Toxicol. Sci. 6 (2009) 611-626 [5] Repetto G.del. P. A. Zurita J.L., Neutral red uptake assay for the estimation of cell viability/cytotoxicity Nat. Protoc. (2008) 1125-1131

27 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 16

Nanocrystals of Medium Soluble Drugs/Actives – a Novel Concept for Improved Skin Delivery

X. Zhai1, C.M. Keck2, R.H. Müller1

1Pharmacy Department, Free University, Berlin, 12169, [email protected]; 2PharmaSol GmbH, Berlin, 12307, Germany

Introduction: Dermal application of nanocrystals was firstly introduced in 2007 with the first cosmetic products appearing on the market, e.g., rutin (Juvena) and hesperidin (La Prairie). Nanonization of poorly soluble coarse drug powders can increase the penetration into skin due to increased specific surface area and saturation solubility, specifically via follicular pathways [1]. The new concept is, to make also nanocrystals from medium soluble actives, and to apply a solution containing additionally nanocrystals (= depot) to the skin. This should enhance the penetration of compounds such as caffeine into the skin, which is mainly a function of the caffeine concentration in the dermal formulation (e.g. anti-cellulite products). However, the production of nanocrystals from medium soluble active is challenging. Reduction of size increases the saturation solubility of nanocrystals during the production process, which can cause pronounced re-crystallization (e.g. formation of asbestos fiber-like microcrystals). Aim of present study was to develop a production process which allows the production of nanocrystals from medium soluble drugs/actives using caffeine as a model drug. Methods: Caffeine nanocrystals were prepared by pearl milling using a PML 2 (Bühler, Germany). 150 ml of caffeine pre-suspension formulated with 20% (w/w) caffeine, 2% (w/w) stabilizer and 78% (w/w) water:ethanol 96% (1:9) was processed by using an Ultra-Turrax T25 (Janke & Kunkel GmbH, Germany). Tween 80, Carbopol® 981 and Polyvinylpyrrolidone (PVP) were applied as stabilizers. Power beads type YSZ (0.4-0.6 mm) were used as milling material. Pearl milling of the pre-suspension was performed at 2,000 rpm, cooled to 5C. The samples were withdrawn at settled time intervals over 180 minutes. The mean particle size (z- average) and polydispersity index (PI) of caffeine nanocrystals were analyzed by photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS (Malvern Instruments, UK). Results: An optimized water-ethanol mixture was employed for nanocrystal production to reduce re-crystallization phenomena. The PCS results showed that particle size fluctuated after milling for 30 minutes in Tween 80-stabilized formulation. This was attributed to some agglomeration phenomena caused by the selection of a suboptimal stabilizer. Investigating the caffeine nanosuspension stabilized with Carbopol® 981 yielded the smallest nanocrystals after milling for 180 minutes (about 200 nm). A steady decrease in particle size as a function of time was observed for the PVP-stabilized formulation. With PVP, the smallest particle size was obtained by after milling for 90 minutes, with a z-average of 560 nm and a PI of 0.3. Conclusions: For the first time, a top-down process was successfully developed for the production of nanocrystals from drugs/actives with medium solubility, by changing the dispersion medium to avoid the re-crystallization phenomena. In vivo studies will be performed to evaluate the validity of the concept of combining dissolved active plus nanocrystals for increased skin delivery.

[1] F. Knorr, J. Lademann, A. Patzelt, et al: Follicular transport route – research progress and future perspectives, Eur. J. Pharm. Biopharm, 71 (2009) 173-180.

28 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 17

Optimization of solvent composition and stabilizer type using a combinative particle size reduction process

Biswadip Sinha1, Rainer H. Müller1, Jan P. Möschwitzer2 1Institute of Pharmacy, Freie University of Berlin, Berlin, Germany,[email protected] 2Pharmaceutical Development, AbbVie Deutschland GmbH & Co. KG, Ludwigshafen, Germany

Introduction: Precipitation was combined with high pressure homogenization process (HPH) by Kipp et al. in 2001 to generate smaller size drug nanocrystal [1]. Later, it was found that particle size reduction could be significantly improved by reduction of the time difference between precipitation and HPH process [2]. Effects of different process parameters on the particle size have already been studied. The purpose of this work was to optimize the solvent type, internal stabilizer type and its content to further reduce the particle size. Resveratrol was used as a model compound due to its poor water solubility. Methods: Solvent phase (S) was prepared by dissolving the drug in one of the 7 water miscible organic solvents such as alcohols (Ethanol, Methanol, Isopropanol), acetone, tertrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide (DMSO) and binary compositions of few solvents. Aqueous solution of SDS (0.2%) was mixed with 0.5% PVP (K40) to constitute the anti-solvent phase (AS). S phase was injected in the AS phase near the homogenization zone of Emulsiflex C5 (Avestin Europe GmbH). Further optimization of the process was carried out with the addition of a range of stabilizers having HLB value from 1 to 29 in the organic phase. Drug nanocrystals were characterized by laser diffractometry, photon correlation spectroscopy and light and electron microscopy. Results: It was found that the use of DMSO or DMSO/Acetone led to smaller size particles compared to the other solvents. The particles obtained by using these solvent types had a D(0.9) size between 897 nm-929 nm and a D(0.5) size between 147-157 nm with 10% particles bellow 70 nm in size. The use of Span 20 as an internal stabilizer further reduced the D(0.9) size to 560 nm. However, D(0.5) value was not changed significantly. This sample had a z-average of 245.8 nm. Alteration of the drug:stabilizer ratio has affected the particle size and the optimum ratio was found to be 1:3. Further investigation by scanning electron microscopy revealed that the particles were fused aggregates of smaller particles having a size ~40-50 nm. It was also found that the HLB value of the stabilizer has a prominent effect on the particle size. Stabilizer with HLB value of 10±2 was found to be the best in this experiment. Conclusion: Optimization of the solvent type and the internal stabilizer was crucial to obtain the smallest particles. [1] Kipp J. et al. (2001) Microprecipitation method for preparing submicron suspensions. U.S. Patent, 6,607,784 B2 [2] Müller RH. et al. (2009) Methods and device for producing very fine particles and coating such particles. U.S. Patent application, 20090297565

29 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 18

Age-specific in vitro investigations with a single dose dry powder inhaler

S. Lindert, A. Below, J. Breitkreutz

Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University 40225 Duesseldorf Germany, [email protected]

Introduction: In the treatment of respiratory diseases there are many differences between children and adults. Huge variations could be found in the mouth-throat region while the pediatric oropharynx is not only smaller than the adult geometry. Furthermore, the therapeutic effect in pediatrics is influenced by low peak inspiratory flow rates (PIF) and small inhalation volumes. In order to consider the pediatric specifics (especially 4-5 year old children) different upper airway models were used. Additionally the inhalation parameters flow rate and inhalation volume were varied and a pediatric inhalation profile was simulated by an electronic lung. Materials and Methods: The experimental setup consisted of the Next Generation Impactor (NGI) according to the European Pharmacopeia. Three upper airway models, a pediatric idealized [1] and realistic [2] model and an idealized adult model [3] were connected to the NGI. An inhalation volume of 1 L and various flow rates (30, 60 and 75 L/min) were adjusted to measure the pulmonary deposition (particles smaller than 5 μm) and the deposition in the upper airway models. In further investigations this setup was extended by using an electronic lung which simulates a time-varying inhalation profile in the model. This profile was generated by a 4-year old child and showed a PIF of 30 L/min and an inhalation volume of 0.6 L. A single dose dry powder inhaler (DPI) containing capsules with salbutamol sulphate (Cyclohaler®, PB Pharma GmbH) was used. The deposited drug was quantified by a validated HPLC method [4]. Results and Discussion: The comparison between the pediatric and the adult idealized model indicated that the anatomy of the oropharynx has an influence on the deposition pattern of the powder. The deposition in the idealized pediatric model was higher (63±2.7% vs. 53±4.5% at 60 L/min) whereas the pulmonary deposition was lower (15±0.1% vs. 22±0.6% at 60 L/min) than in the adult model. By using the simulated inhalation profile with the two pediatric upper airway models no significant difference could be shown. In both the pulmonary deposition was around 15% (±1.1%) and the deposition in the models was about 70% (±5.1%) related to emitted dose. There was no advantage of the inhalation profile over the steady flow rate studying the pulmonary deposition with single dose DPI. Conclusion: With focus on an appropriate in vitro study for single dose dry powder inhaler it is useful to consider the age-specific anatomical differences in the oropharynx and inhalation pattern. The use of idealized upper airway models is sufficient for DPI testing and the realistic model shows no further reduction in predicted pulmonary deposition.

[1] D. Bickmann et al.: Examining inhaler performance using a child`s throat model, RDD, 2 (2008) 565-570. [2] H. Wachtel et al.: Can pediatric throat models and air flow profiles improve our dose finding strategy? RDD, 2 (2010) 195-204. [3] W. Finlay et al.: The flow inside an idealized form of the human extra-thoracic airways, Exp Fluids, 37 (2004) 673-689. [4] M. Lohrmann, N.A. Urabanetz: Adhesion forces in interactive mixtures for dry powder inhalers - Evaluation of a new measuring method, Eur J Pharm Biopharm, 67 (2007) 579-586.

30 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 19

Mixer torque rheometry as a method to identify structural changes during pharmaceutical manufacturing processes

Carmen Stomberg1, Hans-Jürgen Hamann², Venkata-Rangarao Kanikanti², Markus Thommes1, Peter Kleinebudde1

1Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany, [email protected] 2Bayer Animal Health GmbH, Alfred-Nobel-Straße 50, 40789 Monheim am Rhein

Introduction: Within many pharmaceutical production processes, material is exposed to high energy input or shear forces. By means of a mixer torque rheometer these influences can be pre- estimated in a small scale. Additionally, it is possible to detect physical interactions of the substances and modification changes over process time. Methods: In this study, different single substances and substance mixtures were investigated with a mixer torque rheometer (Plastograph W50 EHT®, Brabender, Germany). The kneading chamber holds 55 cm³ and consists of two counter-rotating cam blades. During the process the torque is registered as a measure for the materials resistance. Kneading experiments have been conducted with different adjustments of chamber temperature and kneading speed (Tab. 1): Table 1: Test parameters Start Kneading Wetting and non-wetting liquids were added to a solid substance. temperature speed Torque curves were compared with data from differential 25 °C 90 rpm scanning calometry of the used substances (DSC 1, Mettler 45 °C 120 rpm Toledo, Switzerland). Results: Addition of liquid hydrophilic substances to a mainly hydrophilic mixture leads to an increase of torque (Fig. 1). The wetting liquid results in a torque increase due to a higher liquid saturation. However, the addition of lipophilic liquids like oil induces a sharp torque decrease (Fig. 1). The blades slide through the mixture of hydrophilic paste and non-wetting liquid more easily, therefore the resistance decreases. Furthermore a shift in the slope of torque is observed, if a transition of the material, such as a glass transition of one of the substances occurs (Fig. 1). DSC measurements confirm the glass transition (Fig. 2) Figure 1: Kneading profile, torque, temperature. Figure 2: DSC of same substance used in fig. 1. Conclusion: Kneading experiments in a mixer torque rheometer are a versatile approach to estimate substantial interactions and transformations within pharmaceutical manufacturing processes.

31 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 20

The physicochemical properties of poloxamer 407 hydrogels with benzydamine hydrochloride for vaginal administration

Tomasz Osmaek1, Dorota Wagner1, Marta Kdziora1, Wojciech Biaas2 1Department of Pharmaceutical Technology, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, [email protected]; 2Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences Wojska Polskiego 48, 60-627 Poznan

Introduction: Vaginal infections and vaginitis are the most frequently diagnosed female disorders. The therapy involves local administration of antibiotics, microbicides or anti- inflammatory agents [1]. The thermoreversible hydrogel-based platforms are currently extensively studied because of the ability to achieve an intimate contact with the mucus and extend the residence in the vaginal cavity [2]. Benzydamine hydrochloride (fig. 1) is a locally acting, anti-inflammatory, anaesthetic and antimicrobial drug [3]. It is available in the form of powder which after re-hydration acts as a cleanser to the vaginal area [4].

Methods: The aim of the work was to evaluate the properties of Figure 1. The chemical structure the hydrogels based on poloxamer 407 with various amounts of of benzydamine hydrochloride benzydamine hydrochloride (0,1-1,5 %). The rheological behavior of the hydrogels has been examined on the HAAKE RheoStress 1 (Thermo Scientific) rotational rheometer, equipped with the plate-plate geometry (35 mm Ø). The elasticity and hardness of the samples have been calculated using TA.XT plus (Stable Micro Systems) texture analyzer.

Results: The formulations revealed typical pseudoplastic (shear thining) properties. The flow curves, creep & recovery tests and oscillatory assays showed evidence of the decreased viscosity, lower yield point and reduced sol-gel transition temperature of the gels after addition of benzydamine HCl. The hardness and elasticity of the hydrogels was also decreased after addition of the drug. The formulations have been stable during 30 day storage at room temperature.

Conclusions: In situ forming poloxamer 407 hydrogels seem to be proper candidates for the local vaginal delivery of benzydamine. Thermoreversible properties enable application of the drug in a liquid form, which after exact filling of the vaginal cavity forms a semi-solid to prolong the drug release.

Acknowledgement: This study was partially supported by PUMS grant N0 502-01-03314429- 03439

[1] Valenta C.: The use of mucoadhesive polymers in vaginal delivery. Adv. Drug Delivery Reviews 57 (2005) 1692– 1712 [2] Rathbone M.J., Hadgraft J., Roberts M.S.: Modified-Release Drug Delivery Technology. Marcel Dekker, Inc., New York, 2003, 765-769 [3] Quane P., Graham G.G., Ziegler J.B.: Pharmacology of Benzydamine. Inflammopharmacology 6 (1998) 95-107 [4] www.eipico.com.eg/product.asp?id1=22&id2=0&id4=1

32 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 21

Cilazapril stability in pediatric oral suspension

Sylwia Katarzyna Paszun1, Beata Jadwiga Stanisz1, Anna Zalewska2

1Department of Pharmaceutical Chemistry, [email protected], 2Department of Inorganic and Analytical Chemistry, Pozna University of Medical Sciences, Grunwaldzka 6 St., Pozna

Introduction: Cilazapril is an angiotensin converting enzyme inhibitor, which advantageous therapeutic effect was proven in treatment of congestive heart failure in children population.1 Whilst, the best way of drug administration for children is achieved in form of oral suspensions, presented study aimed at development and stability evaluation of cilazapril oral suspension. Methods: Oral suspensions containing cilazapril in concentration of 1 mg/ml were prepared utilizing cilazapril tablets (original and generic drug) and Ora-Blend vehicle. Suspensions placed in amber glass and PET bottles were stored for 28 days under temperature conditions of 298 K. Suspensions were tested in 7 days intervals for cilazapril content (HPLC method), pH values and rheological properties (measured at the beginning and in the end of the study). Results: Oral suspensions with cilazapril remained stable throughout the study period regardless of sort of bottle used. Cilazapril recovery after 28 days met acceptance criteria of 100 ± 5 %. The pH values did not change statistically, comparing with the suspension pH values at the beginning of the study. Rheological properties remained also not affected.

glass amber bottles PET amber bottles storage time 0 day 28 day 28 day cilazapril concentration O: 102.17 ± 0.72 103.13 ± 1.30 98.85 ± 0.76 G: 102.36 ± 2.10 99.11 ± 3.74 102.43 ± 0.42 pH O: 4.75 ± 0.01 4.61 ± 0.01 4.60 ± 0.02 G: 4.75 ± 0.01 4.65 ± 0.01 4.66 ± 0.00 rheological properties K O: 0.39 0.26 0.26 (Ostwald model) G: 0.44 0.26 0.35

n O: 0.55 0.62 0.62 G: 0.55 0.63 0.59

Conclusions: Cilazapril suspension prepared from, both, original and generic drug tablets were stable, while 28 day long storage in temperature of 298 K. No significant changes in oral suspensions were observed concerning cilazapril concentration, pH values and rheological properties. Acknowledgements: The research was funded by the Polish National Science Centre (Grant No. NN405 050440).

[1] K. Momma: ACE inhibitors in pediatric patients with heart failure, Pediatr Drugs, 8 (2006) 55–69.

33 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 22

Low scale production of hot melt ram extrudates via a high pressure capillary rheometer

Eva Julia Laukamp1, Markus Thommes, Jörg Breitkreutz

Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, 40225 Düsseldorf, Germany, 1Corresponding author: [email protected]

Introduction: Recently, the Solid Dosage Pen was introduced for individual dosing of medicines [1, 2]. To develop drug formulations by hot melt extrusion (HME) for this novel device a screening method was investigated. Low scale production should be achieved using a high pressure capillary rheometer as a hot melt ram extruder. The processability of HME excipients and drug formulations with carbamazepine (CBZ) as a model drug was evaluated. Materials and methods: PEG 2000 (macrogol, Mw = 2000 g/mol, Clariant, NL), polyethylene oxide (Polyox®N12K, Mw = 1.000.000 g/mol, Colorcon, UK), mannitol (Pearlitol®C 160, Roquette, F), isomalt (Galen IQ®720, Beneo-Palatinit, D), xylitol (Danisco, FIN), sucrose palmitate (Surfhope® D-1616, Mitsubishi-Kagaku, J) and HPMC (Pharmacoat® 606, ShinEtsu, J), as well as Poloxamer 407 (Lutrol®F 127), Kollidon® 12 and 17 (PVP), Kollidon®VA 64 (PVP/PVA), and polycaprolactone (Capromer PD1-20, Mw = 2000) all from BASF, D, were tested. CBZ was purchased from BASF, CH. The ram extrudates were produced with a high pressure capillary rheometer (Rosand® RH2000, Malvern, UK) at appropriate temperature after barrel equilibration for 25 min. Approximately 40 g of excipient or -mixture and 10 g of drug formulation were utilized and manually precompressed. The ram extrudates were shaped through a 2.0 mm x 20 mm die. A conveyor belt adjustable for conveying speed (Brabender, D) was used to transport and cool the ram extrudates to room temperature. Results: Ram extrusion was possible for most HME excipients under the adjustment of the piston velocity (50 vPiston [mm/min] 200) and the extrusion temperature (textr [°C] < melting temperature). The conveyor belt velocity was adapted to generate a straight strand (Figure 1). Kollidon VA 64, Kollidon 12 and 17, HPMC and Polyox were not processible for various textr and vpiston showing blockage or agglutination of the die. Twelve CBZ hot melt ram extrudates were produced with a Figure 1: Production of the ram extrudates via the high yield of 50 % due to the dead volume and a diameter between 1.9 mm pressure capillary rheometer and 2.5 mm, dependent on the die swell. and the conveyor belt Conclusion: The production of hot melt ram extrudates with a high pressure capillary rheometer was feasible for various excipients by softening but not by fully melting the material. Based on the preliminary mixtures ram extrudates containing 10 to 50 % CBZ with only 10 g batches were produced enabling future dissolution testing and exploration of mechanical properties. [1] K. Wening, J. Breitkreutz: Novel delivery device for monolithical solid oral dosage forms for personalized medicine, Int J Pharm 395 (2010) 174-181. [2] K. Wening, E.J. Laukamp, M. Thommes, J. Breitkreutz: Individual Oral Therapy with Immediate Release and Effervescent Formulations Delivered by the Solid Dosage Pen, J Pers Med 2 (2012) 217-231.

34 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 23

Modification of release from spray dried drug/polymer microparticles

Sollohub K.1, Cal K.1

1 Department of Pharmaceutical Technology, Medical University of Gdansk, Hallera 107, 80-416 Gdansk, Poland

Introduction: Particle engineering resulting in modification of drugs release is desirable by pharmaceutical industry because of wide range of potential applications. Co-processed initial formulations consisting of pure drug and polymer can be used in different dosage forms – from suspensions to solid oral dosage forms [1, 2]. Published studies show that it is possible to produce microparticles containing drug processed with polymer by spray drying technique. The aim of this study was to use of this method for co-processing of three drugs – verapamil hydrochloride, teophylline and roxithromycin – with well-known polymers, such as Eudragit RL 30D, Eudragit RS 30 D and ethylcellulose (Aquoat ECD).

Methods: Each drug was dissolved in appropriate water based solvent in maximal possible concentration and polymer dispersion was introduced in 0.1, 0.25, 0.5, 0.75 and 1.0 drug/polymer (dry content) ratio. Resulted dispersions were submitted to spray drying using laboratory spray drier equipped with high efficiency cyclone separator. Drying conditions for best dried content yield were chosen for each formulation. Amount of each formulation corresponding to the amount of drug in commercially available products (120 mg for verapamil hydrochloride, 150 mg for teophylline and 100 mg for roxithromycin) were subjected to the release studies in apparatus No 2 (paddle) into the 0.1 HCl and/or phosphate buffer 6.8 as acceptor fluids. The collected samples were analyzed by UV-VIS spectroscopy. Morphology of spray dried microparticles was examined by light microscopy.

Results: Spray dried powders were well flowing and homogenous in size, all under 30 μm. Modification of release was shown, and relationship between drug/polymer ratio and release profile was observed, especially for teophylline/ethylcellulose compositions.

[1] Vehring R.: Pharm. Res. 25, 999 (2008). [2] Sollohub K., Cal K., J. Pharm. Sci. 99:587-597 (2010).

35 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 24

Heuristic modeling of macromolecules release from PLGA microspheres

Szlk Jakub1, Pacawski Adam1, Lau Wai Man Raymond2, Jachowicz Renata1, Mendyk Aleksander1 1 Department of Pharmaceutcial Technology and Biopharmaceutics, Jagiellonian University Medical College, Medyczna 9 st., 30-688 Kraków, e-mail: [email protected] 2 Division of Chemical and Biomolecular Engineering, School of Chemical and Biomedical Engineering, College of Engineering Nanyang Technological University (NTU), 62 Nanyang Drive, Singapore 637459

Introduction: Due to their flexibility and satisfactory safety profile, poly-lactide-co-glycolide (PLGA) microparticles have found their prominent place in the drugs formulation for various administration routes. Their drug-protective properties as carriers are exploited i.e. for peptide drugs, however still no quantitative relationships between microspheres composition and macromolecules release rate was identified. The aim of this work was to create empirical model describing impact of the microspheres composition on the release rate of the macromolecules. Methods: The database was created from the literature data. Initially, literature survey yielded about 200 publications. After careful selection of available information, 68 formulations were included into the database consisting of 745 data records and 320 features. There were 319 independent variables describing formulation characteristics (quantitative/qualitative composition, preparation technology) and dissolution test conditions. For the qualitative composition molecular descriptors were computed (Marvin 5.11, ChemAxon, UK) for the drug substance and excipients as well. A single dependent variable depicted the amount of the drug substance released. Empirical modeling tools were used: artificial neural networks (ANNs) and genetic programming (GP). Crucial variables selection was performed both by sensitivity analysis of ANNs [1] and by caret package from R statistical environment. Results: Crucial variables selection procedure resulted in the 11 variables included into the final model. The best results were obtained for MON-MLP ANNs followed by classical equation created by GP. Generalization errors (RSME) for the models were 15.4 and 16.3 respectively. The equation was characterized by 2 parameters, thus feasible to standard non-linear regression techniques Conclusions: Heuristic modeling led to the mathematical formula describing macromolecules release profiles from PLGA microspheres and with predictive efficiency comparable to the ANNs models. Acknowledgments: This work was funded by Polish-Singapore bilateral cooperation project 2/3/POL-SIN/2012 [1] Mendyk A., Jachowicz R., Unified methodology of neural analysis in decision support systems built for pharmaceutical technology, Expert Syst Appl, 32, (2007) 1124–1131.

36 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 25

Usage of Camsizer XT for measuring thickness of coatings on minitablets and pellets Czajkowska Magdalena1, Sznitowska Magorzata1, Kleinebudde Peter2 1Department of Pharmaceutical Technology Medical University of Gdansk Hallera 107, 80-416 Gdansk, Poland, e-mail: [email protected] 2Institute of Pharmaceutics and Biopharmaceutics in Dusseldorf, Heinrich-Heine-University, 40225 Dusseldorf, Germany

Introduction: Minitablets (1-3 mm) are new oral dosage forms dedicated especially for children and people with oral disorders [1]. Coating of minitablets with various polymers may lead to achieve taste masking or prolonged or enteric release. Polymer film forming on small cores (pellets, minitablets) is performed in the fluid bed systems. Coating of minitablets is more difficult than pellets, because on the minitablets’ edges thinner film is formed and probability of incomplete coating is increased. The objective of this study is to develop a nondestructive technique for determination of the film thickness, which takes into account the geometry of minitablets (surface and edges). The goal of the project is to check if film thickness may be measured by particle size and shape analyzer, based on two-camera system (Camsizer XT) [2]. Methods: Minitablets (2.0 mm and 2.5 mm) and pellets (0.7 mm) were coated with polymer Eudragit E PO in a fluid bed system (Mycrolab, Huttlin). Measurement of film thickness was performed with four different analytical methods using: Camsizer XT (Retsch), microscopy with Leica software QWin Lite V2.3 (Leica Microsystems Imaging Solutions), scanning microscopy (Phenom G2 pro) and stereoscopic microscopy (OPTA-TECH). Results: Measurements made by Camsizer XT clearly showed film thickness of pellets (80- 90 μm) and minitablets (160-170 μm). Good correlation between Camsizer XT and optical microscopy methods was shown for film thicknesses up to 180 μm. However use of Camsizer XT for measurement of minitablets (2.5 mm) coating layer was not reproducible, most probably because of the too large diagonal (3.61 mm), which exceeded the measuring range (1 μm-3 mm). Conclusion: Camsizer XT offers a good nondestructive technique for film thickness measurements of minitablets (2.0 mm) and pellets (0.7 mm) and the results were confirmed by three microscopy systems. This method may be used as a tool for quality control of coated cores up to 2.0 mm in diameter both, spherical and cylindrical.

[1] N. Spomer et al.: “Acceptance of uncoated mini-tablets in young children: results from a prospective exploratory cross-over study.” Archives of Disease in Childhood, 97 (2012) 283-286. [2] http://www.retsch.dk/pdf/pdf_camsizer/brochure_camsizer_xt.pdf

37 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 26

Submicron emulsions stabilized with various phospholipids – stability and compatibility with plastic containers Dorota Watrobska-Swietlikowska, Paulina Zielen Department of Pharmaceutical Technology, Medical University of Gdansk, Hallera Av. 107, 80-416 Gdansk, Poland, Tel: +48583491085, email: [email protected] Introduction: Lecithin as a biocompatible emulsifier is used in submicron dispersed systems intended for parenteral use. Submicron emulsions stabilized with mainly egg lecithin at concentrations 1.2 or 2.4 % are used as drug carriers and for parenteral nutrition. There is no sufficient information about stability of these emulsions with high concentration of lecithins and pure phospholipids.

Methods: Submicron emulsions (10% of soya-bean oil) were prepared with the following phospholipids – type emulsifiers: egg lecithin (1.2 and 5%), soya-bean lecithin (1.2 and 5%), phosphatidylcholine from egg lecithin (1.2%), hydrogenated phosphatidylcholine from egg lecithin (1.2%), hydrogenated phosphatidylcholine from soya-bean (1.2%) and dipalmitoyl- sn-glycero-3-phosphoethanolamine (1.2%). All emulsions were prepared using high pressure homogenization. The emulsions were observed for 6 months at 25 and 40°C. Visual and microscopic observations, pH, zeta potential and size of oily droplets (LD and PCS methods) measurements were performed. Moreover, the stability of prepared emulsions with different plastic containers such as: EVA, PP, LDPE and PE was also examined (immersion test). Influence of thermal sterilization on stability was also investigated with and without plastic materials immersed.

Results: In our studies it was possible obtain two kinds of emulsions: submicron type (droplet size below 1 μm) and traditional (droplet size upper 1 μm). Stable submicron emulsions, which were characterized by size of oil droplets about 300 nm, were prepared using egg lecithin (1.2%) or soya-bean lecithin (1.2 and 5%) as well as dipalmitoyl-sn-glycero-3- phosphoethanolamine (1.2%). Despite the fact that emulsions with 5% of egg lecithin and phosphatidylcholine from egg lecithin was not submicron type (d(90) below 1.76 μm), these emulsions were stable. However emulsions stabilized with hydrogenated phosphatidylcholines (d(90) about 3.81 μm) and very low zeta potetial (-12.5 mV), were unstable due to increasing agglomeration. Despite two emulsions (5% of egg lecithin and 1,2% hydrogenated phosphatidylcholine from egg lecithin), all emulsions were stable when subjected to thermal sterilization. In unstable emulsions after autoclaving size of oily droplets increased from 300 nm up to 800 nm and agglomerates were observed. Lowering the temperature of thermal sterilization to 110°C eliminated this problem. No physical interaction between autoclaved emulsions and investigated plastic containers was observed. Increased oily droplets size in the emulsion stabilized with hydrogenated egg phosphatidylcholine being in contact with EVA material was only noticed.

Conclusions: Our study indicates that there is possibility to obtain stable emulsions with phospholipids but not for parenteral application. Preparing submicron emulsion with pure phospholipids needs further studies.

1. A.G. Floyd: Top ten considerations in the development of parenteral emulsions, PSST, 2 (1992) 134-143 2. D.F. Driscoll, B.R. Bistrian, H. Demmelmair, B. Koletzko: Pharmaceutical and clinical aspects of parenteral lipid emulsions in neonatology, Clin. Nutr., 27 (2008) 497 – 503

38 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 27

Is surface area a useful tool to determine quantity of lubricant for tableting?

Johanna Mosig, Peter Kleinebudde

Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Duesseldorf, Germany [email protected]

Tableting of powder material and granules is usually not possible without addition of small quantities of lubricant in order to reduce wall friction during compression and ejection. It was shown in literature that magnesium stearate adheres on the surface area and forms a lubricant film around the particles during mixing [1] resulting in lower friction as well as in an interference in particle bonding, leading to weaker tablets. In contrast to plastic deforming material lubricant sensitivity of brittle behaving material is minor as lubricant free surfaces are generated during fragmentation. As magnesium stearate coats the particles, determination of surface area could be an important way to find the optimal lubricant amount according to prevent sticking and to obtain a least reduction in strength. Magnesium carbonate, microcrystalline cellulose and powder cellulose were roll compacted with different specific compaction forces. Ribbons were directly dry granulated and sieved to obtain a particle fraction between 315 and 630 μm for every batch. External surface areas for each granule fraction as well as for the raw materials were determined by a Friedrich-Manometer. Granule fractions and powder material were lubricated with an amount of magnesium stearate proportionally to the measured specific external surface area and subsequently tableted. Tablets were characterized 48 hours after production to calculate the tensile strength. Surface area of the granules was smaller as of the raw material. Resulting from this, amount of magnesium stearate added to the granules was much lower (0.1 - 0.2 %) compared to the raw material (0.9 - 2 %). Compaction curves for the granules showed a decrease in tensile strength with increasing specific compaction force during roll compaction. Based on this work hardening phenomenon compression of the raw material should lead to the strongest tablets. In contrast to this compactibility of direct compressed microcrystalline cellulose and powder cellulose is much lower compared to granules. As both materials are lubricant sensitive, added amount of magnesium stearate was too high and prevented particle bonding resulting in weaker tablets. For magnesium carbonate, compactibility of the raw material was still higher compared with the granules in spite of the 10fold lubricant quantity. Further experiments with equal amounts of magnesium stearate for powder and granules of magnesium carbonate were problematic indicating an insufficient lubrication of the raw material in this case. Addition of magnesium stearate proportional to the specific surface area is not suitable as a universal tool for a comparable lubrication. For plastically deforming materials lubrication with magnesium stearate proportionally to the specific external surface area resulted in an overlubrication of the powder material, whereas for the brittle deforming magnesium carbonate surface area seemed to provide an indication for sufficient lubrication. [1] W. A. Strickland, E. Nelson, L. W.. Busse and T. Higuchi: The physics of tablet compression 9: Fundamental aspects of tablet lubrication, J. Am. Pharm. Ass. Sci. Ed., 45 (1956) 51-55

39 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 28

Spray Drying of Nanosuspensions Containing Low Melting Stabilizers

Jan P. Möschwitzer1,2, Maria L.A.D Lestari1, Rainer H Müller1

1Freie Universität Berlin, Institute of Pharmacy Dept. Of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Kelchstraße 31, 12169 Berlin, [email protected]; 2Pharmaceutical Development, AbbVie Deutschland GmbH & Co.KG, Ludwigshafen, Germany

Introduction: Spray drying is a common drying technique used for the solidification of nanosuspensions. In some cases this process can be quite challenging, especially when nanosuspensions contain polymeric stabilizers with a low melting point, such as poloxamer 188. Often, the drug nanoparticles agglomerate and form a sticky mass instead of a nice, dry powder. This research was conducted in order to optimize the bulking agent (type and percentage) and the spray drying parameters to produce dry powders of hesperetin nanosuspensions containing poloxamer 188 as polymeric stabilizer. Materials and Methods: Hesperetin was purchased from Exquim, S.A., Barcelona, Spain and other chemicals were of analytical grades. 5% of coarse hesperetin was suspended in an aqueous poloxamer 188 solution (2%). Nanosizing of this suspension was conducted with an agitated ball mill (Bühler PML 2). The optimization of the spray drying parameters (inlet temperature, aspiration rate) was performed with a Mini Büchi B-190. Mannitol or the polymers (PVA mw 80.000, PVP K25, PVP K12 and HPMC E5) were used as bulking agents at 3 different concentrations, 0%, 3% and 5%. The optimum parameters and the best bulking agent concentration were selected based on the appearance of the spray dried products and the particle size obtained after redispersion (measured with photon correlation spectroscopy and laser diffraction). Results and Discussion: Spray drying of hesperetin nanosuspensions without bulking agent (0% mannitol) resulted in a sticky agglomerated mass. When 3% mannitol was used as bulking agent, a dry product could be obtained. However, redispersion tests revealed a particle size increase of about 2 times compared to the original nanosuspension. Further increase of the mannitol content resulted in even larger particles after redispersion. Similar results were found when PVA was used as bulking agent. In case of PVP, the product was extremely sticky. It could not be removed from the spray dryer; therefore the resulting yield was very low. Spray drying of nanosuspensions together with HPMC E5 as bulking agent resulted in the best products. After redispersion the particle sizes were comparable to the original hesperetin nanosuspensions. The optimal process parameters of the spray drying were strongly depending on the employed bulking agents. In case of mannitol the aspirator rate was only 10%, whereas in case of polymeric bulking agents optimal results have been obtained with higher aspirator rates (45%).

40 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 29

The influence of resveratrol solid state on particle size reduction effectiveness

Tao Liu1; Rainer H. Müller 1; Jan P. Möschwitzer 1, 2 1 Institute of Pharmacy, Dept. of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Freie Universität Berlin, Germany ,email: [email protected];2 Pharmaceutical Development, AbbVie Deutschland GmbH & Co. KG, Ludwigshafen am Rhein, Germany

Introduction: Nanosizing is an established delivery technique for poorly soluble drugs. High pressure homogenization (HPH) is widely used for production of drug nanocrystals. In order to improve the particle size effectiveness, a drug modification step can be performed before HPH, i.e. by spray-drying (H42 technology), rotavapor or quench cooling. This study was focused on the investigation of a potential relationship between drug solid state and the minimal achievable nanoparticle size.

Methods: Different ratios (1:0.75 and 1:0.146) of resveratrol (RVT) and Sodium cholate (SC) were dissolved in ethanol and then rapidly spray-dried (Büchi Labortechnik AG, Switzerland). A rotavapor (Büchi Labortechnik AG, Switzerland) was used to dry RVT/HPMC (1:1 and 1:2) solutions. The obtained powders were dispersed in water and micrcosuspensions were nanosized by running a Micron Lab 40 homogenizer (APV Deutschland GmbH, Germany) for 20 cycles at 1500bar. The solid state of spray-dried powder, rotavapor powder and directly dried nanosuspensions were analyzed by DSC (Mettler Toledo AG, Germany) and X-ray (Philips Industrial & Electro-Acoustic Systems Division, Netherlands). In addition, stabilizers in all nanosuspensions were removed by filter or centrifuge. Solid state of residual RVT was also characterized. Particle sizes in nanosuspensions were analyzed by LD (Malvern Instruments, UK) and PCS (Malvern Instruments, UK).

Results: Amorphous samples were prepared by spray drying (1:0.75) as well as rotavapor (1:2). Amorphous solid from both did not lead to improved particle size effectiveness. In contrast, a predominately crystalline sample obtained via spray-drying resulted in ultrasmall RVT nanocrystals (Z-average, ca. 200nm) after only 1 homogenization cycle. DSC analysis of nanosuspensions produced from unmodified RVT showed reduced crystallinity. However, melting endotherms and no glass transition could be found when the stabilizers were removed from nanosuspensions prior to DSC analysis.

Conclusions: RVT nanocrystals production did not benefit from amorphous solids. The smallest particle size was obtained by using predominately crystalline spray dried RVT. Morphology/microstructure of modified drug will be further studied to find the possible reasons. In order to determine the solid state of drugs, stabilizers in nanosuspensions should be removed before DSC analysis.

41 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 30

NLC Cosmetics: Preserved Argan oil-loaded NLC Concentrates

X.Y., Hu; C.M. Keck; R. H., Müller

Department of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Free University, Berlin, Germany, [email protected]

Introduction: Argan oil-loaded NLC (nanostructured lipid carriers) formulations for cosmetic use were developed in the form of concentrates for admixing them in industrial production of NLC cosmetics. To produce “preservative-free” concentrates, compounds not listed in the regulatory preservative lists but having antimicrobial activity were screened to identify compounds which did not impair the physical stability of the NLC concentrates. Argan oil loaded NLC formulations were preserved by low irritative agents and investigated regarding their physical stability.

Methods: High pressure homogenization (HPH) was used to produce argan oil-loaded NLC suspensions at the conditions of 850C and 800 bar (Cetyl Palmitate 6%, Hydrogenated Castor oil 6%, argan oil 8.0%, TegoCare 450 2.5%). Admix of low irritative preserving agents (10% propylene glycol, 5% pentylene glycol, 10% and 20% ethanol) with NLC concentrate was conducted by gentle stirring at room temperature. To monitor the long term physical stability, light microscopy, photon correlation spectroscopy (PCS, Zetasizer Nano ZS, Malvern, UK) and laser diffraction (LD, Mastersizer 2000, Malvern, UK) have been performed to get optical picture, z-average, polydispersity index (PI) and volume weighted LD diameters. Crystalline polymorphism was determined by Differential Scanning Calorimeter (DSC821e, Mettler Toledo, Germany) and confirmed by x-ray diffractometer (XRD, PW1830, Philips, Netherlands).

Results: Argan oil-loaded NLC admixed with 10% w/w propylene glycol were stable during 2 years storage. No large particles or aggregates > 1 μm could be observed in propylene preserved formulation. LD measurements showed that diameters of 50% particles were all below 0.281μm and 99% particles were below 0.693μm. Concordant results were obtained from PCS and DSC dynamic heating curves by showing similar z-average (about 230nm), PI and melting peaks during 2 years. On the other hand, immediate aggregations were caused by both ethanol concentrations. Formulations with admixed 5% pentylene glycol were unaffected for about 3 months [1], but gelled after 2 years. This gelation was accompanied by a change in the crystalline structure which leads to instability of NLC [2]. X-ray data were in agreement with this observation.

Conclusion: Propylene is suitable preserving agent for argan oil-loaded NLC. Short and long- term destabilizing effects of the other compounds (ethanol, pentylene glycol) are attributed to dehydration of the NLC stabilizer layer and of initiation of crystalline changes leading to gelation.

[1] Hommoss, A. Pharmazie 2011, 66, 187-191. [2] Freitas, C.; Müller, R. H. Eur. J. Pharm. Biopharm. 1999, 47, 125-132.

42 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 31

Comparison of the commercial creams containing 1% of clotrimazole

Magorzata Krzyaniak1, Magorzata Sznitowska1, Katarzyna Centkowska1

1Department of Pharmaceutical Technology Medical University of Gdansk Hallera 107, 80-416 Gdansk, Poland, e-mail [email protected]

The aim: Clinical studies has shown that clotrimazole (CLO) has got a broad spectrum of antifungal (dermatophytes, Candida), antiprotozoal (Trichomonas vaginalis) and antibacterial activity (some of the Gram-positive bacteria). The creams with 1% of CLO are very effective and can be safely used even in the case of infants and women in 2nd and 3rd trimester of pregnancy. The aim of this study was to compare the physicochemical and biopharmaceutical properties of CLO creams available in Polish market and sold as OTC products. Five different creams (A-E) produced by different manufacturers were studied: rheological properties, microscope images and in vitro drug release profiles (determined in two different diffusion apparatus) were taken into consideration.

Methods: Two types of diffusion apparatus were employed in the in vitro release investigation: Mutimer-type (Mc) and VanKel-type (Vkc) with different effective permeation area, i.e. 35.9 cm2 and 3.6 cm2 respectively. Cuprophan® membrane (MWCO 10000Da) was used. The acceptor medium (37°C) was 70 (v/v) solution of propylene glycol. The samples (5 ml) of acceptor fluid were taken for 6 h and analysed using HPLC method. The effect of changes in the speed of rotation and thickness of the cream layer was investigated. The rheological properties (thixotropy and dynamic viscosity) were analysed using rotational viscometer Viscotester 550. The microscopic images were obtained using an optical microscope with a digital camera.

Results: The creams differ in their microscopic images: poor homogenization and large droplet size of the lipid phase of A and E creams was observed, and in the A product crystals of the drug were visible. The area of hysteresis loop has been used as a measure of the thixotropic behaviour. The cream C was characterized the best thixotropic properties and the highest viscosity. In all conditions of the test the highest amount of CLO was released from product E while the slowest release was noted for the cream B (Mc apparatus) and for C cream (Vkc apparatus). Neither change of the paddle rotation speed (Vkc apparatus 20 vs 50 rpm) nor increase in thickness of cream layer (Mc) changed the release profiles.

Conclusions: Large difference between commercial CLO creams were observed. Differences in drug release rates were confirmed in a few experimental models. Faster in vitro drug release was observed from creams poorly homogenized, with larger lipid phase droplets. Only in the case of cream C the slowest drug release can be explained by higher viscosity, while in the case of other creams there has been no obvious correlations noted between dynamic viscosity or thixotropic properties and release profile of CLO.

43 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 32

Sucrose esters in the lipid nanoparticles formulation Cornelia M. Keck1,2 Andjelka Kovaevi3, Snežana Savi3, Jela Mili3, Rainer H. Müller1

1Department of Pharmaceutics, Biopharmaceutics and Nutricosmetics, Institute of Pharmacy, Free University of Berlin, Kelchstrae 31, 12169 Berlin, Germany, [email protected] 2University of Applied Sciences Kaiserslautern Applied Pharmacy Division, Carl-Schurz-Str. 10-16, D-66953 Pirmasen, Germany, [email protected] 3Department of Pharmaceutical Technology and Cosmetology, Faculty of Pharmacy, University of Belgrade, 11000 Belgrade, Serbia, [email protected]

Introduction: Sucrose esters are natural and biodegradable excipients obtained from natural raw materials: sucrose and fatty acids. They are widely used in the cosmetic industry and recently the great interest in their applicability in the pharmaceutical eld was appeared. In this study, sucrose stearate was evaluated for stabilization of aqueous SLN and NLC dispersions. Particles were made from Cutina® CP as solid lipid only (SLN) and its blends with Miglyol® 812 (NLC, the blends contained 20% oil). Methods: Cutina® CP, a commonly used solid lipid for SLN, was provided from Cognis, Germany. Miglyol® 812 was obtained from Caelo, Germany. Surfhope® C-1815, was kindly donated from Mitsubishi Kagakoo, Japan. High pressure homogenization was used as preparation method for lipid nanoparticles [1]. Particles were analysed by photon correlation spectroscopy (PCS), laser diffraction (LD) and zeta potential (ZP) measurements. Thermal analysis of SLN and NLC was also performed. Results: The mean size of the SLN and NLC after production was around 170 nm with polydispersity index below 0.1 (PCS). LD confirmed monomodal size distributions. After 90 days, SLN stayed perfectly stable whereas in the NLC dispersion the presence of microparticles, besides the nanometer bulk population, was observed (LD). After addition of Miglyol® 812 in Cutina® CP the recristalization index (RI) was reduced i.e. distortion of the crystalline lattice of the particles takes place. Interestingly, ZP stays unchanged despite the changes in the lipid matrix structure and size of the particles (Table 1). High ZP of NLC indicates that an increase in the surfactant concentration might lead to an improved physical stability. Conclusions: Sucrose stearate revealed good stabilization ability of SLN dispersions. It can also be considered as interesting representative of this new class of stabilizers for development of NLC formulations.

Table 1. Volume diameters (d(v)0.10 - d(v)0.99), ZP and RI of the formulations after 90 days Formulation Measured parameter d(v) 0.10 d(v) 0.50 d(v) 0.90 d(v) 0.95 d(v) 0.99 ZP (mV) RI (%) SLN 0.085 0.155 0.276 0.325 0.454 -41.6 54.66 NLC 0.108 0.228 10.135 17.349 62.556 -40.1 25.18

[1] R.H. Müller, J.S. Lucks: Arzneistoffträger aus festen Lipidteilchen, Feste Lipidnanosphären (SLN), Medication vehicles made of solid lipid particles (solid lipid nanospheres – SLN), European Patent No. 0605497, (1996).

44 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 33

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45 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 34

The influence of the proper flowability of the tablet mass on minitablets compression process

Anna Kluk1, Magdalena Czajkowska1, Malgorzata Sznitowska1

1Department of Pharmaceutical Technology, Medical University of Gdansk, Hallera 10, 80-416 Gdansk, Poland, [email protected]

In minitablets (diameter 1-3 mm) production, the adequate flowability of the tablet mass is an important factor [1], because due to small diameter of punches, complete and reproducible filling of the dies can be difficult to achieve, even though the tablet mass quality seems to be efficient. These preliminary studies have been undertaken to estimate the quality of ten different placebo tablet masses in the form of granules or powders and its influence on the parameters of compression process and quality of the obtained minitablets. Placebo minitablets (2.5 mm, 13 mg) were obtained by wet granulation method or by direct compression using rotary tabletting machine (Erweka RTP-D8, Germany) equipped with spherical minipunches. Four granules formulations (G1-G4) made with Tablettose 80 and six powders mixtures (P1-P3 made with Tablettose 80 and Vivapur, P4-P6 made with Vivapur, Flowlac and Aerosil) were used. During compression, the changes in process parameters (precompression, compression, ejection force) and minitablets quality (mass, hardness) were monitored in equal time intervals. The physical properties of all investigated blends (moisture content, bulk and tapped density, flowability and for granules - size distribution) and obtained minitablets (mass uniformity, hardness, friability and desintegration time) were evaluated. The lower moisture content (0.78–1,04%) and bigger particles (500-630 um) for G1-G3 formulations in comparison to G4 (1,33% and 315 um, respectively) resulted in better flowability (lower values of Carr’s index and Hausner ratio). Small differences in amount of lubricant as well as in amount of binding solution and final concentration of the binder had no significant influence on flowability of granules. However, long time of compressing process (3 h) was probably the reason for particles segregation in the feeding hopper and as a result, serious problems in stabilization of compression and precompression forces and uniformity of mass and hardness of the obtained minitablets were observed. Flowability of powders mainly depended on their composition. Sodium stearofumarate as a lubricant provided better flow properties than magnesium stearate. Moreover, the change of proportions between amount of lactose and microcrystalline cellulose influenced significantly flowability. In comparison to tabletting of granules direct compression of powders P4-P6 was easier and more reproducible, with better control of the process parameters and minitablets quality. However, the tendency to constant increase of compression and precompression forces was noticed during direct compression process.

[1] J. Flemming, J. B. Mielck: Requirements for the production of micro-tablets: suitability of direct-compression excipients estimated from powder characteristics and flow rates, Drug Development and Industrial Pharmacy, 21, (1995) 2239-2251.

46 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 35

Melt-extruded solid dispersions of a pH-dependent and a neutral poorly soluble drug with Hydroxypropylmethylcellulose-Acetate-Succinate

Robin Meier1,2, Damir Elmar Zecevic2,3, Karl-Gerhard Wagner2,3,

1Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, 40225 Duesseldorf, Germany, [email protected] 2Boehringer Ingelheim Pharma GmbH Co. KG, 88397 Biberach an der Riß, Germany 3Institut für Pharmazeutische Technologie, Eberhard-Karls-University, 72076 Tuebingen, Germany

Introduction: A lot of new drug candidates belong to class II of the BCS and go along with poor bioavailability [1]. As many of those new entities are weak bases the poor solubility in the duodenal fluid (pH≈5.5) represents a major problem. The aim of this study was to produce gastro-resistant solid solutions of a basic and a neutral drug to transfer intact amorphous systems to the duodenal fluid. Materials and Methods: Dipyridamole (Boehringer Ingelheim, Germany) and Griseofulvin (ABCR, Germany) were used as drug substances. Hydroxypropylmethylcellulose-Acetate- Succinate (Aqoat LG, ShinEtsu, Japan), was used as polymer and matrix-forming agent. Powder-mixtures were extruded with a co-rotating twin-screw-extruder (Three-Tec, Switzerland) with a screw diameter of 9 mm and an L/D ratio of 20 at 140 °C. Three-step dissolution tests were performed using a miniaturized USP II paddle apparatus (Boehringer Ingelheim, Germany) and three different media within four hours (hours 0-2: pH 1; hours 2-3: pH 5.5; hours 3-4: pH 6.8). XRD-measurements were performed using a Stadi P (STOE, Germany). Results: Pure Dipyridamole dissolved completely at pH 1 after 1 min and remained solved until the pH was changed to 5.5. It successively recrystallized in the dissolution medium till pH 6.8 when only 10% of the original amount was solved. The melt-extruded matrices with HPMCAS showed completely different behavior. Depending on the drug-load of the matrices only small amounts of Dipyridamole (10-30%) were released within the first two hours. In return from pH 5.5 on the matrix dissolved slowly and provided a stable 5-10-fold supersaturated solution of Dipyridamole even in the weak acidic and the neutral medium. Crystalline Griseofulvin shows a pH-independent poor dissolution profile. Only 10% of the used drug dissolved over the whole measurement. The application of melt extruded HPMCAS- matrices of Griseofulvin led to an improved dissolution behavior, strongly depending on the drug/polymer-ratio. At pH 1 hardly any Griseofulvin dissolved, indicating a gastro-resistance of the matrices. Higher pH-values resulted in a 1.5-5-time improvement of the apparent solubility, compared to pure drug. The XRD-measurements of the Griseofulvin-matrices indicated that the dissolution performance depends on the grade of the matrices’ amorphicity. Conclusions: The acidic polymer HPMCAS is able to form amorphous matrices with different drug substances over a concentration-range, depending on the physicochemical properties of the drug. Furthermore it inverts the pH-depending dissolution-profile of a poorly soluble basic drug and strongly improves dissolution performance of Griseofulvin. [1] C.-Y Wu; L. Z. Benet: Predicting drug disposition via application of BCS: Trans-port/absorption/elimination interplay and development of a biopharmaceutics drug disposition classification system, Pharmaceutical Research, 22 (1) (2005) 11-23.

47 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 36

The physicochemical characteristics of poloxamer 407 hydrogels with photoactive dyes

Tomasz Osmaek1, Anna Froelich1, Anika Mielewczyk1, Dorota Wagner1, Klaudia Baszczyk1, Wojciech Biaas2, Mirosaw Szybowicz3, Robert Skonieczny3 1Department of Pharmaceutical Technology, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, [email protected]; 2Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences Wojska Polskiego 48, 60-627 Poznan 3Chair of Optical Spectroscopy, Poznan University of Technology, Nieszawska 13a, 60-965 Poznan

Introduction: Photodynamic therapy (PDT) is an alternative technique for the treatment of localized tumors. The combination of light and drug results in generation of highly toxic singlet oxygen followed by destruction of the cancer cells [1]. The major drawback of PDT is insufficient selectivity of the photosensitizers after systemic administration, which leads to the photosensitivity of the healthy skin [1,2]. Thus the topical carrier systems for PDT are currently under intense investigation [2]. The hydrogels based on poloxamers seem to be proper candidates for the delivery of the photoactive dyes [3].

Methods: The aim of the work was the characteristics of the hydrogels based on poloxamer 407 containing methylene blue, rose bengal and protoporphyrin IX. Transcutol® and oleic acid were added as the skin absorption enhancers. Benzalkonium chloride was used as a preservative. The rheological parameters of the formulations were calculated. The viscoelasticity was analyzed by oscillatory tests including stress and frequency sweep. The texture analyzer was applied to calculate hardness, elasticity and compressibility of the gels. Raman spectroscopy was utilized as an additional technique to monitor the phase transition temperature. The stability of the photoactive dyes during storage at different temperatures was evaluated by UV-Vis spectroscopy

Results: All the formulations followed non-Newtonian behavior and revealed pseudoplastic properties. The addition of the dyes decreased the viscosity of the hydrogels. Benzalkonium chloride increased the hardness and the elasticity. All the photosensitizers turned out to be stable.

Conclusions: The obtained data revealed that the properties of the poloxamer 407 hydrogels strongly depend on the presence and quantity of additional ingredients. The photosensitizers weaken the structure of the gels and decrease the transition temperature.

Acknowledgement: This study was partially supported by PUMS grant N0 502-14-03314429- 09983

[1] Riemma Pierre M.B., Ricci Jr E., Tedesco A.C., Lopes Badra Bentley M.W.: Oleic Acid as Optimizer of the Skin Delivery of 5-Aminolevulinic Acid in Photodynamic Therapy. Pharm. Res. 13 (2006) 360-366 [2] Donnelly R.F., McCarron P.A., Morrow D.I.J., Sibani S.A., Woolfson A.D.: Photosensitiser delivery for photodynamic therapy. Part 1: Topical carrier platforms. Expert. Opin. Drug. Deliv. 5 (2008) 757-766 [3] Dumortier G., Grossiord J.L., Agnely F., Chaumeil J.C.: A Review of Poloxamer 407 Pharmaceutical and Pharmacological Characteristics. Pharm. Res. 23(2006) 2709-2728

48 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 37

Study of the wettability of selected mucoadhesive polymers M. Olejniczak – Rabinek1, M. Rojewska2, A. Snela1, . Grobelny1, K. Prochaska2, J. Lulek1 1Department of Pharmaceutical Technology, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland; [email protected] 2Institute of Chemical Technology and Engineering, Poznan University of Technology Pl. M. Skodowskiej-Curie 2, 60-965 Poznan, Poland

Introduction: Several theories have been used to explain not fully understood mucoadhesion phenomenon. The surface tension of the mucus and the mucoadhesive polymer in the wetting theory correlates with its ability to spread on the mucus layer [1-3]. The aim of our study was evaluation of surface wettability of selected mucoadhesive polymers showing different physicochemical properties and their mixtures using the methods of contact angle. Methods: Measurements of advancing contact angle of liquid on the polymers and their mixtures were carried out using the sessile drop method with the instrument Tracker I.T.Concept. Contact angle measurement was repeated for each solution–model surface system six times. The curves presented show the average of the six measurements taken. Accuracy of the measurement was ±1.50. For the characterization of the powders and their mixtures, disc of 8 mm diameter were prepared by direct compression of 200 mg powder at 200 psi using hydraulic press (ICL). The compacted discs were placed in a sample holder and a liquid drop with a given volume was placed on each sample and observed by a video camera. Results:

80

70 Kollidon VA 64; contact angle after 1 sec. 60 ] o , [  50

40 HPMC; contact angle after 1 sec. 30 Contact angle angle Contact

20

10 0 5 10 15 20 25 30 HPMC; contact angle after 30 sec. Time, [s]

Fig. 1 Examples of dynamic contact angle of double distilled water on surface: Fig. 2 Examples of static contact angle of double ( ) KollidonVA64, ( ) Sodium alginate, ( ) Carbopol 974 P, ( ) HPMC. distilled water on surface. Conclusions: The results obtained indicate that the examined polymers show different wettability. It depended on the type of material, chemical nature and molecular structure of polymers. All analyzed substances have the contact angle values < 90o confirming their hydrophilic character. Acknowledgement: This study was partially supported by PUMS grants N0 502-14- 03314429-09228 and N0502-14-03314429-09818 and PUT grant N0 DS-PB 32/067/2013

[1] E.A. Kharenko, N.I. Larionowa, N. B. Demina: Mucoadhesive Drug Delivery Systems, Pharm. Chem. J., 43 (2009) 200- 208 [2] V. Khutoryanskiy: Advances in Mucoadhesion and Mucoadhesive Polymers, Macromol. Biosc 11 (2011) 748–764 [3] J. D. Smart: The basic and underlying mechanism of mucoadhesion, Adv Drug Deliver Rev 57 (2005) 1556 – 1568

49 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 38

CapsMorph: >4 Years Long-Term Stability of Industrially Feasible Amorphous Drug Formulations

R. H. Müller1, Q. Wei2, and C. M. Keck2 1Pharmacy Department, Free University, Berlin, 12169, Germany; 2PharmaSol GmbH, Berlin, 12307 Berlin, Germany [email protected] Introduction: CapsMorph was developed to generate amorphous formulation for improving the oral bioavailability of poorly soluble drugs by transferring them into the amorphous state in porous materials. In the CapsMorph process an encapsulation of amorphous drugs is performed in porous materials with a few nm sized ultrafine pores, e.g. in silica such as Neusilin US2 from Fuji [1]. However, the general problem of amorphous formulations is their tendency to re- crystallize. This restricts very much their use in pharmaceutical industry. In this study the ability of the CapsMorph technology to generate long-term stable amorphous state was investigated. After a storage time of at least 4 years, about 30 formulations were investigated. Methods: Model drugs, e.g. candesartan cilexetil (Beta Pharma Shanghai, China) were dissolved in various organic materials and incorporated in porous silica (Neusilin US 2, Fuiji) by adding the solution to the powder. The solvent was evaporated, and the process was monitored by the loss of weight during the evaporation process. The samples were prepared by Capsulution Pharma AG in Berlin (www.capsulution.com) and stored at room temperature. In previous studies it could already be shown that they possessed 3-year stability [2]. After minimum of 4 years of storage time, typically 4.5 years, we evaluated again the samples. The crystalline state was analyzed by differential scanning calorimetry (DSC) using a DSC 821e (Mettler Toledo, Germany) and by x- ray investigation using a PW 1830 (Philips, The Netherlands). Results: The x-ray spectra of candesartan cilexetil after 4.5 years of storage produced using different solvents indicated no crystalline particles in it. The amorphous state remains for such a long time, and that it is independent on which solvent was used for its generation. In all investigated about 30 formulations no re-crystallization was detected, proving the feasibility of the formulation technology for industrial use. Conclusion: The CapsMorph technology is industrially feasible, and cost-effective. The produced amorphous products remained amorphous for at least 4 years, sufficient for the shelf life of a pharmaceutical product (typically 3 years).

[1] González Ferreioro, M, Dunmann, C., Kröhne, L., Voigt, A., EP patent application 2 135 601 A1, priority date 20 June 2008 [2] Dunmann, C., Grabo, M., Müller, R. H., CapsMorphTM: technology for oral derlivery of poorly solubles – 3 year stability of an amorphous oral formulation, #600, Int. Symp. Control. Rel. Bioact. Mater. 38, National Harbor,Maryland, 2011

50 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 39

Dry granulation by roll compaction: Compactability study of various pre- processed mannitol grades

Carl Moritz Wagner, Miriam Pein, Jörg Breitkreutz

Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitätsstraße 1, 40225 Düsseldorf, Germany, [email protected] Introduction: In pharmaceutical industry, dry granulation by roll compaction (RC) gains importance since the process is cost-effective and continuous. Additionally, mannitol as excipient has received more and more attention due to its numerous advantages particularly for orodispersible drug formulations [1]. Therefore, the aim of this study was to investigate the roll compaction behavior of various pre-processed mannitol grades. Granules made of both spray- dried and granulated raw materials were characterized with respect to their particle size distributions (PSD), flow properties and BET surface area. In addition, the compactability of the granules was determined. Materials and methods: As granulated raw materials Mannogem® 2080 (SPI Pharma) and Pearlitol® 300 DC (Roquette) were used. Parteck® M 100 and M 200 (Merck Millipore) were chosen as spray-dried grades. All powders were granulated using an instrumented roll compactor (Mini-Pactor® 250/25, Gerteis). The specific compaction force was set to 2 kN/cm and 10 kN/cm, respectively. The gap between the rolls (1.5 mm) and the roll speed (3 rpm) was kept constant. The PSD of the granules was determined by digital image analysis using the Camsizer® XT (Retsch Technology). Flowability measurements were carried out using a ring-shear cell tester RST_01.pc (Dr. Schulze, Schüttgutmesstechnik). BET surface area was examined by nitrogen adsorption (Tristar, Micromeritics). Drug-free tablets were manufactured using an instrumented rotary tablet press (Pressima MX EU-B/D IMA Kilian) equipped with planar 10 mm punches (Ritter Pharma-Technik). Compression forces of 3 to 18 kN were applied. Results: Free-flowing granules (ffc-value > 10) with acceptable amounts of fine particles ( 90 μm) were produced applying 10 kN/cm. Compared to granules made of granulated qualities, granules of spray-dried mannitol show superior compactability. Higher tensile strength values are reached (1.76 ± 0.19 N/mm2 and 1.73 ± 0.17 N/mm2, respectively compared to 3.08 ± 0.20 N/mm2 and 3.51 ± 0.23 N/mm2, respectively) applying the same compression pressure (12 kN) during tableting. The benefit in compactability of granules made from spray-dried grades can be explained by the considerably larger area available for bonding during compression. This is reflected in the BET surface area of the granules (0.84 ± 0.01 m2/g and 0.80 ± 0.01 m2/g, respectively compared to 3.96 ± 0.02 m2/g and 3.87 ± 0.03 m2/g, respectively). After dry granulation a loss of compactability was observed for every sample, more pronounced at higher compaction forces. This effect can be explained by particle size enlargement and work-hardening phenomenon after roll compaction [2]. Conclusion: Granulated and spray-dried mannitol as pre-processed grades lead to granules with desired properties and to tablets with appropriate tensile strengths and disintegration behavior. Enhanced compactability was observed for both spray-dried mannitol grades.

[1] P. Kleinebudde, Roll compaction/dry granulation: Pharmaceutical applications, Eur. Pharm. Biopharm., 58 (2004) 317- 326. [2] S. Malkowska, K.A. Khan, Effect of re-compression on the properties of tablets prepared by dry granulation, Drug Devel. Ind. Pharm., 9 (1983) 331-347.

51 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 40

Applying QbD tools for Quetiapine XR 400 mg tablets formula composition development

Krzysztof Woyna-Orlewicz1, Renata Jachowicz2, Marta Zakrzewska

1,2Jagiellonian University Medical College in the Faculty of Pharmacy, Department of Pharmaceutical Technology and Biopharmaceutics, ul. Medyczna 9, Kraków 30-688.

e-mail: 1 [email protected]; 2 [email protected]

Introduction: Objective of pharmaceutical development is to design formula composition, manufacturing process and its controls in a way to deliver quality product in a repeatable manner. Knowledge gained during the development phase may support establishment of design space, i.e. the multidimensional combination and interaction of input variables (e.g. types and quantities of materials used) that provide output of desired quality attributes [1]. Aim of the study was to develop formula composition of prolonged release tablets containing quetiapine by applying Quality by Design (QbD) tools. Methods:The extended release tablets were designed as hydrophilic matrix system made by wet- granulation process. Quetiapine fumarate was mixed with buffering agent and agglomerated with binding solution in a low shear processor. The dried granulate was mixed-up with controlled release agents as well as with pre-selected extra-granular excipients. The final blend was compressed into tablets. A simplex lattice design was applied for testing influence of the controlled release agents, i.e. three HPMC grades (viscosities of 50, 100, 4000 mPa·s) on dissolution characteristics of the tablets. The main process outcome was similarity of dissolution profiles of generic and the reference tablets (Seroquel XR 400, AstraZeneca) expressed as f2150 and f1<15. Dissolution was tested in conditions recommended by the FDA for routine quality control of the product (app. II, 50 rpm., water). Results: Process responses were evaluated for each of the considered outcomes (f1 and f2). As a result two individual design spaces were established. Subsequently the two response surfaces were combined into one design space in form of simplex indicating HPMC quantities suitable to produce tablets characterized by similar dissolution profile to the reference drug. Conclusion: QbD approach was successfully achieved by applying Design of Experiments tools. Formula composition was established as design space in form of equilateral triangle. The apexes and sides of the triangle specify boundary levels of the controlled release agents.

[1] Pharmaceutical Development, ICH Harmonized Tripartite Guideline, Q8 (2009) 1–19.

52 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 41

Targeting to the hair follicles by roxithromycin-loaded lipid microparticles

Hanna Wosicka1, Justyna Stefanowska2, Biljana Govedarica3, Stane Srcic3, Krzysztof Cal2

1Department of Pharmaceutical Technology, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland; e-mail: [email protected]

2Department of Pharmaceutical Technology, Medical University of Gdansk, Hallera 107, 80-416 Gdansk, Poland; e-mail: [email protected]

3Department of Pharmaceutical Technology, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia

Introduction: The aim of the study was to develop lipid microparticles for follicular targeting in order to deliver roxithromycin – drug suppressing hair loss and acne symptoms. Methods: Lipid microparticles loaded with the drug and the fluorescent dye were prepared using ultrasonic homogenization. Morphology of the microparticles was determined by Atomic Force Microscope. The mean particle size and zeta potential were measured by photon correlation spectroscopy and microelectrophoresis, respectively. For ex vivo human skin penetration studies, the formulation was massaged into the skin for 5 min using a massage appliance. After a penetration time of 1 h at 37°C, the surplus of the formulation was removed. Vertical and horizontal sections of the skin tissue were obtained using a cryotome and the fate of the particles was observed using a fluorescence microscope. The formulation with drug but without fluorescent dye was used to perform differential stripping in vivo. The follicular biopsies were obtained by cyanoacrylate glue from sural region after 30 min penetration time and were analyzed for the drug content using HPLC-MS technique. Results: Microparticles were spherical in shape and were characterized by approximately 300 nm diameter and polydispersity index below 0.35. Immediately after ex vivo application of the fluorescent microparticles, the superficial aggregations in the follicular openings were clearly visible. No fluorescence was observed deeper in the skin tissue. After 1 h penetration time, skin sections revealed fluorescence only in the areas of the hair follicles deeper in the dermis. Differential stripping performed in vivo demonstrated the drug presence in the hair follicles. Conclusions: The study proved both ex vivo and in vivo that using particulate drug carriers, it is possible to achieve preferential delivery of roxithromycin to the pilosebaceous unit. Acknowledgements: The study was supported by grant No N N405 674740 from the National Science Centre (Poland).

53 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 42

Preparation and physico-chemical characterization of in situ porous implants loaded with a donor of nitric oxide

Aleksandra Gostyska¹, Marianne Parent², Ariane Boudier², Eliza Gówka¹, Janina Lulek1, Philippe Maincent²

¹Department of Pharmaceutical Technology, Poznan University of Medical Sciences, 6 Grunwaldzka Street, 60-780 Pozna, Poland; [email protected]; ²CITHEFOR EA 3452 "Cibles Thérapeutiques, Formulation et Expertise Préclinique du Médicament", University of Lorraine, 5 Albert Lebrun Street, 54001 Nancy, France

Introduction: Nowadays, tissue-engineered biodegradable polymer scaffolds for cell seeding have attracted a great interest, particularly as potential vascular grafts or patches for vascular surgery [1]. Nevertheless, it has been reported that currently used non-biodegradable polymer scaffolds may have several side effects including thrombosis, calcification, infection and lack of cell growth [2]. Therefore, to overcome these side effects, the aim of this work was to develop biodegradable in situ porous implants loaded with one of the nitric oxide (•NO) donors that could be used as a vascular scaffold for growth of cells.

Methods: In situ porous implants were prepared using a poly(D,L-lactic-co-glycolic acid) (PLGA). In the first step, the polymer was dissolved in N-methyl-2-pyrrolidone, then, NaCl crystals were added to the organic solution as a porogen. After injection of this suspension into an aqueous medium, solvent and salt leaching to the medium triggered the solidification of the polymer and formation of the porous implant. Finally, the following •NO donors were loaded into the system: either S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or isosorbide dinitrate (ISDN).

Results: The release of the •NO donors from porous implants was well correlated with their lipophilicity (logP). After 30 h (14 medium changes), GSNO (predicted logP = -2.7) was completely released, while 0.8% of SNAP (predicted logP = 0.08) and 83% of ISDN (predicted logP = 0.87) were not released to the medium and still remained in the system. The surface and interior porosity of the obtained formulations should be suitable for cell seeding as observed by scanning electron microscopy (SEM). Preliminary studies with rat aortic smooth muscle cells (A-10 cell line) showed that the implants were seeded by cells, however, probable inhibition of cell growth was observed after 48 h of incubation.

Conclusions: Overall, it was demonstrated that in situ PLGA porous implants could be efficiently prepared by a simple method using NaCl crystals as a porogen. In vitro release of the incorporated •NO donor was strongly influenced by the lipophilicity of the loaded drug. In conclusion, the obtained in situ porous implants could be used as •NO releasing scaffolds allowing cell seeding. However, further cell culture experiments should be done to determine the impact of the scaffold loaded with •NO donors on cell physiology. Acknowledgement: This study was partially supported by PUMS grant N0 502-01-03314429- 03439

[1] S.W. Cho, O. Jeon, J.E. Lim, S.J. Gwak, S.S. Kim, C.Y. Choi, D.I. Kim, B.S. Kim: Preliminary experience with tissue engineering of a venous vascular patch by using bone marrow-derived cells and a hybrid biodegradable polymer scaffold, J Vasc Surg, 44 (2006) 1329-1340. [2] C. Spadaccio, M. Chello, M. Trombetta, A. Rainer, Y. Toyoda, J.A. Genovese: Drug releasing systems in cardiovascular tissue engineering, J Cell Mol Med, 13 (2009) 422-439.

54 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 43

The influence of hydrochlorothiazide on cilazapril stability in model mixture and fixed dose tablet formulation

Sylwia Katarzyna Paszun1,2, Beata Jadwiga Stanisz2

1Student of full-time Ph.D. Programme, 2Department of Pharmaceutical Chemistry, Pozna University of Medical Sciences, Grunwaldzka 6 St., Pozna, [email protected] Introduction: Cilazapril and hydrochlorothiazide constitute preferred drug combination in hypertension treatment. In many cases, to achieve better compliance, during the combination therapy fixed dose pharmaceutical formulation is applied.1 Nevertheless, what is undoubtedly advantageous from biological point of view that can be disadvantageous, while taking into consideration chemical stability.2 Though, the aim of this study was cilazapril stability assessment in the presence of hydrochlorothiazide. Methods: The kinetic study of cilazapril-hydrochlorothiazide model mixture and fixed dose tablets was carried out by means of isothermal stress testing protocol (T = 333-343 K, RH = 50.9- 74.6%), with subsequent quantitative HPLC analysis. Results: Cilazapril in the presence of hydrochlorothiazide degrades according to autocatalytic reaction kinetic mechanism (Prout-Tompkins equation). Hydrochlorothiazide coexistence with cilazapril in model mixture (Fig. 1) and fixed dose tablet (Fig 2) without blister package accelerated cilazapril degradation in comparison with degradation of pure cilazapril. Increasing values of relative humidity and temperature influent negatively cilazapril stability.

Fig. 1 Fig. 2

Semi-logarithmic relationships: lnk = f(RH) and lnk = f(1/T) are linear. The blister package of commercial fixed dose tablets, protect tablet from moisture and prevents the occurrence of cilazapril – hydrochlorothiazide interaction. Conclusions: Hydrochlorothiazide affects cilazapril stability in model mixture as well as in tablet fixed dose form. The acceleration of cilazapril degradation reaction occurs: shortening of the reaction induction time and the increase of the observed reaction rate constant value. It was also established that elevation of temperature and relative humidity lead to further cilazapril destabilization. It can be concluded that cilazapril-hydrochlorothiazide pharmaceutical formulations should be stored in dry and low temperature environmental conditions. Acknowledgements: The study was financially supported by academic grant for young scientists (No. 502-14- 03305411-50551). [1] A. Prejbisz, A. Januszewicz: Leki zoone, Medical education, Warsaw 2012. [2] S. Yoshioka, V. J. Stella: Stability of drugs and dosage forms. Kulwer Academic Publishers, New York 2002.

55 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 44

In vitro studies of silicone transdermal patches with indomethacin incorporated into matrix or adhesive layer

Barbara Mikolaszek1, Magorzata Sznitowska1

1Department of Pharmaceutical Technology, Medical University of Gdask, Hallera 107, Gdask, Poland e-mail; [email protected]

Dermal patches are considered to be an alternative for other topical preparations. Ointments or cream require daily application, which can be inconvenient for a patient whereas patches provide prolonged contact between active substance and the skin. Silicones are being considered as a suitable material for a patch matrix formulation [1]. Most of elastomers lack of self-adhesiveness and require additional adhesive layer. This layer, however, may affect the drug release from silicone dressing [2]. Most popular method of patch preparation is incorporating an active ingredient into elastomer matrix. Because indomethacin is incompatible with one of the considered matrix-forming silicone materials, possibility of indomethacin incorporation into the adhesive layer was investigated. The effect of drug loading either in matrix or in adhesive layer on the release rate of indomethacin from silicone membranes was evaluated. Two types of transdermal patches were prepared, both made with two-part silicone elastomer blends. Indomethacin was mixed with first component of Gumosil AD-1 (Silikony Polskie), and then the second component was added to obtain matrix-type membrane (1% and 5% w/w drug load). In the second formulation silicone elastomer C6-135 (Dow Corning), incompatible with indomethacin, was used as a drug-free supporting layer and was covered with the adhesive layer components (MG 7-9850, Dow Corning) blended with indomethacin (1% and 2% w/w drug load). In vitro release studies of indomethacin were conducted for 48 h using phosphate buffer pH 7.2 at 37 C as acceptor fluid. Every 12 h samples of acceptor medium were assayed using spectrophotometric method (320nm). Both drug loading methods resulted in attaining visually acceptable silicone films. Indomethacin incorporated in the adhesive layer caused, however, significant decrease of adhesiveness in comparison with placebo adhesive layer. Release rate of indomethacin incorporated into the adhesive layer after 48 h was similar to one achieved for 1% loaded matrix formulations (146.29.4 and 140.731.2 g/cm2, respectively). Whereas both incorporating methods resulted in effective drug release rate, significant loss of adhesiveness caused by drug loading into the adhesive layer was found to limit usefulness of the patch with the drug present in the adhesive silicone layer. Among the studied silicone matrix components only Gumosil can be considered as compatible with the drug.

[1] C. McConville et al.: Reological evaluation of the isothermal cure characteristic of medical grade silicone elastomers, Journal of Applied Polymer Science, 116 (2010) 2320-2327. [2] A. Colas: Silicones in pharmaceutical applications. Dow Corning Publications (1997)

56 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 45

Protein adsorption patterns on i.v. dendritic polyglycerol sulfate nanoparticles for prediction of in vivo fate S. Staufenbiel1,3, D. Gröger2,3, R. Haag2,3, R. H. Müller1,3

1Freie Universität Berlin, Department of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Kelchstr. 31, 12169 Berlin, Germany, [email protected]; 2Freie Universität Berlin, Institute of Chemistry and Biochemistry, Takustr. 3, 14195, Berlin, Germany; 3Helmholtz Virtual Institute (HVI) on „Multifunctional polymers for medicine“, Takustr. 3, 14195, Berlin, Germany

Intravenously administered nanocarriers are on the pharmaceutical market for controlled delivery of drugs (e.g., liposomes like Ambisome®, Doxil® etc.). All these nanocarriers accomplish a controlled release, but are not a targeted application. A simple approach to achieve targeting is the so called “differential protein adsorption” [1]. The proteins adsorbing on the nanocarrier surface after i.v. administration (protein adsorption pattern) differentiate to which cells (organs) the particles are going in the body. To exploit this targeting principle, the correlation needs to be established between patterns and organ distribution, requiring the analysis of the proteins patterns by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) [2]. Knowing the patterns one can predict the in vivo distribution to a certain extent, optimize nanocarriers in vitro and reduce the number of animal studies. For the analysis, the nanocarriers are incubated in plasma and then they need to be separated from unbound proteins, e.g., made by centrifugation or size exclusion chromatography (SEC). However, for ultra-small nanoparticles such as the analyzed dendritic polyglycerol sulfate (dPGS) nanoparticles (M=14.2 kDa) with sizes around 6 nm, these separation methods are not suitable (too long centrifugation times, similar sizes of dPGS and most proteins lead to no clear separation in SEC). Dendritic PGS was synthesized with the aim to develop new synthetic heparin analogues. It has less anti-coagulative, but higher anti-inflammatory properties than heparin [3]. The i.v. injected dPGS nanoparticles should avoid macrophage uptake (liver, spleen), circulate as stealth particles in the blood or accumulate in inflamed regions. To analyze (and optionally optimize) the pattern, a new separation method for ultra-small nanoparticles was developed and the protein analysis was performed. The particles were intentionally aggregated by precipitation with barium chloride, allowing fast separation by applying moderate centrifugation conditions. The aggregates were subsequently subjected to protein analysis by 2-D PAGE. This procedure can be transferred to all ultra-small nanoparticles with appropriate functional groups allowing a precipitation step with a salt. In general, the dPGS nanoparticles exhibited a very low protein adsorption, which supports the relatively weak protein affinity (0.48% of total protein in the sample bound to dPGS). From the little amount of bound protein, the dPGS nanoparticles showed a high adsorption of the dysopsonin albumin. This reduces opsonin adsorption, protecting against uptake by the monocyte phagocytic system (MPS) and promoting circulation in the blood. In addition, a pronounced adsorption of apolipoproteins A1 and A4 was found, promoting uptake by the brain. From this the dPGS nanoparticles appeared suitable for i.v. injection with reduced MPS recognition, and having some affinity to the CNS. The predicted in vivo distribution was well in agreement with the observed data in vivo. This shows that the analysis of proteins adsorption pattern is a helpful in vitro tool for the optimization of i.v. nanocarriers regarding the desired organ distribution.

[1] Müller, R. H., Heinemann, S., in: Bioadhesion - Possibilities and Future Trends (Gurny, R. and Junginger, H. E., eds.), Wissenschaftliche Verlagsgesellschaft Stuttgart, p.202-214, 1989 [2] Blunk, T., et al., Electrophoresis 14(12), 1382-1387, 1993 [3] Türk, H., R. Haag, and S. Alban, Bioconjugate Chemistry, 15(1): p. 162-167, 2003

57 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 46

Development of a flow-through dissolution method for the in-vitro evaluation of diclofenac sodium release from selected rectal dosage forms Bartomiej Milanowski1, Aleksandra Mitka1, Marek Milewski2, Stefan Maycha2, Janina Lulek1 1 Department of Pharmaceutical Technology, Faculty of Pharmacy, Poznan University of Medical Sciences, 6 Grunwaldzka Str, 60-780 Pozna ,Poland [email protected] ; 2 PozLab Ltd. (Contract Research Organization), 2 Parkowa Str, 60-755 Pozna, Poland

Introduction: The flow through method (USP-Apparatus 4) for the determination of the dissolution rate/release rate is a convincing alternative to the known stirrer methods. This method is not only successfully used for tablets and capsules, but with special flow through cells also among other for suppositories, powders and granulates. The aim of our study was to develop a flow-through dissolution method to evaluate sodium diclofenac (DIC) release profiles from selected rectal drug dosage forms. Methods and Results: The in vitro release test was based on the Polish Pharmacopoeia IX (PF IX) regulations regarding testing of release of solid lipophilic drug forms [1]. The following formulations were selected: five suppositories-Diclac, Diclofenac GSK, Dicloratio, Dicloreum and Voltaren as well as one rectal capsule – Olfen. All products contained 100 mg of sodium diclofenac. Preliminary, mass uniformity according to PF VII [2] as well as a disintegration time according to PF IX [1] of both suppositories and a capsule were investigated. Additionally, softening time according to PF IX [1] was determined for lipophilic suppositories. Then, the spectrophotometric method (zero-order and first derivative) of DIC determination in flow-through cell apparatus on/off-line mode was developed. Furthermore, analytical wavelength for DIC and linearity range of spectrophotometric method was established. The precision of UV determinations was calculated and the influence of excipients on the absorption rate of active substance was tested. Moreover, a stability of sodium diclofenac in standard solution was investigated. Next, optimal conditions for the release of API from rectal formulations in the flow-through cell apparatus were established based on “Design of Experiment” theory (DoE) [3]. The conditions for 11 trials were set up based on the complete plan 2(3-0) with three repetition of a centre point. The Gompertz mathematical model, where  parameter represents quality of dissolving process, was applied to the obtained release curves. The statistical analysis of the data was carried out to identify factors having significant effects on the release of API and to select the optimal release conditions of the flow-through method. The final stage of the research included the investigation of in vitro drug release of 12 units of each examined product under optimal conditions in the flow-through cell apparatus using both spectrophotometric and HPLC quantitative analyses of a released drug. Conclusions: The analysis of similarities between the release profiles indicated the lack of pharmaceutical equivalence of Diclofenac GSK and Dicloratio against the reference product – Voltaren.

Acknowledgement: This study was partially supported by PUMS grant N0 502-01-03314429- 03439

[1] Polish Pharmacopoeia IX. Polish Pharmaceutical Society, Warszawa (2011). [2] Polish Pharmacopoeia VII. Polish Pharmaceutical Society, Warszawa (2007). [3] International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonised Tripartite Guideline Pharmaceutical Development Q8(R2), Current Step 4, version dated August 2009.

58 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 47

Nanostructured composite materials HPC-SiO2-CaO-P2O5 as potential carriers for metronidazole

Katarzyna Czarnobaj, Anna Bierek, Wiesaw Sawicki

Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, [email protected] Organic-inorganic composite materials open possibilities of tailoring of the materials with improved mechanical and chemical properties, required toughness as well as elasticity comparing to individual components. One of the most efficient methods of preparation of these materials is the sol-gel process. This technique comprises initial hydrolysis of metal alkoxides (silicon, titanium, phosphorus) or salts and subsequent condensation reactions, resulting in metal oxides. Obtained materials are nontoxic, porous and can act as a carrier of drug [1-3]. The aim of this study was to obtain the porous polymer-modified oxide matrices containing metronidazole (2-methyl-5-nitroimidazole-1-ethanol), as the controlled delivery systems, which could be used in treatment and regeneration of diseased hard tissues. The organic-inorganic composites based on hydroxypropyl cellulose (0, 10, 20 or 30% w/w HPC) were synthesized applying sol-gel process. The inorganic phase was introduced into polymer matrix by hydrolysis and condensation using tetramethoxysilane (TMOS), triethyl phosphate (TEP) and calcium chloride as the precursors of SiO2-CaO-P2O5 oxides, under acidic conditions. Metronidazole (MT), applied in periodontal disease treatment, was chosen as the model drug for preparing the controlled delivery systems. The drug was incorporated into the matrices on the stage of hydrolysis and condensation of matrix precursors. The obtained composite matrices were characterized by different techniques such as FTIR spectroscopy, X-ray powder diffraction (XRD) and the Brunauer-Emmet-Teller (BET) technique. The analysis of the profiles of MT release in SBF solution (SBF – simulated body fluid, pH 7.4) was also evaluated. Based on the presented results it was found that additive of HPC to the oxide matrices affected their mechanical and bioactive properties as well as the drug release from matrices. Increasing the HPC content in matrices improved their mechanical stability in comparison with the brittle oxide matrix, and accelerated the formation of apatite on matrices surface in the test for bioactivity (8 days, 2 weeks and 1 month for matrices with 30, 10 and 0% HPC content, resp.). The study of release of MT to SBF solution showed clear influence of HPC on the prolonged release of drug substance. Total release of MT from matrices with increasing HPC content was observed within 3 h, 8 h, 20 h, 2 days, respectively, whose mechanism was found to be diffusion- controlled. The results of this study emphasize the potential of HPC-SiO2-CaO-P2O5 matrices as controlled drug delivery systems for MT in treatment and regeneration of diseased hard tissues. References [1] F. de Gaetano, L. Ambrosio, M. Raucci, A. Marotta, M. Catauro: Sol-gel processing of drug delivery materials and release kinetics, J Mater Sci Mater Med, 16 (2005) 261-265. [2] R. Shula, S. Falaize, M. Lee, P. Ducheyne: In vitro bioactivity and degradation behavior of silica xerogels intended as controlled release materials, Biomaterials, 23 (2002) 3113-3122. [3] K. Czarnobaj, W. Sawicki: The sol-gel prepared SiO2-CaO-P2O5 composites doped with Metronidazole for application in local delivery systems, Pharm Dev Technol, 17 (2012) 697-704.

59 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 48

Alcohol induced dose-dumping dissolution studies of selected venlafaxine hydrochloride extended-release dosage forms

Bartomiej Milanowski, Micha Skrobaa, Anna Surosz, Janina Lulek Department of Pharmaceutical Technology, Poznan University of Medical Sciences, 6 Grunwaldzka Street, 60-780 Poznan, [email protected]

Introduction: Some modified-release (MR) oral dosage forms contain drugs and excipients which are highly soluble in ethanol, leading to concerns that these products might dose-dump if co-administered with alcoholic beverages. Such dose-dumping could pose a significant risk to patients, either because of increased toxicity or diminished efficacy or both [1-3]. The aim of our study was to assess the performance of an in vitro assay to determine the potential for alcohol induced dose-dumping of some commercially available venlafaxine hydrochloride extended release dosage forms. Methods: The following four extended release products were obtained from local pharmacy: Efectin ER 75 mg (pellets in hard gelatin capsule), Jarvis 75 mg (pellets in hard gelatin capsule), Prefaxine 75 mg (matrix tablets in hard gelatin capsule) and Axyven 75 mg (OROS system). The capsule contents and OROS system were subjected to in-vitro dissolution in 0.1mol/L HCl without and with ethanol concentration ranging from 5-40% v/v. Dissolution was carried out using USP-Type IV apparatus at optimized conditions: closed (500 mL of dissolution media at 37±0.5°C) or open loop (simulation of physiological conditions after ingestion of alcoholic beverages), cell diameter – 22,6 mm; flow rate – 4 mL/min; flow type - turbulent. Drug concentrations were analyzed on-line every 5 min. (closed loop) or every 1- 2.5 min. (open loop) until 2h using UV spectrophotometer at 274 nm. Venlafaxine hydrochloride release profiles were compared according to calculated similarity factor f2. Furthermore, the release rate constants calculated according to Korsmeyer-Peppas kinetic model, were compared. Results and Conclusions: In vitro studies in closed loop system have demonstrated that Efectin ER and Jarvis did show faster rate and complete drug release in 500 mL of 0.1mol/L HCl solutions containing 40 % ethanol (v/v) while Axyven released 50% of the drug and only Prefaxine formulation composition and production technology provide the same release profiles regardless of alcohol concentration in dissolution medium. This perturbed drug release can be explained by the solubility of the polymers in ethanol. Ethylcellulose has a higher solubility than Eudragit in ethanol which explains why alcohol has a more detrimental effect on drug release from Jarvis and Efectin in comparison to Prefaxine. We also found that the rate of drug release from Axyven 75 mg (OROS system) increase significantly in 0.1mol/L HCl after short period testing (30 min.) in the presence of 40% v/v ethanol.

Acknowledgement: This study was partially supported by PUMS grant N0 502-01-03314429- 03439

[1] P. Chandaroy, X. Jiang, C. Lee, C. Thompson, In Vitro Alcohol-induced Dose-dumping Dissolution Studies of Generic Modified-release Oral Drug Products. AAPS National Biotech Conference Abstracts, AAPS Journal. 10 (2008) (S1) [2] Meyer R., Hussain A., FDA’s ACPS Meeting: Mitigating the Risk of Ethanol Induced Dose Dumping from Oral Sustained/Controlled Release Dosage Forms. Location: Food and Drug Administration, Center for Drug Evaluation and Research Advisory Committee Conference Room, October 25-26, 2005, Rockville, USA. [3] Smith A. P., Moore T. W., Westenberger B. J., Doub W. H., In Vitro dissolution of oral modified-release tablets and capsules in ethanolic media. Int. J. Pharm 2010; 398: 93-96.

60 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 49

Influence of hydrophilic matrix tablets composition on sildenafil citrate dissolution profile

Krupa A., Malak E., Mendyk A., Jachowicz R.

Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, Jagiellonian University, Medical College, 9 Medyczna st., 30-688 Cracow, Poland, e-mail: [email protected]

Introduction: Sildenafil citrate is a BCS class I drug used in the therapy of pulmonary hypertension in adults and children. In general the drug should be administered three times a day. Therefore, with the aim to improve the patient compliance, sustained drug release formulations were developed in the present study [1].

Methods: Tablets containing 10 mg of sildenafil citrate, 4 mm in diameter were prepared by compression method. The drug was wet granulated prior tableting with Erweka EK0 press. Hydrophilic matrix was formed of sodium alginate, hypromellose (Metolose® 60 or 90 SH) and silicified microcrystalline cellulose (Prosolv® SMCC HD90). Dissolution studies were performed by the basket method in 900 mL of 0.1 M HCl. Analysis of the dissolution kinetics was carried out with KinetDS 3.0 software [1, 2].

Results: Dissolution results and kinetics studies of sixteen formulations prepared, showed that the amount of Metolose® influenced the most the release of sildenafil citrate. Only 30% of sildenafil was released after 125 min if the tablet matrix was formed of Metolose®. The dissolution process followed zero order kinetics. The incorporation of sodium alginate or silicified microcrystalline cellulose into the tablet mass caused the increase in the amount of sildenafil released. Sildenafil citrate was released immediately from tablets containing Prosolv® SMCC HD90 (Q15>96%).

Conclusions: The incorporation of sildenafil citrate into the hydrophilic matrix composed of both sodium alginate and Metolose® 60/90 SH resulted in tablets characterized by uniform drug distribution and sustained drug release.

[1] P. Costa , J.K. Sousa Lobo: Modeling and comparison of dissolution profiles, European Journal of Pharmaceutical Science, 13 (2001) 123-133. [2] A. Mendyk A., R. Jachowicz, K. Fijorek, P. Doroyski, P. Kulinowski, S. Polak: KinetDS: An Open Source Software for Dissolution Test Data Analysis, Dissolution Technologies, 19 (2012) 6-11.

61 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 50

Optimization of Inter-Tablet Coating Uniformity in an Active Coating Process

S. Just1, G. Toschkoff2, A. Funke3, D. Djuric4, G. Scharrer2, J. Khinast2, K. Knop1, P. Kleinebudde1

1 Institute of Pharmaceutics and Biopharmaceutics, Heinrich Heine University, Duesseldorf, Germany, [email protected]; 2 Research Center Pharmaceutical Engineering GmbH, Graz, Austria; 3 Global Chemical and Pharmaceutical Development, Bayer Pharma AG, Berlin, Germany; 4 L.B. Bohle Maschinen + Verfahren GmbH, Ennigerloh, Germany

Introduction: The objective of this study was to improve the inter-tablet coating uniformity in an active coating process at lab scale by statistical design of experiments (DoE). Methods: A 24 DoE of an active coating process was performed in a side-vented lab pan coater (BFC 5, L.B. Bohle, Germany). The factors pan load, pan speed, spray rate, and the number of spray nozzles were systematically varied. To assess the inter-tablet coating uniformity, the content uniformity of the active pharmaceutical ingredient in the coating layer of the active coated tablets was determined (n=20) by HPLC analysis (Elite LaChrom, VWR Hitachi, Germany). The eluent phosphate buffer (pH 3.0; 3 mM)–methanol (20:80, v/v) was conveyed at a flow rate of 0.6 ml/min on a C18-column (X Bridge, Waters GmbH, Germany) with UV detection at 260 nm. The content uniformity was characterized by the coefficient of variation (CV). Results: A statistically significant regression model (p < 0.001) was obtained. The number of spray nozzles was found to be the most significant parameter to enhance the coating uniformity. The CV was significantly reduced by increasing the number of spray nozzles from two to four. A low spray rate and a high pan speed also significantly improved the coating uniformity. The CV decreased from 13.4% to 6.4% in case of the highest CV, and from 4.5% to 2.3% in case of the lowest CV. Furthermore, by scaling the number of spray nozzles from to two four, the process time can be reduced by half. Conclusions: In this study, the number of spray nozzles was identified as the most influential variable with respect to the coating uniformity. With four spray nozzles, CV values below 6% were achieved as required from regulatory authorities.

62 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 51

Size-dependent Antioxidant Capacity of Tailor-made Quercetin Nanocrystals: a quick look from KRL Test

R. Chen1, M. Prost2, L. Greiner3, R. H. Müller1, and C. M. Keck1,3

1 Department of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Free University of Berlin, Kelchstr. 31, 12169 Berlin, Germany, [email protected]; 2Kirial International/Laboratoires Spiral, 3 rue des Mardors, 21560 COUTERNON, France; 3 Fachhochschule/University of Applied Sciences Kaiserslautern Campus Pirmasens, Carl-Schurz- Str. 10-16, 66953 Pirmasens, Germany

Introduction Nanonization of quercetin can enhance its kinetic solubility, thus improve their proven antioxidant capacity. Nanocrystals with smaller size are expected to have more pronounced solubility improvement, thereby more efficient antioxidant effect. This study produced quercetin nanocrystals with sequential sizes and investigated their antioxidant capacity via the novel KRL Test. Methods Quercetin nanocrystals were processed via high pressure homogenization with formulation of 2% quercetin, 0.2% Poloxamer 188, 95.3% injectable water, and 2.5% Glycerol 85%. Different sizes (sample A to E) were obtained by collecting samples after certain homogenizing cycles. All samples were analyzed by photon correlation spectroscopy and by laser diffraction. The KRL Test was used to investigate the respective antioxidant capacity by correlating the resistance of whole blood and red blood cells to free radical attack. A range of concentration from 0 to 50 mg/L of reaction medium as regards to flavonoid content of nanosuspensions (20 g/L of the liquid sample) was applied. The results were expressed as the time that is required to reach 50% of maximal hemolysis. Results The obtained quercetin nanocrystal sizes ranged from 204 nm (sample E) to 640 nm (A) due to the change of pressure and cycle number of high pressure homogenization (from 2 cycles of 250 bar up to 20 cycles of 1500 bar). It has been proven that increase in the energy input and/or the processing time can break the active’s powder further. The KRL Test results clearly presented that increasing concentration in each sample led to an increase of half-hemolysis time, thus the antioxidant capacity. Owing to the enhanced kinetic dissolution of nanocrystals, the increasing effect with the decreasing of size of quercetin nanocrystals was significant, especially at higher concentrations. Conclusions Tailor-made quercetin nanocrystals with a range of sizes were successfully produced. The results of the novel KRL Test proved that all of them had an increasing antioxidant capacity with increasing of concentration and decreasing of size.

63 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 52

Apparent and intrinsic dissolution rate studies of the glycyrrhizic acid ammonium salt

Bartomiej Milanowski1, Beata Czarczyska-Goliska1, Janina Lulek1, Ewa Tykarska2

1 Department of Pharmaceutical Technology, Faculty of Pharmacy, Poznan University of Medical Sciences, 6 Grunwaldzka Street, 60-780 Pozna, [email protected] 2Department of Chemical Technology of Drugs, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Pozna

Introduction: The dissolution rate of active pharmaceutical ingredients (API) and excipients is one of the most important properties suitable for their characteristics, particularly at the preformulation stage. It depends on a crystalline form, particle size and specific surface area. Biopharmaceutics Classification System (BCS) allows the assessment of the solubility and permeability of API based on the results obtained in in vitro release studies [1]. Glycyrrhizic acid (GA) is a triterpenoid saponin isolated from licorice root. It consists of an aglycone-18ß-glycyrrhetinic acid, and the sugar moiety, which is assembled of two glucuronic acid residues. GA has a significant therapeutic relevance, and according to the recent studies, GA complexes with hydrophobic drugs increase their solubility in water improving their bioavailability [2]. The purpose of our study was to determine solubility class membership of glycyrrhizic acid ammonium salt using disk intrinsic dissolution rate (DIDR) and apparent dissolution rate (ADR). Methods: We employed an Erweka dissolution apparatus fitted with a Wood’s intrinsic dissolution die as well as flow through cell apparatus equipped with powder cells. The DIDRs and ADRs of glycyrrhizic acid ammonium salt were determined in the solutions at pH 1.2, 4.5, and 6.8 as well as in purified water. DIDR was established using rotating disk (100 rpm) of pure API, compressed at 600 psi and immersed in a 500 mL of dissolution medium maintained at 37±0.5°C. During ADR study in flow through cell apparatus medium flow rate, dissolution volume and flow type were 8 mL/min, 500 mL, and laminar, respectively. Drug concentrations were analyzed in dissolution media on-line every minute using UV spectrophotometer at 254 nm. Results and Conclusions: We did not observe any polymorphic form change of API in the compressed disk using Differential Scanning Calorimetry (DSC). We have shown the DIDR of glycyrrhizic acid ammonium salt is above the cut-off value of 0.1 mg/min/cm2 which allows us to classify this API as a high soluble compound in line with solubility classification according to BSC [3]. On the other hand we found ADR is pH-dependent.

Acknowledgement: This study was partially supported by PUMS grant N0 502-01-03314429- 03439

[1]Amidon G.L., Lennernäs H., Shah V.P., Crison J.R.: A Theoretical Basis for a Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability, Pharm. Res. 12 (1995) 413-20. [2] Harwansh R.K., Patra K.C., Pareta S.K., Singh J., Biswas R.: Pharmacological studies on glycyrrhiza glabra: a review, Pharmacologyonline 2 (2011) 1031-1038 [3] Yu L.X., Carlin A.S., Amidon G.L., Hussain A.S.: Feasibility studies of utilizing disk intrinsic dissolution rate to classify drugs, Int J Pharm. 270 (2004) 221-227.

64 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 53

Development of nanostructured lipid carriers (NLC) formulations stabilized with sugar ester

Cornelia M. Keck1,2 Andjelka Kovaevi3, Snežana Savi3, Jela Mili3, Rainer H. Müller1

1Department of Pharmaceutics, Biopharmaceutics and Nutricosmetics, Institute of Pharmacy, Free University of Berlin, Kelchstrae 31, 12169 Berlin, Germany, [email protected] 2University of Applied Sciences Kaiserslautern, Applied Pharmacy, Carl-Schurz-Str. 10-16, D-66953 Pirmasen, Germany, [email protected] 3Department of Pharmaceutical Technology and Cosmetology, Faculty of Pharmacy, University of Belgrade, 11000 Belgrade, Serbia, [email protected]

Sugar esters are nonionic natural surfactants that found many applications in different areas of drug delivery, and some of them are already used in the pharmaceutical industry. NLC are lipid nanoparticles of the second generation which are successfully implemented in the cosmetic industry due to number of positive features [1]. The aim of this study was to develop NLC stabilized with sucrose stearate and to investigate the influence of oil content on the characteristics of the particles obtained. The solid lipid content was defined as 10% (w/w) in all NLC formulations, whereas the percentage of oil in the lipid matrix of the NLC varied between 20% and 40%. The investigated samples are labeled as: S (20), S (30), S (40), where the number in the bracket represents the weight percentage of oil to total lipid. For example, S (20) contains 20 % of the oil in the total lipid. The average particle size and polydispesity index (PI) were measured using a Zetasizer Nano ZS (Malvern Instruments, UK). Laser diffraction measurements were done using a Mastersizer 2000 (Malvern Instruments, UK). Differential scanning calorimetry (DSC) was employed to investigate the effect of the oil on the crystalline structure of the lipid matrix in the NLC. Fig. 1 shows size and polydispersity index (PI) of NLC after production and after 90 days storage at controlled room temperature measured by PCS. No differences in the mean particle population can be seen between different formulations after production. After 90 days in the sample S (30) the main (d(v)0.50)) and large particle population (d(v)0.99) stayed unchanged. In contrast to this in the samples S (20) and S (40) the presence of larger particles beside nanometer bulk population was observed. An increase in the particle size in these samples was followed with the deviation in the melting enthalpy and the width of the melting event (DSC study) (Fig. 2).

1 3000 8 4 0.8 2000 6 0.6

0.4 4 2 1000 z-ave (nm) 0.2 size (micrometer) size 2 0 0 melting enthalpy (J/g)

S (20) S (30) S (40) S (20) S (30) S (40) width of the meliting area (°C) 0 0 S (20) S (30) S (40) day 0 day 90 formulation d(v) 0.50 d(v) 0.90 d(v) 0.95 d(v) 0.99 z-ave w idth of the melting area (°C) melting enthalpy (J/g) Fig. 1. NLC size at day 0 and after 90 days Fig. 2. Results of DSC study at day 90 In conclusion: Sucrose stearate was proven to be a suitable stabilizer for the preparation of NLC. The physical stability of the NLC formulations was affected by the varying ratio between solid lipid and oil.

[1] W. M. Obeidat; K. Schwabe, R.H. Müller, C.M. Keck: Preservation of nanostructured lipid carriers (NLC), Eur. J. Pharm. Biopharm., 76 (2010) 56-67.

65 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 54

INFLUENCE OF MICROCRYSTALLINE CELLULOSE MODIFICATIONS ON PHYSICAL STABILITY OF DIRECTLY COMPRESSED TABLETS

Przemysaw Skocze1, Wiesaw Sawicki2

1 ZF Polpharma SA, Formulation Department of R&D; 19 Pelpliska Street; 83-200 Starogard Gdaski, Poland; ([email protected]) 2 Department of Physical Chemistry, Faculty of Pharmacy, Medical University of Gdask

Introduction: Tablets manufactured using microcrystalline cellulose (MCC) when exposed to high humidity soften due to moisture pickup and loosening of MCC interparticulate hydrogen bonds. The purpose of this study was to evaluate influence of MCC modifications on physical stability of directly compressed placebo tablets after exposition to high relative humidity conditions. Moreover use of dynamic vapor sorption (DVS) and wettability by contact angle (CA) measurement techniques and their potential for prediction of tablets stability was reviewed. Methods: Pure MCC (Avicel® PH 102) and its modifications were tested for flowability, swelling capacity, particle size distribution, wettability (CA measurement) and sorption properties (DVS). Modifications were commercially available (Avicel® HFE 102 – manufactured by co-spray drying of MCC with 10% addition of mannitol) and laboratory prepared (by addition of 5, 10, 20% of stearic acid or cetostearyl alcohol ). MCC was melted with lipophilic ingredient or coated with the organic solution thereof. Hardness, friability and disintegration time of placebo tablets prior and after exposition to high humidity was tested. Results: Hardness of tablets manufactured using pure MCC and laboratory modified MCC was significantly (30 – 65%) reduced after 14 days of exposition to a relative humidity of 75%. In case of tablets prepared with Avicel® HFE 102 decrease of hardness was not significant (3%). Comparison of Avicel® PH 102 and Avicel® HFE 102 showed difference only in CA values (25.0° and 33.5° respectively), suggesting that CA measurement may be a tool for prediction of tablets physical stability. Laboratory modifications of MCC appeared to have higher CA values (up to 56%) and lower sorption tendency, this however does not correlate with stability. Conclusions: Placebo tablets manufactured using Avicel® HFE 102 do not lose their hardness after exposition to high humidity. Proposed laboratory modifications caused decrease in wettability and sorption tendency of MCC but did not prevent softening of placebo tablets after exposition to high humidity. Contact angle measurement and dynamic vapor sorption techniques cannot be considered as a reliable tool for prediction of physical stability of placebo tablets.

66 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 55

Lysozyme Loaded Ultra Small Gelatin Nanoparticles for Dermal Delivery: Physicochemical Characterization and In Vitro Release

X. Zhai1, C.M. Keck2, R.H. Müller1

1Pharmacy Department, Free University, Berlin, 12169, [email protected]; 2PharmaSol GmbH, Berlin, 12307, Germany

Introduction: Many investigations have confirmed the attractive advantages of gelatin nanoparticles (GNPs) regarding controllable drug release, modifiable biodistribution and sub- cellular particle size [1]. In contrast to lipid nanoparticles, they are able to incorporate hydrophilic actives (enzymes, peptides), and are therefore an attractive system to complement the dermal delivery portfolio of our group. The aim of present study was to develop and characterize loaded ultra small GNPs for dermal delivery, using lysozyme as a model enzyme easy to characterize. Methods: A modified two-step desolvation method was employed for the preparation of lysozyme loaded GNPs [2]. The mean particle size (z-average) and polydispersity index (PI) of developed GNPs were determined by photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS (Malvern Instruments, UK). The zeta potential of GNPs was measured by using also the Zetasizer Nano ZS. Morphology of GNPs was analyzed by a transmission electron microscope (Tecnai F20 TEM, FEI Company, USA). The drug loading efficiency and loading capacity of GNPs were estimated by HPLC. In vitro release study of lysozyme from loaded GNPs was performed by using static Franz diffusion cells at 32 ± 1C. The samples were withdrawn at settled time intervals for 36 hours and were analyzed using HPLC. The cumulative amount of lysozyme released as a function of time was calculated. Results: The z-average, PI and zeta potential of lysozyme loaded GNPs were 65 nm, 0.070 and 34.3 mV, respectively, indicating a homogeneous size distribution of the nanoparticles and a good physical stability of the suspension. Lysozyme loaded GNPs were found to be monodisperse with spherical conformation by TEM. The particle size was relatively agreed with the one analyzed by PCS. A drug loading efficiency of 89 ± 1% and a loading capacity of 13.6 ± 1.2 mg (lysozyme per gram GNPs) were obtained by HPLC. In vitro release study showed approximately 67% of lysozyme released from GNPs over a period of 36 hours. Conclusions: Lysozyme was successfully encapsulated into ultra small GNPs (< 100 nm). Gentle production conditions ensured the reproducibility and the drug loading. This process can be transferred to other enzymes, in addition further optimized to get nanoparticles even less than 50 nm.

[1] A. Mahapatro, D.K. Singh: Biodegradable nanoparticles are excellent vehicle for site directed in-vivo delivery of drugs and vaccines, J. Nanobiotechnol, 9 (2011) 55. [2] X.Z. Zhai, R.H. Müller, C. Coester: Production of ultrafine gelatin nanoparticles for dermal delivery, AAPS Annual Meeting, 2011, R6047.

67 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 56

Composition and characterization of powders and granules for preparation of oral gels “on a spoon”

Eliza Wolska, Magdalena Boniecka, Magda Zaraziska, Magorzata Sznitowska

Department of Pharmaceutical Technology, Medical University of Gdansk, al. Hallera 107, Gdansk 80-416, Poland

Introduction: Viscous pulp formed directly on a spoon after powder hydration with small amount of water has recently become increasingly popular. The texture of the gel-like pulp allows easier swallowing of the preparation. Such drug carrier facilitates oral drug administration especially to paediatric patients but also dysphagic patients [1,2]. The aim of the present study was to compare the properties of powder mixtures and granulated powders to form a carrier fast hydrating on a spoon. The effect of APIs (valsartan or candesartan) was also assessed.

Methods and Results: Three-component powder mixtures composed of viscosity increasing substance (guar gum), disintegrant (F-Melt type C or M, Lycatab C, Pharmaburst 500, Pearlitol Flash-Mannitol, Prosolv Easy, Kyron T-314, Prosolv SMCC 50 or 90) and lactose in a 1:1:1 ratio were investigated. The powders were granulated with water. Powders and granules were tested for time-related gel forming ability. Rotary viscosimeter and procedure developed for this purpose were employed. For the resulting gels average viscosity at a shear rate 0,8 s-1 ranged from 140 to 480 Pas. None of the investigated formulations showed thixotropic properties. Viscosity changes within time between the addition of water and drug administration should be considered, and therefore gelation kinetics was evaluated during 10 min after hydration. Obtained results indicate that the proper viscosity was achieved after 1.5 min after preparation and over the next 8.5 min increased only slightly, not more than by 10 – 20 Pas. Granulation of tested powder mixtures was proposed and performed in order to improve powders homogeneity and wettability. Properties of the tested carriers depends mainly on the used disintegrants and the best properties presented granules with Prosolv Easy, F-Melt type M and Kyron T-314. Candesartan and Valsartan (2% and 10% respectively) decreased powders wettability significantly.

Conclusions: Depending on the composition, powder can be easily reconstituted with small amount of water followed by fast swelling. The viscous pulp exhibits acceptable physical properties (semisolid and homogenous consistency, proper viscosity) and is characterized by ease of handling. It would be suitable for paediatric patients as well as geriatric patients or patients with dysphagia, although added active substances may change the application properties significantly. [1] D.A. Satyanarayana, P.K. Kulkarni, H.G. Shivakumar: Gels and jellies as a dosage form for dysphagia patients: a review, Curr Drug Therapy, 6 (2011) 79-86. [2] R.G. Strickley, Q. Iwata, S. Wu, T.C. Dahl: Pediatric Drugs – a review of commercially available oral formulations, J Pharm Sci, 97 (2008) 1731-1774.

68 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 57

Rheological and textural properties of microemulsion-based polymer gel with indomethacin

Anna Froelich1, Tomasz Osmaek1, Pawe Kunstman1, Rafa Roszak1, Wojciech Biaas2, 1Department of Pharmaceutical Technology, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland, [email protected]; 2Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, 60-627 Pozna, Poland

Introduction: Microemulsions are thermodynamically stable, transparent colloidal dispersion systems of two immiscible liquids stabilized by surfactants and co-surfactants. Numerous studies indicate that incorporation of active pharmaceutical ingredients (APIs) in microemulsion-based topical preparation results in the improved absorption and increased therapeutic effect [1-2]. Moreover, microemulsions display excellent solubility properties which make Fig. 1. Indomethacin. them particularly useful as vehicles for Biopharmaceutics Classification System (BCS) class II and IV drugs [3]. In this study we present novel microemulsion-based gel for topical use containing indomethacin, BCS class II non-steroidal anti-inflammatory drug (Fig. 1). The aim of our investigation was to characterize some of physicochemical properties of the novel formulation using rheological and textural analysis.

Methods: The applied microemulsion was prepared according to the procedure described by Nazar et al. [4]. Carbopol 960 was used as a thickening agent. The experiments were conducted with the use of HAAKE Rheostress 1 (Thermo Scientific) rheometer and texture analyzer TA.XT plus (Stable Micro Systems). The results were compared to the placebo gel obtained with the same method.

Results: Yield stress values, thixotropy and viscoelastic properties for both indomethacin and placebo gels have been elucidated and compared. The textural analysis allowed to compare hardness, adhesiveness and cohesiveness of both formulations.

Conclusion: The obtained results reveal that the parameters measured for polymer-thickened microemulsion depend strongly on the concentration of the drug. Indomethacin-loaded gels displayed lower viscosities, lower yield stress values and hardness when compared to placebo gel.

Acknowledgement: This study was partially supported by PUMS grant N0 502-01-03314429- 03439

[1] N. Subramanian, S.K. Ghosal, S.P. Moulik: Enhanced in vitro percutaneous absorption and in vivo anti- inflammatory effect of a selective cyclooxygenase inhibitor using microemulsion. Drug Dev Ind Pharm 31 (2005) 405–416. [2] K.G.H Dessai: Enhanced skin permeation of rofecoxib using topical microemulsion gel. Drug Dev Res 63 (2004) 33–40. [3] Y. Kawabata, K. Wada, M. Nakatani, S. Yamada, S. Onoue: Formulation design for poorly-water soluble drugs based on biopharmaceutics classification system: basic approaches and practical applications. Int J Pharm 420 (2011) 1–10. [4] Nazar M.F., Khan A.M., Shah S.S.: Microemulsion system with improved loading of piroxicam: a study of microstructure. AAPS PharmSciTech 10 (2009) 1286-1294.

69 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 58

Comparison of dynamic image analysis and spatial filter velocimetry for particle growth determination during pellet coating

Friederike Folttmann, Miriam Pein, Klaus Knop, Peter Kleinebudde

Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitätsstr.1, D-40225 Düsseldorf, Germany, e-mail: [email protected]

Spatial filter velocimetry (SFV) has been recently established for in-line particle size monitoring in spray fluidized bed granulation. It was examined that the obtained SFV data could improve the understanding of the impact of process variables [1-3]. However, the usage of SFV methods as continuous analytical tool in pellet coating processes has not been fully explored. In a proof of concept study a SFV probe has been evaluated for at-line analysis of coated pellets with different film thicknesses in order to compare it to an image analysis method. Sugar Spheres were used as non-pareil core material. A taste-masking coating formulation containing Eudragit® EPO (basic butylated methacrylate copolymer) was prepared and applied following the guidelines provided by the manufacturer as an aqueous colloidal solution with 15% incorporated solid content. The coating was performed using a laboratory-scale fluid bed coater (GPCG1, Glatt). Samples of about 5 g were taken during the coating process in equal time intervals in order to monitor coating thickness growth. The particle size of these samples and three samples of the final dried product weighing 50 g were measured using the CamsizerXT image analysis system (Retsch Technology GmbH, X-Fall module (free fall mode)) and a spatial filter velocimetry probe (Parsum IPP 70). The median of the volume distribution was taken for further evaluation and the number of measured pellets per sample/ per batch was counted during measurement. As specifying value of the particle size the mean chord length given by the SFV probe was compared to a chord length value given by the CamsizerXT which is defined as width of the particle projection and is the shortest of measured maximum chords in 32 directions. The absolute particle size and the calculated thickness of the coating (size difference between coated and uncoated pellets) were used for investigation. The absolute particle sizes of the coated and uncoated pellets obtained by the CamsizerXT and SFV probe were comparable. In both methods the final film thickness of the coated pellets was about 30 μm. Between sampling the film grew by approximately 5 μm. It is possible to measure the growth of the coating thickness during fluid bed coating with both methods at-line. Next step will be the in-line implementation of the methods. [1] A. Burggraeve et al.: Evaluation of in-line spatial filter velocimetry as PAT monitoring tool for particle growth during fluid bed granulation, Eur. J. Biopharm., 76 (2010) 138–146. [2] A. Burggraeve et al.: Process analytical tools for monitoring, understanding, and control of pharmaceutical fluidized bed granulation: A review, Eur. J. Pharm. Biophram., 83 (2013) 2-15. [3] S. Schmidt-Lehr et al.: Online-Kontrolle der Partikelgröße während einer Wirbelschichtgranulation, Pharm. Ind. 69, 4 (2007) 478-484.

70 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 59

Organogel with polymeric nanoparticles for roxithromycin delivery to hair follicles

Hanna Wosicka1, Eliza Gówka1, Kinga Hyla1, Justyna Stefanowska2, ukasz Klapiszewski3, Katarzyna Ka`mierska4, Teofil Jesionowski3, Krzysztof Cal2

1 Department of Pharmaceutical Technology, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Pozna, Poland, [email protected] 2 Department of Pharmaceutical Technology, Medical University of Gdansk, Hallera 107, 80-416 Gdask, Poland 3 Institute of Chemical Technology and Engineering, Poznan University of Technology, Skodowskiej-Curie 2, 60-695 Pozna, Poland 4 NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Pozna, Poland

Introduction: Drug delivery into the hair follicles and sebaceous glands is gaining more importance as the number of follicle-associated disorders increases [1, 2]. It was observed that particulate drug delivery systems play an important role in follicular penetration; especially nanoparticles (NPs) accumulate in hair follicle openings [3]. Methods: The aim of the study was to develop and evaluate oranogel with roxithromycin (ROX)-loaded polymeric NPs for follicular targeting. The drug-loaded NPs were prepared by an emulsion/solvent evaporation method using a biodegradable polymer i.e. poly(epsilon- caprolactone) as the matrix. Different formulations were prepared by varying ROX loading. Suspensions of NPs were further evaluated in terms of particle shape, size, zeta potential, encapsulation efficiency and in vitro drug release. Lyophilized NPs were incorporated into pluronic-lecithin organogel (PLO). Rheological measurements of PLO gels with ROX-loaded NPs were done. The fate of the NPs in the skin and skin appendages was traced due to the incorporation of a lipophilic fluorescent dye (Nile Red) into the particles. Results: The obtained ROX-loaded NPs were spherical in shape with irregular surface and were characterized by approximately 300 nm diameter and a negative zeta potential. The encapsulation efficiency and drug release were dependent on the drug loading. All PLO gels showed non- Newtonian, shear-thinning behavior. In ex vivo skin penetration study, horizontal skin sections revealed fluorescence in the areas of the hair follicles deep in the dermis. Conclusions: It was demonstrated that ROX could be efficiently incorporated into polymeric NPs characterized by the appropriate size allowing drug delivery to hair follicles. Although organogel has much higher affinity to the lipidic hair follicle area it does not seem to improve NPs penetration along the hair shaft. However, the obtained results prove that it is possible to achieve preferential targeting to the pilosebaceous unit by using polymeric NPs formulated into the semi-solid form. Acknowledgement: This study was partially supported by PUMS grant N0 502-14-03314429- 09229; This study was partially supported by PUMS grant N0 502-14-03314429-09664

[1] U. Blume-Peytavi and A. Vogt: Human hair follicle: reservoir function and selective targeting, Br J Dermatol, 165 (2011) Suppl 2:13-17. [2] H. Wosicka and K. Cal: Targeting to the hair follicles: Current status and potential, J Dermatol Sci, 57 (2010) 83-89. [3] J. Lademann et al.: Nanoparticles - an efficient carrier for drug delivery into the hair follicles, Eur J Pharm Biopharm, 66 (2007) 159-164.

71 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Technology: I - 60

Fluorescence microscopy as a tool in the studies of the stratum corneum penetration by polysiloxanes

Katarzyna Szymkowska1, Krystyna Mojsiewicz-Piekowska1, Krzysztof Cal2, Justyna Stefanowska2, Dominika Glamowska1

1Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, e-mail: [email protected] 2Department of Pharmaceutical Technology, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland

Introduction: The fluorescence microscopy is one of the tool in the skin imaging. The skin is an important barrier to the penetration and permeation of various substances. However, it can be the insufficient barrier. From this point of view, the study of silicones, including polydimethylsiloxane (PDMS) is an important. Linear and cyclic PDMS are often used in the medical, pharmaceutical and cosmetic industry. The reason for the study were both a lack of sufficient data in the publications on the penetration stratum corneum and the permeation across the layer human skin as well as due to the fact that some reports inform about toxic properties some of the silicones [1,2]. The purpose of the study was to verify the possibility of the stratum corneum (SC) penetration by the low molecular weight cyclic and linear PDMS. These substances are regarded as impermeable through the skin barrier. Methods In study the fluorescence microscope (FM) was used. Two techniques were applied in preparation of the skin samples. These techniques are based on the OECD guidelines, as well as own solutions [3]. The first technique is the "direct method". It is the application of the cyclic and linear PDMS on the skin, then the formulation is placed in an incubator for 20 min. Next the tappe stripping is carried out and added the four fluorescent dyes: fluorescein (FLU), sulforhodamine B (SRB), rhodamine B hexyl ester (RHBE) and porphyrin. After 10 minutes, the images in the magnification: 10, 40 and 100x were viewed. In the second method, the Franz-type diffusion cells were used. On the samples of the skin, the PDMS were placed for 24 h. After separation of epidermis, the individual dyes were put on and observations were carried. Results and Conclusions It was noted the difference between control samples (non application PDMS), and samples with application of PDMS. Based on the results, it was found that FM is a useful tool for the initial assessment the possible penetration of human SC by PDMS. FLU and SRB due to its polar and hydrophilic properties, have affinity to proteins. After staining the corneocytes were clearly visible. After application of the PDMS, the corneocytes were not so evident visible. RBHE has nonpolar lipophilic properties, and exhibited an affinity to lipids (similar as PDMS). The images after application of PDMS demonstrated the stained lipids, located between darkened corneocytes. The control samples had not a glowing spaces. References [1] E. Junngman, C. Laugel, A. Kasselouri,, A. Baillet-Guffroy: Study of the potential of stratum corneum lipids and exogenous molecules interaction by fluorescence spectroscopy for the estimation of percutaneous penetration. International of Journal Pharmaceutical, 434, (2012) 183-190. [2] D. Flassbeck, B. Pfleiderer, R. Gru1mping, A. V. Hirner: Determination of Low Molecular Weight Silicones in Plasma and Blood of Women after Exposure to Silicone Breast Implants by GC/MS. Analytical Chemistry, 73 (2001) 606-611. [3] OECD, Guidance notes on dermal absorption, No 156, Organisation for Economic Co-operation and Development, Paris, 2011.

72 II – Pharmacodynamics and Biopharmacy 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacodynamics and Biopharmacy: II - 1

Ex-vivo human skin permeation studies of low molecular polysiloxanes

Krystyna Mojsiewicz-Piekowska1, Krzysztof Cal2, Katarzyna Szymkowska1, Justyna Stefanowska2, Marzena Jamrógiewicz1, Aleksander Ciesielski3

1Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, e-mail: [email protected] 2Department of Pharmaceutical Technology, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland 3Department of Quality Control, Pharmaceutical Works Polpharma, Starogard Gdaski, Poland Introduction: The skin is an important topic of interest of scientists in the field of medicine, pharmacy, cosmetology, and toxicology. Understanding the relation between the structure of the skin, its components and function is very important in the discussion on the possibility of penetration, or even permeation of the substance. An important object of the study in this area should be polysiloxanes, due to the common use in various fields. Insufficient data in the publications, regarding the possibility of permeation of these polymers across the layer human skin, led to undertake research. This issue is important due to the fact that some reports inform about toxic properties some of the polysiloxanes (commonly name silicones) [1,2]. The aim of this study was to verify the possibility of the penetration and permeation of low molecular linear polydimethylsiloxanes through the human skin layers, which belong to polysiloxanes. Methods: Research methodology was developed based on the OECD guidelines and own modifications [3]. PDMS application to the skin was carried out in a flow-diffusion cell using the acceptor liquid lipophilic properties adequate to the PDMS. The separation of the skin to: the stratum corneum, epidermis and the dermis was performed. Polysiloxane extraction with the use of carbon tetrachloride was carried out from the individual skin layers as well as from the acceptor fluid. Identification of the PDMS was made using a fluorescent microscope and NIR and 1H-NMR spectroscopy. Quantitative study of the PDMS content in the samples was performed by 1H-NMR method using an internal standard, which was the dimethyl sulfoxide DMSO. The control samples were also similarly tested. In the case of the control samples, the purified water instead of the PDMS, were administered.

Results and Conclusions: The results demonstrated the ability of the low molecular weight PDMS to penetrate the stratum corneum of the human skin, as well as to pass through the dermal layer to the acceptor fluid. The presented model indicates the possibility of absorption of the PDMS of molecular weight 236 Da and a viscosity of 1 cSt through the human skin into the bloodstream or lymphatic vessels.

[1] C. Lassen, H. Ch Mikkelsen, S. Maa: Silicones-Consumption, Toxicy and Alternatives, Danisch Ministery of the Environment, Denmark, 2005. [2] A. Baroni, E. Buommino, V. De Gregorio, E. Ruocco, V. Ruocco, R. Wolf.: Structure and function of the epidermis related to barrier properties, Clinics in Dermatology, 30 (2012) 257-262. [3] OECD, Guidance notes on dermal absorption, No 156, Organisation for Economic Co-operation and Development, Paris, 2011.

73 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacodynamics and Biopharmacy: II - 2

Effect of cyclic GMP and dexamethasone on cell-matrix interactions in podocytes exposed to high glucose

Anna Waszkiewicz, Edyta Okruciska, Elbieta Latawiec, Barbara Lewko

Department of Pathophysiology, Faculty of Pharmacy, Medical University of Gdansk, Poland email: [email protected]

Introduction: Podocytes, the cells covering capillaries in kidney glomeruli, are responsible for integrity of filtration barrier. Altered adhesion of podocytes to the basement membrane results in disruption of cell structure and proteinuria. Vasorelaxing mediator cyclic GMP may affect podocyte cytoskeleton and adhesion [1]. Glucocorticoids are widely used in treatment of proteinuric disease and podocytes are their primary target [2]. Podocyte injury in diabetes may be associated with expression and activity of matricellular protein, thrombospondin-1(TSP-1) [3]. The aim of our study was to check (1) if expression of TSP-1 in podocytes is affected by high glucose and/or cGMP (2) if cGMP and glucocorticoids affect motility of podocytes exposed to high glucose. Methods: Conditionally immortalized mouse podocytes were switched to NG (normal glucose, 5.5mM) or to HG (high glucose, 30 mM) media for 5 days. Then, 100 μM IBMX (phosphodiesterase inhibitor), 0.1 μM ANP or 8 bromo-cGMP (non-metabolisable cGMP analog) were added for next 24 hours. Total RNA extracted from the cells was reverse-transcribed and subjected to the PCR analysis. Resulting bands were visualized on agarose gel and gene expression was assessed using densitometry. In motility experiments, the cells were grown in 12- well plates, and the monolayer was scratched with sterile pipette tip to create a cell-free area. Afterward, the media were replaced by NG or HG containing 100 μM 8 bromo-cGMP and/or 1 μM dexamethasone (DEX). Pictures were taken by inverted microscope at 0, 24, 48 and 72 h after scratching. Some cells were cultured on the glass cover slips and TSP-1 protein together with F-actin and vinculin were visualized by immunofluorescence. Results: Podocytes cultured in both, HG and NG media express the TSP-1 gene. High glucose enhanced TSP-1 expression by 55±3%, as compared to NG. Within NG group, stimulation of cGMP did not evoke any significant changes in TSP-1 gene. In HG cells, both ANP and 8 Br – cGMP inhibited TSP-1 expression, ANP by 47±13%, and 8Br-cGMP by 56±11%. High glucose increased the motility of podocytes and the effect was enhanced by 8-Br cGMP. DEX inhibited the glucose- and cGMP-stimulated motility of podocytes. Conclusions: Elevated TSP-1 may trigger signaling pathways leading to podocyte injury in diabetes, while cGMP donors may counteract these effects. However, increased by cGMP motility of podocytes may result in disruption of filtration barrier and proteinuria. Glucocorticoids may protect the integrity of renal filter by stabilizing the structure of podocytes.

[1] A.Greka, P. Mundel: Cell Biology and Pathology of Podocytes, Annu Rev Physiol, 74 (2012) 299–323. [2] CY Xing, MA Saleem, RJ Coward, L Ni, IR Witherden, PW Mathieson: Direct effects of dexamethasone on human podocytes, Kidney Int 70 (2006) 1038-45 [3] C Daniel, K Schaub, K Amann, J Lawler, C Hugo: Thrombospondin-1 is an endogenous activator of TGF-beta in experimental diabetic nephropathy in vivo, Diabetes 56 (2007) 2982-89

74 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacodynamics and Biopharmacy: II - 3

Influence of ketoprofen on propofol pharmacokinetics and pharmacodynamics in oncologic patients

Damian Szczesny1, Pawe Wiczling1, K. Przybyowski2 Agnieszka Bienert2, J. Tyczka3, E. Grzekowiak2, Roman Kaliszan1

1Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdask, Poland, [email protected] 2Department of Clinical Pharmacy and Biopharmacy, University of Medical Sciences in Pozna, Poland 3Department of Anaesthesiology and Intensive Therapy, Wielkopolska Centre for Lung Diseases and Tuberculosis, Pozna, Poland

Introduction: The aim of this study was to evaluate the influence of ketoprofen co- administration on propofol’s pharmacokinetics in lung cancer patients. The problem solved in this study arose on the background of a work of Le Guellec et al. [1], where inhibition of enzymes, involved in propofol’s glucuronidation by ketoprofen, was shown in vitro. Methods: The study was performed based on retrospective data from 23 adult patients scheduled for lobectomy or pneumonectomy, in which some patients received dose of ketoprofen (100 mg) prior to the surgery. Based on bioanalytical data we proposed pharmacokinetic and pharmacodynamic (PK/PD) model for propofol and tested the effect of ketoprofen on PK and PD of this drug. In addition, influence of demographic and physiological parameters, like body weight, age, sex, heart rate and systolic and diastolic blood pressure, on propofol PK and PD, was tested. PK/PD parameters were modeled by use of NONMEM software (Version 7.2.0; ICON Development Solutions, Ellicott City, MD, USA) and graphs were plotted with R software (version 2.14.0; The R Foundation for Statistical Computing) with use of XPOSE4SPECIFIC packages. Results: PK of propofol was best described with a three-compartment model, what is consistent with literature [2]. PK parameters derived were: systemic clearance 2,48 L/min, volume of central compartment 5,32 L, intercompartmental clearances 0,356 and 1,13 L/min, and volumes of peripheral compartments 190 and 21,5 L. In model building, influence of covariates on propofol’s PK was tested. No significant (p<0,001) relationships were found. In preliminary PD analysis, body weight and heart rate appear to influence the rate constant for the distribution of drug to the hypothetical effect compartment. Conclusions: Results of our study suggest that there is no influence of ketoprofen on propofol’s PK and PD. It may be explained by the fact, that propofol is a highly extracted drug and is metabolized in different ways [3]. [1] Le Guellec C, Lacarelle B, Villard PH, Point H, Catalin J, Durand A. Glucuronidation of propofol in microsomal fractions from various tissues and species including humans: Effect of different drugs. Anesthesia and Analgesia 1995;81:855. [2] Schüttler J, Ihmsen H. Population pharmacokinetics of propofol: A multicenter study. Anesthesiology 2000;92:727. [3] Sebel PS, Lowdon JD. Propofol: A new intravenous anesthetic. Anesthesiology 1989;71:260.

75 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacodynamics and Biopharmacy: II - 4

Biotransformation and metabolic stability of new quinoline - and isoquinoline -sulfonamide analogs of aripiprazole in in vitro mouse liver microsmes study

Paulina Kubowicz1, Pawe Zajdel2, Maciej Pawowski,2 Krzysztof Marciniec,3 Elbieta Pkala1

1Department of Pharmaceutical Biochemistry, [email protected] 2Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University, Medical College, 9 Medyczna St., 30-688 Kraków 3Department of Organic Chemistry, Medical University of Silesia, 4 Jagielloska St, 41-200 Sosnowiec

Introduction: Drug metabolism plays a key role in defining the efficacy, stability and toxicity of drugs. Preclinical data on differences in drug metabolism between animals and humans help to predict the pharmacological outcomes in humans [1]. Herein, we investigated the metabolism of aripiprazole and its three azinesulfonamide analogs in in vitro mouse hepatic microsomes assay. In contrast to aripiprazole, all the investigated compounds produced significant antidepressant activity in a mice model of depression (FST). On the other hand, only PZ508, similarly to aripiprazole, displayed remarkable antipsychotic properties in MK-801-induced hyperlocomotor activity in mice [2]. Methods: Mouse pooled liver microsomal preparations were incubated with aripiprazol and its three new derivatives PZ387, PZ508 and PZ549. Microsomes, tested compound and NADPH regenerating system were incubated for 10, 20 and 30 minutes at 37°C. Incubations were quenched by the addition of perchloric acid. The metabolites were determined by LC- MS/MS. Results: The closest analog to aripiprazole, PZ387, was metabolized to four new derivatives. Only one of them – dichloropiperazine – is common with the aripiprazole metabolic in vitro degradation. Among evaluated derivatives, compound PZ549 has the lowest microsomal stability, while PZ508 remained unchanged even after 30 minutes of incubation with mouse liver microsomes. Conclusions: Replacement of arylether moiety in aripiprazole with azinesulfonamide one and different localization of a sulfonamide group in the azine moiety have significantly impacted not only compounds pharmacological properties, but also their stability and metabolism pathways. Our study has confirmed that in vitro assays using commercially available liver microsomes is not only fast, cost-effective and ethically acceptable, but also can be used as a useful tool in early ADME-Tox assays in the process of developing new drug candidates.

[1] P. Fasinu, P.J. Bouic, B. Rosenkranz: Liver-based in vitro technologies for drug biotransformation studies - a review, 13, Curr. Drug Metab. (2012) 215-224. [2] P. Zajdel, K. Marciniec, A. Maslankiewicz, K. Grychowska, G. Satala, B. Duszynska, T. Lenda, A. Siwek, G. Nowak, A. Partyka, D. Wrobel, M. Jastrzebska-Wiesek, A.J. Bojarski, A. Wesolowska, M. Pawlowski: Antidepressant and antipsychotic activity of new quinoline- and isoquinoline-sulfonamide analogs of aripiprazole targeting serotonin 5-HT(1A)/5-HT(2A)/5-HT(7) and dopamine D(2)/D(3) receptors, 60, Eur. J. Med. Chem. (2013) 42-50.

76 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacodynamics and Biopharmacy: II - 5

Formation rate-limited pharmacokinetics of biologically active epoxy-transformers of treosulfan

1Micha Romaski, 1Anna Kasprzyk, 2Agnieszka Karbownik, 1Franciszek Gówka

1Department of Physical Pharmacy and Pharmacokinetics, 6 wicickiego Street; 2Department of Clinical Pharmacy and Biopharmacy, 14 Saint Maria Magdalena Street; Poznan University of Medical Sciences; Poland; e-mail: [email protected]

Introduction: Treosulfan (TREO) is a pro-drug belonging to alkylating agents which has been applied in treatment of platin-resistant ovarian carcinoma since 1980's [1]. Its biologically active epoxy-derivatives (S,S-EBDM and S,S-DEB) are formed under physiological pH and temperature (t0.5 1.5 and 3.2 h, respectively) without enzymes contribution [2]. So far pharmacokinetic studies of the parent drug only have been conducted. Hence, the aim of the study was determination of pharmacokinetic parameters of the epoxy-transformers of TREO in rabbits. Methods: The studies were carried out in 15 New Zealand white rabbits, divided into three equal groups (n = 5). Rabbits from group I and II received the intravenous infusion of pro-drug TREO and the bolus of (±)-DEB, respectively. The reference standard of S,S-EBDM is not commercially available, therefore the compound was administered to group III rabbits as a bolus of the mixture of approximately equimolar amounts of TREO, S,S-EBDM and S,S-DEB, obtained by alkalization of TREO with NaOH. Concentration of TREO in rabbit plasma was determined by the direct RP-HPLC method with refractometric detection, whereas the epoxy- transformers were quantified after derivatization with 3-nitrobenzenesulfonic acid by the more sensitive RP-HPLC-UV method. Pharmacokinetic calculations were performed in WinNonlin 6.2. Results: Changes in plasma concentrations of TREO and its epoxy-transformers were best described by a two-compartment model. The volume of distribution of TREO, S,S-EBDM and S,S-DEB at steady state amounted to 0.46, 0.75 and 0.44 l/kg, respectively. In group I rabbits, which received only the pro-drug, elimination of S,S-EBDM proceeded at the same rate as TREO (t0,5 ~ 1.7 h) but the levels of S,S-EBDM were much lower and S,S-DEB was not quantifiable. The pre-formed epoxides, administered to the group III and II rabbits, were rapidly eliminated (t0,5 for S,S-EBDM and S,S-DEB was 4.1 and 2.8 min, respectively). Conclusion: Elimination of the epoxy-transformers of TREO proceeds much faster than their formation from the pro-drug under physiological conditions. The obtained results prove that after administration of TREO, both S,S-EBDM and S,S-DEB demonstrate a formation rate-limited disposition.

[1] Gówka FK, Romaski M, Wachowiak J: High-dose treosulfan in conditioning prior to hematopoietic stem cell transplantation, Exp Opin Investig Drugs 2010, 19, 1275-1295. [2] Gówka FK, Romaski M, Teyk A, aba C: Direct high-performance liquid chromatography method with refractometric detection designed for stability studies of treosulfan and its biologically active epoxy-transformers, J Pharm Biomed Anal 2013, 72, 145-149.

77 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacodynamics and Biopharmacy: II - 6

Clinical pharmacokinetics of clopidogrel and its metabolites in patients undergoing PCI

Marta Karaniewicz-ada1, Dorota Danielak1, Pawe Burchardt2, ukasz Kruszyna3, Franciszek Gówka1

1Department of Physical Pharmacy and Pharmacokinetics, Faculty of Pharmacy, Pozna University of Medical Sciences, 6 wicickiego Street, 60-781, Pozna, [email protected] 2Division of Cardiology-Intensive Therapy, Department of Internal Medicine, Poznan University of Medical Sciences, 49 Przybyszewskiego Street, 60-355 Pozna, Poland 3Department of General and Vascular Surgery, Poznan University of Medical Sciences, ½ Duga Street, 61-848 Pozna, Poland

Introduction: Clopidogrel (CLP) is a potent antiplatelet drug that significantly reduces occurrence of adverse effects in acute coronary syndromes. However, clopidogrel resistance, characterized by an attenuated response to the treatment and increased risk of death, is present in nearly 20% of patients [1]. Extensive investigation of CLP pharmacokinetics would greatly contribute to determination of risk factors affecting the response to CLP treatment. The aim of this study was to evaluate pharmacokinetic parameters of CLP and its metabolites: inactive carboxylic metabolite (CLPM) and diastereoisomers of thiol metabolite (inactive H3 and active H4) in clinical conditions. Methods: Study included 28 patients who received 75 mg maintenance dose of CLP for at least 7 days prior to sample collection. Blood samples were drawn up to 24 hours after drug administration and stabilized with 2-bromo-3'-methoxyacetophenone [2]. CLP and its metabolites were determined in plasma by a validated HPLC-MS/MS method [3]. Pharmacokinetic parameters were calculated by a non-compartmental analysis using WinNonlin 6.2 software. Results: Pharmacokinetics of CLP and its metabolites varied widely between the studied patients. The values of Cmax for CLP, CLPM, H3 and H4 were 1.18±0.66 ng/mL, 2423±1249 ng/mL, 7.16±6.43 ng/mL and 7.83±6.82 ng/mL respectively, while AUC0t were 4.12±2.61 ng·h/mL, 10565±4599 ng·h/mL, 10.16±7.81 ng·h/mL and 12.37±11.77 ng·h/mL. Median time to reach peak concentration (tmax) was 1 hour and was equal for all analytes. H3 and H4 isomers were rapidly eliminated from patients’ plasma with t0.5 of 0.79±0.68 h and 0.86±0.62 h, whereas t0.5 for CLP and CLPM equaled 2.11±1.57 h and 7.67±3.22 h, respectively. Conclusions: Inter-individual variability in pharmacokinetic parameters of CLP, CLPM, H3 and H4 might be caused by differences in CLP absorption and/or metabolism. Therefore, an influence of the genetic polymorphism of enzymes and proteins contributing to CLP metabolism and transport should be investigated. Acknowledgments: The studies have been supported by Polish Ministry of Science and Higher Education, grant number NN 405 419739.

1. K. Sangkuhl et al. Pharmacogenet. Genomics. 20 (2010) 463–5. 2. G. Tuffal et al. Thromb. Haemost. 105 (2011) 696–705. 3. M. Karaniewicz-ada et al. J. Chromatogr. B 911 (2012) 105–12.

78 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacodynamics and Biopharmacy: II - 7

Effect of mycophenolate mofetil on hematological side effects incidence in renal transplant recipients

Joanna Sobiak1, Jolanta Kamiska1, Maciej Gyda2, Grayna Duda3, Maria Chrzanowska1

1Department of Physical Pharmacy and Pharmacokinetics, Poznan University of Medical Sciences, wicickiego 6, 60-781 Pozna, Poland, [email protected]; 2Department of Transplantology and General Surgery, District Hospital, Juraszów 7/19, 60-479 Pozna, Poland; 3 Department of Bromatology, Poznan University of Medical Sciences, Marceliska 42, 61-354 Pozna, Poland Pozna, Poland

Introduction: Mycophenolate mofetil (MMF) is an immunosuppressant administered after solid organ transplantation. Although it is generally well tolerated, it frequently causes hematological toxicity. The presence of anemia in renal transplant recipients is known to be associated with mortality and graft failure [1]. After oral administration, the pharmacologically inactive MMF is rapidly and entirely hydrolyzed into an active metabolite - mycophenolic acid (MPA). MPA is metabolized into an inactive 7-O-MPA glucuronide (MPAG). In this study, we aimed to assess the relation between the pharmacokinetic (PK) parameters of MMF metabolites (MPA and MPAG) and the adverse effects on hematopoietic system in renal transplant recipients. Methods: The 4-hour pharmacokinetic profiles of MPA and MPAG were determined using HPLC method for MMF-treated patients (n=61) during the late post-transplant period. Additionally, there were 45 renal transplant recipients in the control group. For MPA and MPAG the following PK parameters were calculated: pre-dose concentration (C0), maximum concentration (Cmax), and area under the concentration-time curve from 0 to 4 h (AUC0-4h). In all patients included in the study plasma iron was determined and the information on the iron supplementation was obtained. Results: All MPAG PK parameters correlated negatively with hemoglobin and hematocrit. No statistically significant correlations between MPA PK parameters and hemoglobin or hematocrit, as well as between MPA and MPAG PK parameters and leukocytes or platelets count were observed. All MPAG PK parameters were significantly higher in patients with hemoglobin below the norm. Anemia was more frequently observed in the study group compared with the control group (30.7% vs. 20.0%) and although this difference was insignificant, plasma iron concentrations were significantly higher in patients treated with MMF (32.9±9.4 mol/L vs. 28.7±9.4 mol/L; p=0.032). Iron supplementation was more frequently applied to patients with anemia (48.2%) compared with patients with hemoglobin within the norm (20.3%; p=0.005). Conclusions: MPAG may be the predicting factor of MMF side effects. In renal transplant recipients, especially with deteriorated renal function, extensive iron supplementation may be ineffective as anemia was associated with declined renal function and was not caused by low iron concentration. [1] T. Pile, J. Kieswich, S. Harwood, M.M. Yaqoob: A possible explanation for anemia in patients treated with mycophenolic acid, Transplantation, 92 (2011) 1316-1321.

79 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacodynamics and Biopharmacy: II - 8

Involvement of imidazoline/alpha-adrenergic receptors in effects of selected imidazoline agents on isolated rat heart atria

Justyna Dugokcka, Aleksandra Radwaska, Weronika Sady, Antoni Nasal, Roman Kaliszan Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdask, Gen. J. Hallera 107, 80-416 Gdask, e-mail: [email protected]

Rilmenidine and other „imidazoline-like” centrally acting hypotensive drugs have remarkable antiarrhythmic effects in experimental models of ventricular arrhythmia associated with sympathetic hyperactivity [1]. Clonidine is also capable of producing an significant improvement in hemodynamic parameters in animal models of left ventricular dysfunction [2]. Recently, many attempts have made to elucidate the role of imidazoline receptors in physiology/pathophysiology of the heart. However, no systematic comparative studies were performed nowadays regarding the activity of imidazoline compounds towards imidazoline/2-adrenergic receptors in isolated rat heart atria.

The aim of the study was to determine inotropic and chronotropic effects of selected imidazoline/2-adrenergic receptor ligands: guanabenz, RS 45041-190, agmatine and harman on isolated rat heart atria. The spontaneously beating right atria and the electrically driven left atria were treated with cumulative concentrations of each of the agents studied. The inotropic and chronotropic responses of imidazolines were measured also in the presence of various concentrations of imidazoline I1/I2 receptor (idazoxan), imidazoline I2-receptor (2-BFI) or 2-adrenergic receptor (yohimbine) antagonists. In the case of agmatine and harman two additional receptor antagonists: prazosin (1-adrenoceptor) and cimetidine (histaminergic H2- receptor) were applied. All effects were calculated as per cent of changes of the control value of atria rate or amplitude preceding the administration of each agent studied. The maximum effects observed (Emax) as well as -log EC50 values were considered.

The positive ino- and chrononotropic effects observed were evoked with the rank orders of potency: guanabenz >> agmatine > RS 45041-190 = harman, and guanabenz >> harman = agmatine = RS 45041-190, respectively. The results of the experiments with receptor antagonists lead to the conclusion, that I2-imidazoline and 2-adrenergic receptors may be involved in ino- and chronotropic effects of guanabenz and RS 45041-190. The tropic effects of harman depend mainly not only on I2-imidazoline but also on 1-adrenergic receptors. In the case of agmatine engagement of I2 imidazoline receptors in chronotropic activity of isolated right atria was demonstrated. However the mechanism of inotropic effects exerted by this compound on left atria is more complicated and involves 1/2-adrenergic and I2 receptors as well as histaminergic H2 ones.

[1] J.C. Roegel et al.: Preventive effect of rilmenidine on the occurence of neurogenic ventricular arrrhythmias in rabbits, J. Hypertens., 16 (suppl. 3) (1998) S39-S43. [2] L. Thomas et al.: Hemodynamic and cardiac anti-hypertrophic action of clonidine in Goldblatt one-kidney, one-clip rats, J. Cardiovasc. Pharmacol., 41 (2003) 203-209.

80 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacodynamics and Biopharmacy: II - 9

The influence of diabetes on the pharmacokinetics of sunitinib in rabbit model

Katarzyna Sobaska1, Edyta Szaek1, Agnieszka Karbownik1, Magorzata Lewandowska2, Edmund Grzekowiak1

1Department of Clinical Pharmacy and Biopharmacy, Poznan University of Medical Sciences, 14 Sw. Marii Magdaleny Str., 61-861 Pozna, Poland, 2Clinical Pharmacy Student’s Club, Department of Clinical Pharmacy and Biopharmacy, Poznan University of Medical Sciences, 14 Sw. Marii Magdaleny Str., 61-861 Pozna, Poland e-mail: [email protected]

Introduction: Sunitinib is a new oral multikinase inhibitor , indicated for the treatment of gastrointestinal stromal tumors (GIST), advanced renal cell carcinoma (RCC) and pancreatic neuroendocrine tumors (pNET). There are a few reports suggesting that this agent affect blood glucose levels [1,2,3,4,5]. The mechanism of the hypoglycemic effect remains unclear, but inhibition of c-Kit and PDGFR appears to be involved. This study investigated the influence of the diabetes on the pharmacokinetics of sunitinib. In addition, the hypoglycemic effect in diabetes was assessed. Methods: Rabbits were subjected to one of two groups: diabetic group (n=6) and control (healthy) group (n=6). The rabbits were treated with sunitinib in the oral dose of 25 mg. The blood glucose concentrations were measured at each blood collection. Plasma concentrations of sunitinib and active metabolite SU12662 were evaluated with validated HPLC method with UV detection.

Results: The mean sunitinib Cmax for the diabetic group and for the control group were 149.2 ± 33.7 and 92,1 ± 22,1 ng/ml, respectively. The mean sunitinib AUC0- for the diabetic group and for the control group were 4699.4 ± 1338.3 and 2425.0 ± 1077.5 ng x h/ml, respectively. The mean SU12662 Cmax for the diabetic group and for the control group were 12.8 ± 7.3 and 9.1 ± 3.5 ng/ml. The mean SU12662 AUC0- for the diabetic group and for the control group were 392.7 ± 201.4 and 387.4 ± 136.1 ng x h/ml, respectively. The decline in blood glucose concentrations for the diabetic group was 77-238 mg/dl (14.4-69,6%) and for the control group was 20-51 mg/dl (15,4-33,6%). The highest declines in blood glucose levels in diabetic group were observed between 6 and 12h after the administration of sunitinib. Conclusions: The study proved a significant influence of diabetes on pharmacokinetics of sunitinib. Taking into account the hypoglycemic effect of sunitinib, modification of hypoglycemic agents in diabetes patients may be justified. Of note, the highest declines were observed near the tmax, therefore monitoring of blood glucose around the time of Cmax may be useful to assess the value of antidiabetic effect of sunitinib and to adjust doses of hypoglycemic drugs.

[1] Oh JJ, Hong SK, Joo YM, i wsp. Impact of sunitinib treatment on blood glucose levels in patients with metastatic renal cell carcinoma. Jpn J Clin Oncol. 2012 Apr;42(4):314-7. [2] Billemont B, Medioni J, Taillade L, i wsp. Blood glucose levels in patients with metastatic renal cell carcinoma treated with sunitinib. British Journal of Cancer (2008) 99, 1380 – 1382. [3] Agostino NM, Chinchilli VM, Lynch CJ, i wsp. Effect of the tyrosine kinase inhibitors (sunitinib, sorafenib, dasatinib, and imatinib) on blood glucose levels in diabetic and nondiabetic patients in general clinical practice. J Oncol Pharm Pract. 2011 Sep;17(3):197-202. [4] Templeton A, Brändle M, Cerny T, i wsp. Remission of diabetes while on sunitinib treatment for renal cell carcinoma. Ann Oncol. 2008 Apr;19(4):824-5 [5] Lee Y, Jung HS, Choi HJ et al, Life-threatening hypoglycemia induced by tyrosine kinase inhibitors in a patient with neuroendocrine tumor: A case report, Diabetes Res Clin Pract. 2011; 93(2): 68-70.

81 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacodynamics and Biopharmacy: II - 10

The application of a classical mammillary and physiologically-based catenary three-compartment models in describing propofol pharmacokinetics

Martyna Józefowicz1, Pawe Wiczling1, Agnieszka Bienert2, Roman Kaliszan1

1Department of Biopharmaceutics and Pharmacodynamics, [email protected]; 2 Department of Clinical Pharmacy and Biopharmacy, Karol Marcinkowski University of Medical Sciences

Introduction: The propofol pharmacokinetics is typically described by a three-compartment mammillary model. This model is weakly related to the physiological variables, like cardiac output. Contrary, the physiologically-based pharmacokinetic model (PBPK) of propofol includes all physiological and anatomical characteristic of the body, however typically is too complex to be used for the description of clinically available data. We decided to use the physiological recirculatory model of propofol proposed by Upton [1] and simplify it basing on the principles of mechanistic "lumping" approach [2]. This approach allowed us to transform the original ten- compartment model to a three "lumped" compartments and retain all important features of the original model.. Additional aim of this work was to study relationships between pharmacokinetic parameters of a classical three-compartmental model and the proposed physiologically-based model. The applicability of the new model in describing propofol pharmacokinetics was tested based on the previously published Marsh, Schnider and Schttler data [3]. Methods: The differential equations of three-compartment models were solved by transformation into the Laplace domain. The Laplace transformations and other calculations were done by a computer algebra system that allows manipulating formulas symbolically (Symbolic Math Toolbox in Matlab Software version 7.0, The MathWorks, Inc., Natick, MA, USA). Results: The three-compartmental physiologically-based "lumped" pharmacokinetic model was found to be sufficiently accurate to describe arterial concentrations of propofol. The study revealed that both the classical mammillary and "lumped" catenary three-compartment models lead to the identical three-exponential decline. The AUC, and consequently the total clearance, were the same for both models. The equations were proposed to transform all the clearance and volume terms to physiologically meaningful parameters as the fraction of cardiac output associated with slow exchanging compartment and the permeability surface area product reflecting rate of drug transfer from the slow to the deep compartment. Conclusions: Basing on the physiologically-based three-compartment "lumped" model, it is possible to find pharmacokinetic parameters that are related to human physiology. The proposed model can be used to describe propofol PK data and allows recreating the concentration time profile in all major organs from the classical three-compartment model parameters. [1] Upton RN, Ludbrook G. A physiologically based, recirculatory model of the kinetics and dynamics of propofol in man. Anesthesiology. 2005;103:344-352. [2] Pilari S, Huisinga W. Lumping of physiologically-based pharmacokinetic models and a mechanistic derivation of classical compartmental models. J Pharmacokinet Pharmacodyn. 2010;37:365-405. [3] Masui K, Upton RN, Doufas AG, Coetzee JF, Kazama T, Mortier EP, Struys MM. The performance of compartmental and physiologically based recirculatory pharmacokinetic models for propofol: a comparison using bolus, continuous, and target-controlled infusion data. Anesth Analg. 2010, 111:368- 379.

82 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacodynamics and Biopharmacy: II - 11

The mydriasis model in rats for evaluation of pharmacological properties imidazoline of derivatives

Joanna Raczak-Gutknecht, Joanna Rietz, Magdalena Buszewska-Forajta, Teresa Frckowiak, Antoni Nasal, Roman Kaliszan

Department of Biopharmacy and Pharmacodynamics, Medical University of Gdask, Al. Gen. J. Hallera 80-416 Gdask, Poland, [email protected]

The properties of pharmacologically active imidazoline compounds have been intensively studied recently[1,2]. However our knowledge on selective ligands and the mechanism of action of many substances is still incomplete. It mostly because often one compound binds to different receptor types. Certainly in vitro studies are not sufficient to predict the effect of ligand-receptor interactions. Rat pupil dilation model allows evaluation which receptors (2- adrenergic or imidazoline receptors) take part in mydriatic effect evoked by imidazoline compounds. In this model experiments are carried out in vivo in the whole animal with testing particular imidazoline agents in a wide range of doses, regarding both the agonistic and the antagonistic properties and assessing the strength of action of the ligands studied [3]. The aim of this study was to determine of the mydriatic effect of clonidine and rilmenidine with and without pretreatment with yohimbine, a reference 2-adrenoceptor antagonist.

The studies were performed in male Wistar rats (weight 200-300 g). The agents studied were administered via cannulated femoral vein in cumulative doses. Yohimbine was administered 10 min before applying the first dose of clonidine or rilmenidine. Pupil diameter was measured using a stereoscopic microscope, equipped with a scale, an external light source, and a green filter. All the experiments were performed in a darkened room at controlled light conditions. There were also performed control experiments in which 0,9% NaCl solution was administered to the rats at the same intervals and volumes as the solutions of the imidazolines studied.

2-Adrenomimetic potency of clonidine appeard to be stronger than rilmenidine. Maximum mydriatic effect for clonidine and rilmenidine was found at doses of 30 μg/kg and 1000 μg/kg, respectively. After pretreatment with yohimbine the corresponding doses were 100 μg/kg and 3000 μg/kg, respectively. The administration of 0.9% NaCl did not cause mydriasis. Yohimbine administration caused parallel shift of the dose-effect curve for rilmenidine and clonidine to the right.

It can be concluded that clonidine and rilmenidine exert mydriatic effect having different agonistic affinity for 2-adrenoceptors. The results obtained indicate that rilmenidine elicits about 30 times weaker affinity to central 2-adrenoceptors than clonidine. Yohimbine abolishes pupil dilation evoked by rilmenidine and clonidine, which confirms decisive role of 2-adrenoceptors in the observed mydriatic effects.

[1] B. Szabo: Imidazoline antihypertensive drugs: a critical review on their mechanism of action. Pharmacol Ther. 93 (2002) 1-35 [2] C. Dardonville I Rozas: Imidazoline binding sites and their ligands: an overview of the different chemical structures. Med Res Rev. 24 (2004) 639-661 [3] M.C. Koss: Pupillary dilation as an index of central nervous system 2-adrenoceptor activation. J. Pharmacol. Methods, 15 (1986)1-19

83 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacodynamics and Biopharmacy: II - 12

Modified HPLC method for determination of bupivacaine in plasma; pharmacokinetic application Katarzyna Kosicka1, Iwona Zaporowska-Stachowiak2, Ewa Ryek1, Franciszek Gówka1

1 Department of Physical Pharmacy and Pharmacokinetics, Poznan University of Medical Sciences, 6 wicickiego Street, 61-781 Pozna, [email protected] 2 Department of Pharmacology, Poznan University of Medical Sciences, 5a Rokietnicka Street, 60-806 Pozna Introduction: Bupivacaine, a long-acting local anaesthetic agent, is used to achieve prolonged peripheral anaesthesia and analgesia in postoperative pain control or in labor analgesia [1]. There are several possible routes of bupivacaine’s administration: epidural infusion (EDA), spinal anaesthesia or less common paravertebral blockade (PVB). PVB is a good alternative for EDA, especially in patients with coagulopathy or under anticoagulant treatment, with anatomical abnormalities, as well as with CNS or cardiovascular diseases. Moreover, PVB brings significantly lower risks of serious side effects (hypotension, pulmonary complications, urinary retention, etc.) providing similar pain control [2,3]. However, there is a lack of data presenting bupivacaine’s pharmacokinetics when administered via PVB. The study describes HPLC method with ultraviolet detection for determination of bupivacaine’s concentration in human plasma. Methods: The presented method has been elaborated by modification of previously published HPLC method [4]. Bupivacaine and the internal standard (papaverine), after alkalization, were extracted from plasma samples using n-hexane. The organic layer was separated and evaporated to dryness under the gentle stream of nitrogen at 30°C. The residue was dissolved in the mixture of methanol and mobile phase (1:1, v/v) and injected into HPLC LiChrospher RP-18e column. The mobile phase consisted of acetonitrile and the 30 mM water solution of potassium dihydrophosphate containing 0.16% of triethylamine mixed at the ratio 25:75 (v/v). Results: The calibration curve was linear in the range 25 – 1000 ng/mL, the linearity was confirmed with Mandel’s fitting test. The lower limit of quantitation (LLOQ) for bupivacaine was 25 ng/mL. Intra- and inter-assay precision, expressed as relative standard deviation, was 11.2% and 11.8%, respectively. Intra- and inter-day accuracy of the method, expressed as relative error, was 9.8% and 17.1%, respectively. The analyte was stable in samples stored at room temperature in autosampler for 24 h and in plasma samples for 4 h. The standard solutions (in methanol) were stable for 7 months when stored at 4°C. The method was applied for estimating the bupivacaine’s concentration in plasma samples received from palliative patient after administration of bupivacaine via PVB. The determined concentrations of bupivacaine were in the range of 31.2 – 325.7 ng/mL after PVB boluses and 297.3 – 927.4 ng/mL after PVB continuous infusion. Conclusion: The presented HPLC-UV method fulfills the validation requirements of European Medicines Agency. It is simple and sufficiently sensitive, precise and accurate for pharmacokinetic studies of bupivacaine. The obtained results confirmed the utility of the presented method for the analysis of bupivacaine’s pharmacokinetics. The important advantages are the simplification of extraction procedure and the significantly lower LLOQ.

[1] Drasner K. Chapter 26. Local Anesthetics. In: Katzung BG, Masters SB, Trevor AJ, eds. Basic & Clinical Pharmacology. 12th ed. New York: McGraw-Hill; 2012. [2] Brown DL. Spinal, epidural and caudal anesthesia. In: Miller RD (ed.). Anesthesia 5th ed. New York: Churchill Livingstone Inc., 2000: 1491-1519. [3] Daly DJ, Myles PS. Curr Opin Anaesthesiol 2009;22:38. [4] Qin W, Jiao Z, Zhong M et al. J Chrom B 2010, 878: 1185.

84 III – Analysis

85 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 1

Targeted MS-based proteomic quantitation of osteopontin in healthy and cancerous human breast tissues

Katarzyna Macur1, Lars Hagen2, Tomasz Ciesielski3, Lucyna Konieczna1, Jarosaw Skokowski4, Bjørn M. Jenssen3, Geir Slupphaug2, Tomasz Bczek1

1Department of Pharmaceutical Chemistry, Medical University of Gdask, al. Hallera 107, 80- 416 Gdask, Poland, [email protected]; 2Department of Cancer Research and Molecular Medicine and PROMEC, Proteomics and Metabolomics Core Facility, Norwegian University of Science and Technology, Erling Skjalgssons gt.1, 7491 Trondheim, Norway; 3Department of Biology, Norwegian University of Science and Technology, Høgskoleringen 5, Realfagbygget, 7491 Trondheim, Norway; 4Central Tissue and Genetic Material Bank, Medical University of Gdask, ul. Dbinki 1, 80-211 Gdask, Poland

Introduction: Osteopontin (OPN) is a multifunctional protein, which has been shown to activate cell signaling pathways that lead to cancer development and metastasis. An increased OPN expression has been reported also in breast cancer. However, the levels of this protein typical for healthy and cancerous breast tissues, respectively, have not been defined so far [1]. Here, we present a targeted proteomic approach for OPN determination in this group of clinical samples. Methods: The OPN quantification method, based on measurement of its two characteristic peptides, was developed. Their concentrations were defined applying LC-ESI-QqQ-MS/MS in the multiple reaction monitoring (MRM) mode analysis and stable isotope standards (SIS) [2]. Simultaneously, a novel protocol for targeted OPN peptides isolation from human healthy breast tissues and tumors was elaborated. This method ensured enrichment of the analyzed peptides by capturing them by anti-peptide antibodies (CAPA). Results: The developed SISCAPA-MRM-MS method was applied in the initial study of OPN in 8 normal breast tissues and 6 tumors as a potential biomarker of breast cancer. The PCA analysis of the data indicated that the measured OPN concentrations show similar direction of changes as the clinically applied tumor development scale (pTNM) and the tumor infiltration stage (T). Moreover, the healthy specimens were clustered together, while the tumor samples were spaced in the dispersion. Conclusions: The evaluated SISCAPA-MRM-MS method was successfully applied in OPN determination in the preliminary set of healthy and cancerous breast tissues. The method may therefore be useful for further screening of larger number of clinical samples.

[1] L.R. Rodrigues, J.A. Teixeira, F.L. Schmitt, M. Paulsson, H. Lindmark-Mänsson: The role of osteopontin in tumor progression and metastasis in breast cancer, Cancer Epidemiol. Biomarkers Prev., 16 (2007) 1087-1097. [2] N.L. Anderson, N.G. Anderson, L.R. Haines, D.B. Hardie, R.W. Olafson, T.W. Pearson: Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA), J. Proteome Res., 3 (2004) 235-244.

86 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 2

Analysis of potential biomarkers in pediatric patients with acute leukemia

L. Konieczna1, M. Niedwiecki2, L. Maciejka-Kapuciska2, A. Balcerska2, E. Adamkiewicz-Droyska2, T. Bczek1 1Department of Pharmaceutical Chemistry, Medical University of Gdask, 80-416 Gdask, Hallera 107, Poland, e-mail: [email protected]; 2Department of Pediatrics, Hematology, Oncology and Endocrinology, 80-416 Gdask, Hallera 107, Poland

Introduction: Acute leukemia is a common form of cancer in children, however it is difficult to diagnose because of no specific symptoms of this cancer as well as further heterogeneity in response to therapy and subsequent risk of relapse [1]. There is a clear need to develop techniques to delineate new biomarkers, but it is preferred to analyze no single but several biomarkers simultaneously. Moreover multidimensional approach including both analytical and clinical data, as well as statistical and chemometric tools can routinely assist clinicians in earlier diagnosis and more efficient treatment in cases of pediatric acute myeloid leukemia. Objective: To assess the difference of plasma amino acid concentrations in children with acute leukemia versus healthy control group. Design: Twenty children were studied at diagnosis and after therapy with the prior approval by the local ethics committee. Data were collected by a standardised interview of the mathers. Methods: Hydrophilic interaction liquid chromatography in combination with electrospray mass spectrometry (ESI-MS) on HILIC Atlantis 1 column (4.6 x 250 mm, 5.0 m) was successfully employed to separate underivatized amino acids in biological samples. The investigation of organic acids present in analyzed samples used in mobile phase comprising 20 mM ammonium formate buffer at pH 4 with 0.1% formic acid (phase A) and acetonitrile with 0.1% formic acid (phase B) and mass spectrometric detection in the positive ionization mode. ESI-MS detection in the positive ionization mode provided the necessary information to unambiguously identify analytes of interest. Parameters of detector mass were as follows: gas temperature – 350°C, gas flow – 10L/min, capillary voltage – 6 kV. Data were acquired in single ion monitoring mode to monitor the characteristic pseudomolecular [M+H]+ ion of the analytes. Results: At diagnosis, phenylalanine and glutamic acid concentrations were elevated compared with previously published normal range and remained elevated at all observation times. Arginine, asparagine, aspartic acid, cysteine, histidine, hydroxyproline, lysine, proline, serine, threonine, tyrosine and tryptophan concentrations were significantly higher in control group compared with their concentrations at diagnosis. Glutamine, alanine, glycine and leucine concentrations were significantly lower in control group than at the time of diagnosis. The optimised method was further validated according to the ICH guidelines with respect to linearity and range, precision, accuracy, specificity and sensitivity. The robustness of the method was also determined and experimental confirmed by quantitative responses (peak areas, peak heights) and also qualitative chromatographic parameters (plate count, symmetry factor, resolution, retention times) were measured during the study. Conclusions: These results indicate that the most amino acid concentrations are lower than the normal range at diagnosis in the leukemia patients studied. In summary, the presence of acute leukemia is associated with changes in the amino acid profile.

[1] T. Bczek, L. Konieczna, Mariusz Belka et al., J. Pharm. Biomed. Anal., 70 (2012) 330-336. [2] T. Peng, K.H. Wu, S.J. Lan, et al., Eur. J. Cancer 41 (2005) 1158-1163

87 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 3

Profiling of gene copy number alternations in sporadic breast cancer genome using array-CGH technique Ronowicz A.1, Janaszak-Jasiecka A.1, Madanecki P. 1, Gucwa M. 1, Kochan K. 1, Bogdan A. 1, Skokowski J. 2, Strzelczyk B. 2, Ochocka J.R1, Piotrowski A. 1 1Department of Biology and Pharmaceutical Botany Medical University of Gdansk, Hallera 107, 80-416 Gdansk, Poland, 2Bank of Frozen Tissues and Genetic Specimens, Medical University of Gdansk, Dbinki 1, 80-211 Gdask, Poland

Introduction: Sporadic breast cancer (SBC) is a heterogeneous somatic disease which constitutes a major cause of morbidity and mortality in women and its metastatic spread is the principal reason behind the fatal outcome. SBC differ in many ways, such as in their initiator events, the susceptibility and defenses of the patient, and this makes it difficult to give the most appropriate treatment. Initiation and development of this cancer is inextricably linked with the genomic destabilization and selection of cells with specific DNA copy number aberrations, many of which are recurrent. Recent studies in humans have demonstrated that primary breast tumors displayed 20% higher number of aberrations than metastases as well as frequent differences has been demonstrated between both stage of tumors. That may suggest very early separation of cell lineages during tumor development (parallel progression model). Thus, our aim is evaluation of early structural rearrangements as potential diagnostic and prognostic targets in sporadic breast cancer. Methods: We performed our study using a high-throughput microarray (array-based Comparative Genomic Hybridization, aCGH) technology tailored to the needs of this project as well as whole-genome platforms, along with alternative confirmatory experiments. Results and Conclusions: We carried out whole genome aberration profiling at 135K resolution on samples (peripheral blood leucocytes, healthy mammary tissue, primary tumor, metastasis) from a cohort of sporadic breast cancer patients. To date we have found differentiating changes between primary tumor and metastasis in some patients. This finding supports hypothesis of parallel progression of primary and metastatic tumors and so metastases development has its origin at the beginning of carcinogenesis, that is long time before disease's symptoms. Moreover, our study revealed extensive copy number alterations in the healthy glandular tissue in at least 8% of patients from the same cohort. The most important observation however, is the fact that this aberrations coincide with regions that have been reported as genomic signatures of breast cancer, and we could detect DNA copy number changes in the same loci in the tumors from our cohort.

88 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 4

In search of the sensitivity improvement in capillary electrophoresis…

Szymon Dziomba, Piotr Kowalski, Ilona Oldzka, Tomasz Bczek

Department of Pharmaceutical Chemistry, Medical University of Gdask, 80-416 Gdask, Hallera 107, [email protected]

Introduction: Separation of chemical compounds is one of the basic tasks meet in science and industry laboratories. Chromatographic techniques, especially high-performance liquid chromatography (HPLC), are the most popular and widely used tools for this purpose. Capillary electrophoresis (CE) may be considered as an alternative technique featuring number of advantages over HPLC such as shorter analysis time, unpatrolled separation efficiency, lack or small amounts of organic wastes production, and thus, environmental friendliness and reduction of analysis costs. Unfortunately, according to small sample volume of the injection and short optical path length (spectrophotometric detection), limits of detection in CE are higher than in HPLC. However, application of on-line preconcentration techniques can significantly reduce this inconvenience [1,2]. Methods: Experiments were carried on with a use of selected central nervous system drugs (such as phenothiazines, tricyclic antidepressants, others). Influence of different injection modes were tested in a view of signal amplification of analytes. Head-column field-amplified sample injection (FASI) and field-amplified sample stacking (FASS) with a use of repetitive hydrodynamic injection were compared. Separations were performed using phosphate buffers and fused silica capillaries. Results: All peaks were satisfactorily separated in less than 10 min. Critical parameters of injection methods were designed and optimized. Significant amplification of signals was obtained for both injection modes. Greater signals enhancement was reached with FASI mode. Conclusions: Application of on-line preconcentration techniques can substantially improve the sensitivity of detection in CE. The enhancement of signal in field-amplified techniques is closely related to critical factors, which optimization is necessary for maximized amplification effect. Developed FASS method with repetitive hydrodynamic injection can be applied for low conductivity matrix samples analysis as an extension of normal hydrodynamic injection.

[1] Dziomba, S., Kowalski, P., Baczek, T., Field-amplified sample stacking-sweeping of vitamins B determination in capillary electrophoresis Journal of Chromatography A 1267 (2012) 224-230. [2] Dziomba, S., Kowalski, P., Baczek, T., Micelle to solvent stacking of tricyclic psychiatric drugs in capillary electrophoresis, Journal of Pharmaceutical and Biomedical Analysis 62 (2012) 149-154.

89 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 5

HPLC quantitation of venlafaxine in human saliva

Ewelina Dziurkowska1, Marek Wesolowski2 1Department of Analytical Chemistry, Medical University of Gdansk, Gen. J. Hallera 107, 80-416 Gdansk, Poland, [email protected] 2Department of Analytical Chemistry, Medical University of Gdansk, Gen. J. Hallera 107, 80-416 Gdansk, Poland, [email protected]

Depression is one of the most commonly appearing diseases in the world. Lots of medicaments with different kind of mechanism of action are used for it treatment. One of them is venlafaxine, the bicyclic serotonin-norepinephrine reuptake inhibitor (SNRI), which was introduced to the therapy in 1993. The aim of the study was to develop a sensitive and rapid method for determination of venlafaxine in human saliva. The blank saliva samples were obtained from healthy volunteers. The extraction of the samples was made on SPE columns. The cartridge of the columns were washed with water and adsorbed material was eluted with methanol. The eluate was evaporated and dissolved in acetonitrile. Venlafaxine was analyzed by HPLC with UV detection at 226 nm. The mobile phase was a mixture of acetonitrile and phosphoric buffer (30 : 70 v:v) at a flow rate of 1 mL/min. The linearity of calibration curve was obtained over the range of 10 – 1000 ng/mL of venlafaxine in saliva. The limit of the detection (LOD) was 1 ng/mL (S/N = 3) and the limit of the quantification (LOQ) was 3 ng/mL (S/N = 10). The RSD values for intra-day study varied between 1.66 and 11.5% and for inter-day study did not exceed 8.14%. The developed method can be used for analysis the saliva samples without interference peaks and enables quantitation of venlafaxine in depressed persons. The method can be very useful to control the intaking the drug by patients.

90 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 6

Determination of vitamin C in food samples and dietary supplements by HPLC method with electrochemical detection

Marcin Marsza1, Anna Lebiedziska2, Piotr Szefer2

1Department of Toxicology, 2Department of Food Sciences, Medical University of Gdask, 80-416 Gdask, Al. Gen. J. Hallera 107 [email protected]

Introduction: An accurate and specific determination of micronutrient content in foods is becoming extremely important do describe the relationship between dietary intake and human health. Vitamin C is natural antioxidant in food and biological systems with important nutritional benefits for human health related to the effect against scurvy, cardiovascular disease and cancer [1]. Vitamin C is also used an index of the nutrient quality of fruits and vegetable products. In this work, a sensitive, selective and rapid HPLC-EC method was developed and validated for the identification and quantification of the vitamin C in fortified food products and dietary supplements. Methods: Vitamin C was analysed as L-ascorbic acid (L-AA) by high-performance liquid chromatography with coulometic electrochemical detection (Coulochem II). Prior to HPLC analysis, dehydroascorbic acid (DHAA) was reduced by various sulphydryl-countaining compounds. The method was validated using a Hypersil Gold C18 column packet with 5 μm shell particles (4.6 x 150 mm) and 100 mM phosphate buffer pH 3 with 1 mM EDTA – acetonitrile (85:15 v/v) isoctratic grade at 25 0C. Peak area was used as the analytical response and linearity was studied by the standard additions method in the range 0.001 – 0.5 mg L-1. Good linearity was obtained with correlation coefficient higher than 0.98. Accuracy was evaluated by recovery studies. Good results were obtained and recoveries were in the range 92.5 – 97.5 % for all the matrices analyzed. The relative standard deviations were calculated to be 2.5 and 7.7 % for repeatability and reproducibility, respectively. Results: The method was used for determination of ascorbic acid in fruit juice (n = 26), nectars (n = 10), fruit drinks (n = 37), soft drinks (n = 22) and dietary supplements (n = 20). The experimental results are similar to the label values indicated on the commercial products. Conclusions: The performance of the method was evaluated by analyzing of large number of different fruit drinks, soft drinks and dietary supplements. The proposed method has important advantages such as simplicity and high sample throughput (analysis less than 6 min), indicating than it can be applied in routine analysis. The authors gratefully acknowledge the financial support of grant N N404-270840.

[1] L.M.L. Nollet, F Toldrá. Handbook of Analysis of Active Compounds in Functional Foods. CRC Press, US 2012.

91 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 7

Investigation of interactions between tetrapeptides and enhancers using affinity capillary electrophoresis and frontal analysis

Elfi Busse1, Anja Elpelt2, Yahya Mrestani1, Mike Farwick3, Reinhard H. H. Neubert1

1Institute of Pharmacy, Martin-Luther-University, Halle (Saale), Germany, [email protected]; 2Lausitz University of Applied Sciences, Senftenberg, Germany; 3EVONIK Industries AG, Essen, Germany

The studied tetrapeptides PKEK and GEKG (sequence in one-letter code) possess some adverse properties for dermal penetration especially their high hydrophilicity. Furthermore they have numerous hydrogen bond donors and acceptors, which may lead to interactions with tissue of the skin. However, dermal peptide delivery was realized using oil-in-water emulsions with different enhancer additives like glycerol caprylate/caprate, pentylene glycol and polyglycerol-3-caprate. Thereby the enhancers did not encourage penetration of each peptide to the same extent, but for each peptide another enhancer was favorable. This may be attributed to a particular enhancer mechanism. Specific interactions between enhancer molecule and peptide owing to hydrogen bonds could, for instance, be responsible for the penetration optimization. Therefore, the aim of this work was to 0,0018 investigate possible interactions between the peptide without enhancer applied enhancers and tetrapeptides using 0,0016 peptide with enhancer different capillary electrophoresis methods. 0,0014 0,0012 Affinity capillary electrophoresis (ACE) was 0,0010 employed to find out, if the mixture of 0,0008 peptide and enhancer has changed mo 0,0006 electrophoretic properties compared to the 0,0004 single substances. Molecule interactions are effective0,0002 bility [cm²/Vs] usually associated with a shift in effective 0,0000 GEKG + glycerol PKEK + PKEK + polyglycerol- mobility. The second method applied was caprylate/caprate 2-pyrrolidone 3-caprate frontal analysis continuous capillary (n=3) (n=3) (n=4) 2 electrophoresis (FACCE), wherein sample Fig. 1: Effective mobility μe [cm /Vs] of potential interaction pairs determined with ACE (mean±SD) solution is used as capillary inlet. Since the analytes are continuously present in the capillary, the detection signals of them emerge as ascending plateaus. Interactions between the analyte and ligand are accompanied by a change of the plateau height. The results of the ACE are shown in figure 1. A minor deviation, but no significant difference of the effective mobility between the single peptide and the mixture with enhancer, could only be detected PKEK with polyglycerol-3-caprate. In the electropherograms obtained with FACCE for the mixtures of peptide and enhancer, a plateau with the single peptide level was visible and a second one with the height of the single enhancer. Interactions between peptide and enhancer were therefore not detectable by these methods, although it has been expected because of numerous hydrogen bonding partners in the molecules. Hence, other techniques like NMR or isothermal titration calorimetry have to be used to prove if there are interactions between the peptides and enhancers or if penetration optimization results from another mechanism.

92 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 8

Development of the capillary electrophoretic method for quantification of steroid hormones in human urine samples

Piotr Kowalski, Piotr Szmudanowski, Szymon Dziomba, Ilona Oldzka, Tomasz Bczek

Department of Pharmaceutical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Address, e-mail: [email protected]

Introduction. The use of steroid hormones as doping agents in the disciplines such as bodybilding, strength, endurance and martial arts is now a common and serious problem in sport. It is a threat to the pure and noble competition, it is very important to reliably measure these substances in body fluid of players. A necessery and important diagnostic tool used for this purpose is the analysis of steroid hormones in human urine samples. Many of the steroid hormones can be also considered as potential biomarkers (cortisol, epitestosterone) and their determination in body fluids can create opportunities for rapid diagnosis of many diseases and disorders of the human body. Most existing methods used for the determination of steroids is time-and labor-consuming and quite costly indeed. Therefore, the aim of many analytical laboratories is develop a new, relatively low-cost and rapid implementation methodology for their determination in biological samples. Due to the fact, that there is little literature data on concentrations of steroid hormones in urine samples, it has been attempts to electrophoretic determination of these compounds [1-3]. Methods For this purpose, a extraction procedure for the optimized separation and simultaneous determination of seven steroid hormones (cortisone, hydrocortisone, corticosterone, testosterone, epitestosterone, methyltestosterone, progesterone) in urine samples has been developed. Isolation of analytes was performed by solid phase extraction (SPE) with HLB columns. To separate all analytes a micellar electrokinetic capillary chromatography (MECK) has been selected. The separation buffer solution (pH 9.2) was composed with 10 mM borax, 50 mM sodium dodecyl sulfate (SDS), and 10% methanol. Methodology for the determination of steroid hormones meets all the requirements of the analytical methods; it has been confirmed by validation study. Results and Conclusions Applicability of the method was confirmed for the analysis of biological samples collected from volunteers - both men and women (students, amateur bodybuilding using and not using steroid doping). Concentrations of endogenous steroid hormones, which have been obtained in some urine samples power sports enthusiasts origin, well above the physiological level. The elaborated method may be a useful tool for the routine determination of endogenous and exogenous steroid hormones in human urine samples. Likewise, the obtained data can be used for further research on the analysis of steroid hormones.

[1] L. Konieczna, A. Plenis, I. Oldzka, P. Kowalski, T. Bczek, Talanta 83 (2011) 804-814 [2] A. Plenis, L. Konieczna, I. Oldzka, P. Kowalski, T. Bczek, Mol. Biosyst. 7 (2011) 1487-1500 [3] T. Bczek, I. Oldzka, L. Konieczna, P. Kowalski, A., Sci. World J. (2012) art. nr 268120, 8

93 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 9

The bioinformatic evaluation of urinary metabolites as cancer markers determined using LC-TOF/MS and LC-MS/MS

W. Struck-Lewicka1, D. Siluk1, A. Yumba Mpanga1, M. Markuszewski2, R. Kaliszan1, M. J. Markuszewski1 1) Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdask, Poland 2) Department of Urology, Medical University of Gdask, Poland Email: [email protected] Introduction: Nowadays, in the so-called post genomic era, analysis of metabolic profiles from biological samples can reflect relevant information about human’s health status. Changing levels of various metabolites determined in biological samples like urine or plasma can be an indication of serious disorder like cancer. Hence, the aim of this study was a comprehensive analysis of urine samples with the use of modern analytical techniques like LC-MS/MS as well as LC- TOF/MS from cancer patients and healthy volunteers in order to assess the usefulness of metabolic profiles in cancer diagnosis. Methods: The determination of metabolites from urine samples from cancer patients (n=68) and healthy volunteers (n=61) was performed with the use of HPLC technique coupled with Tripple Quadrupole Mass Spectrometer (LC-MS/MS) or coupled with Time-of-Flight Mass Spectrometer (LC-TOF/MS). Furthermore, the obtained data sets were statistically analyzed with the use of univariate (U-Mann Whitney test) and multivariate statistical analyses (PCA, PLS-DA, K-NN, SVM, logistic regression). Results: After the whole metabolomic procedure including samples pretreatment, methods’ validation and univariate statistical analysis, the obtained data sets were classified using PCA. The results showed that healthy samples were better clustered together than cancer samples. That confirmed higher diversity among samples belonging to different types of cancer. The sensitivity as well as specificity of the statistically significant metabolites calculated with the use of PLS- DA, K-NN, SVM or logistic regression for data obtained from LC-TOF-MS were in the range from 53.9 to 92.3%, and from 37.5 to 85.7%, respectively. The sensitivity and specificity for data obtained from LC-MS/MS were lower and ranged from 61.9 to 88.9% as well as from 27.8 to 66.7%, respectively. Conclusions: LC-TOF/MS as well as LC-MS/MS analyses of metabolic profiles together with the advanced bioinformatics can allow the evaluation of metabolites as cancer markers. However, the larger group of analysed samples is needed to confirm obtained results and drawn conclusions. [1] W. Struck, D. Siluk, A. Yumba Mpanga, M. Markuszewski, R. Kaliszan, M.J. Markuszewski, J Chromatogr A, http://dx.doi.org/10.1016/j.chroma.2013.01.111

94 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 10

Cocaine and benzoylecgonine extraction from water samples with a molecularly imprinted polymers (MIP’s) as a selective sorbents

J. Raczak-Gutknecht1, R.Bujak2, R. Gadzaa-Kopciuch3, A. Nowaczyk4, E. Daghir2, P. Koliski2, B. Buszewski3, M.J. Markuszewski1

1 Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdask, Al. Hallera 107, 80-416 Gdask, Poland; 2Department of Toxicology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toru, dr A. Jurasza, 85-089 Bydgoszcz, Poland; 3 Department of Environmental Chemistry & Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin St., PL-87 100 Toru, Poland 4 Department of Organic Chemistry, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toru, dr A. Jurasza, 85-089 Bydgoszcz, Poland; [email protected]

Abuse of illicit drugs is a major global problem. It may cause diseases, high mortality and many socioeconomic problems [1]. It is difficult to assess how much drugs is really used. The method that provides to obtain realistic estimates of drug use, is monitoring the urinary exerted cocaine and its metabolites in waste waters [2]. Because samples of waste water are complex and impurities could interfere with quantification, solid phase extraction (SPE) with the new, selective sorbents is needed to clean up the sample before the analysis. Molecularly imprinted polymers (MIPs) are highly cross-linked synthetic polymers having molecular recognition properties, towards the template molecules or even group of similar compounds, with specificity and binding selectivity [3]. In this studies, MIPs as a new cocaine and benzoilecgonine selective sorbents were proposed. As a first step, the components of polymers were chosen by computational approach: template, functional monomer, cross-linker and porogen. During MIPs synthesis as the templates, atropine and scopolamine were used. The synthesis based on non-covalent strategy. The synthesis of non-imprinted polymers (NIPs) has been performed simultaneously. The selectivity of new synthesized polymers has been estimated in binding and adsorption study. The concentrations of cocaine and its metabolite benzoylecgonine were measured in liquid phase by HPLC method. Benzoylecgonine is a marker of cocaine use, therefore estimation of these compound is importantin biological and environmental studies. MIP’s used in this experiment appears to be sufficient materials that can be used in extraction step before qualitative or quantitative analysis of cocaine’s metabolite-benziylecgonine from wastewater. Further extraction performance studies are required for estimation of cocaine because of weak recovery from analyzed materials.

[1]. CP O’Brien Drug addiction. In: Brunton LL, Chabner BA, Knollmann BC, editors. Goodman and Gilman’s the pharmacological basis of therapeutics. New York, NY: McGraw-Hill, (2011) 649–68. S. Castiglioni, E. [2] Zuccato , E. Crisci , Ch. Chiabrando ,R. Fanelli ,R. Bagnati, Identification and measurement of illicit drugs and their metabolites in urban wastewater by liquid chromatography-tandem mass spectrometry. Analytical Chemistry 78 (2006) 8421-8429. [3]. M. Lasakova, P. Jandera: Molecularly imprinted polymers and their application in solid phase extraction. J. Sep. Sciences 32 (2009) 799 – 812

95 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 11

Direct injection of biological fluids in determination of steroids

by Micellar Liquid Chromatography (MLC)

Piotr Kawczak1, Marta Denys1, Tomasz Bczek1

1Department of Pharamceutical Chemistry, Faculty of Pharmacy, Medical University of Gdansk, Al. Gen. J. Hallera 107, 80-416 Gdansk, Poland, e-mail: [email protected]

Micellar liquid chromatography (MLC) is an analytical technique belonging to the wide range of reversed-phase liquid chromatographic (RP-LC) separation techniques. MLC with the use of surfactant solutions (anionic, cationic or non-ionic) above its critical micellar concentration (CMC) and the addition of organic modifiers is currently an important analytical tool with still growing theoretical considerations and practical applications in pharmaceutical analysis of drugs and other biologically active compounds. The use of MLC as an alternative, relatively much faster in comparison to conventional chromatographic separation techniques has several advantages, especially as being suitable for screening pharmaceutical analysis. The analytical data received from MLC analysis are considered as an useful source of information to predict passive drug absorption, drug transport and other pharmacokinetics and physicochemical measures of pharmaceutical substances [1,2]. MLC allows biological samples to be analyzed without needing to eliminate proteins and other interfering substances, thus considerably reducing the cost and analysis time. In addition, one of the main applications of MLC is the possibility of direct sample injection of biological material (urine, plasma, serum, milk) into the column due to the ability of micellar aggregates to dissolve sample proteins and other compounds what is particular importance in the biomedical studies [1,3]. In presented study a group of several steroids were identified and separated using a MLC technique. The parameters affecting retention i.e. stationary phase selection or concentration of surfactant and organic modifier in the mobile phase were investigated. One of the most important part of the study was to determine the steroids in urine samples with the application of direct injection method which means there was no previous treatment of the biological samples.

[1] P. Kawczak, T. Bczek , Recent theoretical and practical applications of micellar liquid chromatography (MLC) in pharmaceutical and biomedical analysis, Central European Journal of Chemistry, 10, (2012) 570-584. [2] A. Berthod, M.C. García-Alvarez-Coque, Micellar Liquid Chromatography, vol. 83, Marcel Dekker Inc., New York, (2000). [3] M.C. García-Alvarez-Coque, S. Carda Broch, Direct injection of physiological fluids in micellar liquid chromatography, Journal of Chromatography B, 736 (1999) 1–18.

96 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 12

Influence of Cholesterol and Cholesterol sulfate in Stratum corneum lipid mixtures using Fluorescence spectroscopic methods

J. Mueller1, H.H. Ruettinger1, R.H.H. Neubert 1, A. Schroeter1 1 Institute of Pharmacy, Martin Luther University, Wolfgang-Langenbeck-Str. 4, 06120 Halle Email correspondence to: [email protected]

Introduction: Being the outermost layer of the human skin, the stratum corneum (SC) plays a key role in its barrier function. During the last decades many researches were based on the examination of the SC lipid organization whose main components are ceramides (CER) as the most relevant constituent, free fatty acids (FFA), cholesterol (CHOL) and its derivatives. While many other investigations were focused on the influence of different CER subclasses we con- centrated on characterizing the effect of CHOL and Cholesterol sulfate (ChS) on synthetic lipid mixtures. Materials and Methods: To study primarily the less investigated head group interactions be- tween the different lipids we created liposomes composed of CER[AP], CHOL, stearic acid (SA) and ChS. Analyzing mixtures with and without ChS and varying the concentration of CHOL from 10 % (m/m) to 30 % (m/m) influences of both components on the lipid structure could be observed. Therefore, we applied fluorescence spectroscopy to unilamellar liposomes containing the fluorescence marker Laurdan. While the conventional fluorescence measure- ments disclosed polarity of the head group environment, fluorescence anisotropy provided in- formation concerning the mobility of molecules within the bilayer [1]. Results and Discussion: It has already been ascertained that the influence of CHOL depends on its concentration and on the physical state of the lipid mixture [2]. In our investigated SC lipid model bilayers composed of CER[AP]/CHOL/SA and ChS (66/ 10/ 18/ 6, % m/m) we were able to detect an exceeded solubility of CHOL, which resulted in the formation of so- called lipid rafts containing CHOL and CER [AP]. Moreover, the remaining bilayer structure was formed by all other lipids of the mixture. Our results corroborate with former researches indicating that in gel state CHOL lowers the membrane ordering, while in fluid crystalline state it increases the chain packing. We could also ascertain a compression in the phase transition diagrams when the CHOL concentration is elevated. Thus, we concluded, that this is due to an extensive formation of lipid rafts with higher CHOL content. However, it should be mentioned that CHOL mainly influenced chain packing in the fluid crystalline state, whereas only little changes in gel state ordering could be observed. Our researches concerning ChS led to the sup- position of a more densely packed bilayer structure especially in the gel state, which is probably due to the more polar head group of the CHOL derivative being able to create more hydrogen bonds. Furthermore, there was an evidence suggesting an increased CHOL solubility due to the presence of ChS. These findings coincide with former results according to ChS [3, 4]. Conclusion: It could be demonstrated that liposomes are suitable to serve as a model system for researches concerning SC lipid organization, especially to investigate head group inter- actions. Our results support previous assumptions and illustrate the necessity of additional con- tinuative studies in SC research to clarify the lipid structure in the SC. References: [1] J.R. Lakowicz: Principles of Fluorescence Spectroscopy, Springer Science+Business Media, 3 rd edition (2006) [2] J. Zbytovská: Influence of Cholesterol o the structure of stratum corneum lipid model membrane, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 328 (2008) 90-99 [3] M. Arseneault: Cholesterol Sulfate and Ca21 Modulate the Mixing Properties of Lipids in Stratum Corneum Model Mixtures, Biophysical Journal, 92 (2007) 99-114 [4] J. A. Bouwstra: Cholesterol sulfate and calcium affect stratum corneum lipid organisation over a wide tempera- ture range, Journal of Lipid Research, 40 (1999) 2303-2312

97 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 13

Photostability test of fluphenazine analogue

Agnieszka Sobczak1, Jan Ziarniak1, Anna Jeliska1, Piotr witek2, Wiesaw Malinka2

1Department of Pharmaceutical Chemistry, Poznan University of Medical Sciences, 6 Grunwaldzka Str., 60-780 Pozna, Poland, [email protected]; 2Department of Drug Chemistry, Wroclaw Medical University, 211 Borowska Str., 50-556 Wrocaw

Introduction: Fluphenazine (FPh), a neuroleptic drug, exhibits a significant antimutagenic activity, but its wider application in the treatment of cancer is limited by the serious side effects on the central nervous system. Therefore, the structure of FPh has been modified to obtain more hydrophilic analogue(s) which exhibit similar antimutagenic/chemopreventive activity but less psychotropic effects. One of them is 1-[bis-(2-hydroxyethyl)amino]-3-(2-trifluoromethyl- phenothiazin-10-yl)propan-2-ol hydrochloride (2A). Due to the promising results of the studies of this derivative pharmacological activity, it seems appropriate to perform tests of its photostability [1]. Methods: The evaluation of 2A photostability in solutions and solid state was conducted according to International Conference on Harmonisation Guidelines (ICH Q1B). The HPLC and UV spectrophotometry were applied for the determination of 2A and its degradation products. Both analytical methods were validated (the HPLC technique was described in our previous studies) with respect to selectivity, linearity (0.5018.0 μg/ml; y = (0.0644 ± 0.0005)x, r = 0.9999, n = 11), precision for three concentrations (n = 6; RSD 1.96% for 4.0 μg/ml; 0.74% for 10.0 μg/ml; 0.81% for 16.0 μg/ml), LOD – 0.13 μg/ml and LOQ – 0.39 μg/ml. Results: In the stress test (the exposition - 1.2 million lux hours) the compound 2A did not show decomposition in solid state whereas in solutions it underwent complete degradation. Due to its photosensitivity in aqueous solutions the photostability test was carried out under milder conditions. The degradation of 2A, as a result of photolysis, is a first-order reaction relative to substrate concentration described by the following equations: ln P = ln P0 – kobs × t (HPLC) ln (A – A) = ln (A0 – A) – kobs × t (UV) 1 1 1 1 ln (D – D ) = ln (D 0 – D ) – kobs × t (UV) where: P0, P – the ratio of the peak area of 2A to the peak area of internal standard at time zero 1 1 and t, respectively; kobs – the observed first-order reaction rate constants; A0(D 0), A(D ), 1 A(D ) – the absorbance (the first derivative) values of 2A at time zero, t or , respectively. Conclusions: The rate constants of photolysis reaction, obtained by HPLC and UV (A, D1) methods (k ± k [s-1] = (6.01 ± 0.81) × 10-4; (6.32 ± 2.01) × 10-4; (6.31 ± 1.16) × 10-4, respectively), were compared using parallelism test. The results indicated that there are no statistically significant differences between them and that both methods can be used interchangeably. However, only the HPLC method is selective and does not require determination of measurement at t = .

[1] K. Gsiorowski, W. Malinka, P. witek, A. Jaszczyszyn, Antimutagenic activity of new analogues of fluphenazine, Cell. Moll. Biol. Lett. 8 (2003) 927942.

98 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 14

Mass spectrometric characterization of a new elastin-like biomaterial

C. U. Schräder1*, C. E. H. Schmelzer1, S. Baud2, Suzanne M. Mithieux3, A. S. Weiss3, R. H. H. Neubert1, A. Heinz1

1Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Germany 2Laboratoire de Signalisation et Récepteurs Matriciels, Université de Reims Champagne- Ardenne, France 3School of Molecular and Microbial Biosciences, University of Sydney, Australia *e-mail: [email protected]

Introduction: Elastin is one of the most important proteins in the extracellular matrix of vertebrates and provides elasticity to various tissues. It is generated by cross-linking of its soluble precursor tropoelastin (TE) after oxidative deamination of lysine (K) to reactive allysine (k) residues, which leads to the formation of a variety of characteristic bi- and polyfunctional cross-links including desmosine (DES). Due to its cross-linked structure, elastin is remarkably resistant to enzymatic degradation. Although it is known that so-called KA and KP motifs take part in cross-linking, it remains still unclear which of the 14 possible domains exactly are cross-linked with each other [1]. In this study, a new elastin-like biopolymer composed of in vitro cross-linked, recombinantly produced human TE, is used as a model substrate to detect cross-linked peptides using mass spectrometry (MS). Materials and Methods: Human TE was dissolved in PBS and cross-linked in vitro by adding Aspergillus nidulans amine oxidase (ANAO) [2]. Bovine and human aortic elastin were isolated from tissue biopsies using a method developed by our group earlier [3]. Quantification of DES was performed by LC-MS after a total hydrolysis of the samples. Furthermore, all samples were digested with different proteases including chymotrypsin (CTR), trypsin (TR) and pancreatic elastase. Analysis of the digests was performed using nanoHPLC/MALDI-TOF/TOF-MS/MS. Moreover, the conformational behavior of identified cross-linked peptides was predicted using molecular dynamics (MD) simulations. Results: LC-MS of the totally hydrolyzed biomaterial confirmed that the amino acid DES, which is unique to elastin, was formed during in vitro cross-linking, however, to smaller extent as compared to human elastin. While elastin is highly resistant to cleavage of TR and CTR, cross-linked TE was susceptible to cleavage by the two proteases, which suggests that the biomaterial is less complex in its cross-linking pattern than native elastin. This hypothesis is supported by the presence of non-cross-linked K and k residues in the biomaterial. Two intramolecularly cross-linked peptides were identified and MD simulations of these peptides show possible conformational states which may play a role during cross-link formation. Conclusions: The biomaterial was comprehensively characterized on the molecular level showing that it contains cross-links characteristic of native elastin. The identification of cross- linked peptides and their assignment to the sequence of TE provides a good basis for the identification of the cross-linking pattern in human elastin which can help to understand functional changes in elastin during ageing and disease.

[1] S.M. Mithieux, A.S. Weiss: Elastin. Advanced Protein Chemistry, 70 (2005) 437-461. [2] A.P. McGrath, S.M. Mithieux, C.A. Collyer, J.G. Bakhuis, M. van den Berg, A. Sein, A. Heinz, C.E.H. Schmelzer, A.S. Weiss, J.M. Guss: Structure and Activity of Aspergillus nidulans Copper Amine Oxidase, Biochemistry, 2011, 50, 5718–573. [3] C.E.H. Schmelzer, M.C. Jung, J. Wohlrab, R.H.H Neubert, A. Heinz: Does human leukocyte elastase degrade intact skin elastin? FEBS Journal, 279 (2012) 4191-4200.

99 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 15

The use off-line TLC-HPLC-MS in the qualitative and quantitative analysis of lutein in flour of Triticum species.

1Migas P., 1Stefanowska S.,1Szarko M., 2Wiwart M., 2Suchowilska E., 1Krauze-Baranowska M. 1Department of Pharmacognosy with Medicinal Plant Garden, Medical University of Gdask, Gen. Haller Str 106, 80-416 Gdask, Poland; e-mail: [email protected] 2Department of Plant Breeding and Seed Production, University of Warmia and Mazury in Olsztyn, Plac ódzki 3, 10-724 Olsztyn-Kortowo

Lutein content in flour of three cultivars of Triticum spelta: Sumai, Kamut and Terzino was analyzed by the use of TLC and off-line TLC-HPLC-MS method. The flour was obtained from the native and infected with Fusarium culmorum the above mentioned varieties. The analysis of plant samples was conducted at diminished light. The extraction of carotenoids was carried out with the use of modified method of Frattiani et al [schem 1]. The samples were stored at -20oC for three days. Then they were spotted as 5mm lines (15mm from the edge of Si60 F254S TLC glass plates) with the use of Desaga sampler under nitrogen atmosphere. The bivariant multiple development (BMD) technique was employed for the sample separation. First, constituents of the sample were resolved at a distance of 15mm with n-heptane:ethyl acetate (90:10 v/v) as a mobile phase. The separated zone of accompanied substances was cut off. The exact separation was continued at a distance of 30mm with n- heptane:ethyl acetate:acetone (20:25:55 v/v/v) [schem. 2].The qualitative analysis of the separated spots was performed with the use of co-chromatography with the standards and off- line TLC-HPLC-MS. The densitometric analysis was used for quantification of lutein in the analyzed plant samples.

100 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 16

Application of TLC and chemometric techniques for prediction of log P values of phenanthrene derivatives

Krzesimir Ciura1, Joanna Nowakowska1, Piotr Pikul1, Wiktoria Struck2, Micha J. Markuszewski2

1Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, e-mail: [email protected] 2Department of Biopharmaceutics, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland

Lipophilicity is an important physico-chemical parameter of a compound, and it has a vital role in QSAR and QSRR studies. It is an important factor which plays a role in processes of absorption, distribution, and excretion of drugs and other xenobiotics in the body. The traditional method of determining lipophilicity using n-octanol–water partitioning is laborious and time-consuming. Therefore, currently chromatographic techniques are mostly used to determinate lipophilic character of the molecule. Stock solutions of the phenanthrene derivatives (1 mg/ml) were prepared in methanol. Tested group of compounds included 8 hormones (transandrosterone, methyltestosterone, testosterone, progesterone, cortisone, hydrocortisone, estrone, ß-estradiol), 3 polycyclic aromatic hydrocarbons (1-methylophenanthrene, 1,3-dimethylophenanthrene, 3- methylcholanthrene) and 3 steroid drugs (methylprednisolone, prednisone, norgestrel). Mobile phase was a mixture of acetone-water (30-80%), acetonitrile-water (50-80%, v/v) and acetone-petroleum ether (0-80%, v/v). Stationary phase was RP-TLC (RP-18 WF254s HPTLC) and NP-TLC (silica gel 60 F254 HPTLC) in case of using two organic solvent as mobile phase. Chromatographic analysis was performed on 5 cm × 10 cm plates manufactured by Merck (Darmstadt, Germany), in chromatographic chambers (7 x 11 cm) previously saturated with mobile-phase vapour. Chromatograms were developed to a distance o 0 of 8 cm at room temperature (20 + 2 C). The chromatographic date (RM , S, C0) have been used to designate the Collander equation. Values of experimental log P were derived from ChemSpider database and also calculated by the following softwares: ACD/labs, ChemAxon, ALOGPs, AC LogP, ALOGP, MLOG, KOWWIN, XLOGP 2, XLOGP3, and HyperChem. Correlations between chromatographic data and measures of lipophilicity of compounds are presented as results of established Quantitative Structure-Retention Relationships (QSRRs). Conclusions: 1. The best chromatographic system to predict log P of phenanthrene derivatives is RP-TLC with a mixture of mobile phase consisting of acetone and water ranged from 30%, v/v to 80%, v/v. 2. In case of acetone and petroleum ether mixture as mobile phase, the hydrophobic parameters S and C0 did not correlate with log P. 3. The use of silica gel for prediction of log P is possible, in case of using two organic solvents as mobile phase. That suggested the theory of co-adsorption of organic solvent on surface of silica gel. Probably, alkanes included in petroleum ether, modify the surface on silica gel.

101 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 17

Analysis of thermal properties of paracetamol and naproxen

Anna Froelich1, Ewa Skotarczyk1, ukasz Grobelny1, Marek Pyda2 1Department of Pharmaceutical Technology, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland; [email protected]; 2Department of Chemistry, University of Technology, Al. Powstaców Warszawy 6, 35-959 Rzeszów, Poland.

Introduction: Naproxen (fig. 1) is commonly used non-steroidal anti- inflammatory drug (NSAID) present in many currently marketed products intended for oral and topical use. Paracetamol (fig. 2) display analgesic and antipyretic activity but its anti-inflammatory properties are very weak. Each Fig. 1. Naproxen. of the substances is used to treat mild to moderate pain but they often turn out to be ineffective in treatment of severe pain. There are several studies [1-3] revealing that paracetamol and NSAIDs usually act synergistically. The aim of this study was the investigation of thermal properties of the title Fig. 2. Paracetamol. drugs alone and in mixtures of different ratios.

Methods: The samples of paracetamol and naproxen and their mixtures in weight ratios 1:1, 1:2 and 1:5 were subjected to the analysis with differential scanning calorimetry technique.

Results: The results of the investigations show that both samples were crystalline and paracetamol sample displayed melting point typical for monoclinic polymorph (169.44°C). Cold crystallization of melted paracetamol most probably resulted in orthorhombic form, with observed melting point 157.41°C. Recrystallization of pure melted naproxen during cooling led to the form displaying the same melting point as the initial sample, i.e. 157.20°C. The study of mixtures of both substances revealed the physical interaction between them. In the 1:1 (w/w) mixture simultaneous melting of both substances in 139.08°C was observed. Naproxen recrystallization from the melted mixture during cooling was suppressed by the paracetamol presence.

Conclusion: The presented analyses provide some information which may be useful during the formulation of novel tablets containing both analgesic drugs.

Acknowledgement: This study was partially supported by PUMS grant N0 502-01-03314429- 03439

[1] R.D. Altman: A rational for combining acetaminophen and NSAIDs for mild-to-moderate pain. Clin Exp Reumatol, 22 (2004) 110–117. [2] H.F. Miranda, M.M., Puig, J.C. Prieto, G. Pinardi. Synergism between paracetamol and nonsteroidal anti- inflammatory drugs in experimental acute pain. Pain 121 (2006) 22-28. [3] J.A. Palma-Aguirre, J. Villalpando-Hernandez, G. Novoa-Heckel, I. Oliva, L. Carino, E. Lopez-Bojórquez, V. Burke-Fraga, S. Namur, M.G. de la Parra: Bioavailability of two oral-tablet and two oral-suspension formulations of naproxen sodium/paracetamol (acetaminophen): single dose, randomized, open-label, two-period crossover comparisons in healthey Mexican adult subjects. Clin Ther 31 (2009) 399-410.

102 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 18

Vibrational spectroscopy (UV, FT-IR, Raman) investigations and DFT analysis on the structure of tebipenem. An application for identification of labile drugs

J. Cielecka-Piontek1, B. Barszcz2, K. Lewandowska2, A. Talaczyska1 1University of Medical Sciences, [email protected], Grunwaldzka 6, Pozna, Poland 3Polish Academy of Sciences, Institute of Molecular Physics, [email protected], [email protected], Smoluchowskiego 17, Pozna, Poland

Tebipenem is an active form of tebipenem pivoxyl. Tebipenem pivoxil is the first oral -lactam antibiotic in the group of carbapenems. It is recommended for the treatment of otholaryngologic and respiratory infections of pediatric patients. The instability of tebipenem is connected with the presence of bicyclic 4:5 fused -lactam and heterocyclic rings [1]. The application of UV, FT-IR and Raman spectra was proposed for identification studies of tebipenem. An identification of the tebipenem based on its separation from related substances was achieved after the application of first derivative of direct spectra (FD-UV) in ultraviolet which permitted elimination of overlapping effects. The Raman and FT-IR spectra of tebipenem were recorded in the regions 400–4000 cm-1 and 100–4000 cm-1, respectively. The analysis of experimental spectra was supported by quantum-chemical calculations performed with the use of B3LYP functional and 6-31G(d,p) as a basis set. The geometric structure of molecule, HOMO and LUMO orbitals and molecular electrostatic potential were also determined. The method based on zero-crossing effect of first derivative spectrophotometry ( = 300 nm), which eliminated the overlapping effect caused by related-products degradation products, formed during hydrolysis, oxidation, photolysis and thermolysis, was used for identification of tebipenem. The main characteristic vibrations obtained from the FT-IR and Raman spectra are identified and the main reasons of tebipenem instability were described. The calculated energy values of HOMO was -5.88 eV and LUMO -1.90 eV for tebipenem. The value of the energy separation between the HOMO and LUMO were 3.98eV. The small differences between energy gap of molecule suggests significant instability of tebipenem as the result of intraring stress. Analysis of MEP showed that the positive regions are localized on different sites of molecules. While the negative regions of tebipenem molecule are mainly localized on carbonyl group of - lactam ring and carboxylic group, indicating sites for electrophilic attack. In view of those findings, spectral methods – FD-UV, FT-IR and Raman scattering spectroscopy in particular – may be recommended for quality control analysis of tebipenem as an ecological alternative for the traditional solvent-based analytical techniques.

[1] K. Kazuhiko, S. Yoshiyuki, K. Erika, K. Akihiro, I. Maki, S. Hisashi, S. Shigeki, K. Tohru, T. Ikumi. Molecur. Pharm. 7 (2010) 1747–1752.

103 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 19

Comparison of hyaluronidase activity in different biological samples using newly developed CZE method

Jan Matysiak, Pawe Dereziski, Bartosz Urbaniak, Agnieszka Klupczyska, Anna Zalewska, Zenon J. Kokot

Poznan University of Medical Sciences, Department of Inorganic and Analytical Chemistry, 6 Grunwaldzka Street, 60-780 Pozna, [email protected]

Hyaluronic acid (HA) is a naturally occurring linear polysaccharide. It is a component of the extracellular matrix of the skin, joints, eyes and other tissues and body fluids of vertebrates. Hyaluronidases are the enzymes responsible for degradation of hyaluronic acid. They represent a widespread group of enzymes which occur in mammalian organisms, microorganisms and venoms of snakes, scorpions and bees. Due to the wide use of hyaluronidases in therapy and their high prevalence in biological fluids, tissues, venoms and toxins, it is necessary to use validated methods for determination of hyaluronidase activity. The aim of the study was to analyze and compare hyaluronidase activity in different biological samples. For this purpose a new capillary zone electrophoresis (CZE) method was developed. CZE studies were performed using Agilent G1600 instrument equipped with capillary of 64.5 cm total length, 56 cm effective length and internal diameter of 75 m. Separation was performed in the phosphate buffer (pH=8.10) in the electric field of 20 kV, =220 nm. The procedure was based on mixing of a known quantity of hyaluronic acid and an aliquot of hyaluronidase solution, followed by obtaining CZE profiles after a known period of incubation (0.5 h). The activity of hyaluronidase was calculated using multiple regression analysis in which sizes of the peaks of the main degradation products were used. The newly developed method was fully validated and it is appropriate to evaluate activity of hyaluronidase originating from different sources with high precision and accuracy. T-test’s showed that there were no significant differences between results obtained using turbidimetric, viscosimetric and the new CZE method. The newly developed CZE method allows for determination of enzymatic activity of hyaluronidase from different sources (bee venom and mammalian testes). Hyaluronidase activity in the honeybee venom samples has ranged from 825.2 ± 30.7 U/mg d.m. to 1450.7 ± 101.1 U/mg d.m. Because there have been no significant differences between the results obtained using the newly developed method and the pharmacopoeial tubidimetric and viscosimetric method (p = 0.730) it can be concluded that other honeybee venom components do not influence the hyaluronidase activity. The activity results obtained for the bovine tests hyaluronidase purchased from Sigma have been consistent with the activity given by the manufacturer, which confirms accuracy of the new CZE method. The newly developed method for CZE determination of hyaluronidase activity allows to obtain reliable results in a short time with high precision and accuracy. The new method can be successfully applied to analysis of hyaluronidases of different origin (e.g. from bee venom and mammalian testes). Moreover, the new method involves simple methodology, small volume of the required sample, low amounts of inexpensive chemicals used and, therefore, low cost of analysis. Absence of significant differences between the results obtained using a newly developed method and commonly used turbidimetric and viscosimetric methods indicates that the CZE method is an alternative to classical methods for determining hyaluronidase activity.

104 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 20

Selectivity of chromatographic systems in the application of steroids as a model substances

Krzesimir Ciura, Joanna Nowakowska, Piotr Pikul

Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, e-mail: [email protected]

Chromatographic properties of seven steroids: estrogens (-estradiol and estrone), androgens (testosterone, methyltestosterone, trans-androsterone), progesterone and cholesterol have been studied by normal-, reversed- and hydrophilic neutral cyano-bonded silica stationary phases with five binary mobile phases (acetonitrile-water, acetonitrile-DMSO, acetonitrile-methanol, acetone-petroleum ether, acetone-water) in which the concentration of organic modifier was varied from 0 to 100% (v/v). Generally, higher values of separation factors were observed for all acetonitrile-containing binary mobile phases. On the other hand, very good separation was observed for estrogens with anhydrous eluents in both NP- and RP-chromatography. From the practical point of view, in order to separate: estrogens - androgens and estrogens - progesterone, can be recommended silica gel and RP plates with acetonitrile as the modifier of the aqueous and non-aqueous mobile phase. The best separation of both estrogens was achieved on CN adsorbent by use of mobile phase containing 30% v/v acetone in petroleum ether and on RP- 18 plates by use of mobile phase containing 90% v/v acetonitryle in methanol. Values of slope of correlation lines are higher on normal-phase, in comparison with reversed-phase with aqueous binary mobile phases (acetone-water). On the other hand, on RP TLC plates higher values of slope of correlation lines were achieved for non-aqueous binary mobile phases (acetone-petroleum ether) for estrogens and aqueous binary mobile phases (modifier-water) for androgens and progesterone. PCA does not prove but mathematically indicates the existence of similarities CN stationary phase for NP and RP depending on the type of organic modifier, regardless of whether water is added to the chromatographic system.

105 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 21

Characterization of Sugammadex Inclusion Complexes with Penicillins Using Affinity Capillary Electrophoresis Kinda A. Darwish1, Yahya Mrestani1, Reinhard H.H. Neubert1 ¹Department of Pharmaceutical Technology and Biopharmaceutics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany. E-mail address: [email protected]

Sugammadex is a modified -cyclodextrin (CD) for post-surgery reversing the neuromuscular blockage action of rocuronium by forming strong inclusion complex [1, 2]. The structural modifications, especially the extended lipophilic cavity, could enhance its inclusion complexes with a wide range of drugs at physiological fluid. Affinity capillary electrophoresis (ACE) was defined as versatile analytical tool in analysis and determination of CDs and their derivatives complexation [3]. This work is directed to investigate the interactions between sugammadex and widely used antibacterial agents, the penicillins, (amoxicillin, ampicillin, penicillin G, oxacillin, dicloxacillin, azlocillin and piperacillin) using ACE.

Capillary electrophoresis experiments were performed using diode array detector (190 to 600 nm) coupled to Hewlett Packard model G1600AX (Waldbronn, Germany) 3D CE system. The detection wavelength was 200 nm. The used fused silica capillary was of 50 μm internal diameter and 64.5 cm total length. The length to detector was 56 cm. The temperature was kept at 25° C and the separation voltage was 30 kV. The samples were injected at 50 mbar pressure for 9 s. For each sample, the run was repeated three times.

The electropherograms showed shifting in migration time (tr) of drug peak as sugammadex concentrations increased. The effective motilities changes (inversely related to tr) for amoxicillin, ampicillin, azlocillin, oxacillin and dicloxacillin, as a function of sugammadex concentrations in the backgroundelectrolyte (pH 7.2), were correlated and successfully fitted to nonlinear curve equation [4]. Ka values were calculated. For penicillin G and piperacillin, the effective mobility showed no change at the same range of sugammadex concentrations.

As conclusion, sugammadex inclusion complexes for studied penicillins (except penicillin G & piperacillin) are formed. The affinity strength for each interaction was quantitavely -1 determined (Ka = 383.43, 265.33, 184.54, 95.06 and 13.6 M ) for amoxicillin, dicloxacillin, ampicillin, oxacillin and azlocillin, respectively. Later on, the interruption effect of these complexes formations on the main pharmacological function of sugammadex could be studied.

[1] Bom, A., et al., A novel concept of reversing neuromuscular block: chemical encapsulation of rocuronium bromide by a cyclodextrin-based synthetic host. Angew Chem Int Ed Engl, 2002. 41(2): p. 266-70. [2] Cameron, K.S., D. Fletcher, and L. Fielding, An NMR study of cyclodextrin complexes of the steroidal neuromuscular blocker drug Rocuronium Bromide. Magn Reson Chem, 2002. 40(4): p. 251-260. [3] Larsen, K.L. and W. Zimmermann, Analysis and characterisation of cyclodextrins and their inclusion complexes by affinity capillary electrophoresis. J Chromatogr A, 1999. 836(1): p. 3-14. [4] Ruettinger, H.H., Theory of affinity electrophoresis, in Affinity capillary electrophoresis in pharmaceutics and biopharmaceutics, R.H.H. Neubert and H.H. Ruettinger, Editors. 2003, Marcel Dekker. p. 23-43.

106 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 22

Quantification of the chromatographic process by Soczewiski-Snyder equation and its consequences in retention of ivabradine by TLC

Piotr Pikul, Joanna Nowakowska, Krzesimir Ciura, Kaja Rudnicka, Sandra Maciejewska, Wiesaw Sawicki

Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, e-mail: [email protected]

One of the most well-known processes occurring during the chromatographic process is adsorption, which is described by the Soczewiski-Snyder equation. This competitive adsorption model assumes the competitiveness of the more polar component of the mobile phase and solute for a place on silanol groups on the stationary phase. In this study, several attempts were made to quantify the phenomena responsible for the process of chromatography, optimum applications and further studies of these phenomena, as well as the development of a HPTLC method for the determination of ivabradine. Ivabradine is the first modern drug synthesized to selectively decrease heart rate. It acts by selective inhibition of the If (pacemaker current) in the sinus node cells. Thin-layer chromatography was used to evaluate the behavior of ivabradine in various chromatographic systems. The mobile phases used were aqueous: methanol – water and acetonitrile – water, and non-aqueous: methanol – acetonitrile and methanol – dimethyl sulfoxide (DMSO) in a wide range of concentrations (0 - 100%, v/v). The stationary phases used were HPTLC plates: Kieselgel 60 F254 S, RP-2 F254 S, RP-8 F254 S, RP-18 F254 S, RP-18 WF254 S, Kieselgel 60 CN F254 S, Kieselgel 60 Diol F254 S, Kieselgel 60 NH2 F254 s and TLC plates: Aluminium oxide 150 F254, Cellulose F and Polyamide 11 F254. Single point adsorption combined with the dissociation of the solvation shell consisting of DMSO is explained by the slope of the Soczewniski-Snyder equation curve in the range from 2 to 1 (Silica gel, Aluminium oxide, RP-18). However, for the slope greater than 2 (RP-8 and Diol), it is more likely that multi-point adsorption is also connected to the dissociation of solvation shell. Taking into account H-bonding properties of NH2 and CN groups, as well as the low absolute values of the slope, a more complex mechanism of retention for these stationary phases is assumed. Data obtained using the Soczewiski-Snyder equation suggested the formation of a solvation shell consisting of DMSO. The chromatographic process occurring on the amino-bonded stationary phase is conditioned primarily on the properties of the sorbent. However, physicochemical parameters of the eluent did not significantly affect the retention behavior of ivabradine, as for the application of cyano- and diol-bonded stationary phases.

107 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 23

Insights into the proline hydroxylation of elastin derived from different tissues and species

Marcus B. M. Nagel1, Andrea Heinz1, Sarit-S. Sivan2, Rainer Pankau3, Reinhard H. H. Neubert1,Christian E. H. Schmelzer1

1Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany 2Department of Biomedical Engineering, Israel Institute of Technology, Haifa, Israel 3Finkelstein-Klinik für Kinder- und Jugendmedizin, Heidekreis-Klinikum Soltau-Walsrode, Walsrode, Germany E-mail: [email protected]

Introduction: Elastin is one of few known proteins, which are partially hydroxylated at their proline residues by the enzyme prolylhydroxylase [1]. The amount and function of hydroxyproline (HyP) residues in elastin are almost unknown. Therefore, the aim of this work was to investigate the proline hydroxylation in elastin derived from different species including pig, chicken, cow, carp and human to get an insight into the extent and possible role of this post- translational modification. Methods: Elastin of the previously mentioned species was isolated from skin, aorta, cartilage, ligament and intervertebral disc (IVD) using a gentle method developed by our group [2]. Elastin from IVD samples was, furthermore, isolated by a method described earlier [3]. Moreover, tissue of healthy individuals of different ages and Williams-Beuren syndrome (WBS) patients was analyzed. Isolated elastin was characterized using scanning electron microscopy (SEM) and nanoHPLC-nanoESI-Qq-TOF MS(/MS) after proteolysis by pancreatic elastase. Results: For each tissue and species, several peptides containing one partially hydroxylated proline residue were identified, and the peak areas for the hydroxylated and non-hydroxylated peptides were determined in base peak chromatograms of LC-MS runs (semi-quantiative determination). Two peptides, which exist in all investigated mammalian species, were quantified and showed very different ratios of hydroxylation depending on both tissue and species. Little differences in the ratios of hydroxylated and non-hydroxylated peptides exist between samples from tissues of healthy individuals and WBS patients, and there are almost no differences between samples of the same tissue derived from individuals of different ages. SEM images showed that in particular the thickness of the elastin fibers and their arrangement varied significantly between different samples of different tissues. Conclusion: Proline hydroxylation of elastin differs among different tissues of one species and also between different species. The role of the proline hydroxylation still remains to be identified.

[1] J. Uitto, H.-P. Hoffmann, D. J. Prockop.: Synthesis of Elastin and Procollagen by Cells from Embryonic Aorta, Archives of Biochemistry and Biophysics, 173 (1976) 187-200. [2] C.E.H. Schmelzer, M.C. Jung, J. Wohlrab, R.H.H Neubert, A. Heinz: Does human leukocyte elastase degrade intact skin elastin? FEBS Journal, 279 (2012) 4191-4200. [3] S.-S. Sivan, B. Van El, Y. Merkher, C.E.H Schmelzer, A.-M. Zuurmond, A. Heinz, E. Wachtel, P.-P. Varga, A. Lazary, M. Brayda-Bruno, A. Maroudas: Longevity of elastin in human intervertebral disc as probed by the racemization of aspartic acid, Biochimica et Biophysica Acta, 1820 (2012) 1671-1677

108 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 24

Isolation and Identification of Elastic Fiber Proteins Martin Q. Koehler1*, , Andrea Heinz1, Reinhard H. H. Neubert1, Christian E. H. Schmelzer1 1Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany *e-mail: [email protected]halle.de Introduction: The extracellular matrix (ECM) is localized in between diverse animal cells and consists of polysaccharides and fibrous proteins including collagen and elastin. In addition to the constituents of elastic fibers, elastin and microfibrils, the ECM contains a great amount of fiber-associated proteins like fibrillin-1 and MAGP-5, which affect its structural and functional properties. Elastin and elastic fibers are found in various tissues including aorta, intervertebral disc and skin [1], in which they have to withstand different physical stresses like pressure, tension and elongation stress. It is of great interest to investigate the composition of elastic fibers in diverse tissues using mass spectrometry to identify differences between different tissues and to elucidate the function of a specific protein in the fiber. Therefore, the aim of the project is the development of a method to obtain pure elastic fibers from different tissues followed by molecular-level characterization of the components of the elastic fibers. Materials and Methods: Tissue samples from bovine aorta were used to develop and optimize a method to isolate entire elastic fibers. In a first step, soluble proteins are removed using NaCl (1M) followed by lipid extraction with different organic solutions such as ethanol, chloroform, methanol and ether. Finally, glycoproteins are denatured and eliminated using guanidine hydrochloride (5M) a chaotropic reagent. To investigate the structural constituents of the elastic fibers, the samples were digested using pancreatic elastase, which is able to cleave many proteins including elastin. Subsequently, samples were analyzed using nanoHPLC-ESI-Qq-TOF MS and MALDI-TOF/TOF MS. Furthermore, the morphology of different elastic fiber proteins was studied using scanning electron microscopy (SEM). The samples were compared to elastin samples isolated from cartilage, navel and uterus using a soft isolation method [2]. Results and Discussion: SEM images of cartilage, navel and uterus elastin samples revealed differences regarding the thickness of elastin fibers and the three dimensional structure they adopt. Mass spectrometric investigations on elastic fibers are currently under progress and will give insights into differences in the composition of elastic fibers based on the tissue they are derived from. First results already helped to optimize the isolation method for elastic fibers. Conclusion: Investigating entire elastic fibers will give insight into interactions between different components of elastic fibers and structure-function relationships in the ECM.

[1] S.M. Mithieux, A.S. Weiss: Elastin. Advanced Protein Chemistry, 70 (2005) 437-461. [2] C.E.H. Schmelzer, M.C. Jung, J. Wohlrab, R.H.H Neubert, A. Heinz: Does human leukocyte elastase degrade intact skin elastin? FEBS Journal, 279 (2012) 4191-4200.

109 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 25

Development of an HPLC-APCI-MS/MS assay for vitamin E constituents determination in human serum

Danuta Siluk, Magdalena Buszewska, Wiktoria Struck, Ewa Bartosiska and Roman Kaliszan

Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdask, Al. Gen. Hallera 107, 80-416 Gdask, Poland; [email protected]

Vitamin E naturally occurs in four forms: alpha-, beta-, gamma- and delta-tocopherols and four corresponding unsaturated analogues, tocotrienols. They belong to the most active lipid- soluble antioxidants in vivo. This activity might play a preventive role in diseases associated with oxidative stress like cardiovascular diseases, neurodegenerative diseases or cancer. A majority of conducted studies have been mostly focused on the role of alpha-tocopherol, thought to be the most biologically important form of vitamin E. However, observational studies have failed to consistently support the theory that alpha-tocopherol provides a protection, mainly against cancer disease.

The primary aim of the study was development of a rapid assay for tocopherols and tocotrienols simultaneous determination in human serum with the use of reversed phase high performance liquid chromatography coupled with tandem mass spectrometry (RP-HPLC- MS/MS). The mass spectrometry analysis was carried out by the use of Agilent Technologies system (Palo Alto, CA) 1200 LC with atmospheric pressure chemical ionization interface and Agilent Technology 6430 triple quadrupole. The sample preparation was performed with the use of solid-phase extraction technique.

The separation of four tocopherols and three tocotrienols was successfully achieved with Cosmosil 2.5 NAP column. The chromatographic run was carried out isocratically with mobile phase composed of methanol:water (90:10; v/v) over 13.5 min at a flow rate 0.5 ml/min.

The validation results proved the assay to be sensitive, precise, accurate and specific indicating that it can be applied to the simultaneous determination of tocopherols and tocotrienols in human serum.

110 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 26

Monitoring of ascorbic acid status in urine samples collected from children with cystic fibrosis using capillary electrophoresis method

Ilona Oldzka1, Bogusawa Jachimowicz1, Piotr Kowalski1, Katarzyna Kamierska2, Tomasz Bczek1

1Department of Pharmaceutical Chemistry, Medical University of Gdask, Gdask, Poland [email protected] 2 Department of Pediatrics, Pediatric Gastroenterology, Hepatology and Nutrition, Medical University of Gdask, Gdask, Poland

Introduction. Malnutrition is a major problem in patients with cystic fibrosis (CF). The incidence of malnutrition increases with age and nutritional status and is an important prognostic factor for the disease (poor nutritional status correlates with a poor prognosis for the disease). In CF, increased oxidative stress associated with ongoing inflammatory processes in the respiratory system, may significantly increase the demand for antioxidant vitamins (A, E and C). In cystic fibrosis, attention is also paid to the deficiency other antioxidants and absence of effective reduction of lipid peroxidation as a result of the supplementation with vitamin E. Therefore, an additional supply of vitamin C may prove effective as protection against free radicals' reactions intensity. Vitamin C has an effect on the maintenance of normal oxidative potential in the cell. In addition, vitamin C activates the immune system, which stimulates the growth and efficiency of immune cells and other white blood cells. This results in increasing the amount of interferon that regulates the immune system.

Methods. The primary objective of the proposed project is to evaluate the analytical usefulness of the capillary electromigration techniques as tools to determine vitamin C status in patients with cystic fibrosis (CF). As an analytical method micellar electrokinetic chromatography (MEKC) with UV detection (280 nm) was used, which allowed for the separation and quantitative determination of an analyte in a few minutes in the concentration range of 10-100 ng/ml. The separation buffer consisting of 10 mM sodium tetraborate and 50 mM SDS (pH 9.3) was used. The conditions of extraction procedure of vitamin C from urine samples have also been developed, taking into account the need for a significant reduction in the detection limit of this method. In order to isolate the analyte from the matrix and to remove undesirable contaminants the SPE extraction using SPE column C18 (500 mg) was employed.

Results. In patients with CF, compared to the control group, a significant reduction in levels of vitamin C is observed.

Conclusion. The elaborated method may be helpful in the determination of isolated deficiencies and an indication of their supplementation. This may indirectly improve the nutritional status of patients with CF.

[1] Oldzka, P. Kowalski, T. Bczek, B. Muszyska-Furas, J. Paradziej-ukowicz, M. Taciak, B. Pastuszewska, Determination of water soluble vitamins in laboratory animal feeds by micellar electrokinetic chromatography, Analytical Letters, 45 (2012) 689. [2] I. Oldzka, A. Plenis, L. Konieczna, P. Kowalski, T. Bczek, Micellar electrokinetic chromatography for the determination of cortisol in urine samples in view of biomedical studies, Electrophoresis, 31 (2010) 2356.

111 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 27

Catalytic effect of buffers on the degradation of cefquinome sulphate in aqueous solutions

Agnieszka Doha1, Anna Jeliska1, Judyta Cielecka-Piontek1

1 Department of Medicinal Chemistry Karol Marcinkowki University of Medical Sciences Grunwaldzka Street 6, 60-780 Pozna, [email protected]

Cephalosporins are a class of drugs often used in veterinary medicine in infections with bacterial etiology. Cefquinome sulphate is one of the fourth generation cephalosporins, which applies to a number of infections in farm animals. It is active against many pathogens such as: Actinobacillus spp, Actinobacillus pleuropneumoniae, Haemophilus spp, Clostridium spp, Corynebacterium, Erysipelohtrix rhusiopathiae, Proteus spp, Salmonella spp, Streptococcus spp, Pasteurella spp, Staphylococcus spp, Escherichia coli and other Enterobacteriaceae and Pseudomonas aeruginosa. It is also resistant to the chromosomal and plasmid -lactamase [1,2].

The aim of this work was to evaluate the catalytic effect of buffers on the degradation of cefquinome sulphate in aqueous solutions.

Determination of cefquinome sulphate was performed using, reversed phase high performance liquid chromatography with DAD detection. In this method, the following parameters of separation were used: LiChroCART column RP-18 (5 um, 125 mm × 4 mm), mobile phase: acetonitrile, 0.02 mol L-1 phosphate buffer (pH 7.0) (10:90 V: V), the mobile phase flow rate of 1.0 ml min-1, DAD detector wavelength 268 nm. As an internal standard acetanilide solution of concentration 1 mg ml-1 was used. The developed analytical method was validated in terms of selectivity, linearity, precision, sensitivity. Limit of detection and limit of quantitation were also calculated.

The catalytic effect of buffers (phosphate, acetate, borate) on the degradation of cefquinome sulphate in aqueous solutions was studied at 343 K in the pH range 1.54-10.48 (μ = 0.50 mol L-1) .

Catalytic rate constants of cefquinome sulphate degradation were calculated in tested buffers. The influence of the catalytic effect of the components of phosphate and acetate buffers on the degradation of cefquinome sulphate is similar. The components of a borate buffer have the greatest catalytic effect on the degradation of analysed compound.

Cefquinome sulfate shows greather stability in the pH range 2.5 – 7.0.

1. A. Morioka, T. Asai, K. Ishihara, A. Kojima, Y. Tamura, T. Takahashi: In vitro activity of 24 antimicrobial agents against Staphylococcus and Streptococcus isolated from diseased animals in Japan, Journal of veterinary medical science the Japanese Society of Veterinary Science, 67(2), (2005), 207-210. 2. M. Limbert, D. Isert, N. Klesel, A. Markus, K. Seeger, G. Seibert, E. Schrinner: Antibacterial activities in vitro and in vivo and pharmacokinetics of cefquinome (HR 111V), a new broad-spectrum cephalosporin, Antimicrob Agents and Chemotherapy 1, (1991), 14-19.

112 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 28

Determination of theophylline in commercially available drugs by DSC

Edyta Leyk1, Marek Wesolowski2 1Department of Analytical Chemistry, Medical University of Gdansk, Gen. J. Hallera 107, 80-416 Gdansk, Poland, [email protected] 2Department of Analytical Chemistry, Medical University of Gdansk, Gen. J. Hallera 107, 80-416 Gdansk, Poland, [email protected]

The safety of pharmacotherapy depends in a high degree on the quality of medicinal products. This obliges the drug manufacturers to check the entity and quality of all raw materials used during production of pharmaceutical preparations, as well as to provide the control of the manufacturing process of pharmaceuticals, and of the final products. Particular requirements are provided by pharmacopoeias and they can also be found in regulations of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH, issued to unify the requirements obligatory for new drugs introduced to pharmaceutical market. Differential Scanning Calorimetry (DSC) is a rapid method which does not require complicated sample preparation and that require a small amount of substance. This method allows for qualitative or quantitative analysis of purity, polymorphs content or interactions between components of the drug. For the quantitation of theophylline in selected commercially available drugs, the mixtures of an active pharmaceutical ingredient with different drug matrices (mixtures of excipients) were studied. DSC scans were taken on a DSC 822e heat-flux instrument (Mettler Toledo, Switzerland) with a liquid nitrogen cooling system (Dewar vessel) and a STARe software. Samples of approx. 4.00-4.10 mg were encapsulated in a 40 μL flat-bottomed aluminium pans with pierced lids. The scans over the range of 20 to 300ºC were obtained at a scanning speed of 10ºC/min, using nitrogen as a purging gas at a flow rate of 70 mL/min. Indium and zinc standards were used to calibrate the DSC cell. Relationship between the theophylline content and the peak area depends on matrix composition. For most of drugs, quantitation of theophylline based on the calibration curve correspond with specification. The result obtained by the multiple standard addition method were not satisfying neither for the references mixtures nor for preparations. It is necessary to continue research to evaluate the possibility of determination theophylline in other matrix.

113 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 29

Elucidation of the impact of short-chain ceramides to the nanostructure of Stratum corneum lipid membranes applying neutron diffraction

A. Schroeter1, T. Hauß2, R.H.H. Neubert1 1 Institute of Pharmacy, Martin Luther University, Halle (Saale), Germany 2 Helmholtz Centre Berlin for Materials and Energy GmbH, Berlin, Germany

Email correspondence to: [email protected]

Introduction: The human stratum corneum (SC), the outermost layer of the skin constitutes a most effective barrier. It hinders substances from entering the skin as well as minimizing the transepidermal water loss. The SC consists of dead cells, the corneocytes, which are embedded in an intercellular lipid matrix [1]. The major lipid classes extracted from the SC lipid matrix are ceramides (CER), cholesterol (CHOL) and long-chain free fatty acids (FFA) [2, 3], whereby the CER are known to play a fundamental role for the structural arrangement as well as for the maintenance of the barrier function of the skin [4]. The elucidation of the nanostructure of the lipid matrix of the SC is an extensive and complex task, due to the variability and the complexity of native SC lipid membranes. The employment of lipid model membranes enables a systematic investigation of the influences of the different lipid species to the structural arrangement, the properties of the membrane and its function within the membrane organization. Up to now, the focus was primarily placed on the influence of the hydrocarbon chain length and in particular, the predominant role of the long-chain -acyl CER such as CER[EOS] or CER[EOP]. It was argued that the unique structure of these CER is closely attributed to the existence of the so-called Long-periodicity-phase (LPP) of 130 A ι in the SC lipid matrix [5]. Aim: The goal of this investigation was to define the impact of the long-chain -acyl CER[EOP] to the nanostructure of SC lipid model membranes in the presence of the short- chain CER[AS] or CER[AP]. For CER[EOS] we have already demonstrated that in the presence of the more polar short-chain CER[AP] no LPP is formed, which is attributed to the strong head group interaction created by CER[AP] [6, 7]. As CER[EOP] has an additional OH-group compared to CER[EOS], the question is, whether the LPP can be formed. Materials: The CER were kindly donated by Evonik Goldschmidt GmbH (Essen, Germany) and used without further purification. CHOL and FFA were purchased by Sigma Aldrich GmbH (Taufkirchen, Germany) and used as received. Quartz slides (Spectrosil 2000, 25 x 65 x 0.3 mm3) were procured from Saint-Gobain (Wiesbaden, Germany). For deposition of the lipids an airbrush instrument (Harder & Steenbeck, Norderstedt, Germany) was used. Neutron diffraction: The neutron diffraction measurements were carried out using the membrane diffractometer V1 at the Berlin Research Reactor BER II of the Helmholtz Centre for Materials and Energy (Berlin, Germany). A 2D-position sensitive 3He detector was used. In order to achieve the neutron contrast, the atmosphere in the sample chamber was adjusted to up to 3 different H2O/D2O molar compositions (92/8, 50/50 and 0/100). The structure of the bilayers was analyzed by Fourier construction of the neutron scattering length density (NSLD) profiles in direction normal to the membrane.

114 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 30

Electrophoretic and potentiometric investigations of hyaluronic acid complexation with polyvalent metal ions

Bartosz Urbaniak, Zenon J. Kokot

Pozna University of Medical Sciences, Departament of Inorganic and Analytical Chemistry, 6 Grunwaldzka Street, 60-780 Pozna, [email protected]

Introdction: Hyaluronan (HA) is a heteropolysaccharide, belonging to a group of molecules known as glycosaminoglycans (GAGs). HA plays a role of tensile strength as well as elasticity of cartilage and tendons and as a lubricant in the synovial fluid of joints. It was proved that the injection of highly purified, high molecular weight, and high viscosity HA to patients suffering from osteoarthritis, improves joint mobility and range of motion by restoring elasticity and viscosity of the synovial fluid. HA possess an unique ability to complex polyvalent metal ions. Some of the HA–metallic complexes possess their own biological activity. It was observed that the metal ions exhibit some protective and stabilizing properties that prevent the HA molecule against the degradation to low mass fragments, during the decomposition process by the hyaluronidase enzymes. Methods: The presented study was designed to identify of complexation equilibrium between hyaluronic acid (HA) and polyvalent metal ions: iron(III), aluminum(III), copper(II), zinc(II), calcium(II) and magnesium(II). The quantitative behavior of hyaluronic acid (HA) and its metal ion complexes in aqueous solutions was determined using two different analytical methods: the potentiometric titration method and HPCE method. Results: The HPCE quantification of the complexation reaction between HA and polyvalent metal ions was established using mathematical model, that enables to calculate the complex formation constants (K): 1/r=(1/nK)(1/[M])+1/n. The 1/r fraction described a ratio of complex peak area to the total ligand peak area. The value of K was estimated as a slope of the plotted curve. The values of complex peak area and total ligand peak area were obtained experimentally from electropherograms. The K provides information about the strength of observed interaction between HA and metals. The potentiometric experiments were divided into two separate steps: the protonation studies of free HA and the examinations of complexation of HA with polyvalent metal ions. Both protonation and complexation equilibrium studies were preformed in the same analytical conditions. The experimental data were further analyzed using Hyperquad program. Conclusions: Based on the HPCE experiments it was concluded that complex formation reaction between HA and polyvalent metal ions strongly depends on the pH and metal ions. The highest values of K were estimated for iron(III), copper(II) and zinc(II) ions, respectively; the lowest for aluminum(III) and magnesium(II). As a result of the potentiometric studies the two protonation constants of HA were determined, pKa1 = 3.02 and pKa2 = 11.17. It was stated also that the neutral form of hyaluronic acid (HA) is the most abundant specie in the range of pH from 4 to 9. In strongly acidic conditions (pH < 4), the + positively charged biprotonated form of hyaluronan is also present (H2HA ). Moreover, in basic conditions (pH>10), the negatively charged deprotonated form of hyaluronic acid (HA-) occurs. The overall calculated stability constants for numerous HA-metal complexes shows high affinity of metal ions to the hyaluronic acid molecule. It was estimated also that high protonated species were more stable than deprotonated ones and the stoichiometry of the complex strongly depends on the ratio of metal ions concentration to the concentration of hyaluronic acid. Finally, it can be stated that in the neutral or slightly basic condition the deprotonated (OH-) forms of HA-Me complexes were dominated.

115 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 31

Determination of partition coefficient n-octanol/water for treosulfan and its epoxy-transformers: a surprising negative correlation between lipophilicity and retention in reversed-phase HPLC

Micha Romaski, Anna Siemitkowska, Franciszek Gówka

Department of Physical Pharmacy and Pharmacokinetics, Poznan University of Medical Sciences; 6 wicickiego Street, 60-781 Pozna, Poland; e-mail: [email protected]

Introduction: Since 2000 an alkylating agent treosulfan (TREO) has been increasingly applied in clinical trials as a myeloablative agent prior to hematopoietic stem cell transplantation. Pharmacological activity of the pro-drug depends on its epoxy-transformers, monoepoxide (S,S- EBDM) and diepoxide (S,S-DEB), which are formed in a non-enzymatic consecutive reaction accompanied by a release of methanesulfonic acid [1]. In the present study partition coefficient n- octanol/water (POW) of TREO as well as its biologically active epoxy-transformers was determined for the first time. Methods: In vitro the partition of the compounds was investigated at 37 oC using a classical shake-flask method. The two-phase system was composed of the pre-saturated n-octanol and 0.05 M acetate buffer pH 4.4 adjusted with sodium and potassium chloride to physiological ionic strength of 0.16 M. Concentration of the analytes in the aqueous phase was determined by reversed-phase high performance liquid chromatography (RP-HPLC) method with refractometric detection. In this method the retention times increased from S,S-DEB to TREO. In silico the values of POW were estimated by different computational algorithms provided by ALOGPs 2.1 application, available at www.vcclab.org/lab/alogps. Results: It was shown that the studied compounds underwent neither association nor dissociation in the applied phases. Empirically determined logPOW (TREO: 1.58 ± 0.04, S,S-EBDM: 1.18 ± 0.02, S,S-DEB: 0.40 ± 0.03) proved the hydrophilic character of all the three entities. The same relation of the logPOW values was obtained using the computational algorithms. All the compounds satisfied the Lipinski rule of five. Interestingly, the POW values determined in vitro as well as in silico were inversely correlated with the retention times observed in the RP-HPLC column. It might occur because a cleavage of methansulfonic acid from a small molecule of TREO generates significant changes in the molecular structure. Conclusion: Despite the common chemical origin, TREO, S,S-EBDM and S,S-DEB do not constitute a ‘congeneric’ series of compounds with reference to the relationship between lipophilicity and retention in RP-HPLC. High hydrophilicity of TREO corresponds to its pharmacokinetic parameters described in the literature, such as relatively small volume of distribution, preferential excretion by the renal route and complete bioavailability after oral administration.

[1] Gówka FK, Romaski M, Wachowiak J: High-dose treosulfan in conditioning prior to hematopoietic stem cell transplantation, Exp Opin Investig Drugs 2010, 19, 1275-1295.

116 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 32

Radiation sterilization of anthracycline antibiotics in solid state.

P. Garbacki1, A. Kaczmarek2, J. Cielecka-Piontek1, P. Zalewski1, W. Bednarski3, B. Barszcz3, K. Lewandowska3, W. Kycler4, I. Oszczapowicz5, A. Jeliska1 1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, [email protected], Grunwaldzka 6, Pozna, Poland 2Biofarm Sp. z o.o., [email protected], Wabrzyska 13, Pozna, Poland 3Polish Academy of Sciences, Institute of Molecular Physics, [email protected], Smoluchowskiego 17, Pozna, Poland 4Great Poland Cancer Centre, Department of Oncological Surgery II, [email protected], Garbary 15, Pozna, Poland 5Department of Modified Antibiotics, Institute of Biotechnology and Antibiotics, [email protected], Starociska 5, Warsaw, Poland

Anthracycline antibiotics belong to the group of anticancer drugs. Their low bioavailability necessitates parenteral administration [1,2], which requires sterility obtained mainly by filtration. Given the considerable exposure of medical personnel to anthracycline antibiotics and their adsorption to most surfaces, especially those of sterilization filters, there is a need to find new sterilization methods that do not rely on filtration. The aim of this work was to assess the possibility of applying radiation sterilization (generated by a beam electrons of 25–400 kGy) to doxorubicin and epidoxorubicin as active substances or conversion-induced impurities. Based on ESP results the absence of free radicals was established in doxorubicin after 10 days and in epidoxorubicin after 20 days following irradiation. Radiation-induced structural changes were analysed with the use of spectrophotometric methods (UV-VIS, IR) and electron microscope imaging (SEM). A chromatographic method (HPLC-DAD) was applied to assess changes in the contents of the analogs in the presence of their impurities. The study showed that the structures of the analogs did not demonstrate any significant alterations at the end of the period necessary for the elimination of free radicals. The separation of active substances and related substances (impurities and potential degradation products) allowed determining that no statistically significant changes in the content of particular active substances occurred and that their conversion due to the presence of free radicals resulting from exposure to an irradiation of 25 kGy (prescribed to ensure sterility) was not observed. [1] M.D.Troutman, D.R.Thakker: Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium., Pharm Res, 20 (2003) 1210–1224. [2] S.J.Choi, S.C.Shin, J.S.Choi: Effects of myricetin on the bioavailability of doxorubicin for oral drug delivery in rats: possible role of CYP3A4 and P-glycoprotein inhibition by myricetin., Arch Pharm Res, 34 (2011) 309-15.

117 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 33

Complementary studies on ranitidine hydrochloride photostability

Marzena Jamrógiewicz1, Bartosz Wielgomas2, Wiesaw Sawicki1

1Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, e-mail: [email protected] 2Department of Toxicology, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland

Introduction: This work summarizes existing studies on the complementary determination of ranitidine hydrochloride (RAN) stability test performed mainly in the solid state. Although RAN is a widely used drug substance, a comprehensive determination of its stability profile is lacking. The evaluation of lability of this drug compound was critically compared with the earlier published stability treatment process for RAN. The physicochemical stability profile of RAN and its degradation scheme were initiated in context of waste water analysis [1,2], but in the solid state as a bulk powder, is poorly known. Methods: Studies are based on the use of LC/MS/MS (ToF-MS), HPLC/UV, and HS-SPME- GC-MS. Results: Photoexcitation process of RAN is studied because it causes visible changes of color and emits unpleasant odor. There are detected and identified three volatiles produced during the irradiation of RAN in the solid state. The presence of acetaldoxime, thiazole, dimethylformamide, dimethylacetamide and 5-methylfurfural was confirmed by the analysis of the authentic standards after HS-SPME-GC-MS analysis [3]. Liquid chromatography with ToF- MS allowed to detect compounds known as dimmer, being impurity A or oxime, impurity G [4] and some others. Conclusions: Using the gas chromatography with mass detector there are noticed sixteen major peaks on the chromatograms of photoirradiated RAN and the structures of some compounds were elucidated, Liquid chromatography method with mass spectrometry detection allow to achieve the impurity profile of RAN lability. Proposed molecules’ structures indicate that the identified compounds may be responsible for characteristic odor of ranitidine powder during the storage and irradiation

[1] M. Isidori, A. Parrella, P. Pistillo, F. Temussi, Effects of ranitidine and its photoderivatives in the aquatic environment, Environment International 35 (2009) 821–825. [2] J. Radjenovi, C. Sirtori, M. Petrovi, D. Barceló, S. Malato, Characterization of intermediate products of solar photocatalytic degradation of ranitidine at pilot-scale, Chemosphere 79 (2010) 368-376. [3] M. Jamrógiewicz, B. Wielgomas: Detection of some volatile degradation products released during photoexposition of ranitidine in a solid state, Journal of Pharmaceutical and Biomedical Analysis, 76 (2013) 177- 182. [4] Ph. Eur. 5.0

118 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 34

Simultaneous separation and analysis of nine polyols and sugars in soft drinks by HPLC-CAD

Magorzata Grembecka, Anna Lebiedziska, Agnieszka Dizmen, Agnieszka Opatkowska, Piotr Szefer

Department of Food Sciences, Medical University of Gdask, 80-416 Gdask al. gen J. Hallera 107 [email protected]

Introduction: Sugar is the name of a class of sweet-flavoured substances used as food. There are various types of sugar derived from different sources. The most common are glucose, fructose, sucrose and maltose. Sugar substitutes are considered as any sweetener that can be used instead of regular table sugar (sucrose). Polyols (also known as sugar alcohols) are often used to replace sugar in foods. They can provide varying amounts of sweetness as well as bind varying amounts of moisture, and interact with other components of foods such as fats and proteins. The aim of this work was to develop a new method for a simultaneous determination of erythritol, xylitol, fructose, sorbitol, mannitol, glucose, sucrose, maltitol and maltose in various soft drinks. Methods: The analytes were analysed by high-performance liquid chromatography coupled to a charged aerosol detector (Corona CAD). The method was validated using a ShodexAsahipak, NH2P-50 4E column packed with 5 μm shell particles (4.6 x 250 mm) and acetonitrile – water gradient mobile phase at 25° C. The elaborated method was validated for linearity, precision and accuracy. Each analyte calibration curve had a correlation coefficient of at least 0.999 and was linear in the defined range except for sucrose which linearity was obtained in a logarithmic coordinate system. The analytical results obtained in the validation study were highly satisfying; the recoveries for the analytes studied ranged between 92.6 and 107% and the precision values from 0.86 to 4.97%. Results: By these procedures, the five polyols (erythritol, xylitol, sorbitol, mannitol, maltitol) and four sugars (fructose, glucose, sucrose and maltose) could be well separated and quantitatively determined in varied soft drinks. Conclusions: The work has demonstrated that charged aerosol detector can be successfully used as a single detector in a rapid screening technique for sweetening substances without chromophore groups such as sugars and polyols. Acknowledgments: This study was supported by grant N N404-270840 from the Polish Ministry of Scientific Research and Information Technology.

119 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 35

Assessment of the level of selected toxic metals in patients with floor of the mouth cancer

Anna Woniak1, Maksymilian Kulza1, Monika Seczuk-Przybyowska1, Witold Szyfter2, Krzysztof Szyfter2,3, Wojciech Golusiski4 , Henryk Koroniak5, Ewa Florek1 1Laboratory of Environmental Research, Department of Toxicology, Poznan University of Medical Sciences, Poznan, Poland; [email protected] 2Department of Otolaryngology, Poznan University of Medical Sciences, Poznan, Poland 3Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland 4Head and Neck Surgery Department, Greater Poland Cancer Centre, Poznan, Poland 5Faculty of Chemistry, Adam Mickiewicz University Poznan, Poland

Tobacco smoking is considered to be the etiological factor of many diseases, including floor of the mouth cancers. Another significant stimulant affecting the formation of this disease is ethyl alcohol. It is believed that tobacco and alcohol together explained 75% of HNSCC (Head and Neck Squamous Cell Carcinoma). About 60 known carcinogens are present in tobacco smoke, 40 of them are carcinogenic for humans. It contains, inter alia heavy metals, such as chromium (VI), cobalt, cadmium and lead. Metals in cigarette smoke are a public health problem because of their potential toxicity and carcinogenicity. Cadmium is a metal, which damages the kidneys, lungs and bones. Eating food or drinking water containing very high amounts of cadmium severely irritates the stomach, leading to vomiting and diarrhea [2]. Lead can damage the nervous system, kidneys and reproductive system [1].The main target for lead toxicity is the nervous system, in adults and children. Cobalt toxicity involves many systems, organs and tissues in the human body, including respiratory system, nervous system, sense organs and skin. In experimental studies teratogenic and carcinogenic effect was observed. Chromium VI has proven allergenic effect, but also carcinogenic. The aim of this study was to determine the concentrations of these metals in hair and nail samples taken from 42 patients of Otolaryngology and Laryngological Oncology Clinic of Poznan University of Medical Sciences (SPSK No. 2) and The Head and Neck Surgery Ward of The Greater Poland Cancer Centre in Poznan with floor of the mouth tumors, including the impact on these levels tobacco smoking and alcohol drinking. Samples were collected between December 2012 – February 2013. After appropriate treatment, hair and nail samples underwent the process of digestion, and then metals were determined quantitatively using the methodxofxICP-MS. In the study of cadmium in floor of the mouth tumor there was a statistically significant difference between the concentrations in hair and nails. There was a statistically significant difference between lead concentrations in hair and nail samples in the floor of the mouth tumor. It is not clear, which alternative material – hair or nails, in proportion to serum and urine, is better in the evaluation of exposure to toxic metals in patients with floor of the mouth tumor. Further research is needed.

1. ATSDR: Division of Toxicology and Environmental Medicine ToxFAQsTM, 2007, http://www.atsdr.cdc.gov/tfacts13.pdf 2. ATSDR: Division of Toxicology and Environmental Medicine ToxFAQsTM, 2008, http://www.atsdr.cdc.gov/tfacts5.html A research project funded by the NCN 2011/03/N/NZ7/06266

120 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 36

Quantitative amino acids analysis in serum of beekeepers using liquid chromatography – tandem mass spectrometry

Jan Matysiak, Pawe Dereziski, Agnieszka Klupczyska, Zenon J. Kokot

Poznan University of Medical Sciences, Department of Inorganic and Analytical Chemistry, 6 Grunwaldzka Street, 60-780 Pozna, [email protected]

Allergy to Hymenoptera venom, including honeybee venom, is a serious problem in allergological practice. Stinging by insects of Hymenoptera order represents one of the main causes of anaphylaxis. Beekeepers and their family members are a group of people with high exposure to honeybee stings and with very high risk of allergy to bee venom. Therefore, they are a very appropriate population to study the relationship between clinical symptoms after the sting and results of diagnostic tests or changes in the profile of endogenous compounds, including amino acids. The aim of the study was to compare levels of 44 amino acids in serum of beekeepers directly after a bee sting and after at least six weeks since a last sting. The analysis were conducted using liquid chromatography – tandem mass spectrometry and aTRAQ® reagents.

Studies were performed using triple quadrupole mass spectrometer 4000 QTRAP (AB Sciex) coupled with LC instrument 1260 Infinity (Agilent Technologies). Amino acids were separated on an AB Sciex C18 (5 m, 4.6 mm x 150 mm) column by binary gradient of water (mobile phase A) and methanol (mobile phase B), both containing 0.1% formic acid and 0.01% heptafluorobutyric acids. The flow rate of mobile phase was maintained at 0.8 ml/min, separation temperature 50°C, injection volume 2 l. The analytes were detected in multiple reaction monitoring mode (MRM). Data acquisition and processing were controlled using Analyst 1.5 software (AB Sciex). For statistical analysis the Statistica 10.0 software was used. In all analyzes the level of (p) below 0.050 was taken as indicating statistical significance.

The study involved 27 beekeepers. Blood for the amino acids determination was collected within 3 hours after a bee sting and after a minimum of 6 weeks following the last bee sting. All the samples were stored in -80°C until the analysis. The results obtained were submitted to statistical analysis in order to characterize mutual relations between the levels of amino acids analyzed. Normal distribution of the analyzed data was confirmed by the Shapiro-Wilk test. For comparison of the results obtained directly after a bee sting and after a minimum of 6 weeks following the last bee sting, t-test was used. Statistical analysis showed significant differences between levels of some amino acids in analyzed groups of individuals; at least two amino acids showed differences in analyzed groups.

Applying methodology based on aTRAQ reagents, 44 amino acids can be determined in one run within 18 minutes. The simplicity of the method together with its good precision and accuracy give a new powerful tool in the diagnostic field.

121 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 38

Application of CN-TLC in studies of lipophilic and hydrophobic properties of phenanthrene derivatives

Krzesimir Ciura1, Joanna Nowakowska1, Piotr Pikul1, Wiktoria Struck2, Micha J. Markuszewski2, Wiesaw Sawicki1

1Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, e-mail: [email protected] 2Department of Biopharmaceutics, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland The derivatives of phenanthrene are a huge group of chemical compounds. Tested group of compounds included 8 hormones (transandrosterone, methyltestosterone, testosterone, progesterone, cortisone, hydrocortisone, estrone, ß-estradiol), 3 polycyclic aromatic hydrocarbons (1-methylophenanthrene, 1,3-dimethylophenanthrene, 3-methylcholanthrene) and 3 steroid drugs (methylprednisolone, prednisone, norgestrel). Lipophilicity is one of the important physico-chemical parameters determining the bioactivity of molecules. The most 0 frequently used experimental parameter of lipophilicity in TLC is retention parameter RM . The popular parameter of hydrophobic properties in TLC is C0. 0 C0= RM / S

0 RM - values of RM extrapolated to zero concentration of organic modifier, S – slope Linear relationship between retention parameters and lipophilicity presented the Collander equations. Most frequently, the Collander equation takes the form:

Log POW = aRM + b Relationships between chromatographic parameters and descriptors related to the molecular structure of the analytes describe Quantitative Structure-Retention Relationships (QSRRs). Stock solutions of this aforementioned phenanthrene derivatives (1 mg /ml) were prepared in methanol. Mobile phase was a mixture of acetone-water (30-80%, v/v), acetonitrile-water (50- 80%, v/v) and acetone-petroleum ether (0-80%, v/v). Stationary phase was silica gel 60 CN F254s TLC manufactured by Merck (Darmstadt, Germany). Chromatographic analyses were performed on 5 cm × 10 cm plates, in chromatographic chambers (7 x 11 cm) previously saturated with mobile-phase vapour. Chromatograms were developed to a distance of 8 cm at o 0 room temperature (20 + 2 C). The chromatographic date (RM , S, C0) have been used to set the Collander equation. Values of experimental log P were derived from ChemSpider database and calculated by the following softwares: ACD/labs, ChemAxon, ALOGPs, AC LogP, ALOGP, MLOG, KOWWIN, XLOGP 2, XLOGP3, and HyperChem. Correlations between chromatographic data and measures of lipophilicity of compounds are established based on QSRRs. Conclusions: 1. In case of acetone and petroleum ether mixture as mobile phase, there are no statistically significant correlation with the hydrophobic parameters S and C0 and log P.

2. The hydrophobic parameter C0 has relatively high correlation with log P. The relationship is independent of the mobile phase used, for the mixture of water-organic solvents.

122 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 39

Analysis of lipidomic fraction composition of grasshopper abdomen content using GC-MS

Magdalena Buszewska-Forajta, Joanna Raczak-Gutknecht, Danuta Siluk, Micha Markuszewski, Roman Kaliszan Medical University of Gdask, Department of Biopharmaceutics and Pharmacodynamics, Al. Gen. Hallera 107, 80-416 Gdask, Poland; [email protected]

Most drugs currently used in the treatment of several diseases took their origin from etnopharmacological observations. Folk tales, still popular in certain provinces, carry information about therapeutic properties of numerous substances. Research trends dedicated to the discovery of active substances present in biological material have commonly been acknowledged. Recently, several studies have been focused on insects due to presence in them of high levels of compounds with different biological activities. Biomass of insects contains mainly hydrocarbons, free fatty acids, triacylglicerols, alcohols, waxes, ethyl esters, proteins and sterols[1]. The particular interest paid to insects recently lays in identification of peptides with potential antifungal, antibacterial and myotropic activities[2]. According to etnopharmacological observations an ointment-like material squeezed out from abdomen of grasshoppers was used by villagers of the west central Poland to facilitate healing of wounds and scars. In the present work we focused on of identification of main components of a lipidomic fraction of grasshopper abdomen using GC-MS/MS. Samples were qualitatively analyzed with the use of gas chromatography coupled with mass spectrometry. To cope with the problem of interfering substances, both liquid-liquid extraction and solid-phase extraction (SPE) pretreatment methods were used to concentrate and fractionate compounds from insect matrix. Fractions were analyzed before and after derivatization. The derivatization process was carried out with the use of a mixture of N,O-bis (trimethylsilylo) trifluoroacetamide with trimethylsilyl (Sigma Aldrich, 60 min at 96 °C). Compounds were separated by using a temperature gradient on Phenomenex ZB-MS5 column (30 m x 0.25 mm x 25 μm film thickness). Helium was used as a carrier gas at pressure of 65 kPa. The temperature at the injector was set at 320 °C. The analysis was performed with the use of Shimadzu gas chromatography GCMS-TQ8030 system. The ion source and interface temperature were maintained at 220 °C and 250 °C, respectively. Fractions before derivatization were richer in compounds than derivatized fractions. In derivatized fractions about 350 compounds were determined, whereas non derivatized fractions comprised more than 570 compounds. The analysis of both types of samples enabled us to obtain a full screen of compounds present in the lipidomic fraction of grasshopper abdomen content.

1.Van Huis A., Insect as food in Sub-Saharan Africa, Insect Science Application, (2003), Vol.23, No.3, 163-185 2.Gobiowski M., Malinski E., Bogus M., Kumirska J., Stepnowski P. The cuticular fatty acids of Calliphora vicina, Dendrolimus pini and Galleria mellonella larvae and their role in resistance to fungal infection. Insect Biochemistry and Molecular Biology 38, 619-627 (2008).

123 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 40

Evaluation of a column classification system based on the KUL method using the separation of moclobemide and its two metabolites Alina Plenis, Natalia Mikus, Tomasz Bczek Department of Pharmaceutical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland [email protected]

Introduction: The popularity and commercial availability of reversed-phase liquid chromatographic (RPLC) stationary phases cause analysts to be often confronted with the problem of column selection. Therefore, general test methods to characterize RPLC columns have been extensively studied since the 1970s. Among them, interesting approach is chromatographic test procedure developed at the Katholieke Universiteit Leuven (KUL method) which allows to rank columns due to the values of four parameters and the calculation of specific F-values for a reference column [1]. Because the F-values is a function of four different contributions to column selectivity, all columns can be arranged in order to the relative selectivity. It is important to verify, whether the KUL test procedure gives reliable results by checking whether columns having similar parameters produce similar separations in pharmaceutical and biomedical practice [2-4]. Methods: The study focused on correlating the column classification based on the KUL method with the selectivity obtained for a real separation. The analysis of moclobemide and its two metabolites (Ro12-5637 and Ro12-8095) in human plasma was carried out according to the method described by Plenis et al. [5,6]. This separation was performed on 18 new RPLC stationary phases which had been previously characterized chromatographically. For deeper comparative analysis of KUL classification of the stationary RPLC brands and their column performance in pharmaceutical practice factor analysis (FA) has been used. Results: The obtained FA results confirmed that stationary phase classes closely related by KUL method gave comparable separation for moclobemide and its two metabolites. Conclusions: The column ranking system based on the evaluation of F-values can be considered as a helpful tool in the selection of a suitable column for pharmaceutical and biomedical analyses.

[1] P. Dehouck, D. Visky, Y. Vander Heyden, E. Adams, Z. Kovács, B. Noszál, D.L. Massart, J. Hoogmartens, J. Chromatogr. A 1025 (2004) 189-200. [2] A. Plenis, E. Balakowska, T. Bczek, The comparison of two column classification systems during the chromatographic analysis of steroids, J. Sep. Sci., 34 (2011) 3310-3321. [3] J. Szulfer, A. Plenis, T. Bczek, Evaluation of a column classification method using the separation of alfuzosin from its related substances, J. Chromatogr. A, 1229 (2012) 198-207. [4] J. Szulfer, A. Plenis, T. Bczek, Application of a column classification method in a selectivity study involving caffeine and its related impurities, Talanta, 99 (2012) 492-501. [5] A. Plenis, A. Chmielewska, L. Konieczna, H. Lamparczyk, A validated high-performance liquid chromatographic method for the determination of moclobemide and its two metabolites in human plasma and application to to pharmacokinetic studies, Biomed. Chromatogr., 21 (2007) 958-966 [6] A. Plenis, I. Oldzka, T. Bczek, Classification of the LC columns based on the QSRR method and selectivity towards moclobemide and its metabolites, (2013) J. Pharm. Biomed. Anal., in press.

124 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 41

The validation of the HPLC method in the determination of the solubility of telmisartan in crystalline and amorphous form

Kamil Wodarski, Przemysaw epek, Wiesaw Sawicki

Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, e-mail: [email protected]

The amorphization is one of methods improving the solubility of poorly soluble drug substances. This process, destroying the arranged crystalline structure of a drug, leads to the amorphous form, characterized by the chaotic arrangement of molecules in the solid state. This form frequently improves the physical properties, such as the solubility and the dissolution rate, while maintaining the chemical structure and therefore the pharmacological activity of a drug substance [1]. The aim of this study was to compare the solubility of amorphous and crystalline Telmisartan (TLM), an angiotensin II receptor antagonist used in the management of hypertension, and to validate the applied HPLC method. The amorphization of TLM was carried out using two independent processes: the vitrification and the cryogenic grinding. The existence of an amorphous form was confirmed by differential scanning calorimetry and X-ray diffraction analysis. Afterwards, a simple and selective HPLC method was developed and validated for the quantitative evaluation of the solubility of crystalline and amorphous forms of TLM in water, 0.1 M hydrochloric acid and phosphate buffer pH 6.8 (25 C and 37 C). The analysis was performed using Phenomenex C-18 column (250x4.6 mm, 5 m) in the gradient flow (1-15 min: solvent A 35% and solvent B 65%, 15-17 min: solvent A 95% and solvent B 5%; where solvent A was acetonitrile/methanol in the ratio 60/40 and solvent B was 0,1% acetic acid) with a flow rate of 1 ml/min. The run time was 17 min and the retention time of TLM was 12.26 min, detected at the wavelength 230 nm. The method has been validated on the basis of ICH Q2, Eur. Ph. and USP guidelines for the specificity, limit of detection and quantification, linearity, accuracy, precision, robustness and rudgeness [2]. Both amorphous forms of TLM revealed a higher solubility than their crystalline counterpart in all media at 25 C and 37 C (except the vitrified TLM in water at 37 C). The highest ninefold improvement of the solubility was observed in the case of vitrified TLM in phosphate buffer at 37 C. All parameters of validation, i.a. accuracy (RSD less than 2%, recovery between 95% and 105%), intermediate precision (RSD less than 5%) and linearity (R=0.998; the average balance of the residuals less than 10%) were found to be satisfactory. The results confirmed the improvement of the solubility of the amorphous form of TLM, obtained by two methods, in three different media in comparison with the crystalline form. This provides the basis for further studies on utilization of amorphous drug substances for the manufacture of modern solid dosage forms. The applied conditions in HPLC method turned out to be effective and reliable for the evaluation of the content of TLM in a sample.

[1] L. Yu: Amorphous pharmaceutical solids: preparation, characterization and stabilization, Advanced Drug Delivery Reviews, 48(1) (2001) 27-42. [2] P. epek, W. Sawicki, K. Wodarski, Z. Wojnarowska, M. Paluch, L. Guzik: Effect of amorphization method on telmisartan solubility and the tableting process, European Journal of Pharmaceutics and Biopharmaceutics, 83(1) (2013) 114-21.

125 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 42

An LC-MS metabolomics method for analysis of urine sample from patients with urogenital tract cancer

A. Yumba Mpanga1, W. Struck1, M. Buszewska-Forajta1, D. Szczsny1, M. Markuszewski2, R. Kaliszan1, M. J. Markuszewski1 1) Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdask, Al. Hallera 107, 80-416 Gdask, Poland 2) Department of Urology, Medical University of Gdask, Mariana Smoluchowskiego 17, 80-214 Gdask, Poland Email: [email protected]

Introduction: Metabolomics is a complex study of small molecules that represent the end point of biological processes in cells, tissues or biofluids such as blood or urine. One of the two approaches used in the metabolomics field is an untargeted fingerprint approach where all detectable metabolites found in the sample are analyzed in order to highlight the differences between two physiological states. In this study, an LC-MS metabolomics method was used to compare patients with urogenital tract cancer to healthy persons. Method: An LC-ESI-TOF/MS method was performed to analyze urine sample from 14 patients with urogenital tract cancer and 16 healthy persons constituting a control group. The analysis of urine was done using gradient elution consisted of 1 % formic acid in water and 1 % formic acid in methanol on a Zorbax Eclipse Plus C-18 RRHD (2.1 x 150 mm 1.8 μm) at 15 °C with a flow rate of 0.5 ml/min. Prior analysis urine samples were centrifuged, diluted and filtrated. Statistical methods such as U Mann Whitney, principal component analysis (PCA), partial least-squares discriminant analysis (PLS-DA) have been used to analyze the obtained data. Results: The statistically significant differences among eight metabolite levels in groups of cancer patients and healthy controls have been found. The PCA analysis showed a good separation between the two groups. Two dataset were analyzed by PLS-DA: dataset A for statistically significant metabolites present in all samples and dataset B for all statistically significant metabolites present in at least one of two groups. The PLS-DA presented a good sensitivity (75 % for both datasets) and a good specificity (80 % for dataset A and 100 % for dataset B). Conclusion: The combination of high performance liquid chromatography with TOF/MS detection is particularly attractive for analysis of multicomponent mixtures of biological samples such as urine. LC-TOF/MS analysis and advanced statistical methods can allow the identification of potential biomarkers based on the differences in metabolites levels.

126 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 43

Development and optimization of analytical method for quantification of stratum corneum lipids by LC-MS and FID techniques

S.Steinbach1, R.H.H. Neubert1 1Institute of Pharmacy, Martin Luther University Halle- Wittenberg Halle/ Saale, Germany, [email protected]

In order to understand the human skin barrier it is necessary to identify the role of free fatty acids (FFA) in the stratum corneum (SC). The aim of the present work is to develop a highly selective, precise and sensitive analytical method to quantify low concentrations of FFA in human SC samples. We focus our attention on odd numbered FFA, because it was found that CER[NP] subspecies and CER[EOS] have an odd number of carbon atoms in both chains of the molecule, one in the sphingosine or phytosphingosine base and the other one in the esterfied fatty acids[1]. Gas chromatography with mass spectrometric detection for qualification and flame ionization detection for quantification are the preferred methods to analyze saturated odd- numbered FFA with a chain length varying from 15 to 26 carbon atoms. The applied capillary column Optima FFAPPlus from Macherey Nagel (Düren, Germany) was found to be suitable for all types of FFA investigated here. Linoleic acid as monounsaturated fatty acid is also used as standard to better comprehend the entire human skin barrier. For a high volatility of the FFA during the GC measurement, we use trimethylsulfoniumhydroxide (TMSH, 2 mol, solved in ethanol, Macherey Nagel, Düren, Germany) as derivatization reagent. TMSH is easy to handle as the reaction proceeds at room temperature and the by-products (dimethylsulfide and methanol) do not superimpose the desired detection signals. Currently we achieve a good separation of the peaks. However there is a problem concerning the oxidation of linoleic acid during the storage time. Also we are testing different solvent mixtures like methanol, chloroform, hexane or tert-butyl methyl ether for the best solubility of the FFA. Moreover, we are using an alternative derivatization reagent to ensure complete chemical conversion of the FFA to fatty acid methyl esters (FAME). And we are examining the application of antioxidants for an improved storability of linoleic acid. Unfortunately, we have many by- products such as ceramides and cholesterol in our samples. They can reduce the robustness and repeatability. Consequently we have to develop and include a preseparation step such as solid-phase extraction. After dealing with the above-mentioned difficulties, appropriate HPLC experiments of the human skin samples are planned to analyze the ceramides and to identify the composition of odd numbered amid- bound fatty acids. As a result, we are able to present an overview about the odd numbered FFA and the amid-bound fatty acids of the ceramides in human SC.

[1] Hinder, A., et al. 2011. Investigation of the Molecular Structure of the Human Stratum Corneum Ceramides [NP] and [EOS] by Mass Spectrometry. Skin Pharmacology and Physiology. 24: 127-135

127 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 44

Stability-indicating HPLC method for the determination of cefpirome sulfate

Przemysaw Zalewski, Judyta Cielecka-Piontek, Paulina Marczak

Department of Pharmaceutical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6 60-780 Pozna Poland [email protected]

CH3 COO O O N N N H 3 H SO N 2 4 N S H N HH 2 O S Introduction: Cefpirome sulfate is a new, parenteral, fourth generation cephalosporin highly resistant to -lactamases. It has a has a broad spectrum of antibacterial activity against Gram- positive and Gram-negative bacteria, especially against S. aureus and S. epidermidis as well as P. aeruginosa.

Methods: An isocratic HPLC method with DAD detection was developed and validated for the determination of cefpirome sulfate. A LiChrospher RP 18 e column (125 mm × 4 mm, particle size 5 m, Merck, Germany) was used as the stationary phase. The mobile phase consisted of acetonitrile: 12 mM ammonium acetate (10:90 V/V) with a flow rate of 1.0 mL/min were used. The detection wavelength was 270 nm and temperature was 30oC. The method was validated with regard to selectivity, linearity, precision, limit of detection, limit of quantitation and robustness and did not require any preliminary treatment of samples.

Results: The validation of the HPLC method demonstrated its selectivity for cefpirome sulfate (tR = 6.6 min) in the presence of its degradation products (tR = 1.0 – 5.0 min). The method was linear (y = 40412 x; r = 0.9999) in the concentration range 40 μg/mL to 200 μg/mL, precise (RDS < 2%) and repeatable. The LOD was 3.63 g/mL and the LOQ was 11.01 g/mL.

Conclusions: The isocratic RP-HPLC method developed for the analysis of cefpirome sulfate is selective, precise, and accurate. The method is useful for routine analysis (kinetic studies) due to short run time. This method can be used for determining the stability of cefpirome sulfate in its pharmaceutical preparations.

128 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 45

Binding study of drug substances with 1-acid glycoprotein using high-performance liquid chromatography

Aleksandra Chmielewska1, Tomasz Bczek2

1,2Department of Pharmaceutical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, [email protected]; [email protected]

Essential for well-conducted pharmacotherapy and the development of new classes of drugs are: knowledge of the degree of drug/protein binding, possibility of determining the actual concentrations of drugs and their metabolites in plasma, furthermore the knowledge of the mechanisms of protein binding of xenobiotics. Among the many techniques used to study drug- protein binding advantageous for determining the parameters of binding appears to be high performance liquid chromatography [1-3]. The aim of this work was to study drug-protein binding in the chromatographic system with immobilized 1-acid glycoprotein (AGP). To the study was selected a set of standard therapeutic substances for which literature data exist concerning the degree of protein binding. Tested compounds were from the group of central nervous system (CNS) drugs and drugs of the cardiovascular system. Chromatographic analysis was performed on a chiral stationary phase Chiral-AGP (100 × 4.0 mm, 5 m) using spectrophotometric detection. The analytical wavelength () for each compound corresponds to the maximum absorbance. The study used two types of binary mobile phases: phase I 2-propanol - 10 mM phosphate buffer pH 7 (10:90, v/v) and phase II 2-propanol - 20 mM phosphate buffer pH 7 (6:94, v/v). Flow rate in both cases was 0.9 ml / min; temperature of analysis was 25 ° C. Obtained chromatographic data were allowed us to determine the binding values P (% protein binding) of the drug from 1-acid glycoprotein. Comparative analysis of values P obtained from chromatography and the data from literature was conducted within each group of compounds covered by the experiment. The correlation between the observed and predicted degree (%) drug binding to proteins was obtained for 20 compounds of the group of CNS drugs and for 13 compounds with a group of drugs of cardiovascular system. Higher R2 value was obtained for the chromatographic data based on mobile phase II.

[1] T. Bczek, R. Kaliszan: Quantitative structure / retention relationships in affinity chromatography, Journal of Biochemical and Biophysical Methods 49 (2001) 83–98. [2] A. Buciski , M. Wnuk, K. Goryski, A. Giza, J. Kochaczyk, A. Nowaczyk, T. Bczek, A. Nasal: Artificial neural networks analysis used to evaluate the molecular interactions between selected drugs and human 1-acid glycoprotein, Journal of Pharmaceutical and Biomedical Analysis 50 (2009) 591–596. [3] A. Chmielewska, T. Bczek, Comparative Analysis of Chiral Drugs in View of Chemometrics, Journal of AOAC International, 95 (3) (2012) 624-635.

129 7th Polish-German Symposium on Pharmaceutical Sciences Analysis: III - 35

Assessment of the level of selected toxic metals in patients with floor of the mouth cancer

Anna Woniak1, Maksymilian Kulza1, Monika Seczuk-Przybyowska1, Witold Szyfter2, Krzysztof Szyfter2,3, Wojciech Golusiski4 , Henryk Koroniak5, Ewa Florek1 1Laboratory of Environmental Research, Department of Toxicology, Poznan University of Medical Sciences, Poznan, Poland; [email protected] 2Department of Otolaryngology, Poznan University of Medical Sciences, Poznan, Poland 3Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland 4Head and Neck Surgery Department, Greater Poland Cancer Centre, Poznan, Poland 5Faculty of Chemistry, Adam Mickiewicz University Poznan, Poland

Tobacco smoking is considered to be the etiological factor of many diseases, including floor of the mouth cancers. Another significant stimulant affecting the formation of this disease is ethyl alcohol. It is believed that tobacco and alcohol together explained 75% of HNSCC (Head and Neck Squamous Cell Carcinoma). About 60 known carcinogens are present in tobacco smoke, 40 of them are carcinogenic for humans. It contains, inter alia heavy metals, such as chromium (VI), cobalt, cadmium and lead. Metals in cigarette smoke are a public health problem because of their potential toxicity and carcinogenicity. Cadmium is a metal, which damages the kidneys, lungs and bones. Eating food or drinking water containing very high amounts of cadmium severely irritates the stomach, leading to vomiting and diarrhea [2]. Lead can damage the nervous system, kidneys and reproductive system [1].The main target for lead toxicity is the nervous system, in adults and children. Cobalt toxicity involves many systems, organs and tissues in the human body, including respiratory system, nervous system, sense organs and skin. In experimental studies teratogenic and carcinogenic effect was observed. Chromium VI has proven allergenic effect, but also carcinogenic. The aim of this study was to determine the concentrations of these metals in hair and nail samples taken from 42 patients of Otolaryngology and Laryngological Oncology Clinic of Poznan University of Medical Sciences (SPSK No. 2) and The Head and Neck Surgery Ward of The Greater Poland Cancer Centre in Poznan with floor of the mouth tumors, including the impact on these levels tobacco smoking and alcohol drinking. Samples were collected between December 2012 – February 2013. After appropriate treatment, hair and nail samples underwent the process of digestion, and then metals were determined quantitatively using the method-of-ICP-MS. In the study of cadmium in floor of the mouth tumor there was a statistically significant difference between the concentrations in hair and nails. There was a statistically significant difference between lead concentrations in hair and nail samples in the floor of the mouth tumor. It is not clear, which alternative material – hair or nails, in proportion to serum and urine, is better in the evaluation of exposure to toxic metals in patients with floor of the mouth tumor. Further research is needed.

1. ATSDR: Division of Toxicology and Environmental Medicine ToxFAQsTM, 2007, http://www.atsdr.cdc.gov/tfacts13.pdf 2. ATSDR: Division of Toxicology and Environmental Medicine ToxFAQsTM, 2008, http://www.atsdr.cdc.gov/tfacts5.html A research project funded by the NCN 2011/03/N/NZ7/06266

130 IV – Pharmacognosy

131 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 1

Comparative assessment of extraction, acid hydrolysis and TLC procedure for quantitation of diosgenin in fenugreek seeds

Król-Kogus B.1, Migas P.1, Krauze-Baranowska M. 1

1Department of Pharmacognosy Medical University of GdaLsk; Al. Gen. J. Hallera 107, 80-416 GdaLsk, Poland; e-mail address: [email protected]

Semen Foenugraeci is classified in European Pharmacopoeia as a mucilaginous herbal drug. Nowadays, it is very often a constituent of dietary supplements with antidiabetic, nutritious, strengthening and anti-acne properties. Besides polysaccharides, fenugreek seeds contain a rich complex of steroidal saponins, of which the most important sapogenin is diosgenin [3] possesing anticancer [4], hypolipidemic [3], prebiotic [1], and antidiabetic [2] activity. The aim of the work was to establish the quantitative HPTLC-densitometric method for analysis of diosgenin in fenugreek seeds. The analysed plant material were the seeds of fenugreek of polish origin obtained from three different suppliers (Flos, Kruszynek, Kawon). For the assessment of the effectiveness of different procedures for sample preparation, several methods of extraction and acid hydrolysis conditions were tested. In TLC analysis of steroidal aglycones, different adsorbents and mobile phases were tested together with several derivatisation reagents, with assessments of their usefulness in both qualitative and quantitative analysis. As a result the HPTLC-densitometric quantitative method for determination of diosgenin after acid hydrolysis was established. Fenugreek seeds were extracted exhaustively in Soxhlet apparatus with three solvents (petroleum ether, chloroform, methanol). Methanol extract was hydrolysed (2M sulfuric acid; 2h, 80ºC) and extracted with chloroform (3 x 50mL). Pooled chloroform extracts were evaporated under vacuum and diluted to 10mL with chloroform. Analysis was performed on HPLTC silica gel F254 plates, using mobile phase n-heptan:ethyl acetate 7:3 (v/v). The densitometric quantification was carried out at (=590nm after the derivatisation with modified anisaldehyde reagent followed by heating (100˚C, 30 sec.). The determined content of diosgenin was similar and ranged between 0,12% (Kruszynek, Kawon) and 0,13% (Flos).

1. Huang CH, Cheng JY, Deng MC, Chou CH, Jan TR (2012) Prebiotic effect of diosgenin, an immunoactive steroidal sapogenin of the Chinese yam. Food Chemistry 132:428-432 2. Lu FR, Shen L, Qin Y, Gao L (2008) Clinical observation of Trigonella foenum-graecum saponin combining sulphanylureas on 36 cases of type 2 diabetes mellitus. Zhongguo Zhongyao Zazhi 33:184-187 3. Petit PR, Sauvaire YD, Hillaire-Buys DM, Leconte OM, Baissac YG, Ponsin GR, Ribes GR (1995) Steroid saponins from fenugreek seeds: Extraction, purification, and pharmacological investigation on feeding behavior and plasma cholesterol. Steroids 60:674-680 4. Srinivasan S, Koduru S, Kumar R, Venguswamy G, Kyprianou N, Damodaran C (2009) Diosgenin targets Akt-mediated prosurvival signaling in human breast cancer cells. International Journal of Cancer 125:961-967

132 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 2

Accumulation of free and bound phenolic acids in Aronia melanocarpa callus culture cultivated on the Linsmaier and Skoog medium variants

Agnieszka Szopa, Halina Ekiert

Chair and Department of Pharmaceutical Botany Jagiellonian University, Collegium Medicum Medyczna 9,30-688 Kraków, Poland email:[email protected], [email protected]

Introduction: Phenolic acids, derivatives of cinnamic and benzoic acid and depsides possess many therapeutically important properties, e.g. antioxidant, anticancer and immunostimulating activities Plant in vitro cultures can be a rich source of this group of metabolites. These compounds occur in plant cells free and bound as, for instance, glucosides or esters [1]. Our earlier studies demonstrated that callus cultures of Aronia melanocarpa cultivated on Linsmaier and Skoog (L-S) medium [2] variants could be a good source of free phenolic acids [3].

The aim of the present study was to analyze the contents of free and bound phenolic acids in biomass cultivated on the same L-S medium variants.

Methods: The callus culture was maintained on five L-S medium variants differing in the contents of plant growth regulators (PGRs): BAP (cytokinin) and NAA (auxin), in the concentration range from 0.1 to 3.0 mg/l. Methanolic extracts of calluses and fruits were analyzed after acid hydrolysis (2 M HCL, 2 x 2h) by RP-HPLC method [4] for the contents of eleven phenolic acids and cinnamic acid.

Results: All analyzed extracts were shown to contain five metabolites. Derivatives of benzoic acid: salicylic acid (SA), p-hydroxybenzoic acid (p-HBA) and syringic acid (SyA) were the main compounds in the callus extracts. The contents of individual compounds and their total content depended on the concentration of PGRs in the L-S medium. The total content ranged from 63,73 to 102.31 mg%, the content of SA from 9.22 to 20.52 mg%, p-HBA from 23.43 to 27.71 mg% and the content of SyA from 22.28 to 46.97 mg%. The highest total content was obtained on L-S medium variant containing 3 mg/l BAP and 1mg/l NAA. The total content of the analyzed compounds in fruit extract analyzed for comparison, was 90.60 mg%. The main metabolites in this extract were: p-hydroxybenzoic acid (p-HBA) – 51.55mg% and SA – 22.44 mg%.

Conclusions: The concentration of the tested PGRs in the L-S medium variants have a vital influence on the accumulation of phenolic acids. In order to obtain greater amounts of the investigated metabolites, other variants of the L-S medium should be tested. The cells of undifferentiating callus culture of A. melanocarpa can be a potential source of SyA, p-HBA and also SA.

1. H. Ekiert, F.Ch. Czygan: In: Biotechnology-Secondary Metabolites. Plants and Microbes, K.G. Ramawat, J.M. Merillon, ed., Science Publishers, Enfield, New Hampshire, USA (2007) 445-482. 2. E.M. Linsmaier, F. Skoog: Physiol. Plant, 18 (1965) 100-127. 3. A. Szopa, H. Ekiert: Plant Cell Tiss. & Organ Culture, (2012) DOI 10.1007/s11240-012-0272-0 4. S. Tian, K. Nakamura, T. Ciu, H. Kayahara: J. Chromatogr., 1063 (2005) 121-128.

133 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 3

Accumulation of free and bound phenolic acids in Aronia melanocarpa callus culture cultivated on the Murashige and Skoog medium variants

Agnieszka Szopa, Halina Ekiert

Chair and Department of Pharmaceutical Botany Jagiellonian University, Collegium Medicum Medyczna 9,30-688 Kraków, Poland email:[email protected], [email protected]

Introduction: Phenolic acids are a very attractive object of phytochemical, pharmacological and biotechnological studies due to very interesting and therapeutically important biological activities of this group of plant metabolites, including anticancer, antioxidant, immunostimulating activities. Phenolic acids occur in plant cells free and bound as, e.g. glucosides or esters [1]. Earlier we documented that undifferentiating callus culture of A. melanocarpa cultivated on Murashige and Skoog (M-S) [2] medium variants accumulated interesting amounts of some selected free phenolic acids [3]. Now, we decided to analyze the contents of free and bound phenolic acids in biomass cultured on the same M-S medium variants.

Methods: The callus culture was maintained on seven M-S medium variants differing in the contents of plant growth regulators (PGRs): BAP (cytokinin) and NAA (auxin), in the concentration range from 0.1 to 3.0 mg/l. Methanolic extracts of calluses and fruits were analyzed after acid hydrolisis (2 M HCL, 2 x 2h) by RP-HPLC method [4] for the contents of eleven phenolic acids and cinnamic acid.

Results: The callus extracts were confirmed to contain five phenolic acids. Derivatives of benzoic acid: salicylic acid (SA), p-hydroxybenzoic acid (p-HBA) and syringic acid (SyA) dominated quantitatively. The concentration of PGRs in the tested M-S medium variants had an important influence on the accumulation of the analyzed metabolites. The total content of phenolic acids differed from 80.36 to 96.55 mg%, while the contents of SA were from 10.50 to 31.80 mg%, p-HBA from 18.23 to 35.33 mg% and SyA from 14.26 to 40.93 mg%. The M-S medium variant containing 2 mg/l BAP, and 2 mg/l NAA was the best “productive” medium. The obtained maximum total contents of phenolic acids were higher than in fruits analyzed for comparison (90.60 mg%).

Conclusions: The concentration of the tested PGRs in the M-S medium variants is very important for the accumulation of the analyzed phenolic acids. The practical application of these results can be expected after subsequently optimization of PGRs contents in the M-S media. The cells of A. melanocarpa cultured in vitro can be a potential source of SyA, p-HBA and also SA.

1. H. Ekiert, F.Ch. Czygan: In: Biotechnology-Secondary Metabolites. Plants and Microbes, K.G. Ramawat, J.M. Merillon, ed., Science Publishers, Enfield, New Hampshire, USA (2007) 445-482. 2. T. Murashige, F. Skoog: Physiol. Plant., 15 (1962) 473-496. 3. A. Szopa, H. Ekiert: Plant Growth Regulators, (2012), under review. 4. S. Tian, K. Nakamura, T. Ciu, H. Kayahara: J. Chromatogr., 1063 (2005) 121-128.

134 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 4

Evaluation of commercial herbs of lemon balm by HPLC fingerprint and quantitation of phenolic acids

Agnieszka Arceusz1, Marek Wesolowski2 1Department of Analytical Chemistry, Medical University of Gdansk, Gen. J. Hallera 107, 80-416 Gdansk, Poland, [email protected] 2Department of Analytical Chemistry, Medical University of Gdansk, Gen. J. Hallera 107, 80-416 Gdansk, Poland, [email protected]

To evaluate the quality consistency of commercial medicinal herbs, a simple and reliable method of high-performance liquid chromatography (HPLC) with UV-Vis detector was developed, both for fingerprint analysis and quantitation of some pharmacologically active constituents (marker compounds). Melissa officinalis L. (lemon balm) was chosen for this study because it is widely used as an aromatic, culinary and medicine remedy. About fifty peaks were found in each chromatogram of a lemon balm extract, including twelve well-resolved characteristic peaks. A reference chromatographic fingerprint for the studied medicinal herb was calculated using Matlab 9.1 software as a result of analysing all the 19 lemon balm samples obtained from 12 Polish manufacturers. The analysis of similarity values revealed that all the samples were highly correlated with the reference fingerprint. Quantitation of selected phenolic acids in the studied herbal samples was also performed. The results have shown that the levels of phenolic acids: gallic, chlorogenic, syringic, caffeic, ferulic and rosmarinic were as follows (mg/g of dry weight): 0.001 – 0.067, 0.012 – 0.333, 0.007 – 0.553, 0.047 – 0.705, 0.006 – 1.589 and 0.158 – 48.608, respectively. Statistical analysis indicated that rosmarinic acid occurs in M. officinalis at the highest level, whereas gallic acid in the lowest. A detailed inspection of these data has also revealed that regardless of reference chromatographic fingerprints, monitoring of the lemon balm quality consistency should be supported by quantitation of principal pharmacologically active constituents of the plant.

135 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 5

Biotransformation of hydroquinone into arbutin in in vitro cultures of Aronia melanocarpa – preliminary results

Inga Kwiecie, Judyta Majcher, Halina Ekiert

Chair and Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, 9 Medyczna St., 30-688 Kraków, Poland, e-mail: [email protected]

The cells of many plant species cultured in vitro have been shown to transform the exogenous precursor, hydroquinone into its -D-glucoside, arbutin. The previous experiments from our laboratory with Ruta graveolens and R. graveolens ssp. divaricata in vitro cultures show that the obtained contents of this product (up to 13.6%), could be very interesting from practical point of view [1,2]. This compound is used for pharmaceutical (urinary tract disinfectant) and cosmetic purposes (skin whitener) [3]. The aim of this study was to confirm the ability of cells from Aronia melanocarpa in vitro culture to glucosylation of hydroquinone into arbutin.

The Aronia melanocarpa agitated shoot cultures were maintained in Murashige-Skoog medium [4], supplemented with 2 mg/l NAA and 2 mg/l BAP. Different concentrations of hydroquinone (96, 144,192, 288 and 384 mg/l of medium) were administered into the culture flasks in one, two or three portions. The products of the biotransformation were quantitatively determined in methanolic extracts from dry biomass and lyophilized media by an HPLC method [5].

Arbutin was the main product of biotransformation in the extracts from biomass. The product was present also in the media up to 28.09 mg per flask when the highest doses (288 and 384 mg/l) of hydroquinone were administrated. Portioning of the precursor doses had a significant impact on arbutin content and on the efficiency of biotransformation process, especially in the case of the highest hydroquinone concentration. The maximum product content in biomass - 60.7 mg/g d.w. (76.67 mg/g d.w. per flask) was obtained in the case of 384mg/l hydroquinone concentration, divided into 3 portions. The maximum yield of hydroquinone biotransformation was 57.42%.

After optimization of biotransformation process Aronia melanocarpa in vitro culture could be proposed as a potential rich source of arbutin.

[1] Piekoszewska A., Ekiert H., Zubek S. 2009. Acta Physiol. Plant. 32: 223-9. [2] Zubek S., Janas E., Ekiert H. 2009. 5th German-Polish Symposium “New challenges for pharmaceutical sciences”, Pozna, Abstracts, p.126. [3] Ekiert H., Kwiecie I., Szopa A., Muszyska B. 2012. Pol. J. Cosmetol. 15: 151-162. [4] Murashige T., Skoog F. 1962. Physiol. Plant 15: 473-97. [5] Štambergová A., Supiková M., Leifertová I. 1985. eskoslov Farm 34:179–182.

136 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 6

The arbutin production via biotransformation of hydroquinone in in vitro cultures of Schisandra chinensis – preliminary results

Inga Kwiecie, Judyta Majcher, Halina Ekiert

Chair and Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, 9 Medyczna St., 30-688 Kraków, Poland, e-mail: [email protected]

Arbutin, -D-glucoside of hydroquinone, is a well-known plant metabolite possessing both therapeutic and cosmetic values [1]. The previous research carried out in the Department of Pharmaceutical Botany JU CM revealed that in vitro cultures of Ruta graveolens and R. g. ssp. divaricata are a good source of this compound. The maximum content of this product, obtained so far is 7.8 % d.w. and 13.6 % respectively [2,3]. The aim of this study was to confirm the ability of Schisandra chinensis in vitro cultures established in our laboratory to perform glucosylation of exogenous hydroquinone into arbutin. Earlier such experiments with positive results were performed by Czech team [4].

Schisandra chinensis agitated shoot cultures were maintained in Murashige-Skoog liquid medium [5], supplemented with two variants of growth regulators 2 mg/l NAA + 2 mg/l BAP and 1 mg/ml NAA + 3 mg/l BAP. Different concentrations of the hydroquinone (96 - 384 mg/l of medium) were administered into the culture flasks in one, two or three portions. The products of the biotransformation were qualitatively determined in methanolic extracts from dry biomass and in lyophilized media by HPLC method [6].

Cells of Schisandra chinensis shoots cultures successfully transformed hydroquinone into arbutin. This compound was accumulated mostly in the biomass. Different concentration as well as portioning of the precursor have influence on the biotransformation reaction. In the case of medium supplemented with 2 mg/l NAA and BAP, the elevation of precursor dose caused 1.26- to 3.13-fold rise in arbutin amounts. The maximum arbutin content obtained in shoots was 28.14 mg/g d.w. (maximum tested concentration, 2 portions of precursor). In the case of the second medium variant (1 mg/ml NAA, 3 mg/l BAP) the elevation of precursor dose caused 1.72-, 3.56- and 1.04-fold rise in arbutin amounts. The maximum product content in the shoot amounted to 25.2 mg/g d.w. The maximum yield of hydroquinone biotransformation on the tested medium variants was 20.7% and 16.09%, respectively.

After optimization of biotransformation process Schisandra chinensis in vitro culture could be proposed as a potential rich source of arbutin.

[1] Ekiert H., Kwiecie I., Szopa A., Muszyska B. 2012. Pol. J. Cosmetol. 15: 151-162 [2] Piekoszewska A., Ekiert H., Zubek S. 2009. Acta Physiol. Plant. 32: 223-9. [3] Zubek S., Janas E., Ekiert H. 2009. 5th German-Polish Symposium “New challenges for pharmaceutical sciences”, Pozna, Abstracts, p.126. [4] Dušková J., Dušek J., Jahodár L. Poustka F. 2005. eska Slov Farm. 54;78-81 . [5] Murashige T., Skoog F. 1962. Physiol. Plant 15: 473-97. [6] Štambergová A., Supiková M., Leifertová I. 1985. eskoslov Farm 34:179–182.

137 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 7

Caryopteris incana in vitro root cultures for selective production of volatile compounds

Anna Dampc1*, Maria Luczkiewicz1, Adam Kokotkiewicz1, Zbigniew Jaremicz1 and Marek Mardarowicz2 1 Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Gdask, al. Gen. J. Hallera 107, 80-416 Gdansk, Poland 2 Department of Chemical Physics, Faculty of Chemistry, Maria Curie-Sklodowska University, sq. M. Curie-Sklodowska 3, 20-031 Lublin, Poland * e-mail address: [email protected]

Plant cell cultures are considered to be an interesting source of valuable secondary metabolites, which allows for the production of biologically active compounds in a controlled way, independent of climatic conditions and seasonality [1].

Caryopteris incana was chosen to establish excised root cultures with the aim of developing in vitro plant material rich in essential oil. The herb of this plant contains abundant volatile oil and polyphenolic fractions. Due to this property it is used in traditional Chinese medicine to make anti-inflammatory, anti-rheumatic and antiseptic preparations [2].

The initial phytochemical and biotechnological studies carried out at the Department of Pharmacognosy, Medical University of Gdansk revealed that not only the herb but also roots of C. incana contained a rich volatile fraction. Unlike to the herb, C. incana roots were characterised with a large content of sesquiterpenoids, among which was abietatriene, a compound of moderate anticancer activity [3]. In order to check the possibility of volatile oil production in in vitro conditions, excised root cultures derived from C. incana microshoots were established on liquid Schenk-Hildebrandt medium supplemented with 1 mg/L indole-3- butyric acid. The biosynthesis of volatile compounds was improved in comparison to the roots of the maternal plant. Chromatographic analysis of plant biomasses was carried out using gas chromatograph coupled with mass spectrometer and the resulting spectra were compared with the data from the library. The analysis revealed the differences between in vitro-derived roots and roots of the intact plant in terms of volatile compounds.

A prototype basket-bubble bioreactor constructed at the Department of Pharmacognosy, Medical University of Gdansk [4] was used to grow the roots and to upgrade the scale of the C. incana excised root cultures. With immobilized roots and a new aeration method, large -1 amounts of biomass were obtained (FWmax 1014.5 g l ) which produced three times more volatile compounds (3.44%) than the roots of the intact plant. The in vitro plant material included significant quantities of abietatriene and 1-octen-3-ol, a compound which is absent in the roots of the maternal plant.

[1] F. Bourgaud, A. Gravot, S. Milesi, E. Gontier: Production of plant secondary metabolites: a historical perspective, Plant Sci., 161 (2001) 839-851. [2] J.-J. Gao, K. Igalashi, M. Nukina: Radical scavenging activity of phenylpropanoid glycosides in Caryopteris incana. Biosci. Biotechnol. Biochem., 63 (1999) 983-988. [3] M. Luczkiewicz, W. Dembinska-Migas, P. Migas, W. Cisowski, A. Parchowska, R. Zarate, M. Mardarowicz: Volatile fraction from in vitro and in vivo biomass of several Caryopteris plants. Paper presented at the 35th International Symposium of Essential Oils, Messina, Italy, 29 Sept.-2 Oct. 2004. [4] M. Luczkiewicz, A. Kokotkiewicz: Co-cultures of shoots and hairy roots of Genista tinctoria L. for synthesis and biotransformation of large amounts of phytoestrogens, Plant Sci., 169 (2005) 862-871.

138 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 8

The isolation and identification of flavonoids from Phyllanthus multiflorus obtained in vitro biomasses

Barbara Sparzak, Mirosawa Krauze-Baranowska

Department of Pharmacognosy, Al. Gen. J. Hallera 107, e-mail; [email protected]

Genus Phyllanthus (leafflower) contains about 800 species, which are commonly used in traditional medicine of Asia and South American in the treatment of liver and genitourinary disorders. Plants from the genus Phyllanthus are rich source of secondary metabolites belonging to groups of lignans, securinega-type alkaloids, flavonoids, flavan-3-ol derivatives and sterols. However, chemical composition of many species remains unknown [1]. The aim of the present study was to isolate and identify two unknown compounds which were chromatographically revealed in Phyllanthus multiflorus biomasses obtained in vitro, next to previously recognized -sitosterol and flavan-3-ol derivatives [2]. As the plant material, the shoot culture harvested on SH0 medium and callus culture harvested on SH IAA 3.0 mg/L, 2,4-D 3.0 mg/L medium were used. Preliminary TLC and HPLC analysis showed the presence of compound 1 in methanol extracts from the shoot culture and compounds 1 and 2 in the extracts from callus culture. For isolation of compounds preparative chromatography methods were applied – preparative TLC, HPLC, column chromatography on Sephadex G-10 and SPE- Gigatubes. As a result, both compounds were obtained as amorphous powder. Preliminary NMR and MALDI-MS data revealed that investigated compounds might be derivatives of flavone – acacetin (5,7-dihydroxy-4-methoxyflavone) with sulphonate group in position C-7-OH or C-8 of a flavones skeleton. HPLC-DAD analysis showed that compounds 1 and 2 possess similar UV spectra with maxima of absorption at max 267, 341 and max 267, 343, respectively. In ESI and MALDI mass spectra of compound 1 an intensive signal of molecular ion at m/z 365 was present while in mass spectra of compound 2 the presence of molecular ion at m/z 351 was revealed, which may show that both compounds 1 and 2 differ with CH3- group (14 Da). Until now, the both compounds have not been described in the plant kingdom [3].

[1] J. B. Calixto et al. A review of the plants of the genus Phyllanthus: their chemistry, pharmacology, and therapeutic potential. Medicinal Research Review 18 (1998) 225 – 258. [2] B. Sparzak et al. High-Performance Thin Layer Chromatography Densitometric Determination of -sitosterol in Phyllanthus Species. Journal of AOAC International 92 (2009) 1343-1348. [3] J. B. Harborne, The Handbook of Natural Flavonoids Volume 1, (1999): John Wiley&Sons.

139 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 9

A new flavonoid - tricin glucuronide methyl ester of Axyris amaranthoides L. herb Paulina Znajdek – Awie1, ukasz Marczak2, Wiesawa Bylka1 1 Department of Pharmacognosy, Poznan University of Medical Sciences, 4 Swiecickiego St., 60-781 Poznan, Poland [email protected]

2 Institute of Bioorganic Chemistry PAS, 12/14 Noskowskiego St., 61-704 Poznan, Poland

Introduction A. amaranthoides L. is a herbaceous annual plant, commonly known as “Russian pigweed”. It comes originally from Asia but it is also naturally widespread all over the USA as well as in some European countries, e.g. in France. According to the literature the species has been the subject of only a few phytochemical studies so far. From the seeds of A. amaranthoides 1,20R-dihydroxyecdysone, 20-hydroxyecdysone and polypodine B were isolated [1]. In addition, phytochemical studies carried out on A. amaranthoides herb indicated that plant is a rich source of flavonoid compounds, mainly tricin acylated glucuronides [2]. The aim of this study was to identify individual compounds of flavonoid fractions.

Methods From the aqueous fraction of the methanol extract from the herb of A. amaranthoides L. almost one hundred subfractions were obtained using column chromatography (cellulose, Sephadex LH-20). After TLC control, the most proper fractions were then characterized. Phytochemical study was executed using high-performance liquid chromatography combined with high-resolution tandem (quadrupole-time of flight, QToF) mass spectrometry, which allows identification of the chemical constituents in the mixture.

Results The studied fractions of A. amaranthoides herb contained 6 different tricin glucuronides. Moreover, one of the identified compounds is a new structure, previously not described: tricin 7 – O – [2’-O-feruloyl-glucuronopyranosyl-(1-2)-O-glucuronide methyl ester].

Conclusion The results indicate that A. amaranthoides L. herb is a rich source of rare flavonoid tricin glucuronides. However, the attempts at isolation of individual compounds failed, therefore the constituents were characterized using HPLC- QToF – MS, previously demonstrated to be a suitable method for identification of acylated flavonoid glucuronides [3]. One of the identified compounds is a new tricin diglucuronide methyl ester.

References [1]Sarker SD, Sik V, Rees HH, Dinan L. 1, 20 R-Dihydroecdysone from Axyris amaranthoides. Phytochem. 1998;49 (8): 2305-10. [2]Bylka W. Acylated tricin glucuronides from Axyris amaaranthoides L. (Chenopodiaceae). 46th Annual Congress of the Society for Medicinal Plant Research, Vienna 1998. [3]Marczak , Stobiecki M, Jasiski M, Oleszek W, Kachlicki P. Fragmentation pathways of acylated flavonoid diglucuronides from leaves of Medicago truncatula, Phytochem Anal. 2010 May-Jun;21(3):224-33.

140 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 11

Development and validation of HPLC-DAD method to determination of cinarin and luteolin in artichoke leaf (Cynara scolymus L.) extract

Maksymilian Kulza1, Anna Woniak1, Anna Wachowiak1, Monika Seczuk-Przybyowska1, Gerard Nowak2, Ewa Florek1

1Laboratory of Environmental Research, Department of Toxicology, Poznan University of Medical Sciences, Poznan, Poland, e-mail: [email protected], 2Department of Medicinal and Cosmetic Natural Products, Poznan University of Medical Sciences, Poznan, Poland

One well-known for centuries for its beneficial properties of plant materials is the artichoke (Cynara scolymus L.). In addition to the widely known properties to facilitate digestion, used in all kinds of digestive disorders, preparations of artichoke with proven cholesterol lowering, hepatoprotective, antioxidant and hypoglycemic properties. With all these favorable profiles of responsibility is contained in the plant, the active compounds belong to two main groups: dikawoilchinone acids (cinarine) and flavonoids (luteolin). The aim of this study was to develop and validate the method of cynarin and luteolin, the main constituents of artichoke leaf and its determination in artichoke leaf (Cynara scolymus L.) extract. The compounds were separated using the high-performance liquid chromatography technique with diode array detection (HPLC-DAD). The HPLC separation was performed on C18 column (Waters Allsphere ODS-2 – 5μm/250 mm with Agilent Eclipse XDB-C18/12,5 mm precolumn) under gradient conditions using a mobile phase – 0,05% trifluoroacetic acid in water and methanol (the gradient condition was: in first 15 min – 10 to 90% of methanol linearity increase and next 5 minutes was return to 10% of methanol and next column condition after 6 minutes). The detector vawelenght was set at =330 nm. In this study we used Agilent Technologies 1200 SL chromatograph with vacuum degaser, autosampler, quaternary pump and column thermostat (20°C). The first stage of the study was to determine the optimal separation conditions of the both substances using HPLC. Tests were carried out using standards solutions of luteolin and cynarin in methanol (1mg/ml). For cynarin we give retention time 9,56. For luteolin 15,13 min. The validation was related to linearity, sensitivity (LOD and LOQ), accuracy and repeatability. In the validated method the linearity was achieved within concentration range 1,5625 – 50,0 μg/cm3 for the cynarin (R2=0,999) and 1,5625 – 200,0 μg/cm3 for the luteolin (R2=0,998). The limits of detection for cynarin and luteolin was: 0,75 μg/cm3 and 0,1 μg/cm3 and the limits of quatification: 2,25 μg/cm3 and 0,2 μg/cm3, respectively (determined based on signal/noise ratio). Coefficient of variation for the inter-day and the intra-day analysis, which is a precision and accuracy parameter, do not exceed 10 %. The practical application of this method was proved by analysis of artichoke leaf (Cynara scolymus L.) extract produced by Martin Bauer Group in Germany, in 2010, a 2,5% minimum content of caffeoylquinic acid. Weighed 200 mg of the extract and dissolved in 2 ml of methanol and the solution thus obtained was injected into the chromatographic column. Calculated concentrations of the both compounds was: cynarine - 4600 g/cm3, luteolin – 5,4 g/cm3. After converting our result in to 1g of the extract we obtained 23 mg/g of cinarine and 0,0252 mg/g luteolin in artichoke leaf (Cynara scolymus L.) extract. The presented method is suitable for the assessment of active substances in artichoke leaf (Cynara scolymus L.) extract.

141 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 12

The influence of some medium components on the accumulation of phenolic acids in Exacum affine Balf. f. in vitro culture

Ewa Skrzypczak-Pietraszek, Magdalena Bartman

Chair and Department of Pharmaceutical Botany Jagiellonian University Collegium Medicum Medyczna 9,30-688 Kraków, Poland. e-mail: [email protected]

Introduction Phenolic acids are an important group of plant secondary metabolites with different, valuable therapeutic properties (e.g. antiphlogistic, immunostimulating, antioxidant, antiseptic and cholagogic [1]). Besides plants growing in the open air, tissue cultures can be an alternative source of secondary metabolites. Yield of their accumulation in in vitro cultures can be increased by different methods, including culture medium supplementation with precursors and changing the standard amounts of the medium components. The purpose of this study was to investigate the influence of selected medium components (sucrose and phosphates) and addition of precursor (L-phenylalanine) on phenolic acids accumulation in agitated shoot cultures of Exacum affine Balf. f. Methods Cultures were maintained in conical flasks with Murashige and Skoog medium [2] supplemented with BAP (1 mg/l), NAA (0,5 mg/l) and GA3 (0,25 mg/l). After two weeks modifications of medium were prepared and added to cultures yielding nine groups. Variants of the medium varied in phosphate content (standard or half amount, or absence of phosphates) and sucrose content (standard, double or tripled amount). After another week, the precursor (L-phenylalanine) solutions were added, to give the final concentration of 1,6 and 3,2 g/l of medium. Plant materials were collected two days after the addition of L- phenylalanine. Control samples were collected too. Phenolic acids were assayed in the collected biomass before and after acid hydrolysis (2 M HC1). Qualitative and quantitative analysis of phenolic acids in methanolic extracts from biomass were conducted by an HPLC method [3]. Results Eight phenolic acids (protocatechuic, chlorogenic, p-hydroxybenzoic, caffeic, syringic, p-coumaric, ferulic, rosmarinic) and cinnamic acid were found in all samples before hydrolysis. After hydrolysis additionally the ninth phenolic acid – vanillic was observed. The total content of free phenolic acids increased from approximately 0,056% to 0,182% (3-times) and the total content of the whole phenolic acids (free and bound) – from 0,132% to 0,568% (about 4–times). The maximum content of total phenolic acids has been obtained on a medium containing a standard amount of phosphates, double amount of sucrose and the addition of precursor (L-phenylalanine) of 3,2 g/l of medium. Conclusions Analysis of the results in the described experiment showed that it is possible to increase the accumulation of phenolic acids in Exacum affine cultures – increasing sucrose concentration, reducing the amount of phosphate ions in the medium, and adding the precursor (L-phenylalanine).

[1] S. Khadem, R. Marles: Molecules, 15 (2010) 7985-8005. [2] T. Murashige, F. Skoog: Physiol. Plant, 15 (1962) 473-497. [3] M. Ellnain-Wojtaszek, G. Zgórka: J. Liq. Chrom. & Rel. Technol., 22 (1999) 1457-1471.

142 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 13

HPLC-MS Analysis of Polyphenols from Some Species of the Genus Populus

Loretta Pobocka-Olech, Daniel Gód, Jolanta Zarembska, Mirosawa Krauze-Baranowska

Department of Pharmacognosy with Medicinal Plants Garden , Medical University of Gdask, Gen. J. Hallera 107, 80-416 Gdask, Poland, [email protected]

Poplars (Populus) have been used for a long time as diuretic and anti-rheumatic herbal remedies [1,2]. Literature data [1,2] described leaves, buds and bark as medicinal plant material. According to the 6th Polish Pharmacopoeia monograph [3] a source of medicinal plant material from the genus Populus are leaves from the Black Poplar (Populus nigra) contained not less than 1,0% of flavonoids expressed as quercetin. Flavonoids and salicylic compounds are probably responsible for the pharmacological activity of Populus, especially anti-inflammatory [1,4]. Besides P. nigra, species P. alba (White Poplar) and P. tremula (Common Aspen) naturally grow in Poland, and both could be a source of plant material for pharmaceutical purposes. The chemical composition of these poplars are poorly recognized [1,2]. The aim of our work was to optimize the separation of salicin and its derivatives, procyanidin B1, catechin and flavonoids by HPLC-MS method to verify the presence of these compounds in different plant material originating from Populus. The developed chromatographic separation was carried out on a Discovery HS C-18 (7,5  2,1 mm, 3 μm) column under gradient elution with increasing concentration of methanol from 10% to 72% A in A+B (A – water/trifluoroacetic acid (99,9+0,1, v/v), B – acetonitrile/water/ trifluoroacetic acid (50+50+0,1, v/v/v); (tG 30 min).

[1] Z. Bach-Olszewska, A. Dugosz, B. Kowal-Gierczak, E. Lamer-Zarawska, J. Niedworok: Fitoterapia i leki rolinne. PZWL, Warszawa (2007) 385-386. [2] N. Bisset, M. Wichtl: Herbal Drugs and Phytopharmaceuticals. CRC, London (2001). [3] Farmakopea Polska VI PTFarm, Warszawa, (2002) 885-886. [4] S. Dudonné, P. Poupard, P. Coutière, M. Woillez, T. Richard, J.-M. Mérillon, X. Vitrac: Phenolic composition and antioxidant properties of poplar bud (Populus nigra) extract: Individual antioxidant contribution of phenolics and transcriptional effect on skin aging. J. Agric. Food Chem. 59 (2011) 4527-4536.

143 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 14

Analysis of chemical composition and antioxidant activity of Lonicera caerulea and Rubus ideaus

Marta Kula1, Daniel Gód1, Magdalena Majdan1, Adam Matkowski2, Mirosawa Krauze- Baranowska1

1Department of Pharmacognosy with Medicinal Plant Garden, Faculty of Pharmacy, Medical University of Gdask, Al. Gen. J. Hallera 107, 80-416 Gdask, Poland, [email protected]; 2Department of Pharmaceutical Biology and Botany, Medical University in Wroclaw, ul. Borowska 21, 50-556 Wrocaw, Poland.

Introduction: Due to the link between antioxidant potential of plant foods and their health promoting properties, much interest has been given to berries, which stand out as one of the best plant antioxidants [1]. The red raspberry (Rubus idaeus) and the blue-berried honeysuckle (Lonicera caerulea) are common examples of berries that are popular dietary ingredients and folk remedies in Central and Eastern Europe. Berry fruits and stems have a long tradition of being used in traditional medicine, for example in treatment of the common cold and flu-like infections [2]. The aim of the study was to evaluate the antioxidant potential of the fruit of L. caerulea and young shoots of R. idaeus in relation to their chemical composition. Methods: The phenolic composition of L.caerulea var. edulis ‘Wojtek’ and the shoots of R. idaeus ‘Heritage’ was evaluated using HPLC-DAD-ESI-MS analysis as well as two-dimensional “comprehensive” LCxLC. The free radical scavenging ability (FRS) of the extracts was tested using DPPH assay, and their reducing power using the phosphomolybdenum assay. Results: The L. caerulea extract contained several anthocyanins, the predominant one being cyanidin 3-glucoside, but also cyanidin 3,5-diglucoside, cyanidin 3-rutinoside, pelargonidin 3- glucoside, peonidin 3-glucoside, peonidin 3-rutinoside and peonidin 3,5-diglucoside. Additionally, procyanidin B1, catechin, epicatechin, chlorogenic acid, and neochlorogenic acid have also been identified in the fruit, with procyanidin B1 being reported for the first time. In the shoots of R. idaeus, an ellagitanin – sanguiin H6 and ellagic acid were found to be the predominant compounds besides several other phenolic acids and flavonoids detected. In the DPPH assasy EC50 was established after 30 min. at 109,5 μg/ml for L. caerulea fruits and at 19,4 μg/ml for R. idaeus shoots, while AAE in the phosphomolubdenum assay was 87,3 mg/g for L. caerulea and 427,9 mg/g for R. idaeus. Conclusions: The shoots of R. idaeus proved to be a much more effective antioxidant than the fruits of L. caerulea. It can suggest that ellagitannins and ellagic acid present in large quantities in the shoots of R. idaeus are responsible for their excellent antioxidant activity, as opposed by the anthocyanin rich fruits of L. caerulea.

[1] Heinonen et al.: Antioxidant activity and antimicrobial effect of berry phenolics-a Finnish perspective, Mol Nutr Food Res, 51 (2007) 684-91. [2] Samochowiec: Kompendium zioolecznictwa, Urban and Partner, Wrocaw (2002).

144 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 15

Estimation of cytotoxic activity of Cyclopia sp. and Paris quadrifolia L. extracts against human cell lines

Stefanowicz – Hajduk Justyna, Hering Anna, Bartoszewski Rafal, Ochocka J. Renata

Department of Biology and Pharmaceutical Botany, Medical University of Gdansk, Hallera 107, 80-416 Gdansk, Poland , e-mail: [email protected]

Although progress had been made in anticancer drugs development, cancer is still one of main causes of death worldwide. Thus, the search for novel cytotoxic agents continues to be important in the advancement of modern anticancer drugs. The use of plant-derived compounds as anticancer agents contributed to establishing of several important drugs currently used in chemotherapy. The structural diversity of plant compounds and their bioactivity potential, makes them very promising source of novel anticancer agents. Therefore, it is necessary to develop reliable and fast methods to analyze the cytotoxicity of plant extracts in in vitro human cell cultures.

This study objective was to investigate cytotoxicity of extracts from: Paris quadrifolia and Cyclopia sp. in HeLa and HaCaT cell lines, using the real time and label free Roche xCELLigence system. The system consist of the electrical impedance cell sensor array integrated into the bottom of cell culture microtiter plates (E-plates) which provides continuous, quantitative information about the biological status of attached cells. Impedance measurements are displayed as Cell Index (CI) values, that correspond to cell number and cell viability. Briefly, xCELLigence cell index impedance measurements were performed according to the instructions of the supplier. After seeding 20 000 cells per well of the E- plate, cell proliferation was monitored every 15 min for a period of up to 48 h by the xCELLigence system. For each extract, following proliferation real time analysis, we determined, base on dose response curves, the half maximal inhibitory concentration (IC50) as well as the time range of the cytotoxic activity. Vinblastine was used as a positive control. Our data showed that the xCELLigence system can be successfully used for dynamic and rapid monitoring of cellular viability and therefore is a suitable tool for testing toxicity of plant extracts against human cell lines.

145 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 16

Two-dimensional HPLC techniques in separation of C- glycosylflavones from Fenugreek seeds Król-Kogus B.1, Gód D.1, Krauze-Baranowska M. 1, Matawska I.2

1Department of Pharmacognosy Medical University of Gdask; Al. Gen. J. Hallera 107, 80-416 Gdask, Poland; e-mail address: [email protected]. 2 1Department of Pharmacognosy Medical University of Poznan; wicickiego 4; 60-781 Pozna, Poland

Fenugreek seeds are a rich source of polysaccharides, steroidal saponins [4] and flavonoids [10]. Among these compounds, the most important group are flavone C-glycosides, possessing anti-inflammatory [5,6], antioxidant [5], neuroprotective [3], anti-cancer [7,8,11], alfa-glucosidase inhibitory [2] and anti-depresant like [1] activity. The complex of C- glycosyloflavones in fenugreek seeds is complicated, including flavone mono- and di-C- glycosides, derivatives of both apigenin and luteolin as well as of their ester forms [9,10]. The composition of C-glycosyloflavones with a use of HPLC-DAD-ESIMS method was elucidated by Prati et al. [9] and Rayyan et al. [10]. However, a separation of all compounds was not achieved. The aim of our work was to establish the conditions for the analysis of C- glycosyloflavones by the use of different two-dimensional HPLC techniques in fenugreek seeds of polish origin. In two-dimensional systems different techniques were used: heart cutting off-line and comprehensive in both off-line and on-line mode. For all LCxLC analyses the Nucleodur Sphinx RP-18-Phenyl (100mm x 1mm x 5μm) column was used in the first dimension. In heart-cutting off-line mode 1D separations were performed with the use of gradient elution with increasing concentration of the mixture of methanol: water: trifluoroacetic acid (50:50:0.1, v/v/v) in 0.1% aqueous TFA from 40% to 100% in tG=25 min. The obtained nine first-dimensional fractions were subsequently analysed in the second dimension on Kinetex (100mm x 4.6mm x 2.6m) column. The mobile phase consisted of the mixture of acetonitrile:water:TFA (50:50:0.1; v/v/v) in 0.1% aqueous TFA. Each fraction was analysed under experimentally established isocratic elution. For 1D separations in off-line comprehensive mode different mobile phases composing of methanol or acetonitrile or tetrahydrofuran and water with the addition of 0.1% TFA were tested. For 2D analysis the Discovery HS C18 (75mm x 2.1mm x 3μm) column was chosen and the eluent was the mixture of acetonitrile:water:trifluoroaceticatic (50:50:0.1; v/v/v) in 0.1% aqueous TFA in different concentrations. The same columns as for off-line comprehensive analyses, were used in on-line comprehensive mode. The 1D eluent was the mixture of methanol:acetonitrile:water:trifluoroacetic acid (330:40:80:0.45, v/v/v) in 0.1% aqueous TFA at concentration increasing from 26% to 55% according to gradient program. Finally, 18 flavones were separated and identified in the methanol extract from fenugreek seeds. The best separation was achieved in heart-cutting mode. The presence of two new compounds in fenugreek seeds was revealed, namely: vicenin-3 and schaftoside. Furthermore, five unknown C-glycosides, probably derivatives of apigenin were revealed.

[1]. Can O, Demir Özkay U, Üçel UI (2012) Anti-depressant-like effect of vitexin in BALB/c mice and evidence for the involvement of monoaminergic mechanisms. European Journal of Pharmacology [2]. Choo CY, Sulong NY, Man F, Wong TW (2012) Vitexin and isovitexin from the Leaves of Ficus deltoidea with in-vivo -glucosidase inhibition. Journal of Ethnopharmacology 142:776-781

146 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 17

Phytochemical analysis and antimicrobial activity of Myosotis arvensis L.

Wiesawa Bylka1, Paulina Znajdek – Awie1, Jolanta Dugaszewska2

1 Department of Pharmacognosy, Poznan University of Medical Sciences, 4 Swiecickiego St., 60-781 Poznan, Poland [email protected]

2 Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, 4 Swiecickiego St., 60-781 Poznan, Poland

Introduction Myosotis arvensis, forget-me-not is one of the species of Boraginaceae family. The aerial parts of the plant is claimed to be useful in the treatment of malignant tumor of the oral cavity and sex organs and tuberculosis [1]. According to the literature, the species of genus Myosotis contain alkaloids, saponins and higher fatty acids [2]. However, detailed reports on the chemical constituents and biological activities of M. arvensis are scarce so far. Therefore, the aim of this study was phytochemical investigation as well as determination of antibacterial activity against Staphylococcus aureus strains.

Methods Air-dried herb of M.arvensis were twice extracted with methanol. The combined extracts were evaporated to dryness and partitioned between H2O, Et2O and AcOEt. Preliminary analysis were made using paper chromatography as well as TLC. AcOEt fraction was chromatographed by CC on celullose. The selected fractions were combined and then chromatographed on Sephadex LH-20, due to purification and separation individual compounds. In addition, antimicrobial activity of methanolic, AcOEt and aqueous extracts was tested against Staphylococcus aureus (ATCC 4163) strains using the method of serial dilutions.

Results Preliminary analysis using PC as well as TLC have confirmed the presence of at least three flavonoid compounds probably quercetin derivatives and large amount of phenolic acids, mainly protocatechuic acid. Among tested extracts, the AcOEt fraction presented the strongest antibacterial activity against S. aureus (MIC = 0.23 mg/ml, MBC = 0.47 mg/ml).

Conclusions The study indicate that M. arvensis herb is a source of polyphenolic compounds, mainly phenolic acids and flavonoids. Further studies on the chemical composition in progress. Preliminary studies on the antimicrobial activity suggest that compounds responsible for the strongest antibacterial effect are present in the AcOEt fraction.

References [1]Shinkarenko Yu.V., Vasil’ev V.G. Phenolcarboxylic acids from Myosotis krylovii and M. palustris, Chemistry of Natural Compounds. 44 ( 2008) 632-633. [2] Shinkarenko Yu.V. Content of flavonoids in plant species of genus Myosotis L., Chemistry of Sustainable Development 16 (2008) 593-598.

147 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 18

Total antioxidant capacity and total phenolic compounds content in herbs used as drugs and spices

Beata Ulewicz-Magulska1, Marek Wesolowski2 1Department of Analytical Chemistry, Medical University of Gdansk, Gen. J. Hallera 107, 80-416 Gdansk, Poland, [email protected] 2Department of Analytical Chemistry, Medical University of Gdansk, Gen. J. Hallera 107, 80-416 Gdansk, Poland, [email protected]

Plants that are rich alternative sources of natural antioxidants, can be complementary to the antioxidants produced by human organism. Various studies have shown that plants constitute rich source of antioxidants. Compounds with antioxidant properties are found in plants including vitamins A, E and C and phenolic compounds. The aim of the study was to evaluate the antioxidant activity and total phenolic compounds content in 66 samples of medicinal plant raw materials and spices. The content of phenolic compounds in methanolic and aqueous extracts was determined spectrophotometrically using Folin-Ciocalteu reagent. Total phenol content in plant extracts was quantified as gallic acid equivalent (mg GAE/g d.w.). The antioxidant activity was assessed by the DPPH (2,2’- diphenyl-1-picrylhydrazyl) radical scavenging method. The total phenolic compounds content in herbal drugs varied from 13.85 to 120.67 mg GAE/g d.w., whereas the corresponding value obtained for spices was 9.70-120.68 mg GAE/g d.w. Detailed inspection of these data shows that both plant species and morphological parts of plant containing various quantities of phenolic compounds. The total phenolic compounds in herbs and leaves extracts is higher than that in extracts from roots and fruits. Generally, the phenolic compounds level in water extracts is higher than that in methanol extracts of the same plant part. The DPPH radical scavenging activity varied from 2.04 to 50.36% and the mean values were 30.5 and 28.81% for herbal drugs and spices, respectively. Methanolic extracts were the most effective DPPH radical scavengers. The results have indicated a positive correlation between DPPH radical scavenging activity and phenolic compounds content in methanolic (r=0.869) and aqueous (r=0.949) extracts. The results have also shown that the plants, which had strong antioxidant activity, had high total phenolic compounds content as well. These were Melissa officinalis, Origanum vulgare, Rosmarinus officinalis.

148 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 19

Experimental model in the evaluation of hepatoprotective and hepatoregenerative action of Cynara Scolymus

Monika Seczuk-Przybyowska1, Maksymilian Kulza1, Anna Woniak1, Anna Wachowiak1, ,Gerard Nowak2, Ewa Florek1

1Laboratory of Environmental Research, Department of Toxicology, Poznan University of Medical Sciences, Poznan, Poland, e-mail: [email protected], 2Department of Medicinal and Cosmetic Natural Products, Poznan University of Medical Sciences, Poznan, Poland Artichokes have been used for centuries as a food, but until in the fourteenth century, were discovered for their prohealth properties. Permanently appeared in medicine in the eighteenth century the other hand, in the 30’ twentieth century was the first study conducted pharmacological and clinical experiments with extracts of artichoke and isolated from their constituents. Medicinal products containing extracts of artichoke traditionally recommended in disorders of the production and flow of bile in the gallbladder and inflammation of the bile ducts in liver failure and hypercholesterolemia. Artichoke extract have hepatoprotective properties by increasing blood flow through the liver, the activation energy reserves, increased dinucleus liver cells and RNA content in the hepatocytes, as well as enhancing the biosynthesis of proteins. It also causes the stimulation of cell division [1, 2]. In the present study we examined the protective and regenerative effects on the liver extract of artichoke leaves in an experimental model. In our experiment 165 adult rats were used - male and female Wistar Rats (body weight 350-500 g). The animals were divided into 3 groups: I – rats received artichoke leaf extract in different doses, II – rats received carbon tetrachloride (CCl4) to induce liver damage, III – rats received of two substances (artichoke leaf extract and CCl4), last group of animals was control group. For the evaluate of the hepatoprotective performance of an artichoke extract determined in plasma of animals following parameters: reduced glutatione, superoxide dismutase, Total protein, glutatione s-transferase, nitrites, lipid peroxidation marked as thiobarbituric acid reactive substances and asparagin aminotransferase (AST) and alanin aminotransferase (ALT) as main merker of liver damage. In this study has been shown a reduction in AST, ALT in animals after liver damage, compared with groups that received carbon tetrachloride after the artichoke (Cynara scolymus L.) leaf extract. In females with a liver damaged we observed reduction in ALT and AST activity compared to animals that received only carbon tetrachloride. Another observation was the reduction of superoxide dismutase activity after administration of the artichoke – Cynara scolymus L. – leaf extract to rats after liver injury in both sexes (antioxidatve properties of artichoke – Cynara scolymus L. – leaf extract) as well as the reduction of lipid peroxidation with increasing doses of the extract in the plasma of females with hepatocytes damaged.

[1] Speroni E., Cervellati R., Govoni P., Guizzardi S., Renzulli C., Guerra M. C.: Efficacy of different Cynara scolymus preparations on liver complaints. J. Ethnopharmacol., 2003, 86, 203-211. [2] Zapolska-Downar D., Zapolski-Downar A., Naruszewicz M., Siennicka A., Krasnodbska B., Koodziej B.: Protective properties of artichoke (Cynara scolymus) against oxidative stress induced in cultured endothelial cells and monocytes. Life Sci., 2002, 71, 2897-2908.

The project was funded by the 502-14-03315431-50520

149 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 20

Tropane alkaloid production in the bioreactor cultures of selected Solanaceae biomasses

Zbigniew Jaremicz1, Maria uczkiewicz1, Adam Kokotkiewicz1, Rafael Zàrate2

1 The Chair and Department of Pharmacognosy, Medical University of Gdask, al. gen. J. Hallera 107, 80-416 Gdask, Poland, [email protected]; 2 Instituto Universitario de Bio-Orgánica AG González, University of La Laguna, Avenida Francisco Sánchez 2, 38206 La Laguna, Tenerife, Spain

Tropane alkaloids such as scopolamine and hyoscyamine are widely used in medicine (i.e. in cardiology, ophthalmology and gastroenterology) due to their anticholinergic activity. Chemical synthesis of this compounds is very expensive because of their stereochemical structure, thus they are still obtained from plants cultivated under natural conditions which is associated with many drawbacks. Therefore, from many years, efforts have been made to developed plant in vitro system capable of producing high amounts of this valuable compounds [1]. In the presented studies Hyoscyamus niger and Atropa baetica hairy root cultures and adventitious roots of H. niger were cultivated in different bioreactor systems. In the experiments two types of bioreactors were used: bubble-column bioreactor and hybrid submerged-spray bioreactor. Aeration, medium mixing and immobilization systems were modified in order to achieve optimal biomasses growth and production of tropane alkaloids. Moreover, to increase culture productivities, elicitation with methyl jasmonate was introduced. MD-HPTLC with densitometric detection was used in quantitative and qualitative phytochemical analyses of biomasses obtained at all stages of the biotechnological experiments [2]. Modification in culture scale and cultivation conditions significantly influenced growth and metabolic profiles of all biomasses. The best production of hyoscyamine (8,88 mg/g DW) was achieved in A. baetica hairy roots cultivated in submerged-spray bioreactor, whereas the highest accumulation of scopolamine (5,16 mg/g DW) and cuscohygrine (25,94 mg/g DW) was obtained in H. niger transformed roots cultivated in bubble-column bioreactor. Application of methyl jasmonate substantially increased (2-fold) biosynthesis of scopolamine and cuscohygrine in H. niger hairy roots cultivated in submerged-spray bioreactor. It was proven that it is possible to grow A. baetica and H. niger in vitro roots in various bioreactor systems. Additionally, aforementioned cultures are capable of producing tropane alkaloids in quantities significantly exceeding the amounts present in respective intact plants. Therefore, the developed systems can be considered as a new, alternative source for production of these valuable compounds. [1] G. Grynkiewicz, M. Gadzikowska: Tropane alkaloids as medicinally useful natural products and their synthetic derivatives as new drugs, Pharmacology Reports, 60 (2008) 439-463. [2] Z. Jaremicz, M. uczkiewicz, M. Kisiel, R. Zàrate, P. Migas: Multi-development (MD) – HPTLC method for quantification of hyoscyamine, scopolamine and their biosynthetic intermediates in the selected Solanaceae plants grown in natural conditions and as in vitro cultures, 8th International Symposium on Chromatography of Natural Products "The application of analytical methods for the development of natural products" Lublin (Poland), May 17- 20, 2012.

150 7th Polish-German Symposium on Pharmaceutical Sciences Pharmacognosy: IV - 21

Accumulation of deoxyschizandrin and -schizandrin in shoot– differentiating and undifferentiating callus cultures of Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine)

Agnieszka Szopa, Halina Ekiert

Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, ul. Medyczna 9, 30-688 Kraków, Poland, e-mail: [email protected], [email protected]

Introduction: Schisandra chinensis (Turcz.) Baill. is a medicinal plant species which is known mostly for adaptogenic, hepatoprotective, antioxidant and anticarcinogenic actions. Biological activity of the plant is attributed mostly to dibenzocyclooctadien lignans [1]. In an earlier study in our laboratory, we analyzed S. chinensis in vitro cultures for their ability to accumulate two lignans, schizandrol A and B [2]. Encouraged by the proven biosynthetic potential of both cultures, in this study we undertook to examine the accumulation of two other major lignans – deoxyschizandrin and -schizandrin.

Materials and methods: Experimental shoot-differentiating callus cultures were maintained on six Murashige-Skoog (MS) medium [3] variants differing in the concentrations of plant growth regulators, BA and NAA (mg l-1): 0.1 and 2.0; 0.5 and 2.0; 2.0 and 0.5; 2.0 and 1.0; 2.0 and 2.0; 3.0 and 1.0. The experimental callus cultures were cultivated on two MS medium variants, containing 2 mg l-1 BA and 2 mg l-1 NAA, and 3.0 mg l-1 BA and 1.0 mg l-1 NAA. Both cultures (three series) were grown under constant artificial light at 25 ± 2 °C for four weeks. The plant material, harvested in Poland in 2010, was analyzed for comparison; it comprised the leaves and fruits of S. chinensis (Rogów Arboretum – Warsaw University of Life Science). In the methanolic extracts from lyophilized biomass, chromatographic quantification of two lignans: deoxyschizandrin and -schizandrin, was performed using the HPLC method developed by Zhang et al. [4].

Results: Deoxyschizandrin contents in extracts from the shoot-differentiating callus cultures were high and differed from 70.14 to 308.51 mg/100 g d.w. -Schizandrin contents were also diverse, ranging from 1.07 to 22.09 mg/100g d.w. The maximum deoxyschizandrin and - schizandrin contents were obtained on MS medium containing 3 mg/l BAP and 1 mg/l NAA and 2 mg/l BAP and 2 mg/l NAA, respectively. Extracts of the undifferentiating callus contained lower amounts of lignans, 18.75 and 1.03 mg/100 g d.w., respectively. The contents of the analyzed lignans in extracts of leaves and fruits from plants amounted to 41.01 mg/100 g d.w. and 60.72 mg/100g d.w. for deoxyschizandrin, and 22.27 mg/100 g d.w. and 66.50 mg/100g d.w. for -schizandrin, respectively.

Conclusions: In this context, deoxyschizandrin contents obtained in the shoot-differentiating callus culture should be considered to be substantial and interesting from a practical perspective.

[1] J.L. Hancke, R.A. Burgo, F. Ahumada: Schisandra chinensis (Turcz.) Baill. Fitoterapia, 70 (1999) 451-471. [2] A. Szopa, H. Ekiert: Lignans in Schisandra chinensis in vitro cultures. Pharmazie, 66 (2011) 633-634. [3] T. Murashige, F. Skoog: A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiol Plant, 15 (1962) 473-497. [4] H. Zhang, G. Zhang, Z. Zhu, L. Zhao, Y. Fei, J. Jing, Y. Chai: Determination of six lignans in Schisandra chinensis (Turcz.) Baill.. Fruits and related Chinese multiherb remedies by HPLC. Food Chem, 115 (2009) 735- 739.

151 V – Synthesis and Biological Activity

152 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 1

Novel derivatives of 2-(2-cyclohexylethyl)-1H-benzo[d]imidazole: synthesis, characterization and tuberculostatic activity

Katarzyna Gobis1, Henryk Foks1, Ewa Augustynowicz-Kope2, Agnieszka Napiórkowska2, and Andrzej Skadanowski3

1Department of Organic Chemistry, Medical University of Gdask, 107 Gen. Hallera Str. 80-416 Gdask, Poland, [email protected]; 2Department of Microbiology, Institute of Tuberculosis and Pulmonary Diseases, 26 Pocka Str. 01-138, Warsaw Poland; 3Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, 11/12 Narutowicza Str., 80-233 Gdask, Poland

Introduction: Previously we described a significant tuberculostatic activity of 2-phenylalkyl- and 2-cyclohexylalkylbenzimidazoles [1]. Then we reported synthesis and activity of 2-(2- cyclohexylethyl)-1H-benzo[d]imidazoles analogues with benzimidazole type of structure. Their tuberculostatic activity in vitro was at the level appropriate for the administrated chemotherapeutics [2]. These findings prompt us to extended our studies on the development of novel tuberculostatic agents. Here we disclosed the synthesis of novel 2-(2-cyclohexylethyl)-1H- benzo[d]imidazoles. We synthesized structures with different substituents at the benzene ring of benzimidazole system, including the methyl or nitro group, fluorine, chlorine or bromide atoms.

R1 N

N R2 H

1 2 R :Me,F,Cl,Br,NO2 ;R :H,Me,Cl Methods: Target compounds were obtained by the heating of 3-cyclohexylpropanoic acid with respective diamines in polyphosphoric acid (PPA). All the newly synthesized compounds were characterized by IR, 1H NMR, and 13C NMR spectra. They have been also tested for tuberculostatic activity against M. tuberculosis sensitive and resistant strains, as well as H37Rv standard strain. Their cytotoxic activity towards eukaryotic cells was also evaluated. Results: As a result of the synthesis ten novel derivatives of 2-(2-cyclohexylethyl)-1H- benzo[d]imidazole have been obtained. The compounds exhibited an excellent activity towards M. tuberculosis sensitive and resistant “wild” strains and the standard strain. Cytotoxicity studies indicated no toxicity of these compounds against eukaryotic cells. Conclusion: Obtained results suggest that the synthesized compounds are good candidates for tuberculosis drugs. These compounds have a very good therapeutic potential. In a further phase of the study we plan to test them in vivo in mice infected with tuberculosis strains. [1] H. Foks, D. Pancechowska-Ksepko, W. Kumierkiewicz, Z. Zwolska, E. Augustynowicz-Kope, M. Janowiec: Synthesis and Tuberculostatic Activity of New Benzimidazole Derivatives, Chem. Heterocycl. Compd., 42 (2006) 697–700. [2] K. Gobis, H. Foks, K. Bojanowski, E. Augustynowicz-Kope, A. Napiórkowska: Synthesis of novel 3- cyclohexylpropanoic acid-derived nitrogen heterocyclic compounds and their evaluation for tuberculostatic activity, Bioorg. Med. Chem. 20 (2012) 137-144.

153 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 2

New lipopeptide analogues as a perspective in therapy of bacterial and fungal infections

Anna Karafova, Sylwia Bartoszewska, Maciej Jaskiewicz, Wojciech Kamysz

Department of Inorganic Chemistry, Faculty of Pharmacy, Medical University of Gdansk, Gdansk, Poland, [email protected] Introduction: Native lipopeptides present a structurally diverse class of products of bacterial and fungal cultivation. Due to a wide spectrum of their antimicrobial and surfactant activity are useful in pharmaceutical and cosmetic industry, respectively. In recent years the increasing interest in this group of compounds is related to the problem with treatment of multi-drug resistant infections [1]. Many studies, focused on mechanism of action, showed that lipopeptides are capable of producing some modifications in cell walls. Moreover, lack of selectivity and high toxicity towards mammalian cells were confirmed [2].

Methods: The purpose of this study was to synthesize on polystyrene AM-RAM resin, using Fmoc/tBu chemistry a new group of synthetic lipopeptides preserving their amphiphilic character and to investigate an antimicrobial activity. In order to determine the cytotoxicity evaluation, the MTT assays were performed on human keratinocytes (HaCaT). Minimal inhibitory concentration (MIC) for four new linear synthetic lipopeptides composed from one fatty acid residue and two or three amino acids: Y-KK1, Y-KK2, Y- KK3, Y-KK4 (sequences not shown, intended to patent application) were determined on 10 reference strains. All MIC’s and the half maximal inhibitory concentration (IC50) values were compared to the model lipopeptide such as Pal-KK-NH2. This peptide is known for its high antimicrobial activity as well as extremely high cytotoxicity.

Results and Conclusions: The obtained results showed comparable antimicrobial activity of all synthetic compounds with model lipopeptide, while cytotoxic effect on HaCaT keratinocytes was lower for Y-KK analogues (IC50 = 18.1 ± 5.6) than for Pal-KK-NH2 (IC50 = 0.12 ± 0.03) in the ranged concentrations used in this study. These results suggest that those synthetic peptides could be promising in therapy of infectious diseases.

[1] J. M. Raaijmakers, I. De Bruijn, O. Nybroe, M. Ongena: Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactancts and antibiotics, FEMS Microbiol Rev., 34 (2010) 1037-62.

[2] Y. Shai, A. Makovitzky, D. Avrahami: Host defense peptides and lipopeptides: modes of action and potential candidates for the treatment of bacterial and fungal infections, Curr Protein Pept Sci. 7 (2006) 479-86.

154 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 3

Synthesis and characterization of novel iron(III) porphyrazines containing peripheral 2,3,5-substituted-pyrrol-1-yl and dimethyloamino groups

Koczorowski T.1, , Szczoko W.1, Dawidowska M.1, Sobotta L.2, Orzechowska A3, Burda K.3, Mielcarek J.2, Goliski T.1 1Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, Poznan; 2Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, Poznan; 3AGH University of Science and Technology in Krakow, Faculty of Physics and Applied Computer Science, al. Mickiewicza 30, Krakow; e-mail: [email protected]

Introduction: Porphyrazines (Pzs) constitute separate group of macrocycles related to phthalocyanines, which unlike porphyrins possess meso nitrogen atoms in the macrocyclic core [1]. Properties of Pzs can be modified by insertion of various metal ion entities or peripheral functionalization with N-, O-, S-, aryl, alkyl substituents [2]. Pzs have been investigated as potential photosensitizers for photodynamic therapy (PDT), building blocks for material chemistry, nanotechnology, and as components of optical devices, fuel cells [3]. Iron is oneof twelve metals with known physiological functions and plays a crucial role in variety of metabolic processes [4]. Nowadays, there are many iron chelators currently used in clinics, like deferoxamine and its derivatives [5]. Moreover, there are many macrocycles considered as potential chelators [6]. R Mg(BuO) N 2 TFA R: MgPz 2HPz N N Cl N N NC NH2 NC R R N Fe N R N N N N N N NC NH NC N N 2 N DMAE 2HPz N R 1 2 3 4 Results: The synthesis was based on Paal-Knorr reactions of commercially available diaminomaleonitrile with hexa-2,5-dione, 1-phenyl-1,4-pentanedione, 1,4-diphenyl-1,4- butanedione and 1,2,4-triphenyl-1,4-butanedione, respectively. Maleonitrile derivatives were further methylated and utilized in macrocyclization reactions. Two synthetic approaches were considered. In the first approach Linstead macrocyclization led to magnesium Pz, which was subsequently demetallated with TFA and remetallated with iron salt. In the second approach macrocyclization reaction with dimethylethanolamine led directly to a free base Pz, which was again remetallated with iron salt. Novel Pzs 1-4 were purified by column chromatography and characterized. Their purity was analyzed by HPLC. Further studies including Mössbauer spectroscopy and electrochemistry will be presented in the due course. Mössbauer spectroscopy is a very sensitive method to valence and spin state of a Mössbauer probe, in our case 57Fe, and to the arrangement and type of its ligands. It should determine the homogeneity of the iron binding sites and electron delocalization within Pzs.

This study was supported by the National Science Centre under Grant No. N N404 069440.

[1] Michel S.L.J., Hoffman B.M., Baum S.M., Barrett A.G.M. In Progress in Inorganic Chemistry; Karlin K.D., Ed.; J. Wiley & Sons: New York, 50 (2001) 473–590. [2] Rodriguez-Morgrade M.S., Stuzhin P.A., J. Porphyrins Phthalocyanines, 8 (2004) 1129-1165. [3] Allison R.R, Sibata C.H. Photodiagn. Photodyn. Ther. 7 (2010) 61-75. [4] Farina M., Silva Avila D., Teixeira da Rocha J.B., Aschner M., Neurochemistry International, (2013) in press [5] Liu Z.D., Hider R.C., Coordination Chemistry Reviews, 232 (2002) 151-171. [6] Liao M.-S., Watts J.D., Huang M.-J., J. Phys. Chem., 109 (2005) 7988-8000.

155 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 4

The Structures of Products of 2,5-Dinitroidazole with Thiomorpholine

Marcin Kowiel1, Andrzej K. Gzella1,2

1Department, Department of Organic Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Pozna, Poland, [email protected]; 2Faculty of Pharmacy, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, A. Jurasza 2, 85-089 Bydgoszcz, Poland;

The title compounds with potential anti-inflammatory activity were obtained in the reaction of 2,5-dinitroindazole 1 with thiomorpholine. The reaction of 1 with thiomorpholine led to several products 2-5. The structures of two compounds: 5-nitro-3-thiomorpholino-1H-indazole 2 and molecular complex of 3,5-dinitroindazole with thiomorpholine 3 were determined previously [1,2,3]. The present work deals with the crystallographic investigation on the two remaining products. Our investigations have shown that compound 4 exist as oxidized form of 5-nitro-3- thiomorpholino-1H-indazole and the second one as nitrosothiomorpholine 5. The results have indicated that the reaction of 2,5-dinitroindazole with thiomorpholine occurs via a cine-type nucleophilic substitution of N2-nitro group and a subsequent redox reaction of 5-nitro-3- thiomorpholino-indazole 2.

[1] A. Gzella and U. Wrzeciono: Azoles. 30. Structure of the molecular complex of 3,5-dinitroindazole with thiomorpholine, Acta Cryst, C47 (1991), 980-982 [2] A. Gzella, U. Wrzeciono and Z. ukaszewski: Molecular complex of 3,5-dinitroindazol and their electron affinity. Part 37: azoles, Pharmazie, 49 (1994) 319-322. [3] A. Gzella, U. Wrzeciono and W. Pöppel: Azole. 47. Über 3-Thiomorpholino- und 3-(4-Methylpiperazino)-5- nitroindazole, Acta Cryst, C57 (2001) 1189-1191

156 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 5

Methyl oleanolate derivatives as active cytotoxic agents

Barbara Bednarczyk-Cwynar1, Piotr Ruszkowski2, Lucjusz Zaprutko1, Teresa Bobkiewicz-Kozowska2

1Department of Organic Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka Str. No. 6, 60 – 780 Pozna, Poland, [email protected]; 2Department of Pharmacology, Faculty of Pharmacy, Poznan University of Medical Sciences, Rokietnicka Str. 5, 60–806 Poznan, Poland, [email protected]

Introduction: The cytotoxic properties of oleanolic acid are known for at least 30 years. First tests of this type were conducted for plant extracts in inhibiting of tumor-promoting effects by TPA [1]. As it is known from literature data, the derivatives of oleanolic acid that possesses double-bounded group at C-3 position belong to a class of the most active species. They exhibit cytotoxic activity towards cancer lines which were resist to many anticancer drugs [2–4]. Methods: Mother compound, oleanolic acid (see picture below) was transformed into derivatives within A or C ring as well as OH or COOH group. The compounds were obtained on the basis of multistep set of reactions with the use of reactions such as: transformation of COOH group into amide or methyl ester, acylation, hydrolysis, Jones or allylic oxidation, oxime formation, Beckmann rearrangement. The derivatives were COOH subjected to sulforhodamine B tests with the use of HeLa, KB, MCF-7 and Hep-G2 cell lines in order to evaluate their HO anticancer activity. Results: As a result 12 derivatives of oleanolic acid were obtained. The methods of the synhesis of the above derivatives and the optimization of reaction conditions were performed. The structures of the resulted compounds were elucidated on the basis of spectral data. IC50 values for the tested compounds were determined. Conclusions: The cytotoxic activity of semi-synthetic oleanane-type derivatives has been investigated. The overall results suggest that some of the received oleanolates can effectively inhibit the growth of HeLa, KB, MCF-7 and Hep-G2 cancer cell lines at microgram concentrations and could be promising new anticancer agents. The lactam derivatives of oleanolic acid with changed or unmodified carboxyl group turned to be more potent anticancer agents in comparison to mother compound – oleanolic acid.

[1] Y. Ito, S. Yanase, H. Tokuda et al.: Epstein-Barr virus activation by tung oil, extracts of Aleurites fordii and its diterpene ester 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate, Cancer Lett., 18 (1983) 87 – 95. [2] A. Paszel, B. Rubi, B. Bednarczyk – Cwynar et al.: The oleanolic acid derivative methyl 3,11-dioxoolean-12- en-28-oate targets multidrug resistance related to ABCB1, Pharmacol. Rep., 63 (2011) 1500 – 1517 [3] B. Bednarczyk–Cwynar, L. Zaprutko, P. Ruszkowski et al.: Anti-cancer effect of A-ring or/and C-ring modified oleanolic acid derivatives on KB, MCF-7 and HeLa cell lines, Org. Biomol. Chem., 10 (2012) 2201–2205. [4] D. Kaminskyy, B. Bednarczyk – Cwynar, O. Vasylenko et al.: Synthesis of new potential anticancer agents based on 4-azolidinone and oleanane scaffolds, Med. Chem. Res., 21 (2012) 3568 – 3580.

157 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 6

Hamacanthins as lead for the development of novel PDGFR kinase inhibitors

Eugen Johannes1, Joachim Schlosser1, Christian Peifer1*

1Institute of Pharmacy, University of Kiel, Gutenbergstraße 76, D-24118 Kiel, Germany, [email protected];*[email protected]

In this study we report on pyrazin-2(1H)-ones as lead for the development of potent ATP competitive protein kinase inhibitors with implications as anti-cancer drugs. Initially, we identified the pyrazin-2(1H)-one scaffold from Hamacanthins (marine sponge alkaloids)[1] by Molecular Modeling studies as core binding motif in the ATP pocket of receptor tyrosine kinases (RTK) which are validated drug targets for the treatment of various neoplastic diseases.[2] Structure-based design studies on a human RTK member PDGFR (plateled- derived growth factor receptor) suggested a straight forward lead optimization strategy. Accordingly, we focused on a Medicinal Chemistry project to develop pyrazin-2(1H)-ones as optimized PDGFR binders. In order to reveal Structure-Activity-Relationships (SAR), we established a flexible synthetic route via microwave mediated ring closure to asymmetric 3,5-substituted pyrazin-2(1H)-ones and produced a set of novel compounds.[3] Herein, we identified highly potent PDGFR binders with IC50 values in the nM range showing interesting properties for their further development as PDGFR-inhibitors.

[1] Zoraghi, R., et al.: Methicillin-resistant Staphylococcus aureus (MRSA) Pyruvate Kinase as a Target for Bis- indole Alkaloids with Antibacterial Activities. Journal of Biological Chemistry, 52 (2011) 44716-44725.

[2] Dar, A.C., Shokat, K.M.: The Evolution of Protein Kinase Inhibitors from Antagonists to Agonists of Cellular Signaling. Annu. Rev. Biochem., 80 (2011) 769–95.

[3] Miyake, F.Y., K. Yakushijin, and D.A. Horne: Synthesis of marine sponge bisindole alkaloids dihydrohamacanthins. Org Lett, 4 (2002) 941-943.

158 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 7

Interaction of novel synthetic chalcone derivatives with tubulin in vitro and in tumor cells

Marcin Serocki1, Micha Sabisz1, Anita Bulakowska2, Marek T. Konieczny3

and Andrzej Skladanowski1

1) Laboratory of Molecular and Cellular Pharmacology, Gdansk University of Technology, Gdansk, Poland, e-mail:[email protected]; 2) Department of Organic Chemistry, Medical University of Gdansk, Gdansk, Poland; 3) Department of Pharmaceutical Technology of Drugs, Medical University of Gdansk, Gdansk, Poland

Chalcone derivatives exhibit a broad range of biological activities, with antioxidant and cytotoxic properties among the most studied. Despite numerous reports on chalcones as cytotoxic, and potentially anticancer agents, very little is known on their actual mechanism of activity. We have recently developed several groups of novel chalcones with high cytotoxic activity toward different tumor cell types in vitro and bearing potent anticancer properties. The objective of this study was to identify molecular target(s) of these compounds and characterization of drug- target interactions that lead to tumor cell killing. Generally, chalcones are considered as antimitotic agents, which interfere with tubulin assembly [1], therefore, we first evaluated potential interaction of or compounds with purified tubulin in vitro by tubulin assembly assay. We found that these compounds inhibit tubulin polymerization in vitro at concentrations similar to vinblastine and combretastatin. Unexpectedly, relationship between chemical structure of studied chalcones and their ability to inhibit tubulin polymerization in vitro is unclear as similar structural features can be found in both biologically active and inactive chalcones. Inhibition of tubulin polymerization by selected chalcones was confirmed in living cells. Exposure of A549 cells to growth inhibitory concentrations of selected chalcones, including two potential lead compounds, at (0.5-1 μM) resulted in G2/M arrest. Arrest in mitosis induced by chalcones was further confirmed by immunofluorescence analysis of the mitotic epitope MPM-2 in drug-treated cells. The MPM-2 epitope is characteristic of mitotic cells and is present from prophase until anaphase. Quantitative analysis showed that the potential lead compound, chalcone AMG-332, induced accumulation of cells with MPM-2 epitopes and after 24h this fraction amounted to 72.4%. Interestingly, after prolonged treatment of tumor cells (A549) with studied chalcones we observed accumulation of dead cells with features of mitotic catastrophe. We also found that there is a reasonable correlation between the cytotoxic activity of the studied chalcones and their ability to inhibit tubulin polymerization with correlation coefficients are approx. 0.7-0.8 for two different sets of the cytotoxicity data (activity toward A549 and HeLa cells). Together, we provide experimental evidence that one of the cellular targets of new biologically active chalcones is tubulin, as shown both in in vitro studies as well as in tumor cells. [1] S. Ducki: Antimitotic Chalcones and Related Compounds as Inhibitors of Tubulin Assembly, Anti-Cancer Agents Med. Chem., 9 (2009) 336-347.

159 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 8

In vitro cytotoxic activity of (+)-usnic acid and (-)-usnic acid

Elbieta Studziska-Sroka1, Magorzata Kuciska2, Hanna Piotrowska2, Wiesawa Bylka1, Marek Murias2 Department of Pharmacognosy, Poznan University of Medical Sciences, 4 Swiecickiego St., 60- 781 Poznan [email protected] Department of Toxicology, Poznan University of Medical Sciences, 30, Dojazd Str., 60-631 Poznan

Introduction: Usnic acid is the optically active dibenzofuran derivative occurring abundantly in lichens especially in generas: Alectoria, Cladonia, Usnea, Lecanora, Ramalina and Evernia. Usnic acid is also one of the best known and extensively studied lichen secondary metabolite [1]. It has been demonstrated that the compound presents a broad spectrum of pharmacological activities including antibacterial, antiviral, antiprotozoal, antiinflamatory, analgesic and anticancer properties [1, 2, 3]. The aim of the present study was to evaluate cytotoxic activity of (+)- and (-)-usnic acid on six cancer (MDA-MB-231, T47D, MCF-7, LnCaP, HepG2, CaLu-1) and one non-tumorigenic (MCF-10A) human cell lines of different tissue origin.

Methods: The (-)-usnic acid was isolated from Cladonia uncialis (L.) Weber ex Wigg. (Cladoniaceae) collected in Lubnia, X 2009 and the (+)-usnic acid originated from the collection compounds of the Department of Pharmacognosy Poznan University of Medical Sciences. The chemical structure was confirmed by its 1H and 13C NMR data and optical rotation of both compounds was measured polarimetrically. The cytotoxic properties of (+)- and (-)-usnic acid was determined using MTT test [4]. Concentration–effect curves were fitted by a Hill equation and used for IC50 calculation, for this purpose GrapPad Prism software was used.

Results: The (+) and (-)-usnic acids were reported to have different activities against a variety of cancer cell lines [3]. The results obtained in our study indicated that the (+)-usnic acid is generally a compound of higher toxicity in cells compared to the (-) isomer (IC50 = 9.8, 35.1, 13.1, 6.7, 64.5, 15.7 μM, 69.3 (+)-usnic acid and 17.4, 57.1, 42.2, 9.4, 62.7, 37.3, 20.6 μM (-)- usnic acid for MDA-MB-231, T47D, MCF-7, LnCaP, HepG2, CaLu-1, and MCF-10A, respectively).

Conclusion: This study demonstrated that both isomers of usnic acids have cytotoxic activity. It should be stressed that in this study the most sensitive was MDA-MB-231 (estrogen independent, highly matastaic human breast epithelial cell line) followed by LnCaP (human prostate cancer cell line). The cytotoxic effect in LnCaP, CaLu-1, HepG2 (for both isomers of usnic acid) and MDA-MB-231 (for (-)-usnic acid), was not reported before in available literature.

[1] Ingólfsdóttir K. Usnic acid. Phytochemistry. 2002;61:729-36. [2] Cocchietto M, Skert N, Nimis PL, Sava G. A review on usnic acid, an interesting natural compound. Naturwissenschaften. 89 (2002) 137-46. [3] Galanty A, Koczurkiewicz P, Burakowska D, Janeczko Z. Aktywno biologiczna i farmakologiczna kwasu usninowego. Postpy Fitoterapii. 13 (2012) 162-72. [4] Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev. 11 (2005) 127–52.

160 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 9

Fragrant advantageous structures of jasmone like 1,3-heterocyclic ketones

Anna Paweczyk, Lucjusz Zaprutko

Department of Organic Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences Grunwaldzka 6, 60-780 Pozna, Poland, [email protected]

Introduction: Jasmone type compounds (Z-jasmone, E-jasmone, dihydrojasmone) are well known component of plant volatiles and are a very important substances determining the odor of jasmine flowers [1]. Many of jasmone like structure heterocyclic compounds were obtained [2]. The most of heterocyclic analogues of jasmone, especially those based on pyrrolidin-2-one, oxazolidin-2-one and thiazolidin-4-one system and their appriopriate thiocarbonyl derivatives are fine fragrant compounds both from the point of odor as well as odor durability [3].

Methods: Considering structure-odor relationship in this heterocyclic series it was observed that oxazolidinones are one of the most interesting fragrant compounds which are characterized by very intensive sweet odor with coconut, vanilla, aniseed and caramel note with definite floral note sometimes [3]. To confirm the favorable heteroatoms location in the five- membered cycles of these fragrant molecules it have been decided to prepare following 1,3- heterocyclic 2-ketones and 2-thioketones azole systems structurally similar to 4-methyl-2- oxazolidinone (1) which contain heteroatoms in both alpha positions relative to the carbonyl or thiocarbonyl group in the five-membered ring and prepared succeeding series of 4-methyl-2- thiazolidinone (2) and 4-methyl-2-imidazolidinone (3) analogues (Fig. 1). Heterocyclic system obtained were reacted remarkably fast with appropriate saturated or unsaturated alkyl bromides under microwave irradiation [4].

O(S) O(S) O(S)

R R R O N S N HN N

123 CH3 CH3 CH3 R = -n-pentyl, -2-Z-pentenyl, -2-E-pentenyl, -2-pentynyl;

Figure 1 Results and conclusions: Optimal microwave synthesis and fragrant properties of some 1,3-heterocyclic derivatives of natural fragrances were reported. Results obtained indicate that 1,3-heteroanalogues prepared are fragrant compounds and their odor properties were similar to those of the corresponding 1,3-heterocyclic moiety oxazolidinones.

[1] K. Bauer, D. Garbe, H. Surburg, Common Fragrance and Flavor Materials: Preparation, Properties and Uses, Wiley-VCH, Germany, 1997, 85–86, 200. [2] A. Kurek, L. Zaprutko: Jasmine-odor substances, Pol. J. Cosmet. 3 (2004) 140-153. [3] A.Paweczyk, L. Zaprutko, Flavour Fragr. J. 26 (2011) 101-106. [4] D. Bogdal, J. Pielichowski, K. Jaskot, Heterocycles 45 (1997) 715–722.

161 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 10

Identification of structural features of novel synthetic chalcone derivatives which are essential for their substrate specificity toward phase II drug metabolizing enzymes

Michal Sabisz1, Marcin Serocki1, Anita Bulakowska2, Marek T. Konieczny3

and Andrzej Skladanowski1

1) Laboratory of Molecular and Cellular Pharmacology, Gdansk University of Technology, Gdansk, Poland, e-mail:[email protected]; 2) Department of Organic Chemistry, Medical University of Gdansk, Gdansk, Poland; 3) Department of Pharmaceutical Technology of Drugs, Medical University of Gdansk, Gdansk, Poland

Chalcone derivatives exhibit a broad range of biological activities, with antioxidant and cytotoxic properties among the most studied. Despite numerous reports on chalcones as biologically active agents, very little is known on their metabolic transformations and bioavailability [1,2]. We have recently developed a new class of chalcones (see exemplary structure below) with high cytotoxic activity toward different tumor cell types in vitro and bearing potent anticancer properties. O OH S O O

O O The objective of this study was to determine whether new chalcone derivatives can be conjugated with glutathione, glucuronic acid and sulfate moieties that may lead to rapid elimination of these compounds from the organism, and low bioavailability. To this end, the in vitro phase II metabolism of selected chalcone derivatives was investigated. We showed that several biologically active chalcone derivatives are good substrates of GSTs and are efficiently conjugated with GSH, glucuronic acid as well as sulfate groups, as revealed by HPLC-DAD analysis. Interestingly, some chalcones can bind glutathione in a non-enzymatic process although with much lower efficiency. Together, efficient conjugation with GSH by GSTs required enone moiety, whereas for conjugation with both glucuronic acid and sulfate groups the presence of free hydroxyl group in chalcone molecule was essential. With this knowledge we will be able to design new compounds with improved pharmacological properties, including better bioavailability, and increased antitumor activity in vivo. [1] S. Das and J. P. N. Rosazza: Microbial and Enzymatic Transformations of Flavonoids, J. Nat. Prod., 69 (2006) 499-508. [2] E. Hijova: Bioavailability of Chalcones, Bratisl. Lek. Listy, 107 (2006) 80-84.

162 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 11

Experimental and Theoretical Investigations on Hydrogen Bonding in 1-(2- Phenylsulfonylisopropyl)benzotriazole

Marek K. Bernard, Jacek Kujawski, Andrzej Gzella, Krzysztof Lorenc

Department of Organic Chemistry, Pozna University of Medical Sciences, ul. Grunwaldzka 6, 60-780 Pozna, Poland; e-mail: [email protected]

The hydrogen bond is an important phenomenon in structural chemistry and biology. This electrostatic interaction is pivotal to the structure of proteins and nucleic acids and is also responsible for binding of many biological substances and drugs in the receptor active site.

The title compound 1 was investigated for its cytotoxic activity and, albeit it showed only moderate potency against several cell lines, we chose this compound for the studies due to its relatively simple structure and the presence of structural fragments that seemed to be responsible for the biological action in another series of compounds [1].

The X-ray diffraction has shown that the molecules of 1 are connected through weak hydrogen bonds involving the methyl group hydrogens as donors and the sulfonyl oxygens as acceptors into centrosymmetric dimers. The DFT calculations confirmed the existence of such weak hydrogen bonding. However, the theoretical calculations dealing with interactions between compound 1 and DNA bases or basic aminoacids (arginine) revealed that the hydrogen bonding does not involve the methyl groups as hydrogen donors. The sulfonyl oxygens, however, still may act as hydrogen acceptor for the NH group of arginine or DNA bases.

N N N 1

CH3 O2S CH3 Ph

[1] M. Skonieczna, J.Kujawski, M. K. Bernard unpublished data.

163 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 12

Synthesis of isomeric isopropoxynitroimidazoles

Justyna wawiak, Beata Janiszewska, Piotr Pikosz, Lucjusz Zaprutko

Department of Organic Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Pozna, Poland

mail: [email protected]

Introduction: Nitroimidazole derivatives show a wide spectrum of biological activity [1,2]. With the objective of discovering new pharmacologically interesting derivatives, we have studied the reactions of 3-isopropoxy-1-(2-methyl-4,5-dinitroimidazol-1-ilo)-2-propanol (1) and bicyclic 2- isopropoxy-methyl-7-nitroimidazo[5,1-b]-2,3-dihydrooxazole (2) with nucleophiles, such as: primary amines, secondary cyclic amines and -aminoacids in alcoholic solvents. Methods: Treatment of the substrate (1) with 2 equivalents of appropriate amine/-aminoacid in room/elevated temperature provided the products with amine/-aminoacid rest substituted at C-4 position of nitroimidazole ring (3). In contrast, reactions of bicyclic substrate 2 with mentioned nucleophiles gave mainly amino-products of substitution at C-5, after ring opening reaction (4).

1 2 3 4

Results and conclusions: As a result, two series of isomeric N-alkyl-4-amino(-aminoacid)-5- nitro- and N-alkyl-4-nitro-5- amino(-aminoacid)imidazole has been obtained. Analysis of PASS results revealed interesting potential biological activities of obtained compounds. Also, these products have favorable log P and PSA values. In conclusion, we have developed the synthetic method of obtaining 4- and 5-nitro- isomers, depending on the structure of substrate. In 4,5- dinitroimidazoles C(4) nitro group is privileged in the SN reaction because of the steric effect. On the other hand, NO2 group in bicyclic system (2) has strong withdrawing effect. It causes the deficit of electrones on the C-5 atom of the imidazole ring. This effect is heightened by the neighbourhood of electronegative oxygen atom in dihydrooxazole system. Hence, the C-5 atom of imidazole ring is particularly susceptible to nucleophilic attack.

[1] A. Mital: Synthetic Nitroimidazoles: Biological Activities and Mutagenicity Relationships., Sci. Pharm., 77 (2009) 497–520. [2] H. Sasaki et al.: Synthesis and Antituberculosis Activity of a Novel Series of Optically Active 6-Nitro-2,3- dihydroimidazo[2,1-b]oxazoles, J Med Chem 49(26) (2006) 7854-7860.

164 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 13

Influence of counterion on antimicrobial activity of amphiphilic lipopeptides

Katarzyna Greber1, Magorzata Dawgul2, Wojciech Kamysz2, Wiesaw Sawicki1

1Department of Physical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, e-mail: [email protected], 2Department of Inorganic Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland

Introduction. Solid phase peptide synthesis frequently requires trifluoroacetic acid (TFA) for detachement of peptide from the solid support. Additionally the TFA is used in low concentrations to improve peptides solubility and HPLC separation. Such procedure is resulting that synthetic product will contain acidic ion-pair reagent. The purpose of this study was to investigate antimicrobial activity of lipopeptides containing TFA counterion and compare it with compounds without acidic ion-pair. Methods. The lipopeptides were synthesized by the solid-phase procedure using 9- fluorenylmetoxycarbonyl (Fmoc) metodology and purified by high performance liquid chromatography (RP-HPLC) [1]. Removing of TFA counter-ions from lipopeptides was achieved by ion-pair extraction on VariPure IPE devices. To confirm the absence of acidic ion- pair the FT-IR spectra were recorded. The lipopeptides were subjected to microbiological tests: MIC (Minimum Inhibitory Concentration) and MBC/MFC (Minimum Bactericidal Concentration/ Minimum Fungicidal Concentration) on reference strains of Staphylococcus aureus ATCC 6538, Rhodococcus equi ATCC 6939, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Klebsiella pneumoniae ATCC 700603, Candida albicans ATCC 10231, according to the procedures outlined by the Clinical Laboratory Standards Institute. Results. The FT-IR spectra of the investigated lipopeptides confirm complete removal of trifluoroacetic acid counterion. Synthetic lipopeptides with net charge from +2 to +4 containing TFA counterion (Pal-KK-NH2, Pal-KKK-NH2, Pal- KKKK-NH2) were most active against Gram positive and less active against Gram negative bacteria. It was noticed that mentioned above lipopeptides without TFA counterion reveal slightly higher antimicrobial activity.

Conclusion. Our data show that trifluoroacetic acid counterion has no significant influence on antimicrobial activity of tested lipopeptides. It can be assumed that its presence does not affect on the mechanism of action of cationic lipopeptides.

[1] G.B. Fields, R.L. Noble, Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids, Int. J. Pept. Protein. Res., 35 (1990) 161–214.

165 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 14

Design, Synthesis and Biological Evaluation of 3,4-Diarylmaleimides as Vascular Endothelial Growth Factor Receptor (VEGF-R) Inhibitors

† Rebecca Horbert1 , Boris Pinchuk1,2, Janina Rahlff2, Sheraz Gul2, and Christian Peifer1*

1Institute of Pharmacy, University of Kiel, Gutenbergstraße 76, D-24118 Kiel, Germany, † [email protected], *[email protected]; 2European Screening Port GmbH, Schnackenburgallee 114, D-22525 Hamburg, Germany

Protein kinases (PKs) phosphorylate key proteins involved in signal transduction cascades. Overexpression and/or misregulation of PKs are closely related to many diseases such as cancerogenesis. The tyrosine kinases VEGF-Rs (vascular endothelial growth factor receptors) are relevant for the formation of new blood vessels and thus the blood supply of solid tumors. Blocking VEGF-R kinases as antiangiogenic concept illustrates why PKs are among the most promising targets in current drug discovery.

We report on the successful molecular design and synthesis of two novel 3,4-diarylmaleimides 1 and 2 as selective VEGF-R inhibitors [1]. The binding mode was investigated by molecular modeling studies which revealed the unique architecture of the hydrophobic pocket I of VEGF-R that determines selectivity [2]. 1 and 2 have shown good antiangiogenic effects in the chick embryo model in terms of inhibiting vessel growth [3]. PK assays showed a selective inhibition of VEGF-R 2 and 3 with an IC50 value in the nanomolar range for compound 1 [2]. Interestingly the corresponding carbazole 2 appears to be less active in the kinase assays but both compounds were found to be cytotoxic in the micromolar range for most tested cell lines. In addition 2 showed a high selectivity for RPMI-8226, a myeloma cell line. To investigate the site of action in detail we are currently applying fluorescence microscopy. Further examinations e.g. fluorescence-activated cell sorting (FACS) will follow.

[1] Peifer et al.: Design, Synthesis, and Biological Evaluation of 3,4-Diarylmaleimides as Angiogenesis Inhibitors, J. Med. Chem. 49 (2006), 1271–1281. [2] Peifer et al.: Profile and Molecular Modeling of 3-(Indole-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-dione as a Highly Selective VEGF-R2/3 Inhibitor, J. Med. Chem. 49 (2006), 7549–7553. [3] Peifer; Dannhardt: A novel quantitative chick embryo assay as an angiogenesis model using digital image analysis, Anticancer Res. 24 (2004), 3A, 1545-1552.

166 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 15

Synthesis and photochemical properties of novel porphyrazine bearing 2-adamantyl-5-phenylpyrrolyl substituents

Micha Kryjewski1, Ewa Tykarska2, Maria Gdaniec3, Tomasz Goliski2, Jadwiga Mielcarek1

1 Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780, Poznan, Poland, [email protected]; 2 Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780, Poznan, Poland 3Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland

Porphyrazines (pzs) are synthetic aza-analogues of naturally occurring porphyrins. Pzs are promising candidates for photodynamic therapy (PDT), which is a relatively novel treatment method of various diseases, especially tumors. PDT requires photosensitizer, which after irradiation with light of an appropriate wavelength generates reactive oxygen species (ROS), particularly singlet oxygen that leads to necrotic and/or apoptotic cell death. There is a constant interest in synthesis of novel photosensitizers.

OO O OO O N Br O NH2 O + O O + NH N 2

1 2 3 4 5

R N

N N N N R N N N N N Mg N R = N R N N N N NH N N 2 N

N R 6 7 8

Fig. 1 Alkylation reaction of 1 with bromoketone 2 gave 3 (Fig. 1). The next step in the synthetic procedure leading to 4 was hydrolysis and oxidative decarboxylation of 3 [1]. Compound 4 was subjected to the Paal-Knorr type reaction with diaminomaleonitrile 5, leading to derivative 6. Product 6 was subjected to methylation reaction to 7 and utilized in the Linstead macrocyclization towards magnesium porphyrazine 8. Novel macrocycle was subjected to photochemical studies, including singlet oxygen quantum yield measurements. This study was supported by the National Sciences Centre under grant No. N N404 069440. [1] D.C. Cole et al., J. Med. Chem. 49 (2006) 6158-6161.

167 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 16

Antistaphylococcal activity of amphibian peptides: citropin 1.1 and temporin A against isolates from patients with atopic dermatitis

Malgorzata Dawgul1, Wioletta Baranska-Rybak2, Maciej Jaskiewicz1, Anna Karafova1, Wojciech Kamysz1

1 Faculty of Pharmacy, Department of Inorganic Chemistry, Medical University of Gdansk, Gdansk, Poland, [email protected] 2Faculty of Medicine, Department of Dermatology, Venereology and Allergology, Medical University of Gdansk, Gdansk, Poland

Introduction. Skin colonization with Staphylococcus aureus (SA), which is confirmed in ca. 80% cases of patients with atopic dermatitis (AD), leads frequently to exacerbation of the basic disease. Suppressed levels of ceramides, superficial polar lipids and endogenous antimicrobial peptides (AMPs), as well as the pH shifted to alkaline region are responsible for SA skin colonization in AD. As they are essential part of innate immunity of the skin AMPs seem to be an interesting alternative to conventional antibiotics. Their strong antimicrobial activity against broad spectrum of microorganisms has been confirmed. Moreover, owing to the mechanism based on altering of the permeability properties of the microbial cell plasma membrane, the risk to develop bacterial resistance to AMPs is relatively low. [1, 2] Methods. SA strains were isolated from 12 patients with AD. The peptides were synthesized by the solid-phase method using Fmoc chemistry and purified by high performance liquid chromatography (RP-HPLC). Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentration (MBEC) were determined for peptides (citropin 1.1, temporin A) and conventional antistaphylococcal agents (erythromycin, mupirocin, fusidic acid, vancomycin). Afterwards 10 consecutive passages of tested strains in liquid medium supplemented with peptides and antibiotics (below their MIC values) were performed in order to evaluate the progressive reduction in susceptibility to the tested compounds. Results. We have identified strains resistant to the majority of conventional antibiotics. AMPs turned out to be effective against all the clinical isolates. After the passages of strains with peptides (0.5 MIC) no significant decrease of their activity was observed. The susceptibility of biofilm to erythromycin, fusidic acid and mupirocin was reduced by up to 1000 fold in comparison to planctonic cells, while for AMPs and vancomycin a slight decrease of activity was observed. Conclusion. Citropin 1.1 and temporin A turned out to be very promising antistaphylococcal agents as they were active against both living forms of clinical SA isolates. Obtained results encourage to continue the research on antimicrobial peptides and their potential application in therapy of staphylococcal infections.

[1] K. Yamasaki, R.L. Gallo: Antimicrobial peptides in human skin disease, Eur. J. Dermatol. 18 (2008) 11- 21. [2] R. Bunikowski, M.E.A. Mielke, H. Skarabis et al.: Evidence for a disease-promoting effect of Staphylococcus aureus – derived exotoxins in atopic dermatitis. J. Allergy Clin. Immunol. 105 (2000) 814-818.

168 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 17

Synthesis, determination of lipophilicity and validation of method for the determination of selected esters of 6-(4-MeOPh)-TACV

Monika Leniewska1, Izabela Muszalska1, Tomasz Ostrowski2, Joanna Zeidler2

1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Pozna, Poland, [email protected]; 2Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Pozna, Poland Introduction: Acyclovir (ACV) is a selective antiviral drug and has a low toxicity. It is active primarily against HSV-1, -2 and VZV viruses. It has a low bioavailability after oral administration, low infiltration by the blood-brain barrier and a fast metabolism and elimination from the body[1]. Therefore, new ACV derivatives with more favorable pharmacokinetic parameters are searched. The test compound – O 6-(4-MeOPh)-TACV (3,9-dihydro-3-[(2- N hydroxyethoxy)methyl]-6-(4-methoxy-phenyl)- N 9-oxo-5H-imidazo[1,2-a]purine) – is a tricyclic OCH3 derivative of aciclovir, which has slightly N N N RO O H higher activity in comparison to the parent substance[2]. Introduction of the ester group R=H 6-(4-MeOPh)-TACV in the structure of the compound can modify the R=CH -CO Ac-6-(4-MeOPh)-TACV physicochemical and pharmacokinetic 3 R=(CH3)2-CH-CO iBut-6-(4-MeOPh)-TACV parameters, so it was decided to investigate the R=(CH3)3-C-CO Piv-6-(4-MeOPh)-TACV effect of some ester groups on the lipophilicity. R=CH3-CH2-O-CO Et-6-(4-MeOPh)-TACV N Five esters of 6-(4-MeOPh)-TACV were R= CO Nic-6-(4-MeOPh)-TACV synthesized and their lipophilicity was investigated [acetyl (Ac), isobutyryl (iBut), pivaloyl (Piv), ethoxycarbonyl (Et), nicotinoyl (Nic)]. Methods and Results: Esterification was carried out using the appropriate acid anhydrides, acid chlorides, and 1,1’-carbonylimidazole/EtOH. The parent compound and the prepared esters were analyzed by methods: 1H-NMR, the elemental analysis, HPLC and measurement of the melting point, to confirm their identity and purity. Their lipophilicity was determined using HPLC (LiChrospher RP-18, 250x4 mm, 5 μm; mobile phase: acetonitrile 30-60%; flow rate 1,0 ml/min; UV detection: 262 nm). The logk0 values were found for test compounds based on the linear correlation of logk as a function of concentration of ACN in the mobile phase. The HPLC method for the determination of all compounds in the presence of degradation products was developed and validated. The parameters were: LiChrospher RP-18, 250x4 mm, 5 μm; mobile phase: phosphate buffer pH 6 – ACN 65:35; 1,0 ml/min; UV: 262 nm; i.s.: ethyl and methyl 4- hydroxybenzoates. Conclusions: On the basis of the obtained values of logk0 it was concluded, that the test compounds have different lipophilicity: 6-(4-MeOPh)-TACV < -Ac < -Nic < -Et < -iBut < -Piv. Developed HPLC method is useful for the determination of the test compounds in the presence of degradation products, it can be used in the tests of chemical stability and it is selective both in respect of the parent compound and acyclovir and impurities from the synthesis.

[1] C. Fletcher, B. Bean: Evaluation of oral acyclovir therapy, Drug Intell Clin Pharm, 19 (1985) 518-524. [2] B. Golankiewicz, T. Ostrowski: Tricyclic nucleoside analogues as antiherpes agents, Antiviral Res, 71 (2006) 134-140.

169 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 18

Synthesis of azaheterocyclic jasmolactone derivatives

Krystyna Majewska, Lucjusz Zaprutko, Agnieszka Jankowska

Department of Organic Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka Str. No. 6, 60 – 780 Pozna, Poland, [email protected]

Introduction: The fragrant compounds are great significance in daily life. Accordingly chosen give the impression of authenticity and specificity of products. The right fragrance increase the value of articles and support to their purchase. Because of these, the great demand for new, more excellent and cheaper odorous substances on many branches of industry and trade is observed. As a result of our investigation the syntheses of azaheterocyclic jasmolactone derivatives, that comprise ingredient of natural jasmine flower extract are presented.

Methods: Two (2, 3) of three heterocyclic structures were obtained by modification of basic method [1] with the use of dimethyl carbonate and N-methyl-1,3-propylenediamine or 1-hydroksypropylene-3-amine in mol ratio 1:1. First structure (1) was catalog reagent. In the second stage of our researches all of the above cyclic compounds were alkylated by alkyl bromide of different length of carbon chain (a, b). The microwave assistant organic synthesis (MAOS) technique was applied. The reactions were performed in a microwave reactor on solids, without solvents, using mixture of K2CO3 and KOH as a carrier and catalytic amount of TBAB as a phase transfer catalyst [2]. O O O

N R H3C NNR ONR

1, 1a, 1b 2, 2a, 2b 3, 3a, 3b

R= H

a CH2 CH3

b CH2 CH3 (CH2)8

Results: Six alkyl derivatives of azaheterocyclic ketones with different odors (fruity, herbal flavoures and pleasurable candle) were obtained. The structures of syntheshized products were confirmed on the basis of spectral analysis. Conclusions: Among different fruity scented derivatives, none of them was similar to characteristic jasmine and fruty- coconut note odor of jasmolactone model. However the obtained compounds can be applied in a future as a subtle fragrance additives in cosmetics.

[1] P. Alewod, M. Benn, R. Reinfried, Can. J. Chem., 1974, 52, 4083-4089 [2] D. Bogdal, J. Pielichowski, K. Jaskot, Heterocycles 1997, 45, 715-722

170 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 19

Methyl oleanolate derivatives as antitubercular agents

Barbara Bednarczyk-Cwynar1, Scott G. Franzblau2, Lucjusz Zaprutko1

1Department of Organic Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka Str. No. 6, 60 – 780 Pozna, Poland, [email protected]; 2Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago (UIC), 833 S. Wood St., Rm 412, M/C964, Chicago, IL 60612, USA, [email protected]

Introduction: As literature data shows, one third of the world’s population is infected with Mycobacterium tuberculosis [1]. The presence of new multi-drug resistant strains of M. tuberculosis increased the interest of scientists in natural products, also in triterpenes, in the hope of discovering new antitubercular drugs. Only sparse tests were conducted for oleanolic acid derivatives [e.g. 2,3]. Methods: Multistep set of reactions for mother compound, oleanolic acid (see picture below) was performed. This set of reactions consisted of: transformation of COOH group into amide or methyl ester, acylation, hydrolysis, Jones or allylic oxidation, oxime formation, Beckmann rearrangement. The derivatives were subjected to microplate Alamar Blue assay (MABA) and Low Oxygen Recovery Assay (LORA) tests with H37Rv strain COOH for the determination of anti-tubercular activity of the tested triterpenes. HO Results: As a result 14 derivatives of oleanolic acid with transformations within A or C ring as well as with 3-oxo or 3- hydroxyimino function were obtained. The methods of the synthesis of the above derivatives and the optimization of reaction conditions were performed. The structures of the resulted compounds were elucidated on the basis of spectral data. MIC values in MABA and LORA tests for the obtained compounds were determined. Conclusions: The anti-tubercular activity of semi-synthetic oleanane-type derivatives has been investigated. The obtained results suggest that some of the received oleanolates can effectively inhibit the growth of H37Rv strain at microgram concentrations and could be promising new anti-tubercular agents. Some of the oxo and hydroxyimino derivatives of oleanolic acid with changed or unmodified carboxyl group turned to be more potent anticancer agents in comparison to mother compound – oleanolic acid.

[1] A. García, V. Bocanegra-García, J.P. Palma-Nicolás et al.: Recent advances in antitubercular natural products, Eur. J. Med. Chem., 49 (2012) 1 – 23. [2] C.G. Caldwell, S.G. Franzblau, E.Suarez et al.: Oleanane Triterpenes from Junellia tridens, J. Nat. Prod. (2000) 63, 1611 – 1614. [3] J.Q. Gu, Y. Wang, S.G. Franzblau, G. Montenegro et al.: Constituents of Quinchamalium majus with Potential Antitubercular Activity, Z. Naturforsch. (2004) 59c, 797 – 802.

171 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 20

Evaluation of 2-adrenergic receptors contribution in hypotensive effect of new imidazoline derivatives: AK-71 and TCS-213 Joanna Kasprzyk1, Marta Kunczyska1, Magdalena Wróblewska1, Artur Lehmann1, Konrad Boblewski1, Anita Kornicka2, Jarosaw Sczewski3, Franciszek Sczewski2, Apolonia Rybczyska1 1Department of Pathophysiology, 2Department of Chemical Technology of Drugs, 3Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Gdask, [email protected] Hypertension is the third leading death cause worldwide. Central nervous system, via 2- adrenergic and I1-imidazoline receptors, plays significant role in pathogenesis of hypertension. Centrally acting compounds are clonidin and marsanidin [1, 2], both being imidazoline derivatives. Recently, we have proven that imidazoline derivatives TCS231 [3] and AK71 [2] possess hypotensive properties and that they decline heart rate in rats. The goal of the study was to assess if the hypotensive and heart rate decreasing effects of TCS213 and AK71 are mediated by 2 receptors. Wistar rats were anaesthetized intraperitoneally with thiopental (70 mg/kg body wt). The studied compounds were given in a single dose (100 g/kg body wt, i.v) , while 2 receptor antagonist (RX 821002) was administered i.v twice in doses of 5 and 10 g/kg body wt. Control rats received a corresponding volume of saline. Mean arterial pressure (MAP) and heart rate (HR) constant measurements were conducted directly in the carotid artery using BIOPAC Systems, Inc., Model MP100 equipment.

Obtained results are shown in the table below. Control TCS213 AK71 TCS213 AK71 TCS213 AK71 + RX 5 + RX 5 + RX 10 + RX 10 MAP -1,27±1,97 -56±5,55 -47±3,6 -52±3,8 -53±4,1 -32±3,5 -51±4,3 HR -8,64±2,74 -116±9,32 -147±5,3 -111±10,9 -139±12,9 -81±15,0 -144±7,9 Results from 4-6 experiments are shown as a mean ±SE. – difference between results measured before and 30 minutes after administration of studied compounds.

Our results suggest that the hypotensive and heart rate decreasing effects of TCS-213 are mediated by 2 receptors. However, involvement of 2 receptors could not be observed in case of AK-71. It seems likely that AK-71 acts via different mechanism, in which probably I1- imidazoline receptors may be involved.

[1] Bousquet P.: I1 receptors, cardiovascular function, and metabolism. Am J Hypertens. 2001, 14, 317S-321S. [2] Sczewski F, Kornicka A, Rybczyska A, Hudson AL, Miao SS, Gdaniec M, Boblewski K, Lehmann A.: 1- [(Imidazolidin-2-yl)imino]indazole. Highly alpha 2/I1 selective agonist: synthesis, X-ray structure, and biological activity. J Med Chem. 2008, 51(12):3599-3608. [3 ] Marta Kunczyska. Praca Magisterska 2012.

172 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 21

Amino Derivatives of 3-Tosylpyrazolo[5,4-c]indazole as Potential Serotonin Antagonists

Marek K. Bernard, Jacek Kujawski, Anna Myka, Beata Drabiska, Joanna Kruk, Joanna Adamus, Kornelia Czaja, Elbieta Jodowska, Remigiusz Klimaszewski, Anna Zacharzewska, Patryk Kamierczak

Department of Organic Chemistry, Pozna University of Medical Sciences, ul. Grunwaldzka 6, 60-780 Pozna, Poland; e-mail: [email protected]

Compound 1 represents one of the recently synthesized lead compounds that act as a 5-HT6 receptor ligands and may be useful for the treatment of schizophrenia and Alzheimer's disease [1].

Previously we obtained a series of compounds of the general formula 2. However, the preliminary tests showed that these compounds, devoid of the amino residue, were weak serotonin antagonists. Attempts to attach an amino residue via the Mannich reaction failed due to stereoelectronic reasons. We thus designed and synthesized several new compounds 3 containing a secondary amino moiety within their structure. The synthesis involved five steps, namely the vicarious nucleophilic substitution of hydrogen, hydrogenation of the nitro group, diazotization-cyclization of the resulted amino derivatives, N-nitration and cine-substitution of the N-nitro group by a secondary amine. The compound are currently undergoing biological screening.

N Ar N Ar H O2S O2S O2S N X HN N N N HN N N N H 1 H 2 H 3

[1] K. G. Liu, A. J. Robichaud, A. A. Greenfield, J. R. Lo, C. Grosanu, J. F. Mattes, Y. Cai, G. M. Zhang, J. Y. Zhang, D. M. Kowal, D. L. Smith, L. Di, E. H. Kerns, L. E. Schechter, T. A. Comery Bioorg. Med. Chem. 2011, 19, 650-662.

173 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 22

Metabolic stability study of novel arylpiperazine derivatives – potent antidepressive drugs

Mariusz Belka1, Szymon Ulenberg1, Weronika-Hewelt-Belka2,3, Marek Król4, Franciszek Herold4, Tomasz Bczek1

1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of Gdansk, Hallera 107 Str., 80-416 Gdansk, Poland; 2Department of Analytical Chemistry, Chemical Faculty, Gdansk University of Technology, Narutowicza 11/12 Str., 80-233 Gdask; Poland; 3Mass Spectrometry and Chromatography Laboratory, Pomeranian Science and Technology Park, Zwycistwa 96/98 Str., 81-451 Gdynia, Poland; 4Department of Drug Technology and Pharmaceutical Biotechnology, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1 Str., 02-097 Warszawa, Poland Within the ADME processes, metabolism certainly covers the largest, and still poorly understood aspect and consequently the most difficult to evaluate and to predict. Among available model enzymatic systems suitable for in vitro metabolism simulation, liver microsomes are most widely used for high throughput screening of newly synthesized drug candidates. Their main advantage is easy preparation procedure and a possibility of long-term storage, thus cost are dramatically reduced in comparison with cell- and animal-based assays. Moreover, microsomes are a rich source of all main metabolizing enzymes types, from different families (I phase – CYPs, FMOs; II phase – UGTs), depending on a used cofactor In this work metabolic stability of selected arylpiperazine derivatives (Fig.1) was measured with particular emphasis on elucidation of metabolites structures using LC-MS and LC-MS/MS techniques. Human and rat liver microsomes were chosen as a model enzymatic systems to provide an approximation of phase I metabolism. Chromatographic analyses were performed at different time points, till incubation end at 120 min. Poroshell EC120 C18 column (3 mm x 100 mm, 2.7 m) was used to obtain high selectivity and sufficient resolution for all of studied compounds.

R1

R2 O

NN N O N R3 Fig. 1 Chemical structure of studied compounds. Differences in metabolic stability between particular arylpiperazines are discussed in view of their chemical structure. Moreover, a MS/MS fragmentation study is presented leading to preliminary identification of possible metabolite types (C-hydroxylation, N-oxidation) and the site of biotransformation.

174 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 23 Synthesis and structure of novel imidazoline-containing derivatives and their copper (II) complexes with potential biological activities

ukasz Balewski1, Franciszek Sczewski1, Maria Gdaniec2, Patrick Bednarski3

1Department of Chemical Technology of Drugs, Medical University of Gdask, Hallera 107, Gdask, [email protected]; 2Faculty of Chemistry, A. Mickiewicz University, Pozna; 3Institut für Pharmazie der Ernst-Moritz-Arndt-Universität Greifswald, Germany

It is well known that copper, which is found in living organisms, is an essential co-factor in a number of enzymes and physiological processes [1]. Copper complexes have been investigated as potential antitumor [2] and antiinflammatory [3] agents or as superoxide dismutase (SOD) mimicking compounds [4]. In continuation of our previous research [5] devoted to the synthesis of novel biologically active substances bearing imidazoline moiety, we have elaborated a series of novel imidazoline derivatives 3 and their corresponding copper(II) complexes of type 4 and 5, according to the presented Scheme:

NNH R R=4-OCH3, R 4-CH2CH3, Cl 2 3 ,4-di-CH3, N N 4-OCH2CH3 + other products N N 4-OCH3,6-CH3, N 4-OCH2CH(CH3)2 O 1 4-OCH2C6H5 O NH 3

. . CuCl2 2H2O/DMF CuCl2 2H2O/DMF or EtOH

R R

Cl N N N N R=4-CH2CH3, 4 ,5-di-CH3, Cl Cu Cl NH Cu N 4-OCH2CH3 R=4-OCH3, N N 4-OCH ,6-CH, O 3 3 4-OCH2CH 3, NH Cl 4-OCH2CH(CH3)2 2 4 4-OCH2CH( CH3)2 NH 5 4-OCH2C6H5

The structure of novel compounds was confirmed by IR, NMR spectroscopic data and elementary analysis. The structure of corresponding Cu(II) complexes was confirmed also by single crystal X-ray analysis. In vitro antitumor activities of both the ligands 3 and copper (II) complexes 4 and 5 have been investigated at the Department of Medicinal Chemistry, University of Greifswald, Germany.

[1] H. Tapiero, D.M. Townsend, K.D. Tew: Trace elements in human physiology and pathology. Copper, Biomed. Pharmacother., 57 (2003) 386-398. [2] S. Adsule, V. Barve et.al.: Novel Schiff base copper complexes of quinoline- 2 carboxaldehyde as proteasome inhibitors in human prostate cancer cells, J. Med. Chem., 49 (2006) 7242-7246. [3] J.E. Weder, C.T. Dillon, T.W. Hambley et al.: Copper complexes of non-steroidal anti-inflammatory drugs: an opportunity yet to be realized. Coord. Chem. Rev., 232 (2002) 95-126. [4] I. Szilágyi, I. Labádi et.al.: Synthesis and IR spectroscopic characterization of immobilized superoxide dismutase (SOD) mimicking complexes, J. Mol. Structure, 744-747 (2005) 495-500. [5] F. Sczewski, E. Dzimidowicz-Borys, J.P. Bednarski, M. Gdaniec: Synthesis, crystal structure and biological activities of copper(II) complexes with chelating bidentate 2-substituted benzimidazole ligands, J. Inorg. Biochem., 100 (2006) 1389-1398.

175 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 24

In vitro studies of the safety of aripiprazole derivatives

1Beata Powronik, 2Pawe Zajdel, 2Maciej Pawowski, 3Krzysztof Marciniec, 1Elbieta Pkala

1Department of Pharmaceutical Biochemistry, [email protected], 2Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University, Medical College, 9Medyczna St., 30-688 Kraków; 3Department of Organic Chemistry, Medical University of Silesia, 4 Jagielloska St.,41-200 Sosnowiec

Introduction: Drug safety evaluation plays an important role in the early phase of drug development. One of the regulatory requirements for the new drug substances is assessment the mutagenicity potential. Actually, the results of mutagenicity tests decide whether a compound can be introduced into the clinical trials. Therefore, evaluation of mutagenic activity of the drug substances is extremely important. The aim of this work was evaluated the anty/mutagenicity new azinesulfonamide derivatives of the long-chain arylpiperazines which are potential psychotropic agents for the treatment of anxiety, depression, and/or schizophrenia [1].

Methods: Mutagenic potential of tested compounds was estimated using alternative to Ames test microbiological Vibrio harveyi assay [2]. In this assay, a series of genetically modified strains (named BB7, BB7M, BB7X and BB7XM) of marine bacterium V. harveyi is employed. The V. harveyi mutagenicity test consists of the detection of neomycin-resistant mutants on Petri dishes after incubation of bacterial cultures in a liquid medium, in the presence of tested compounds or in the presence of reference mutagen. For compounds without mutagenic potential also the antymutagenicity study will be performed (modified V. harveyi test). Modification is based on the incubation of the tested substances together with a reference mutagen in a culture medium.

Results: The experimental data were estimated by comparing the number of neomycin- resistant colonies mutants grown in the presence of tested compounds with the number of colonies formed on solid medium with the reference mutagen (NQNO). The experimental results showed that the new aripiprazole derivatives do not cause mutations in any strains of V. harveyi. This means that they can be qualified into further stages of pre- and clinical studies. Results from the antimutagenicity studies demonstrated that in the case of BB7 and BB7XM, molecule PZ508 exhibited strong antimutagenic effect and may be potential chemopreventive agent.

Conclusions: V. harveyi assay is an excellent alternative to traditional methods of mutagenicity and genotoxicity studies. Carrying out the tests estimating of mutagenicity after the preliminary pharmacological screening tests allow rationally plan further investigation for potential drugs. The modified version allows to estimate the beneficial antimutagenic activity of the test compounds.

[1] P. Zajdel , K. Marciniec, A. Malankiewicz, G. Sataa, B. Duszyska, A.J. Bojarski, A. Partyka, M. Jastrzbska-Wisek, D. Wróbel, A. Wesoowska, M. Pawowski: Quinoline- and isoquinoline- sulfonamide derivatives of LCAP as potent CNS multi-receptor-5-HT1A/5-HT2A/5-HT7 and D2/D3/D4-agents: the synthesis and pharmacological evaluation. Bioorg Med Chem. 2012, 15;20(4):1545-56

[2] B. Podgórska, E. Ch, K. Ulanowska, G. Wgrzyn: Optimisation of the microbiological mutagenicity assay based on genetically modified Vibrio harveyi strains, Journal of applied Genetics 2005, 46(2) 241-246

176 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 26

3,4-Diaryl-isoxazole and -imidazole Derivatives as Potent and Selective Inhibitors of Casein Kinase 1

Jakob Halekotte1*, Andreas Luxemburger2, Joachim Bischof3, Uwe Knippschild3, and Christian Peifer1†

1Institute of Pharmacy, University of Kiel, Gutenbergstraße 76, 24118 Kiel, Germany;*[email protected]; †[email protected] 2Callaghan Innovation Research Ltd., 69 Gracefield Road, Lower Hutt 6009, New Zealand 3Department of General, Visceral and Transplantation Surgery, University of Ulm, Steinhoevelstraße 9, 89075 Ulm, Germany

Isoform of casein kinase family 1 (CK1) phosphorylates serine and threonine residues of proteins that act as key mediators in a vast range of signaling transduction cascades. Their overexpression and misregulation are closely related to different diseases such as tumorigenesis, neurodegenerative events and inflammatory disorders [1,2]. Especially CK isoforms and show a high degree of similarity and it remains unclear whether CK1/ possesses a predominant role in diverse cellular processes [3]. Hence, isoform selective inhibitors are urgently needed to investigate the exact pharmacological importance for different medical or therapeutic applications.

1 2

We report on the identification and synthesis of 3,4-diaryl-isoxazole 1 and –imidazole 2 derivatives as potent dual inhibitors of CK1 and p38 with IC50 values in the low micromolar range [4]. Further optimization performing molecular modeling strategies yielded isoxazole compounds that show a distinct increase of potency and CK1 selectivity in cell based assays. Regarding the imidazole series, our modeling approaches suggest optimized analogues adapted from scaffold 2 to be even more potent and selective for CK1.

[1] Knippschild U; Gocht A, Wolff S, Huber N, Löhler J, Stöter M. The casein kinase 1 family: participation in multiple cellular processes in eukaryotes. Cellular Signaling, 17 (2005), 675-689 [2] Aud DM, Peng SL. Methods of treating inflammatory diseases with selective inhibitors of the casein kinase 1 isoforms. [F. Hoffmann-La Roche A.-G.]. Patent WO.2007-EP63339[2008071605] 2007 [3] Fish KJ, Cegielska A, Getman ME, Landes GM, and Virshup DM. Isolation and Characterization of Human Casein Kinase I (CKI), a Novel Member of the CKI Gene Family. J. Biol. Chem., 270 (1995), 14875-14883. [4] Peifer C, Abdaleh M, Bischof J, Hauser D, Schattel V, Hirner H, Knippschild U, Laufer S. 3,4-Diaryl-isoxazoles and –imidazoles as Potent Dual Inhibitors of p38 Mitogen Activated Protein Kinase and Casein Kinase 1. J. Med. Chem., 52 (2009), 7618-7630

177 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 27

Anticancer Activity of Dimeric Heterocylic Compounds Containing Indazole Ring

Jacek Kujawski1, Magdalena Skonieczna2, Joanna Kruk1, Hanna Popielarska1, Marek K. Bernard1

1Department of Organic Chemistry, Pozna University of Medical Sciences, ul. Grunwaldzka 6, 60-780 Pozna, Poland; e-mail: [email protected] 2Silesian University of Technology, Institute of Automatic Control, Department of Engineering of System, ul. Akademicka 16, 44-100 Gliwice, Poland

Compounds containing the indazole ring, like Axitinib, constitute a new class of anticancer drugs that act as Tyrosine kinase inhibitors [1].

In our search for anticancer agents, we obtained a series of dimeric heterocycles 1 containing the indazole moiety. The multi-step synthesis included N-hetarylation of hetarene, vicarious nucleophilic substitution of hydrogen, catalytic hydrogenation of the nitro group, and diazotization-cyclization to the final products 1. The preliminary tests on cell lines K562 (human leukemia) and HCT116 (human colon carcinoma) showed that the most potent in both tests were compounds 1c and 1f.

Het

N O 2 Het = N N N S N N N N NN HN N 1a 1b 1c 1d 1e 1f 1

[1] C. Avendaño, J. C. Menéndez Medicinal Chemistry of Anticancer Drugs, Elsevier 2008, Ch. 9

178 7th Polish-German Symposium on Pharmaceutical Sciences Synthesis and Biological Activity: V - 28

Embellicines A and B - Absolute Configuration and Potent NF-B Transcriptional Inhibitory Activity

Abdessamad Debbab1*, Weaam Ebrahim1, Amal H. Aly1, Marc Diederich2, Tibor Kurtán3, Peter Proksch1*

1Institut für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, Geb. 26.23, 40225 Düsseldorf, Germany, [email protected]; 2Department of Organic Chemistry, University of Debrecen, POB 20, 4010 Debrecen, Hungary; 3Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg 9, rue Edward Steichen L-2540 Luxembourg, Luxembourg

Two new metabolites, embellicines A and B (1 and 2), were isolated from the EtOAc extract of the fungus Embellisia eureka, an endophyte of the Moroccan plant Cladanthus arabicus (Asteraceae). The structures of these new compounds were determined on the basis of extensive one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configuration of embellicine A (1) was determined by TDDFT ECD calculations of solution conformers, whereas that of embellicine B (2) was deduced based on ROESY correlations and on biogenetic considerations in comparison to 1. Both embellicines (1 and 2) exhibit pronounced cytotoxic and cytostatic potential and strongly inhibit NF-B transcriptional activity, indicating that inhibition of NF-B may be a possible mechanism of action of these compounds. Embellicine B (2) was found to be tenfold more effective than 1 in inhibiting K562 cell growth and TNF-induced NF-B transcriptional activity in the same cell line, and exhibited IC50 values in the nM range.

H H H H

H H H H O O HO O

HO H O O NH NH HO HO

1 2

179 VI – Pharmaceutical Practice

180 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Practice: VI - 1

Polish students’ readiness for interprofessional learning – a trial Cerbin Magdalena1, Michalak Michal2

1Department of Pharmaceutical Technology, Poznan University of Medical Sciences, 70 Bukowska Street,60-812 Poznan (Poland) [email protected]; 2Department of Computer Science and Statistics, Poznan University of Medical Sciences 79 Dabrowskiego Street, 60-529 Poznan (Poland)

Introduction: There are many studies showing possibilities of team work training and its effect on quality of service delivery and patient care [1]. Although there were few activities for interprofessional education (IPE) implementation in Poland, varied barriers are difficult to overcome [2]. Considering challenges which need to be confront during IPE initiation policymakers need to be sure that students are ready to participate in interprofessional education model. The aim of the study was to determine differences between pharmacy and medicine students’ readiness for interprofessional learning (RIPLS) in relation to the participation of both groups in seminars about pharmacist-physician partnership.

Methods: Anonymous questionnaire was used for collecting data. It included 19 statements determining student attitudes toward interprofessional learning and healthcare professionals’ cooperation [3]. The form was filled by two groups of students: those who participated in interprofessional seminars and those who did not (as a control group). Analyzed data was given in ordinal measurement scale that is why data were compared using non-parametric tests. Comparison between two groups was performed using Mann-Whitney test. All tests were considered significant at p<0.05. Analysis was performed by Statistica 10 (StatSoft Inc.) software.

Results: Survey covered 19 students (47,37% of pharmacy, 52,63% of medicine) who participated in seminars and 28 students (39,29% of pharmacy, 60,71% of medicine) as the control group. Students’ attitudes toward few statements differ between both groups. Moreover, at the beginning of the survey statistically significant differences between students opinions according to their faculty were found. Pharmacy students were more likely to believe that learning with other students can help them become a more effective member of a health care team and that communication skills should be learned with other health care students.

Conclusions: Collected results confirmed that students’ attitudes can differ according to their faculty. Furthermore, this trial showed that after participating in interprofessional classes students RIPLS can increase. Further studies with larger groups of students are needed.  Acknowledgement: This study was partially supported by PUMS grant N0 502-01- 03314429-09985

[1] E. Suter, S. Deutschlander: Can Interprofessional Collaboration Provide Health Human Resources Solutions? A Knowledge Synthesis, Journal of Interprofessional Care, 26 (2012) 261-268. [2] M. Cerbin, J. Lulek: Edukacja interprofesjonalna – zachodni trend czy sposób na podniesienie jakoci opieki zdrowotnej?, Farmacja Polska, 69 (2013) 86-89. [3] G. Parsell, J. Bligh: The development of a questionnaire to assess the readiness of health care students for interprofessional learning (RIPLS), Medical Education, 33(1999): 95-100.

181 7th Polish-German Symposium on Pharmaceutical Sciences Pharmaceutical Practice: VI - 2

Individual Medication Management System implementation in pharmacists’ opinion in Poznan

Magdalena Cerbin1, Magdalena Waszyk-Nowaczyk1, Marek Simon2, Sebastian Lawicki3, Michal Michalak4

1 Department of Pharmaceutical Technology, Poznan University of Medical Sciences, Poland, 70 Bukowska Street, 60-812 Poznan, [email protected]; 2Department of Pathophysiology, Poznan University of Medical Sciences, Poland, 6 Swiecickiego Street, 60-781 Pozna, 3Student’s Scientific Society of Pharmaceutical Care, Poznan University of Medical Sciences, Poland, 70 Bukowska Street, 60-812 Poznan; 4Department of Computer Science and Statistics, Poznan University of Medical Sciences, Poland, 79 Dabrowskiego, 60-529 Poznan

Introduction: Over the past years, many countries of the world including Poland, have been taking actions for improving the role of the pharmacist as a health care professional. One of those was implementation of pharmaceutical care (PC), as a documented specialist medical service, which also includes pharmacist interventions, such as preparing Individual Medication Management System (IMMS), to enhance patient’s adherence [1]. Because of the chance to monitor the dosage and to detect and prevent drug problems occurrence, IMMS is thought to be an opportunity for individualized, effective and safe patient’s pharmacotherapy [2,3,4]. The aim of the study was to determine pharmacists’ attitudes toward IMMS. Methods: The survey was conducted in Poznan, between June 2011 and March 2012. An anonymous questionnaire was delivered either electronically or personally to community pharmacists. The survey covered 129 pharmacists (99 women and 30 men) who answered questions about their view of IMMS effectiveness, it’s funding sources and possible impact on PC implementation. The study included also the evaluation of pharmacist-physician cooperation to determine whether IMMS can improve partnership among health care professionals for proper patient’s care. Each questionnaire was provided with a short information brochure attached and presentation of demos how to use IMMS. Results: Survey confirmed that pharmacist are interested in implementation IMMS. 85.3% of respondents would recommend the use of IMMS to their patients and 74.2% are willing to extent their pharmacy business scope with this service (p<0,05). 88.4% of pharmacists found IMMS as an effective tool to increase patients’ adherence (p<0,05). 88.5% of them pointed out that IMMS should be refund by the National Fund of Health (p<0,05). Although collected data indicate that most pharmacists (64,8%) do not interact with physicians for elaborating patients’ pharmacotherapy, they emphasized that implementation of IMMS can lead to PC development resulting in establishing pharmacist-physician partnership (p<0,05). Conclusions: According to the scale of non-compliance, implementation of IMMS as a part of PC can be a chance both for patients and their physicians to increase the safety and effectiveness of therapy and for pharmacists who are intent to highlight their role as a part of health care system. Acknowledgement: This study was partially supported by PUMS grant N0 502-14-03314429- 09415 [1] M. Waszyk-Nowaczyk: Patients and physicians expectations about pharmacist’s role in pharmaceutical care implementation, Doctoral Thesis (2012). [2] Kanvelage J, Salditch I 2011: Medicine-on-time®. www.medicine-on-time.com (cited 2013, February 17). [3] Shavelsky E, Helliwell L 2011: MedMinder TM. www.medminder.com (cited 2013, February 17). [4] P. Rivers: Compliance aids-do they work? Drugs Aging, 2 (1992) 103-11.

182 Index D A Daghir, 95 Dampc, 138 Adamkiewicz-Droyska, 87 Danielak, 78 Adamus, 173 Darwish, 106 Aly, 179 Dawgul, 165, 168 Arceusz, 135 Dawidowska, 155 Augustynowicz-Kope, 153 Debbab, 179 Denys, 96 B Dereziski, 104, 121 Baisaeng, 22 Diederich, 179 Balcerska, 87 Dizmen, 119 Balewski, 175 Djuric, 62 Baranska-Rybak, 168 Dugaszewska, 147 Barszcz, 103, 117 Dugokcka, 80 Bartman, 142 Doha, 112 Bartosiska, 110 Drabinska, 173 Bartoszewska, 154 Duda, 79 Bartoszewski, 145 Dziomba, 89, 98 Baud, 99 Dziurkowska, 90 Bczek, 86, 87, 89, 93, 96, 111, 124, 129, 174 E Bednarczyk-Cwynar, 157, 171 Bednarski, 117, 175 Ebrahim, 179 Belka, 174 Ekiert, 9, 133, 134, 136, 137, 151 Below, 30 Elpelt, 92 Bernard, 163, 173, 178 F Biaas, 32, 48, 69 Bienert, 75, 82 Farwick, 92 Bierek, 59 Florek, 120, 141, 149 Bischof, 177 Foks, 153 Baszczyk, 48 Folttmann, 70 Bobkiewicz-Kozowska, 157 Franzblau, 171 Boblewski, 172 Frckowiak, 83 Bogdan, 88 Froelich, 48, 69, 102 Bogucewicz, 20 Funke, 62 Boniecka, 68 G Boudier, 54 Breitkreutz, 5, 30, 34, 51 Gadzaa-Kopciuch, 95 Brniak, 26 Garbacki, 117 Bujak, 95 Gdaniec, 167, 175 Bulakowska, 159, 162, Gód, 143, 144, 146 Burchardt, 78 Gówka, 54, 71, 77, 78, 84, 116 Burda, 155 Gyda, 79 Busse, 92 Gobis, 153 Buszewska-Forajta, 83, 110, 123, 126 Gohla, 21 Buszewski, 95 Golusiski, 120 Bylka, 140, 147, 160 Gostomczyk, 18 C Gostyska, 54 Goliski, 6, 13, 155, 167 Cal, 11, 35, 53, 71, 72, 73 Govedarica, 53 Centkowska, 23, 43 Greber, 165, Chen, 63 Greiner, 63, Chmielewska, 129 Grembecka, 119 Chrzanowska, 79 Grobelny, 49, 102 Cielecka-Piontek, 103, 112, 117, 128 Gröger, 57 Ciesielski, 73, 86 Groth, 7 Ciura, 101, 105, 107, 122 Grzekowiak, 75, 81 Czaja, 173 Gucwa, 88 Czajkowska, 37, 46 Gul, 166 Czarczyska-Goliska, 64 Gzella, 156, 163 Czarnobaj, 59

183 H Konieczna, 86, 87 Konieczny, 159, 162 Haag, 57 Kornicka, 172 Hagen, 86 Koroniak, 120 Halekotte, 177 Kosicka, 84 Hamann, 31 Koliski, 95 Hauß, 114 Kotowska, 14 Heinz, 99, 108, 109 Kovaevi, 44, 65 Hering, 145 Kowalski, 89, 93, 111 Herold, 174 Kowiel, 156 Hewelt-Belka, 174 Krauze-Baranowska, 100, 132, 139, 143, 144, 146 Homernik, 18 Król, 174 Horbert, 166 Król-Kogus, 132, 146 Hu, 42 Kruk, 173, 178 Hyla, 71 Krupa, 26, 61 J Kruszyna, 78 Kryjewski, 167 Jachimowicz, 111 Krzyaniak, 43 Jachowicz, 26, 36, 52, 61 Kubowicz, 76 Jamrógiewicz, 73, 118 Kuciska, 27, 160 Janaszak-Jasiecka, 88 Kujawski, 163, 173, 178 Janiszewska, 164 Kula, 144 Jankowska, 170 Kulza, 120, 141, 149 Jaremicz, 138, 150 Kunczyska, 172 Jaskiewicz, 154, 168 Kunstman, 69 Jeliska, 98, 112, 117 Kurtán, 179 Jenssen, 86 Kwiecie, 9, 136, 137 Jesionowski, 71 Kycler, 117 Jodowska, 173 Johannes, 158 L Józefowicz, 82 Latawiec, 74 Junmahasathien, 19 Lau, 36 Just, 62 Laukamp, 34 K Lebiedziska, 91, 119 Lehmann, 172 Kaczmarek, 117 Lesniewska, 169 Kaliszan, 4, 75, 80, 82, 83, 94, 110, 123, 126 Lestari, 40 Kamiska, 79 Lewandowska, 81, 103, 117 Kamysz, 154, 165, 168 Lewko, 74 Kanikanti, 31 Leyk, 113 Karafova, 154, 168 Lindert, 30 Karaniewicz-ada, 78 Liu, 41 Karbownik, 77, 81 Lorenc, 163 Kasprzyk, 77, 172 Luczkiewicz, 138 Kawczak, 96 Lulek, 16, 27, 49, 54, 58, 60, 64 Kamierczak, 173 Luxemburger, 177 Kamierska, 71, 111 Keck, 10, 19, 22, 28, 42, 44, 45, 50, 63, 65, 67 Kdziora, 32 epek, 125 Khinast, 62 uczkiewicz, 150 Klapiszewski, 71 Kleinebudde, 24, 31, 37, 39, 62, 70 M Klimaszewski, 173 Kluk, 46 Maciejewska, 107 Klupczyska, 104, 121 Maciejka-Kapuciska, 87 Knippschild, 177 Macur, 86 Knop, 24, 62, 70 Madanecka, 14 Kochan, 88 Madanecki, 88 Koczorowski, 155 Maincent, 54 Koehler, 109, Majcher, 136, 137 Majdan, 144 Kokot, 104, 115, 121 Kokotkiewicz, 138, 150 Majewska, 170 Malinka, 98

184 Maycha, 58 Paweczyk, 161 Man, 36 Pawowski, 76, 176 Marciniec, 76, 176 Peifer, 8, 158, 166, 177 Marczak, 128 Pein, 51, 70 Mardarowicz, 138 Peters, 22 Markuszewski, 94, 94, 95, 101, 122, 123, 126, 126 Pkala, 76, 176 Marsza, 91 Pikosz, 164, Malak, 61 Pikul, 101, 105, 107, 122 Matkowski, 144 Pinchuk, 166 Matawska, 146 Piotrowska, 160 Matysiak, 104, 121 Piotrowski, 88 Meier, 47 Plenis, 124 Mendyk, 36, 61 Paczek, 20 Mielcarek, 13, 155, 167 Pobocka-Olech, 143 Mielewczyk, 48 Poom, 15 Mikus, 124 Popielarska, 178 Mitka, 58 Powronik, 176 Migas, 100, 132 Prochaska, 49 Mikolaszek, 56 Prokopowicz, 17, 25 Milanowski, 58, 60, 64 Proksch, 179 Milewski, 58 Prost, 63 Mili, 44, 65 Przybyowski, 75 Mishra, 21, Pyda, 102 Mithieux, 99 R Mojsiewicz-Piekowska, 18, 72, 73 Möschwitzer, 29, 40, 41 Raczak-Gutknecht, 83, 95, 123 Mosig, 39 Radwaska, 80 Mrestani, 92, 106 Rahlff, 166 Mueller, 97 Rietz, 83 Müller, 10, 19, 21, 22, 28, 29, 40, 41, 42, 44, 45, 50 Rojewska, 49 Murias, 27, 160 Romaski, 77, 116 Muszalska, 169 Romero, 45 Muszyska, 9 Ronowicz, 88 Myka, 173 Roszak, 69 N Rudnicka, 107 Ruettinger, 97 Nagel, 108 Ruszkowski, 157 Napiórkowska, 153 Rybczyska, 172 Nasal, 80, 83 Ryek, 84, Neubert, 2, 92, 97, 99, 106, 108, 109, 114, 127 S Niedwiecki, 87 Nowaczyk, 95 Sabisz, 159, 162 Nowak, 141, 149 Sady, 80, Nowakowska, 101, 105, 107, 122 Savi, 44, 65 O Sawicki, 15, 18, 25, 59, 66, 107, 118, 122, 125, 165 Sczewski, 172, 172, 175 Ochocka, 88, 145 Scharrer, 62 Okruciska, 74 Schlosser, 158 Olejniczak-Rabinek, 49 Schmelzer, 99, 108, 109 Oldzka, 89, 93, 111 Schneider, 16 Opatkowska, 119 Schräder, 99 Orzechowska, 155 Schroeter, 97, 114 Osmaek, 32, 48, 69 Seczuk-Przybyowska, 120, 141, 149 Ostrowski, 169 Serocki, 159, 162 Oszczapowicz, 117 Shegokar, 21 P Siemitkowska, 116 Siluk, 94, 110, 123 Pacawski, 36 Sinha, 21, 29 Pankau, 108 Skadanowski, 153, 159, 162 Papavramidou, 45 Skocze, 66 Parent, 54 Skokowski, 86, 88 Paszun, 33, 55 Skonieczna, 178

185 Wachowiak, 141, 149 Skonieczny, 48 Wagner, 32, 47, 48, 51 Skotarczyk, 102 Waszkiewicz, 74 Skórka, 26 Watrobska-Swietlikowska, 38 Skrobaa, 60 Wei, 50 Skrzypczak-Pietraszek, 9, 142 Weiss, 99 Slupphaug, 86 Wesoowski, 90, 113, 135, 148 Snela, 49 Wiczling, 75, 82 Sobaska, 81 Wielgomas, 118 Sobczak, 98 Wierzchowski, 13 Sobiak, 79 Wiwart, 100, Sobotta, 13, 155 Wodarski, 125 Sollohub, 35 Wojewódzka, 16 Sparzak, 139 Wolska, 68 Srcic, 53 Wosicka, 53, 71 SSivan, 108 Woyna-Orlewicz, 52 Stanisz, 33, 55 Woniak, 120, 141, 149 Staufenbiel, 57 Wrotyski, 13 Stefanowicz-Hajduk, 145 Wróblewska, 27, 172, Stefanowska, 53, 71, 72, 73, 100 Yumba-Mpanga, 94, 126 Steinbach, 127 Z Stomberg, 31, Struck, 101, 110, 122, 126 Zacharzewska, 173 Struck-Lewicka, 94 Zajdel, 76, 176 Strzelczyk, 88 Zakrzewska, 52 Studziska-Sroka, 160 Zalewska, 33, 104 Suchowilska, 100 Zalewski, 117, 128 Sukowska-Ziaja, 9 Zaporowska-Stachowiak, 84 Surosz, 60 Zaprutko, 157, 161, 164, 170, 171 Szaek, 81 Zàrate, 150 Szarko, 100 Zaraziska, 68 Szczesny, 75 Zarembska, 143 Szczsny, 126 Zecevic, 47 Szczoko, 13, 155 Zeidler, 169 Szefer, 91, 119 Zhai, 28, 67 Szewczyk, 9, 25 Ziarniak, 98 Szlk, 36 Zielen, 38 Szmudanowski, 93 Znajdek-Awie, 140, 147 Sznitowska, 14, 20, 23, 37, 43, 46, 56, 68 Szopa, 9, 133, 134, 151 Szybowicz, 48 wawiak, 164 Szyfter, 120, 120 Szymkowska, 72, 73 witek, 98

wieczkowski, 18 T

Talaczyska, 103 Thommes, 31, 34 Toschkoff, 62 Tyczka, 75 Tykarska, 64, 167 U

Ulenberg, 174 Ulewicz-Magulska, 148 Urbaniak, 104, 115, 130 W

186