Second World Conference on Physico-Chemical Methods in ...2nd World Conference on Physico-Chemical...

Second World Conference on Physico-Chemical Methods in Drug Discovery and Development

Programme &

Book of Abstracts Zadar, Croatia, September 18-22, 2011

Second World Conference on Physico-Chemical Methods in Drug Discovery and Development Zadar, Croatia, September 18-22, 2011

Programme & Book of Abstracts Published by International Association of Physical Chemists E-mail: [email protected], URL: http://www.iapchem.org For Publisher Zoran Mandić Editor Zoran Mandić Design, Page Making and Computer Layout Aleksandar Dekanski Circulation 150 Copies Printing DENONA d.o.o., Getaldićeva 1, 10000 Zagreb, Croatia

Scientific Committee and Advisory Board

Alex Avdeef, pION, Woburn, USA Biserka Cetina-Čižmek, PLIVA, Zagreb, Croatia Gregg Siegal, University of Leiden, The Netherlands Jasna Peter Katalinić, University of Münster, Germany Kin Tam, AstraZeneca, UK Klara Valko, GlaxoSmithKline, Stevenage, UK Michael Przybylski, Unversity of Konstanz, Germany Pierre Parot, CEA, France Rolf Hilfiker, Solvias AG, Switzerland, Head of the Scientific committee Slobodan Šašić, Pfizer, Groton, USA Vesna Gabelica, Galapagos, Zagreb, Croatia Zoran Mandić, University of Zagreb, Croatia

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CONTENTS

CONFERENCE PROGRAMME _______________________________________________ I

ORAL PRESENTATIONS __________________________________________________ 1

The importance and different approaches for the determination of permeability across an artificial membrane

Kin Y. Tam ____________________________________________________________ 3

Mechanistic prediction of oral drug absorption from physicochemical property Kiyohiko Sugano _______________________________________________________ 4

Applications of bio-mimetic HPLC methods for the estimation of in vivo distribution of early drug discovery compounds

Klára Valkó ____________________________________________________________ 5

Implementation of in vitro permeability studies for evaulation and improvement of generic drug formulations before bioequivalence studies

Seval Korkmaz _________________________________________________________ 6

Dynamic dissolution testing in R&D Martin Čulen, Jiři Dohnal, Josef Jampílek , Anna Řezáčová ______________________ 7

GLP determination of equilibrium solubility with special interest of ionization and polymorphism

Krisztina Takács-Novák __________________________________________________ 8

Techniques for investigating supersaturation and the enhancement of solubility John Comer ___________________________________________________________ 9

Dissolution of nanocrystalline particles for IV route: in vitro studies and in silico modelling

Guillaume Louit, Xavier Pepin, Morgane Vacher _____________________________ 10

Structure-based QSAR studies on 5-HT2A partial agonists Stefan Dove, Maria E. Silva, Ralf Heim, Andrea Strasser, Sigurd Elz _______________ 11

Zadar, Croatia, September 18-22, 2011

VI

Modeling Biorelevant Solubility and Dissolution: Effects of Molecular Ionization Michael B. Bolger, Robert Fraczkiewcz, Marvin Waldman, Dechuan Zhuang, Walter S. Woltosz ___________________________________________________________ 12

The relationship study of Clog P and aphicidal activity of cyantraniliprole analogs HuiBin Yang, Hua Chen, JunFeng Wang, Bin Li _______________________________ 13

IS-CE: A fast high-throughput method for pKa determination by capillary electrophoresis

Martí Rosés, Joan Marc Cabot, Elisabet Fuguet, Clara Ràfols ___________________ 14

NMR studies of peptides and proteins and their interactions: implications for drug development

Horst Kessler _________________________________________________________ 15

Functional mechanisms of drugs studied by NMR: methodology and applications Teresa Carlomagno ____________________________________________________ 16

Determining the orientation of drugs in receptors by solvent paramagnetic relaxation NMR spectroscopy

Simone Kosol, Christoph Göbl, Katarina Čuljak, Walter Hohlweg, Predrag Novak, Klaus Zangger ________________________________________________________ 17

The importance in drug design of resolving structural and dynamic details for membrane drug targets at the nanoscale

Anthony Watts ________________________________________________________ 18

Statistically optimized Raman microscopy for detecting low levels of undesired polymorphic forms in low dose active tablets

Slobodan Šašić, Mark Whitlock, Shawn Mehrens _____________________________ 19 14N NQR study of polymorphism of piroxicam and nifedipine; characterization of their polymorphs and solid-solid transformations

Zoran Lavrič, Janez Pirnat, Janko Lužnik, Zvonko Trontelj, Stane Srčič _____________ 20

Challenging characterization of polyphasic drugs. Use of combined physicochemical techniques in pharmaceutical development

Danielle Giron ________________________________________________________ 21

Hydrates: Thermodynamics and Determination of Relevant Parameters Rolf Hilfiker __________________________________________________________ 22

The determination of dehydratation mechanism of lactose monohydrate by using thermogravimetric methods

Petar Tudja, Ernest Meštrović ____________________________________________ 23

Solid-state chemistry and new drug forms Elena V. Boldyreva _____________________________________________________ 24

Study of the Role of Molecular Mobility in Physical Stability of Amorphous of Anti-Inflamantory Drug: Celecoxib

Marian Paluch, Katarzyna Grzybowska, Żaneta Wojnarowska, Kia L. Ngai _________ 25

2nd World Conference on Physico-Chemical Methods in Drug Discovery and Development

VII

Tailor-made batch crystallisation of Entacapone through the use of self-assembled layers on gold single crystal surface

Ana Kwokal, Kevin J. Roberts ____________________________________________ 26

Physical insight into tautomerization reaction in some pharmaceutically important molecules

Żaneta Wojnarowska, Marian Paluch ______________________________________ 27

The influence of glycosylation to biological activity of proteins Gottfried Pohlentz, Kristina Neue, Anne Zeck, Jörg Thomas Regula, Jasna Peter-Katalinić ___________________________________________________ 28

Mass spectrometric approaches for structural elucidation of “misfolding”: aggregating neurodegenerative proteins: Ion mobility- MS and affinity- MS

Camelia Vlad, Claudia Cozma, Marilena Manea, Marius Iurascu, Kathrin Lindner, Jieun Jung, Martin Scheffner, Marcel Leist, Michael Przybylski __________________ 29

Biophysical methods for identifying and characterizing protein-ligand interactions Geoff Holdgate _______________________________________________________ 30

Dynamic Force Spectroscopy for studying high affinity ligand receptor complexes Pierre Parot, Jean-Luc Pellequer __________________________________________ 31

TRANSIL High Sensitivity Binding Assay: A novel tool for assessing plasma binding of hardly soluble and sticky drugs

Hinnerk Boriss, Kathleen Boehme _________________________________________ 32

Chromatographic and spectroscopic studies on the chiral recognition of sulfated β-cyclodextrin as chiral mobile phase additive: Enantiomeric separation of a chiral amine

Shengli Ma, Sherry Shen, Nizar Haddad,Wenjun Tang, Jing Wang, Heewon Lee, Nathan Yee, Chris Senanayake, Nelu Grinberg _______________________________ 33

Automated Patch Clamp in Drug Discovery – from the bench to the midfield Morten Sunesen _______________________________________________________ 34

Synergistic and antagonistic effects between selected phenolic and polyphenolic antioxidants

Ewelina Kowalewska, Grzegorz Litwinienko _________________________________ 35

Compressibility investigation of chewing gum bases used for API containing direct compressed chewing gum tablet preparation

Peter Kása jr., Imre Jojart, Klára Pintye-Hódi ________________________________ 36

Dynamics of enzymes-inhibitor complexes from protein crystallography J. Hašek, K. Zimmerman, T. Skálová, J. Dušková, J. Dohnálek 37

Online SAW-Bioaffinity- Mass Spectrometry: New Bioanalytical Application in Detection, Structure Determination and Quantification of Protein-Ligand Interactions from Biological Material

Michael Przybylski _____________________________________________________ 38


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POSTER PRESENTATIONS _______________________________________________ 39

Characterization of topical delivery systems by in vitro release and permeability testing

Maja Radić, Marjana Durrigl, Biserka Cetina-Čižmek __________________________ 41

Investigating alkyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxoazepan-1-yl)hexanoates as potential transdermal penetration enhancers

Kateřina Brychtová, Josef Jampílek, Lenka Dvořáková, Radka Opatřilová, Jiři Dohnal 42

A Chromatographic Method to determine the 1-Octanol/Water Partition Coefficient (log Po/w) of Drugs

J.M. Pallicer, M. Rosés, C. Ràfols, E. Bosch, R. Pascual, C. Calvet, A. Port __________ 43

Setup and validation of a shake-flask procedure for the determination of the partition coefficient log D at pH 7.4 using less than 2 mg of drug

Axel Andrés, Martí Rosés, Clara Ràfols, Elisabeth Bosch, Sonia Espinosa, Víctor Segarra, Josep Mª Huerta __________________________________________ 44

Physico-chemical profiling of antibacterial fluoroquinolones Gábor Vizserálek, Gergely Völgyi, Kin Y. Tam, Krisztina Takács-Novák ____________ 45

Correlation between the lipophilicity and the antioxidant activity of the parabens Trifon M. Angelov, Elena I. Korotkova, Ana Vlasenko __________________________ 46

Zwitterionic characteristics of piroxicam and a novel carboxy methylated pyridoindole derivative as expressed in the retention/pH behavior on reversed phase and ion interaction HPLC

Nikos Triantos, Milan Stefek, Anna Tsantili-Kakoulidou ________________________ 47

Study of pH-dependent drugs solubility in water Aneta Pobudkowska, Urszula Domańska, Aleksandra Pelczarska ________________ 48

Solubility – pH Profiles of Ionizable Compounds Elham Shoghi, Clara Ràfols, Elisabet Fuguet, Elisabeth Bosch ___________________ 49

Increasing of drugs water solubility using pectines and their derivatives Zbyněk Oktabec, Josef Jampílek, Jiři Dohnal, Martin Čulen, Vladimir Král __________ 50

Development of biorelevant dissolution method for sparingly water soluble drug product with different polymorphs

Jasmina Turkalj, Anamarija Tomljenović Gluhak, Biserka Cetina-Čižmek __________ 51

Crystal structures and dissolution behaviour of co-crystals of meloxicam with carboxylic acids

Svetlana A. Myz, Tatyana P. Shakhtshneider, Nikolay A. Tumanov, Elena V. Boldyreva _____________________________________________________ 52

Development and validation of dissolution tests and analysis methods for Cetraxate HCl capsules and Sodium picosulfate in Korean Pharamceutical Codex

Juyoung Jung, Chungsik Min, Minjung Kim, Sheenhee Kim, Soonhan Kim, Dongsup Kim,Youngja Lee, Rackseon Seong _________________________________ 53


IX

Validation of Analysis Method to Study the Dissolution Behavior of Acetylcysteine Capsules and Sodium Loxoprofen Tablets

Raeseok Jung, Sanghyeok Sohn, Oksoon Heo, Miae Na, Minchul Kim, Wooyoung Jung, Soyoung Wang, InKyu Kim _________________________________ 54

Development of Dissolution testing method for Sobrerol capsules in Korean pharmaceutical codex

E. J. Kim, J. H. Lee , Y.M. Jung, K. H. Sohn, L. Choi, I. K. Kim, D. S. Kim _____________ 55

Particle characterization in the pharmaceutical industry Iva Tunjić, Biserka Cetina-Čižmek _________________________________________ 56

Effect of the physico-chemical properties of materials on the properties of direct compressed bi-layer tablets

Tamás Sovány, Kitti Papós, Péter Kása jr., Ilija Ilič, Stane Srčič, Klára Pintye-Hódi ___ 57

An investigation on the polymorphism of racemic betaxolol Teresa M. R. Maria, Ricardo A. E. Castro, António O. L. Évora, Suse S. Bebiano, M. Ramos Silva, Hugh D. Burrows, M. Luísa Ramos, Licínia L. G. Justino, João Canotilho, M. Ermelinda S. Eusébio ___________________________________ 58

Deciphering Interactions of Macrolide Antibiotics with Membrane-Mimetics by NMR Spectroscopy

Evelyne Schrank, Simone Kosol, Katarina Čuljak, Christoph Göbl, Klaus Zangger, Predrag Novak ________________________________________________________ 59

Highly sensitive NMR system for discovery and structure elucidation of natural drug leads

Masashi Tsuda, Keiko Kumagai ___________________________________________ 60

Investigation of the Entericidins EcnA/EcnB – a membrane-bound toxin-antitoxin system

Gabriel Wagner, Simone Kosol, Klaus Zangger _______________________________ 61

The role of cation - π interactions in stabilizing charged peptide residues inside of biological membranes

Walter Hohlweg, Christoph Göbl, Klaus Zangger _____________________________ 62

Influence of Risperidone on Pharmacokinetics of Caffeine in Rabbits Nino Lominadze, Manuchar Gvaramia, Manoni Kurtanidze, Maka Alexishvili, Marina Rukhadze ______________________________________________________ 63

Interaction structure of the complex between neuroprotective factor humanin and Alzheimer’s β-amyloid peptide revealed by affinity-mass spectrometry and molecular modelling

Madalina Maftei, Xiaodan Tian, Marilena Manea, Thomas E. Exner, Daniel Schwanzar, Christine A.F. von Arnim, Michael Przybylski _______________________ 64

Binding parameters involved in the interactions between some acidic drugs with human and bovine serum albumin

Sílvia Zarza, Clara Ràfols, Elisabeth Bosch __________________________________ 65


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Brain Tissue Binding of Drugs: evaluation and validation of solid supported porcine brain membrane vesicles (TRANSIL) as a novel high -throughput method

Hinnerk Boriss, Kathleen Boehme _________________________________________ 66

Effect of chicken egg white cystatin on proliferation of cancer cells Renata Brykner, Łucja Cwynar-Zając, Katarzyna Juszczyńska, Agnieszka Sosnowska, Krzysztof Gołąb, Jakub Gburek, Piotr Dzięgiel, Antoni Polanowski, Tadeusz Trziszka ______________________________________ 67

Polyethylenimine-functionalized single-walled carbon nanotube for efficient delivery of plasmid DNA

Mohammad Ramezani, Behzad Behnam, Khalil Abnous _______________________ 68

Supercritical CO2 extraction and large-scale separation of natural drug leads Keiko Kumagai, Masashi Tsuda ___________________________________________ 69

Development of Analytical Method of Compounds in Drugs Using HPLC instead of UV Spectrophotometer Method

JaeYong Song, SeungEun Jang, SunHoi Kim, GilBong Lee, JeaMan Lee, YongHee Kim 70

Chromatographic characteristics of inorganic analytes in nonionic micellar liquid chromatography with ion-pair additives

Nino Lominadze , Levan Akhalkatsi, Manuchar Gvaramia , Marina Rukhadze, ______ 71

DFT Calculations on pregabalin as a pharmaceutical compound for the treatment of neuropathic pain

E. Vessally, M. Rezaei, M. Saber __________________________________________ 72

Ab initio Calculations on Galantamine as a pharmaceutical compound for the treatment of Alzheimer’s Disease

Maryam Motallebzadeh, Mehdi Rezaei, Esmail Vessally _______________________ 73

A new method of producing monoclinic paracetamol suitable for direct compression Andrey G. Ogienko, Elena V. Boldyreva, Andrey Yu. Manakov, Vladimir V. Boldyrev, Alexander S. Yunoshev, Anna A. Ogienko, Svetlana A. Myz, Alexei I. Ancharov, Andrey F. Achkasov, Tatyana N. Drebushchak _______________________________ 74

DFT Calculations on Quetiapine Hemifumarate as a pharmaceutical compound for the treatment of schizophrenia

E. Vessally, A. A. Jafari, M. Rezaei _________________________________________ 75

AUTHOR INDEX ________________________________________________________ 79

Conference Programme


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Sunday, September 18th 10:00 – 12:00 &16:00 – 18:00

Course 1, Lecture Room “Mala dvorana”, Hotel DonatPolymorphs and other solid forms: identification, properties and analytical principles Rolf Hilfiker, Solvias AG, Switzerland

10:00 – 12:00 &16:00 – 18:00

Course 2, Lecture Room “Bura”, Hotel FunimationChiral recognition and separation mechanisms Nelu Grinberg, Boehringer-Ingelheim, USA

10:00 – 12:00 &16:00 – 18:00

Course 3, Lecture Room “Novi Park”, Hotel FunimationQSAR analysis - principles, methods and applications Stefan Dove, University of Regensburg, Germany

09:00 – 10.30 15:00 – 18.30

Conference Desk openParticipant registration, Hotel Donat

20:00 Welcome party, Hotel Adriana

All Conference Lectures, as well as Oral Presentations will be given in the Conference Hall, Hotel Donat

Monday, September 19th 08:00 Poster mounting, in front of the Conference Hall, Hotel Donat

09:00 Opening Ceremony

Chair: Klara Valko

09:20 OP 01

The importance and different approaches for the determination of permeability across an artificial membrane Kin Y. Tam, AstraZeneca, UK

10:05 OP 02

Mechanistic prediction of oral drug absorption from physicochemical property Kiyohiko Sugano, Pfizer, UK

10:50 Coffee break

Chair: Kiyohiko Sugano

11:10 OP 03

Applications of bio-mimetic HPLC methods for the estimation of in vivo distribution of early drug discovery compounds Klara Valko, GlaxoSmithKline, UK


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11:55 OP 04

Implementation of In Vitro Permeability Studies for Evaluation and Improvement of Generic Drug Formulations Before Bioequivalnce Studies Seval Korkmaz, Abdi Ibrahim, Turkey

12:30 OP 05

Dynamic dissolution testing in R&DMartin Čulen, Jiři Dohnal, Josef Jampílek , Anna Řezáčová Universiy of Veterinary and Pharmaceutical Sciences, Czech Republic

13:00 Lunch

Chair: Kin Tam

15:00 OP 06

GLP determination of equilibrium solubility with special interest of ionization and polymorphism Krisztina Takács-Novák, Semmelweis University, Hungary

15:40 OP 07

Techniques for investigating supersaturation and the enhancement of solubility John Comer, Sirius-analytical Ltd. UK

16:20 OP 08

Dissolution of nanocrystalline particles for IV route: in vitro studies and in silico modelling Guillaume Louit, Xavier Pepin, Morgane Vacher, Sanofi-Aventis, France

16:45 Coffee break

Chair: John Comer

17:00 OP 09

Structure-based QSAR studies on 5-HT2A partial agonistsStefan Dove, Maria E. Silva, Ralf Heim, Andrea Strasser, Sigurd Elz University of Regensburg, Germany

17:40 OP 10

Modeling Biorelevant Solubility and Dissolution: Effects of Molecular Ionization Michael B. Bolger, Robert Fraczkiewcz, Marvin Waldman, Dechuan Zhuang, Walter S. Woltosz, Simulations plus Inc. USA

18:20 OP 11

The relationship study of Clog P & aphicidal activity of cyantraniliprole analogs HuiBin Yang, Hua Chen, JunFeng Wang, Bin Li Shenyang Research Institute, China

18:45 OP 12

IS-CE: A fast high-throughput method for pKa determination by capillary electrophoresis Martí Rosés, Joan Marc Cabot, Elisabet Fuguet, Clara Ràfols University of Barcelona, Spain


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Tuesday, September 20th Chair: Predrag Novak

09:00 OP 13

NMR studies of peptides and proteins and their interactions: implications for drug development Horst Kessler, Technical Unversiy of Müncheh, Germany

09:40 OP 14

Functional mechanisms of drugs studied by NMR: methodology and applications Teresa Carlomagno, EMBL, Germany

10:20 OP 15

Determining the orientation of drugs in receptors by solvent paramagnetic relaxation NMR spectroscopy Simone Kosol, Christoph Göbl, Katarina Culjak, Walter Hohlweg, Predrag Novak, Klaus Zangger, Universiy of Graz, Austria

10:50 Coffee break

Chair: Danièle Giron

11:10 OP 16

The importance in drug design of resolving structural and dynamic details for membrane drug targets at the nanoscale Anthony Watts, Oxford University, UK

11:50 OP 17

Statistically optimized Raman microscopy for detecting low levels of undesired polymorphic forms in low dose active tablets Slobodan Šašić, Mark Whitlock and Shawn Mehrens, Pfizer, USA

12:30 OP 18

14N NQR study of polymorphism of piroxicam and nifedipine; characterization of their polymorphs and solid-solid transformations Zoran Lavrič, Janez Pirnat, Janko Lužnik, Zvonko Trontelj, Stane Srčič University of Ljubljana, Slovenia

13:00 Lunch

Chair: Biserka Cetina Čižmek

15:00 OP 19

Challenging characterization of polyphasic drugs. Use of combined physico-chemical techniques in pharmaceutical development Danielle Giron, Consultant, France

15:40 OP 20

Hydrates: Thermodynamics and Determination of Relevant Parameters Rolf Hilfiker, Solvias AG, Switzerland

16:20 OP 21

The determination of dehydratation mechanism of lactose monohydrate by using thermogravimetric methods Petar Tudja, Ernest Meštrović, PLIVA d.o.o, Croatia

16:40 Coffee break

Chair: Rolf Hilfiker

17:00 OP 22

Solid-state chemistry and new drug formsElena V. Boldyreva, Novosibirsk State University, Russia


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17:40 OP 23

Study of the role of molecular mobility in physical stability of amorphous of anti-inflamantory drug: Celecoxib Marian Paluch, Katarzyna Grzybowska, Żaneta Wojnarowska, Kia L. Ngai Institute of Physics, Poland

18:10 OP 24

Tailor-made batch crystallisation of Entacapone through the use of self-assembled layers on gold single crystal surface Ana Kwokal, Kevin J. Roberts, PLIVA d.o.o. Croatia

18:35 OP 25

Physical insight into tautomerization reaction in some pharmaceutically important molecules Zaneta Wojnarowska, Marian Paluch, Institute of Physics, Poland

19.00 Poster session (until 23:00)

Wednesday, September 21st Chair: Pierre Parot

09:00 OP 26

The influence of glycosylation to biological activity of proteins Jasna Peter Katalinić, University of Münster, Germany

09:45 OP 27

Mass spectrometric approaches for structural elucidation of “misfolding” - aggregating neurodegenerative proteins: Ion mobility- MS and affinity- MS Camelia Vlad, Claudia Cozma, Marilena Manea, Marius Iurascu, Kathrin Lindner, Jieun Jung, Martin Scheffner, Marcel Leist, Michael Przybylski University of Konstanz, Germany

10:30 Coffee break

Chair: Jasna Peter Katalinić

10:50 OP 28

Biophysical methods for identifying and characterizing protein-ligand interactions Geoff Holdgate, AstraZeneca, UK

11:35 OP 29

Dynamic Force Spectroscopy for studying high affinity ligand receptor complexes Pierre Parot, Jean-Luc Pellequer, CEA Marcoule, France

12:00 OP 30

TRANSIL High Sensitivity Binding Assay: A novel tool for assessing plasma binding of hardly soluble and sticky drugs Hinnerk Boriss, Kathleen Boehme, Sovicell GmbH, Germany

12:25 Lunch

13:30 Excursion

20:30 Conference Dinner


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Thursday, September 22nd Chair: Vesna Gabelica

09:00 OP 31

Chromatographic and spectroscopic studies on the chiral recognition of sulfated β-cyclodextrin as chiral mobile phase additive: Enantiomeric separation of a chiral amine Shengli Ma, Sherry Shen, Nizar Haddad,Wenjun Tang, JingWang, Heewon Lee, Nathan Yee, Chris Senanayake, Nelu Grinberg, Boehringer-Ingelheim, USA

09:45 OP 32

Automated Patch Clamp in Drug Discovery – from the bench to the midfield Morten Sunesen, Sophion Bioscience, Denmark

10:30 Coffee break

Chair: Nelu Grinberg

10:50 OP 33

Synergistic and antagonistic effects between selected phenolic and polyphenolic antioxidants Ewelina Kowalewska, Grzegorz Litwinienko, University of Warsaw, Poland

11:15 OP 34

Compressibility investigation of chewing gum bases used for API containing direct compressed chewing gum tablet preparation Peter Kása jr., Imre Jojart, Klára Pintye-Hódi, University of Szeged, Hungary

11:40 OP 35

Dynamics of enzymes-inhibitor complexes from protein crystallography J. Hašek, K. Zimmerman, T. Skálová, J. Dušková, J. Dohnálek Institute of Macromolecular Chemistry, Praha, Czech Republic

12:05 S 1

Seminar/Workshop: Bioaffinity - Mass Spectrometry,Michael Przybylski, University of Konstanz, Germany

13:00 Conference closing

Zoran

Highlight

Oral Presentations


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The importance and different approaches for the determination of permeability across an artificial membrane

Kin Y. Tam

AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK

Permeability is one of the important parameter that is routinely determined in pharmaceutical research. It describes the ease of a drug molecule across a membrane. Poor permeability makes the molecules difficult for future development as an oral drug, and it is of great interest to identify these compounds in a drug discovery programme. Over the last decade, parallel artificial membrane permeability assay (PAMPA) has been evolved into one of screening toolkits in many research laboratories to support the early phase drug discovery projects. In this presentation, we will highlight the important developments/advancements, including new membrane models, control of hydrodynamics in the assay, and mathematical model for correcting paracellular transport. We will discuss our recent work on the determination of permeability across artificial membranes. The method is illustrated using two sets of marketed drugs, one of which is amphoteric drug predominately charged at the physiological pH, with known absolute bioavailability data in human. We will demonstrate that the permeability models developed in this study correlate well with the fraction absorbed data in human.

OP 01 – Invited Lecture


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Mechanistic prediction of oral drug absorption from physicochemical property

Kiyohiko Sugano

Pfizer, Pharmaceutical Science, Sandwich. UK

Oral drug absorption is a determined by the dissolution, precipitation and membrane permeation of a drug. Recently, the gastrointestinal unified theoretical framework (GUT framework) has been proposed 1-18. The GUT framework is intended to fully describe each process of oral drug absorption from molecular property of a drug. In this presentation, the mechanistic relationship between the molecular property of a drug and in vivo oral absorption will be discussed.

References: [1] Kiyohiko Sugano, Introduction to computational oral absorption simulation, Expert. Opin. Drug

Metab. and Tox, 2009, 259-293 [2] Kiyohiko Sugano, Arimichi Okazaki, Shohei Sugimoto, Sumitra Tavornvipas, Atsushi Omura, Takashi

Mano, Solubility and dissolution assessment, DMPK, 2007, 225-254 [3] Sugano, K., Aqueous Boundary Layers Related to Oral Absorption of a Drug: From Dissolution of a

Drug to Carrier Mediated Transport and Intestinal Wall Metabolism. Mol. Pharm. 2010, 1362-1373. [4] Kiyohiko Sugano, Yoshiyuki Shirasaka, Shinji Yamashita, Estimation of Michaelis Menten constant of

efflux transporter considering asymmetric permeability, Int. J. Pharm., Accepted [5] Kiyohiko Sugano, Fraction of a dose absorbed estimation for structurally diverse low solubility

compounds, Int. J. Pharm., 2010, 79-89 [6] Kiyohiko Sugano, Computational oral absorption simulation of free base drugs, Int. J. Pharm., Int. J.

Pharm., 2010, 73-82 [7] Kiyohiko Sugano, Makoto Kataoka, Claudia da Costa Mathews, Shinji Yamashita, Prediction of food

effect by bile micelles on oral drug absorption considering free fraction in intestinal fluid, Eur. J. Pharm. Sci, 2010, 118-124.

[8] Kiyohiko Sugano, Possible reduction of effective thickness of intestinal unstirred water layer by particle drifting effect, Int. J. Pharm., 2010, 103-109

[9] Kiyohiko Sugano, Fraction of dose absorbed calculation: comparison between analytical solution based on one compartment steady state approximation and dynamic seven compartment model, 2009, 75-93

[10] Kiyohiko Sugano, A simulation of oral absorption using classical nucleation theory, Int. J. Pharm., 2009, 142-145

[11] Kiyohiko Sugano, Oral absorption simulation for poor solubility compounds, Chemistry & Biodiversity, 2009, 2014-2029

[12] Kiyohiko Sugano, Theoretical investigation of passive intestinal membrane permeability using Monte Carlo method to generate drug like molecule population, Int. J. Pharm., 2009, 55-61

[13] Kiyohiko Sugano, Estimation of effective intestinal membrane permeability considering bile micelle solubilisation, Int. J. Pharm., 2009, 116–122

[14] Kiyohiko Sugano, Theoretical comparison of hydrodynamic diffusion layer models used for dis-solution simulation in drug discovery and development. Int. J. Pharm., 363(1-2) (2008) 73-77.

[15] Okazaki Arimichi, Takashi Mano, Kiyihiko Sugano, Theoretical dissolution model of poly–disperse drug particles in biorelevant media, J. Pharm. Sci., 2008, 1843-1852.

[16] Obata K, Sugano K, Saitoh R, Higashida A, Nabuchi Y, Machida M, Aso Y., Prediction of oral drug ab-sorption in humans by theoretical passive absorption model. Int J Pharm. 293(1-2) (2005) 183-92.

[17] Kiyohiko Sugano, Yoshiaki Nabuchi, Minoru Machida, Yoshinori Aso, Prediction of human intestinal permeability using artificial membrane permeability, Int. J. Pharm., 257 (2003) 245-251

[18] Kiyohiko Sugano ,Noriyuki Takata, Minoru Machida, Kimitoshi Saitoh , Katsuhide Terada, Prediction of passive intestinal absorption using bio-mimetic artificial membrane permeation assay and the paracellular pathway model, Int. J. Pharm., 241(2) (2002) 241-251

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Applications of bio-mimetic HPLC methods for the estimation of in vivo distribution of early drug discovery compounds

Klára Valkó

Computational and Structural Chemistry - Analytical Chemistry, Platform Technology

Sciences, GlaxoSmithKline, Stevenage, UK High Performance Liquid Chromatographic (HPLC) retention time measurements can be used to determine a compound’s distribution between an aqueous mobile phase and a bio-mimetic stationary phase. Thus, it provides an easily automated high throughput technique to determine important characteristics of drug discovery compounds that can be used to predict in vivo drug disposition. HPLC methods have been developed1 to measure compounds lipophilicity (using C-18 stationary phase), plasma protein binding (using commercially available Human Serum Albumin2 and α-1-acid-glycoprotein stationary phases) and membrane partition (using Immobilised Artificial Membrane3 stationary phase). Quantitative structure – retention relationships were established that describe the binding characteristics of compounds to proteins and phospholipids using the linear solvation equation model4. The solvation equations are also used to describe various biological distribution processes using simple molecular descriptors, such as H-bond acidity and H-bond basicity, dipolarity/polarisability, size and excess molar refraction. The bio-mimetic binding properties could be used to build mechanistic models for the estimation of human in vivo volume of distribution, and various tissue bindings such as brain and lung. Recent results revealed that the lung tissue and mucus binding can be strongly related to a compound’s retention on α-1-acid-glycoprotein stationary phase, as mucus and lung tissue also contain highly glycosilated proteins. The brain tissue binding could be modeled by the sum of phospholipid and albumin binding. The bio-mimetic HPLC binding data of over hundred thousand research compounds for several respiratory and neuroscience programs are compiled in company database and used to aid the lead optimization and drug design efforts. References: [1] K. Valko, J. Chromatogr. A 1037 (2004) 299 [2] K. Valko, C. M. Du, C. Bevan, D. P. Reynolds, M. H. Abraham, Curr. Med. Chem. 8 (2001) 1137 [3] K. Valko, S. Nunhuck, C. Bevan, M. H. Abraham, D. P. Reynolds, J. Pharm. Sci. 92 (2003) 2236 [4] M. H. Abraham, Chem. Soc. Rev. 22 (1993) 73



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Implementation of in vitro permeability studies for evaulation and improvement of generic drug formulations before bioequivalence studies

Seval Korkmaz

Abdi Ibrahim Ilac A.S., Sanayi Mh. Tunc Cd No:3, Esenyurt, Istanbul, Turkey [email protected]

The primary role of bioequivalence (BE) studies is to show that two formulations of a drug have similar bioavailabilities. A bioequivalence study compares the bioavailability between a test and a reference drug product in terms of the rate and extent of drug absorption. Most of generic pharmaceutical companies apply only in vitro solubility test before sending their new generic formulations for bioequivalence studies. According to BCS (Biopharmaceuticals Classification System), there are two important parameters for bioavailability which are solubility and permeability. In vitro permeability studies of generic drugs have been neglected in routine studies so far. This presentation was prepared to prove the importance of permeability studies for a generic drug before bioequivalence studies. In spite of successfulness of in vitro solubility tests, any oral solid generic drug formulation under the development can be failed in bioequivalence tests. The reason could be related to their permeability charecteristics. Those types of permeability studies we currently developed were designed to prove availability of Caco-2 studies for improvement of formulations before bioequivalence studies. In our cell culture laboratory, Caco-2 studies are performed to test permeability charecteristics of generic oral solid drug formulations and different polymorphs of APIs by comparing with their original drug formulations. Xcelligence real time cell analyzer is used to show the effect of our drug concentrations (100uM) on viability of Caco-2 cells. TEER (Transepithelial Electrical Resistence) and Lucifer Yellow tests were applied as integrity tests and followed by the use of cells for permeability tests. Test groups are comprised active pharmaceutical ingradients (APIs), physical powder mixtures, generic drug tablets and original tablets. Caco-2 permeability studies are performed under the FeSSIF conditions (pH 6,5). After Caco-2 permeability studies, samples are evaluated by LC/MSMS method. Availability of in vitro permeability studies to improve generic formulation and to support for bioequivalence studies will be explained with this presentation. Based on thse studies, it can be proposed that in vitro permeability studies chould be performed for improvement of a generic formulation and it should be appeared in pharmacopoeia as in vitro bioavailability tests for development of generic drugs.

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Dynamic dissolution testing in R&D

Martin Čulen, Jiři Dohnal, Josef Jampílek , Anna Řezáčová

Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences,

Palackeho 1-3, 612 42 Brno, Czech Republic, E-mail: [email protected]

Dissolution testing in R&D often requires more creative approach than the classical USP methods. For these purposes, our team has deisgned an artificial digestive system – Golem, for biorelevant dissolution testing. The apparatus simulates physiological human gastrointestinal conditions and it constitutes a powerful tool for simulation of physiological dissolution of oral dosage forms, especially when used with biorelevant dissolution media. The lecture provides a brief description of the Golem system and an example of its use in practice.

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GLP determination of equilibrium solubility with special interest of ionization

and polymorphism

Krisztina Takács-Novák

Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary

The solubility is one of the key parameters determining the gastrointestinal absorption, suitable aqueous solubility is a precondition of oral administration of drugs. Determination of solubility is inevitable part of physico-chemical profiling of drug candidates in DD&D. Remarkable development of different experimental methods has been achieved in the last decade including both kinetic and equilibrium solubility measurement. Primary goal was to develop HT capacity, material saving and cost effective methods and less attention was devoted to the reliability, precision, reproducibility of the measured data, all in all to the GLP of the measurement. This presentation gives an overview of the state-of-the-art of methods, summarizes the actual guidelines of the authorities and points out the shortcomings of the present practice. Talk will review the recent experiences in solubility measurements using a new protocol of saturation shake-flask method [1]. Examples will be presented about the pH-dependent solubility determination of ionizable drugs, about the validity of HH equation [2] and the molecular interactions may cause deviations from the theoretical curve. The solubility of different salt forms (hydrochloride, mesylate, fumarate, salicylate, etc) of organic bases and the common ion effect will be discussed. Through interesting examples the importance and difficulties of solubility determination of polymorphs will be introduced. Finally some useful guidelines for good laboratory practice of logS measurements will be presented. References: [1] E. Baka, J.E. Comer, K. Takács-Novák. Study of equilibrium solubility measured by saturation

shake-flask method using hydrochlorothiazide as model compound. J. Pharm. Biomed. Anal. 46 (2008) 335-341.

[2] G. Völgyi, E. Baka, K. Box, J.E. Comer, K. Takács-Novák. Study of pH-dependent solubility of organic bases. Revisit of Henderson-Hasselbalch relationship. Anal. Chim. Acta 673 (2010) 40-46.

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Techniques for investigating supersaturation and the enhancement of solubility

John Comer

Sirius Analytical Ltd., UK At the IAPC Conference in 2009, we presented results from our techniques for determining the extent and duration of supersaturation of ionisable drugs, and for dissolution experiments under simulated GI conditions. This talk will build on these earlier results, focusing on new research. Studies will be described in which precipitation of a number of drugs was delayed, and supersaturation was enhanced by the addition of polymers. The studies will show how a suitable proportion of drug: polymer can be identified to provide a good balance between solubility enhancement and the avoidance of heavy precipitation. The results have led to further developments in the theory of chasers and non-chasers to characterise compounds with a tendency to amorphous behaviour. GI Dissolution experiments will be described in which concentration of a dissolving (or precipitating) drug is monitored during several, increasing pH bands to simulate passage through the GI tract. These will include new experiments in which lipids were used to simulate the gut’s capacity for absorption, in the presence of simulated GI fluids, and with polymers added to simulate drug formulations. These experiments provide a practical in vitro method for modelling drug dissolution behaviour in the human or animal GI tract.

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Dissolution of nanocrystalline particles for IV route: in vitro studies and in silico modelling

Guillaume Louit, Xavier Pepin, Morgane Vacher

Sanofi-Aventis R&D, Vitry sur Seine, France

In the last decades, nanocrystalline suspensions have had a growing share in commercial oral formulations as they can increase the bioavailability for poorly soluble drugs. This approach is also of interest for IV route, and even though no product is on the market yet, examples of these applications can be found at the preclinical or clinical stage [1]. Sterile Solid Nanosuspensions for IV have the potential of administering high drug loads to humans in a package that contains very small amounts of excipients, thereby avoiding dose limitations due to classical solubilizer related toxicity experienced with this route of administration. The underlying biopharmaceutical aspects of nano- or polydisperse solid drug formulations have been well studied for the oral route [2-3], but are much less documented for IV. At least two phenomena can be anticipated with suspensions: (1) the particles must be stable in blood (no aggregation) and (2) the particles should dissolve fast enough to avoid capture by the immune system. In this study we have focused on nanocrystalline suspension dissolution, performing in vitro measurements and in silico calculations. Evaluation of dissolution in real blood flow is extremely challenging, as there are no fully satisfying separation and analysis methods to date that would allow good separation of small crystals and released active molecule in the presence of proteins and cells in a timeframe of seconds. As a consequence, by analogy with the oral route our first step was to measure and explain solubilisation in simpler and well controlled media. Firstly we will introduce the model we have chosen for dissolution based on stagnant film layer theory and the principles of ab initio numerical resolution for a suspension for which solubility & granulometry is known. We will discuss parameters that will influence dissolution of nanoparticles. Secondly, we will present results of in vitro dissolution of nanosuspensions for 3 different poorly water soluble molecules; along with different size distributions and formulations, in simple media constituted of water-cosolvent or albumin containing phosphate buffer. While we have observed very different dissolution kinetics depending on the molecule properties and particle size distribution, the correlation of in silico and in vitro models was reasonably good. Discrepancies between the models will also be discussed. As it is the ultimate aim of dissolution studies, we will finally discuss how to anticipate the solubilisation in blood.

References: [1] J.Wong, A. Brugger, A. Khare, M. Chaubal, P. Papadopoulos, B. Rabinow, J. Kipp, J. Ning,

Suspensions for intravenous (IV) injection: A review of development, preclinical and clinical aspects, Adv. Drug Deliv. Rev. 60 (2008) 939–954

[2] M. T. Crisp a, C. J. Tucker b, T. L. Rogers, R. O. Williams III, K.P. Johnston, Turbidimetric measurement and prediction of dissolution rates of poorly soluble drug nanocrystals, J. Contr. Rel. 117 (2007) 351–359

[3] A. Okazaki, T. Mano, K. Sugano, Theoretical Dissolution Model of Poly-Disperse Drug Particles in Biorelevant Media, J. Pharm. Sci. 97 (2008), 1843-1852

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Structure-based QSAR studies on 5-HT2A partial agonists

Stefan Dove, Maria E. Silva*, Ralf Heim, Andrea Strasser, Sigurd Elz

Univ. Regensburg, Inst. Pharm., 93040 Regensburg, Germany *Paracelsus Med. Univ., Inst. Pharmacol. Toxicol., Strubergasse 21, 5020 Salzburg, Austria

Following a hierarchic approach, fragment regression analysis (FRA), homology modeling of receptor structures, docking and 3D-QSAR methods were applied to a series of 51 5-HT2A receptor partial agonists, arylethylamines (primary or benzylamines) from different structural classes (indoles, methoxybenzenes, quinazolinediones). The data, pEC50 values and intrinsic activities (Emax) on rat arteries, show high variability of pEC50 from 4 to 10 and of Emax from 15% to 70%. FRA identifies substructures which affect potency or intrinsic activity. The high contribution of halogens in para position of phenethylamines to pEC50 indicates a specific hydrophobic receptor pocket. Furthermore it can be concluded that hydrogen bonds of the aryl moiety account for activation and that benzyl groups increase affinity and reduce intrinsic activity. Results from FRA and data on all available mutants were considered for the prediction of a common binding site at the rat 5-HT2A receptor. A homology model based on the β2-adrenoceptor structure was generated and checked by MD simulations. Typical derivatives serving as templates for the structural classes were docked into this model. The docking poses indicate common interactions, e.g., with serines in transmembrane segments TM3 and TM5 and with a cluster of aromatic amino acids in TM5 and TM6. Subsequently all other compounds were docked in poses corresponding to the templates. The complexes were minimized, leading to a structure-based alignment of the whole series. The pEC50 values correlate with the docking energies and the hydrophobicity of the aryl moieties. Based on this alignment, 3D-QSAR approaches (CoMFA and CoMSIA) were performed with a training set of 36 and a test set of 15 compounds. The correlation of pEC50 with steric, electrostatic, hydrophobic and H-bond acceptor fields is reflected by sufficient fit (q2: 0.75 - 0.8, r2: 0.92 - 0.95) and predictive power (r2

pred: 0.85 - 0.88). The important interaction regions resulting from the 3D-QSAR analyses largely reflect the patterns provided by the putative binding site. Most notably, the fit of the aryl moieties and benzyl substituents to two distinct hydrophobic pockets becomes evident. In conclusion, the application of different QSAR and modeling approaches has led to common and reasonable hypotheses on agonist 5-HT2A receptor interactions.

This work was supported by the Graduate Training Program (Graduiertenkolleg) GRK 760, “Medicinal Chemistry: Molecular Recognition - Ligand-Receptor Interactions”, of the Deutsche Forschungsgemeinschaft.

Reference: [3] M.E. Silva, R. Heim, A. Strasser, S. Elz, S. Dove, Theoretical studies on the interaction of partial

agonists with the 5-HT2A receptor, J. Comput. Aided Mol. Des. 25 (2011) 51-66.

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Modeling Biorelevant Solubility and Dissolution: Effects of Molecular Ionization

Michael B. Bolger, Robert Fraczkiewcz, Marvin Waldman, Dechuan Zhuang, Walter S. Woltosz

Simulations Plus, Inc., 42505 10th Street West, Lancaster, CA 93534, USA

Purpose To build in silico models based on our chemically diverse database of solubilities for 160 drugs and drug-like molecules in simulated gastrointestinal fluids for the stomach, fasted state small intestine, and fed state small intestine. Such models should have greater relevance to in vivo conditions used in gastrointestinal simulations. Methods All but two of the 160 drugs and drug-like compounds were obtained in their free base or free acid form. Atorvastatin calcium and butoconazole nitrate were the two exceptions. Equilibrium molar solubilities in fasted state simulated intestinal fluid (FaSSIF), fed state simulated intestinal fluid (FeSSIF) and fasted state simulated gastric fluid (FaSSGF) were determined using the familiar 24 hour shake-flask method at 37o C. We compared the accuracy of literature-based equations for the influence of octanol/water partition coefficient (logP) on the change in equilibrium water solubility due to the addition of sodium taurocholate (S. Mithani, 1996) to our models built using the model-building functions of ADMET Predictor™ (Simulations Plus, Inc.). We also tested the influence of molecular descriptors for ionization calculated at the experimentally observed final pH value. Descriptors tested for their ability to improve the accuracy of the literature-based equations were fraction cationic (FCation), anionic (FAnion), zwitterion (FZwitter), and average net formal negative charge (QAveNeg). Results The original Mithani equation for the log of solubilization ratio (log SR = 2.234 + 0.606 log [P]) provided reasonably good estimates of solubility in biorelevant media (for all molecules: RMSE = 0.61, r2 = 0.80). Addition of the descriptors FCation, FAnion, and QAveNeg to a multiple linear regression resulted in an improved model of log SFaSSIF (RMSE = 0.44, r2 = 0.88). Application of the model-building methods in ADMET Predictor resulted in a purely in silico artificial neural network ensemble (ANNE) model (S+FaSSIF) with similar performance (average test set RMSE = 0.48, r2 = 0.77). Conclusions A rich and chemically diverse database of solubilities for drugs and drug-like molecules can provide a good training set for in silico estimates of solubility in biorelevant media. Hydrophobicity, degree of ionization, and charge type have a significant influence on the ability of biorelevant media to increase the solubility of drug molecules. These effects can be incorporated into the dissolution equations used in oral absorption simulation programs like GastroPlus™. Reference: [1] Mithani S., Pharm. Res. 13(1) (1996) 163

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The relationship study of Clog P and aphicidal activity of cyantraniliprole analogs

HuiBin Yang, Hua Chen, JunFeng Wang, Bin Li

Shenyang Research Institute of Chemical Industry Co., Ltd, State Key Laboratory of the Discovery and Development of Novel Pesticide, Shenyang 110021 China

Cyantraniliprole, as a ryanodine receptor activator, is the second anthranilamide insecticide developed by DuPont. Cyantraniliprole is characterized by its excellent insecticidal activity, broad pest spectrum across numerous crops and low toxicity to mammals. It is known that certain ranges of simple parameters (molecular mass, logP, hydrogen-band donors and acceptors) have been correlated with pesticidal activity. Based on Clog P calculated by computer, a series of cyantraniliprole analogs were designed, synthesized and screened for aphicidal activity. The relationship of Clog P and aphicidal activity was summarized as Figure 1. The Clog P of compounds with potent aphicidal activity was between 2.0 and 2.4.

Figure 1. Structure-aphicidal activity relationships of cyantraniliprole analogs

Acknowledgement: We gratefully acknowledge the support of this work by Key Projects in the National Science & Technology Pillar Program during the 12th Five-Year Plan Period (Grant No. 2011BAE06B01), the National Program on Key Basic Research Project (973 Program) (Grant No. 2010CB735601) and the LiaoNing Province Natural Science Foundation (Grant No. 20102181).

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IS-CE: A fast high-throughput method for pKa determination by capillary electrophoresis

Martí Rosés, Joan Marc Cabot, Elisabet Fuguet, Clara Ràfols

Departament de Química Analítica, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona

The determination of acidity constants is of main interest in the pharmaceutical industry. Many potential drugs are weak acids or bases, and their use in further studies strongly depends on the value of certain physicochemical parameters such as the acidity constant, hydrophobicity, and solubility. In most instances, these parameters must be determined in a fast way for many substances. Thus, high-throughput methods are required. The internal standard (IS) is a fast high-throughput method for determination of acidity constants by capillary electrophoresis (CE). The IS-CE method is based in the use of internal standards of pKa similar to that of the test compound. Mobilities of standards and test are measured in two pH buffers, where they are partially or totally ionized, respectively. The difference between the pKas of test and standard is easily calculated from the ratios of the difference mobilities between test and standard. The IS-CE method has all the advantages of CE methods: low sample and solvent consumption, sample impurities allowed, not exactly known sample concentration required, and universal measurement and high-throughput technique. In addition, the number of measuring points is much lower in the IS-CE method and the pH does not need to be exactly known. In fact, two CE runs are enough for each pKa. With appropriate standards, the method also minimizes the interactions of test compound with the buffers. This communication describes the routine implementation in physicochemical measurement laboratories of the IS-CE as a high-throughput method for pKa determination of drugs. References: [1] E. Fuguet, C. Ràfols, E. Bosch, M. Rosés, A Fast Method for pKa Determination of Drugs by

Capillary Electrophoresis, Chem. Biodiversity 6 (2009) 1822-1827 [2] E. Fuguet, C. Ràfols, E. Bosch, M. Rosés, A fast high throughput method for the determination

of acidity constants by capillary electrophoresis. 1. Monoprotic weak acids and bases, J. Chromatogr. A, 1216 (2009) 3646–3651

[3] J.M. Cabot, E. Fuguet, C. Ràfols, M. Rosés, Fast high-throughput method for the determination of acidity constants by capillary electrophoresis. 2. Acidic internal standards, J. Chromatogr. A 1217 (2010) 8340-8345

[4] E. Fuguet, C. Ràfols, M. Rosés, A fast high throughput method for the determination of acidity constants by capillary electrophoresis. 3. Basic internal standards, J. Chromatogr. A (2011), doi: 10.1016/j.chroma. 2011.04.054

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NMR studies of peptides and proteins and their interactions: implications for drug development

Horst Kessler

Institute for Advanced Study and Center of Integrated Protein Science, Technische Universität München, Department Chemie, Garching, Germany

and Chemistry Department, Faculty of Science, King Abdul Aziz University,

Jeddah 21589, Saudi Arabi

NMR is one of the most powerful methods in drug development [1,2]. It can be used for screening (e.g. using 19F NMR for identification of hits) [3] but also for determination of structures in solution (constitution, configuration, conformation) under nature-like conditions and especially for the investigation of dynamic interactions. Peptides are perfect starting points for the development of drugs targeting proteins or nucleic acids as peptides are already natural signaling molecules (e.g. peptide hormones) or can be easily identified from peptide libraries. After introduction of conformational restraints via cyclization their NMR structure is used for the rational development of peptidomimetic drug candidates. NMR is especially suited to investigate protein-protein interactions under native-like conditions. Inhibitors of these interactions are potential lead structures for development of candidates. In the presentation the modern methodology of the conformational analysis of cyclic (bioactive) peptides is presented. Pitfalls in the application of NMR to smaller molecules are indicated. Protein NMR allows the identification of internal flexibility and of intermolecular interactions sides. Often, X-ray structures of proteins facilitate structure determination, but not all proteins are stable enough to yield crystals of sufficient quality for X-ray, whereas NMR can solve structures of labile proteins or protein domains, can identify dynamic parts within the protein for engineering stable proteins and allows a direct study of protein complexes. A recent example is given by the interaction of the tumor suppressor p53 with the chaperon Hsp90 [4].

References: [1] M. Pellecchia et al., Perspectives on NMR in Drug Discovery: A Technique Comes of Age, Nat.

Rev. Drug Disc. 7 (2008) 738-745. [2] H. Kessler, J. Klages, NMR in Drug Discovery (Vol.3, p.901-920) in “Comprehensive Medicinal

Chemistry II, Elsevier, Oxford 2006. [3] J. Klages, M. Coles, H. Kessler; NMR-Based Screening: A Powerful Tool in Fragment-Based Drug

Discovery; in “Exploiting Chemical Diversity for Drug Discovery” (Eds.: P. A. Bartlett, M. Entzeroth); RSC Publishing, Cambridge, 2006, Chapter 12; p. 263-290 and Mol.BioSyst., 2 (2006) 318-332 or Analyst 132 (2007) 693-705.

[4] F. Hagn, S. Lagleder, M. Retzlaff , J. Rohrberg, O. Demmer, K. Richter, J. Buchner, H. Kessler, Structural analysis of the interaction of the heat shock protein 90 with the tumor suppressor protein p53, Nature Struct. Mol. Biol. 2011 in press.



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Functional mechanisms of drugs studied by NMR: methodology and applications

Teresa Carlomagno

European Molecular Biology Laboratory, Heidelberg, Germany

Small molecules play a fundamental role in the regulation of the function of proteins, nucleic acids and molecular machines. The development of specific binders that selectively alter the function of only one or a few cellular targets relies on the availability of structural information for the target active site and its mode of interaction with low affinity ligands, identified for example in screening experiments. When this structural information is not available, the rationale design of a selective drug is impossible and the process of drug development has to rely on the screening of large libraries of molecular fragments accompanied by many, lengthy, parallel routes of chemical synthesis. Recently we have developed a new NMR methodology, INPHARMA[1,2] which provides access to the relative binding mode of low-affinity ligands to a common target. The method is based on the observation of interligand, spin diffusion mediated, transferred-NOE data, between two ligands A and B, binding competitively and weakly, to a macromolecular receptor T. In accordance with existing SBDD workflows, the experimental information derived from the INPHARMA NOEs is currently used to select the correct binding mode among many possible binding orientations obtained by molecular docking [3]. The binding poses of L1 and L2 result from docking the bound conformations of the ligands, obtained by transferred-NOEs or transferred-CCR rates, to a structural model of the apo-receptor T. These binding poses are ranked by evaluating the agreement between the theoretical INPHARMA NOEs calculated for each pair of TL1 and TL2 complex models with the experimental values. The method was originally developed to derive the binding mode of L2 when the binding mode of L1 is known (relative binding mode). However, we have recently demonstrated that the INPHARMA approach is much more powerful and, in favourable cases, can allow the de novo description of the binding mode of both L1 and L2 (absolute binding mode). In [3] we have shown that the methodology is precise enough to unambiguously select one docking mode per ligand and accurate enough to select the correct docking mode. In this work, the INPHARMA method was applied to a pair of ligands binding the catalytic subunit of the Protein Kinase A (PKA). The availability of crystal structures for this system has allowed us to verify that the INPHARMA methodology is able to select the correct docking poses (those corresponding to the crystal structures) from a pool of 200 docking poses per ligand generated by docking the ligands to the structure of the apo-protein. The INPHARMA method allows closing a gap in structure based drug discovery, where, commonly, crystal structures of some but not all interesting chemical lead series are accessible. The bound ligand structure, easily obtainable from transferred-NOE data, and a structural model of the protein, which are used to generate plausible docking modes, are the only requirements for the application of the method. Here we will show the principles of INPHARMA and benchmark its performance with respect to the parameters and information used in the evaluation of the data. In addition, we will demonstrate the application of INPHARMA to the design of ligands targeting membrane proteins and tubulin. [1] V. M. Sanchez-Pedregal et al., Angew. Chem. Int. Ed. Engl. 44 (2005) 4172. [2] M. Reese et al., Angew. Chem. Int. Ed. Engl. 46 (2007) 1864. [3] J. Orts et al., Angew. Chem. Int. Ed. Engl. 47, (2008) 7736.

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Determining the orientation of drugs in receptors by solvent paramagnetic relaxation NMR spectroscopy

Simone Kosol, Christoph Göbl, Katarina Čuljak*, Walter Hohlweg, Predrag Novak*, Klaus Zangger

Institute of Chemistry, University of Graz, Austria *Department of Chemistry, University of Zagreb, Croatia

A detailed knowledge of receptor-ligand interactions is important in the process of rational drug design. NMR spectroscopy is often employed for screening compound libraries and, in cases of weak ligand-receptor interactions, for the determination of binding epitopes. Here we present a new solution state NMR approach for obtaining the orientation of a drug in a receptor. Upon the addition of an inert paramagnetic agent to the solvent, relaxation enhancements on ligand nuclei depend on the insertion depth in the protein. If the ligand is in fast exchange between the free and bound state, these solvent paramagnetic relaxation enhancements (solvent PREs) are partly transferred to the free ligand where they can be observed with high resolution and without any size limitation of the receptor. The information obtained about the orientation of the ligand with respect to the receptor could be used for guiding docking calculations, provided that the three-dimensional atomic resolution structure of the receptor is available. Identifying which parts of a drug are buried deepest in the receptor can provide clues about the site on the ligand where chemical modification most likely results in an altered binding strength. Solvent PREs have also been used to study the binding and orientation of macrolide antibiotics to membrane mimetics. These studies could rationalize a side effect of macrolide antibiotics- phospholipidosis. Macrolide antibiotics induce phospholipidosis by reducing the activity of phospholipase A1 towards phosphatidylcholine in membranes. Our results indicate that macrolides inhibit this function via reducing the amount of free phosphatidylcholine through membrane binding.

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The importance in drug design of resolving structural and dynamic details for membrane drug targets at the nanoscale

Anthony Watts

Biomembrane Structure Unit, Biochemistry Department, Oxford University, Oxford, OX1 3QU, UK.

It is now possible to resolve local dynamics within a membrane bound protein at near physiological conditions in natural membrane fragments or in reconstituted complexes, using solid state NMR approaches [1]. This information is obtained by isotopically (2H, 13C, 19F, 15N, 17O) labeling selective parts of either a ligand or the protein understudy, and observing the nucleus in non-crystalline, macromolecular complexes.

Ligands with complex structure have differential mobility at their binding sites. Substituted imidazole pyridines, for example, which inhibit the H+/K+-ATPase and have therapeutic use, are constrained in the imidazole moiety, but shows significant flexibility at the pyridine group (see figure). It is this group which has a direct interaction with an aromatic (phe198) residue, with the potential for π-electron sharing. Similarly, the steroid moiety of ouabain undergoes motions which are similar to those of the target protein, the Na+/K+-ATPase, but the rhamnose undergoes a high degree of flexibility at fast rates of motions whilst interacting with Tyr198. For acetyl choline

when bound in the nicotinic acetyl choline receptor (nAChR), the quaternary ammonium group undergoes fast rotation at an aromatic binding site, which is driven by thermal fluctuations which may be functionally significant. Our current focus is on GPCRs, specifically the brain neurotensin receptor (NTS1) for which the structure (by single molecule cryo-EM) and ligand binding interactions are being studied. Reference: [1] Watts, A. Solid state NMR in drug design and discovery, Nature Drug Discovery, 4 (2005) 555-

568 See also: www.bioch.ox.ac.uk/~awatts/

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Statistically optimized Raman microscopy for detecting low levels

of undesired polymorphic forms in low dose active tablets

Slobodan Šašić, Mark Whitlock*, Shawn Mehrens

Pfizer Global Research and Development, Groton CT 06340 USA

*Pfizer Global Research and Development, Sandwich CT13 9NJ UK Many active pharmaceutical ingredients (APIs) have excellent Raman responses that can allow for identification of low levels of undesired polymorphic forms in finished solid-dosage products. By mapping the entire surface of the tablet with a Raman microscope (Raman mapping instrument) through a grid with a very large number of points, one may be able to detect even significantly less than 1% w/w of a sought API polymorph in a tablet. When the laser beam hits a region containing the undesired form, a Raman spectrum having detectable spectral signature of that form should emerge. However, the number of points in the grid should not be arbitrarily set: if it is too large, unnecessarily long experiments are carried out, and if it is too small, the particles of interest may be missed due to insufficient number of scans. This study proposes a statistically based sampling method to determine the optimal Raman mapping experiments for the described purpose. It uses two statistical charts to determine the probability of detecting an event as a function of the number of attempts (i.e. number of spectra / pixels in the image in this case), and assess the number of experiments needed to confirm a hypothesis. An investigation has been carried out with an active formulation with the undersired polymorphic form varying from 0.36 to 1.04% w/w and with the total API at approximately 5% w/w. The number of Raman spectra collected in each of these experiments was determined by using the first chart above and in all of the evaluated samples the undesired API form was clearly recognized. Repeating the experiments confirms the hypothesis that proposed mapping with the pre-determined number of spectra is a reliable approach for the optimal use of the instrument with minimal risk of erroneous results. The described sampling method is then applied to the real tablets with only 1% w/w API that are spiked with 2.5 to 10% of various polymorphs relative to the desired form. All of these polymorphs have been detected, with identifying 0.025% w/w of the amorphous form in the tablet being particularly worth mentioning. The commercial packaged tablets on stability are then analyzed by the same method and none of the polymorphs has been detected.



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14N NQR study of polymorphism of piroxicam and nifedipine; characterization of their polymorphs and solid-solid transformations

Zoran Lavrič, Janez Pirnat*, Janko Lužnik*, Zvonko Trontelj*, Stane Srčič

University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia *Institute of Mathematics, Physics and Mechanics, Ljubljana, Slovenia

NQR spectroscopy with its specific advantages is on the way to become an analytical tool in pharmaceutical application. The method is based on coupling of local electric field gradient tensor (EFG) with electric quadrupole moment tensor of nucleus, like for instance 14N. These nuclei are present in most active pharmaceutical ingredients (API) [1]. Since the EFG is a result of the ordering of molecules with distribution of their electrons in solid lattice, the method was proved to be very suitable for the study of APIs polymorphism [2]. The first objective of the presented study was to use 14N NQR as a way to qulitatively and quantitatively characterize different solid state forms of piroxicam and nifedipine [3]. Thermodynamically stable forms (piroxicam I and nifedipine I) of said substances were obtained on the market. Subsequently, samples of unstable amorphous nifedipine and piroxicam II, III and X (newly discovered) were prepared. DSC, ATR FT-IR, Raman and XRPD were used to identify differences between the samples. 14N NQR method was then used with the same samples to (re)confirm the already established polymorphic states [3]. 14N NQR method proved itself capable of accurately and precisely determining the solid-state forms of studied pure compounds. The possibility of accurately determining the mixture composition of different forms was also demonstrated. Mixtures of piroxicam form I and monohydrate in different ratios were prepared and then measured with 14N NQR. So determined mixture ratios were in close agreement with the known values. In the second part the dynamics of solid-solid transformations of said prepared forms was examined. Piroxicam II, III and X were exposed to incresed temperatures (140-160°C). Sub-sequent transformations were then followed with 14N NQR. Results indicate that form II transformed directly to form I, wheras form III and X transformed to form II and then to I. Nifedipine glassy state was heated for a definite time at different temperatures (from 60 to 160 °C) and the transformation to the crystalline form I was followed with 14N-NQR. Contrary to the expectations, by lower temperatures the glassy state was not 100% transformed to the crystalline form. The reason for this incomplete recrystallization is the subject of ongoing work. Results show that 14N NQR could be used not only for a determination of a specific crystalline state of a compound, but also as a tool that provides information about the kinetics involved in the solid-solid transformation processes.

References: [1] S.C. Pérez, L. Cerioni, A.E. Wolfenson, S. Faudone, S.L. Cuffini, Utilization of pure nuclear

quadrupole resonance spectroscopy for the study of pharmaceutical crystal forms, Int. J. Pharm. 298 (2005) 143-152.

[2] Z. Lavric, J. Pirnat, J. Luznik, J. Seliger, V. Zagar, Z. Trontelj, S. Srcic, Application of 14N NQR to the Study of Piroxicam Polymorphism, J. Pharm. Sci. 99 (2010) 4857-4865.

[3] F. Vrecer, M. Vrbinc, A. Meden, Characterization of piroxicam crystal modifications, Int. J. Pharm. 256 (2003) 3-15.

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Challenging characterization of polyphasic drugs. Use of combined

physicochemical techniques in pharmaceutical development

Danielle Giron

Consultant, 3614 route de Montoulieu, F- 33190 St Bauzille de Putois, France As a result of the advance in high throughput technologies and modeling, it is now possible to evaluate a large number of drug candidates. The interface between drug discovery and development is very important to optimize the candidate selection considering the physicochemical properties needed for an optimal development of the targeted drug product. The selection of a proper salt for ionizable drugs increases the possibility to obtain the desired solid state physicochemical properties. The ability of organic substances to uptake solvent or water in their crystal lattice lead to different compounds. Depending of water activity in the solvent of crystallization or of granulation, in the formulation , in the air, depending of temperature, pressure, several stable hydrates have to be considered. Organic substances may show supersaturation behaviour and metastable solid phases outside the domain of stability fixed by phase diagrams may occur. Conversions of stable forms and metastable forms during processing, and ageing with kinetic factors lead to concomittant phases. Therefore API are polyphasic solid phases with stable thermodynamic windows according to phase diagrams which can be studied and predicted and metastable states which need to be properly detected. Basic thermodynamic rules are needed for solid state physico chemistry [1]. Thermoanalytical methods [2,3] are the best for this challenging task. Modern instrumentation allows the combination of these techniques with spectroscopic, gravimetric, X-ray diffraction even in situ with temperature, humidity. Case studies will show the complementarity of several techniques to detect the potential forms, to select a specific form and monitor it for its introduction to the market in a stable formulation. References: [1] D. Giron, Solid-State Physicochemistry, in Comprehensive Medicinal Chemistry, Volume 5,

ADME-Tox Approaches, B.Testa, H.van de Waterbeemd, Elsevier, 2007 , pp. 509-530. [2] D. Giron, Thermal Analysis and calorimetric methods in the characterization of polymorphs

and solvates, Thermochimica Acta, 248 (1995) 1-59. [3] D. Giron, Thermal Analysis and microcalorimetric methods in the industry: essential

techniques for proper development of pharmaceuticals, European Pharmaceutical Review, 5 (2006) 84-90.



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Hydrates: Thermodynamics and Determination of Relevant Parameters

Rolf Hilfiker

Solvias AG, Department of Solid-State Development, Römerpark 2,

4303 Kaiseraugst, Switzerland Many compounds can crystallize both as hydrates and anhydrous forms. Often the hydrate form is the optimal form for pharmaceutical development. It is a delicate decision whether the anhydrate or the hydrate should be developed to a drug product, though, and both the kinetics and thermodynamics of hydrate formation have to be investigated. In this presentation we will focus on methods which allow a precise determination of thermodynamic parameters (critical water activity) and show how they relate to practically relevant properties such as solubility, intrinsic dissolution rate and methods to produce highly pure forms.

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The determination of dehydratation mechanism of lactose monohydrate by using thermogravimetric methods

Petar Tudja, Ernest Meštrović*

Pliva-Croatia Ltd., Generics Research and Development, Zagreb *Pliva-Croata Ltd., TAPI Research and Development, Zagreb

The stability of pharmaceutical product is a prerequisite for the application that must be met unconditionally. Degradation of the drug can trigger many external conditions (temperature and humidity in which the mixture is kept, radiation, chemical incompatibility with excipents) or may be the result of presence of thermodynamically less stable form of the pharmaceutical substance in final dosage form. Kinetic studies help us to understand the substance decomposition processes. The dehydratation processes are part of degradation processes. In this process compounds are losing water and changing its physical and chemical properties. In this work lactose monohidrate is used as a model substance for research of dehitratation procesesses. Lactose monohydrate is one of most used excipeient in preparation of pharmaceutical dosage form. On the basis of measurements included in this study, comprehensive physicochemical characterizations are made on lactose monohydrates which include thermal methods (DSC and TGA), XRPD, IR and dynamic water sorption (DVS). Furthermore, lactose monoydrate was subjected to isothermic and non-isothermic thermogravimetric experiments for understanding the process of dehydration. The four different methods (including modulated thermogravimetry MTGA) are used to determine activation energy. The lactose monohydrate was found to belong to hydrate with an isolated lattice sites (classification according to K. Morris [1]), WET type 4 (classification according to Galway[2]) and show the best fit to models F1 (zero-order reaction model), B1(Prout-Tompkins nucleation model) and D3 (3-D diffusion- Jander equation). References: [1] K. R. Morris,”Structural aspect of hydrates and solvates in “Polymorphism in Pharmaceutical

Solids” (H.G.Brittain Ed.). Drugs and the Pharmaceutical Sciences, Vol 95 Marcel Dekker, New York (1999) 125-181

[2] A.K.Galwey, Thermochim. Acta 355 (2000) 181-238.

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Solid-state chemistry and new drug forms

Elena V. Boldyreva

Institute of Solid State Chemistry & Mechanochemistry SB RAS, Novosibirsk, Russia;

Research and Education Centre REC-008 “Molecular Design and Ecologically Safe Technologies”, Novosibirsk State University, Novosibirsk, Russia;

[email protected]

The contribution reviews the results of using the concepts and achievements of solid-state chemistry for design and preparation of new drug forms, with a special emphasis on obtaining new forms without modifying the molecular formula of the active pharmaceutical ingredient. Examples will be based mainly on the research done in REC-008 Novosibirsk State University and the Institute of Solid State Chemistry and Mechanochemistry SB RAS and cover the following topics: 1) control of polymorphism of pharmaceutical substances; 2) pharmacetical co-crystals; 3) obtaining pharmaceutical compounds as amorphous solids; 4) solubilization of drugs; 5) control of particle size and shape distribution for pharmaceutical solids; 6) "dry" synthesis of pharmaceutical compounds, and their composites with excipients; 7) "dry technologies" of extracting biologically active compounds from natural resources and/or preparing their composites with excipients. Acknowledgements: This work was supported by grants from CRDF (RUX0-008-NO-06), RF Ministry of Education and Science (2.2.2.2/10470), RFBR (10-03-00252, 11-03-00684, 11-03-12114-оfi-m-2011), Projects 5.10, 21.44 and 5.6.4 of the Russian Academy of Sciences. .

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Study of the Role of Molecular Mobility in Physical Stability of Amorphous of Anti-Inflamantory Drug: Celecoxib

Marian Paluch, Katarzyna Grzybowska, Żaneta Wojnarowska, Kia L. Ngai*

Institute of Physics, UniVersity of Silesia, ul. Uniwersytecka 4,

40-007 Katowice, Poland *3CNR- IPCF Associate, Dipartimento di Fisica, Università di Pisa,

Largo Bruno Pontecorvo, 3, I-56127 Pisa, Italy Broadband dielectric spectroscopy was employed to monitor the molecular mobility both in superccoled and glassy state of celecoxib. A number of relaxation processes were detected and their molecular origin and temperature characteristics were established. We found that the structural relaxation time at the temperature of storage of amorphous celecoxib, T=293 K, correspond to the time of maximum rate of recrystallization of amorphous celecoxib at this temperature. Thus, it was concluded that physical stability of the studied amorphous drug is controlled mainly by the structural relaxation process. Herein we also indicate that the secondary relaxation process which originates from intermolecular interactions might be responsible for devitrification of celecoxib. Finally, we also provide a method how to efficiently improve a stability of amorphous celecoxib. Acnowledgment: The authors are deeply thankful for the financial support of their research within the framework of the project entitled “From Study of Molecular Dynamics in Amorphous Medicines at Ambient and Elevated Pressure to Novel Applications in Pharmacy” (Contract No. TEAM/2008-1/6), which is operated within the Foundation for Polish Science Team Programme cofinanced by the EU European Regional Development Fund.

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Tailor-made batch crystallisation of Entacapone through the use of self-

assembled layers on gold single crystal surface

Ana Kwokal, Kevin J. Roberts*

PLIVA Croatia, Ltd., Prilaz baruna Filipovića 29, 10000 Zagreb *Institute of Particle Science and Engineering and of the Process Research and

Development, University of Leeds, LS2 9JT Leeds, UK

The solution crystallisation of the pharmaceutically active ingredient Entacapone; (E)-2-Cyano-N,N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl) propenamide has been examined with the aim of controlling the process through the use of a defined surface as nucleation template and in-line analytics tools. A Self-Assembled molecular Layer of Entacapone (SALE) on a gold single crystal surfaces, has been prepared in order to serve as defined surface nucleating agent ("seed") for tailor-made batch crystallisation. The SALE has been characterised in regard to the chemical identity, the thickness and the surface organisation. The basic crystallisation parameters in acetone as well as in the mixed acetone/water solvent system were determined. The solid state behaviour of Entacapone polymorphs were examined revealing acetone solution yielded Entacapone form A (thin plate crystal), while acetone/water solvent system yielded form D as a needle like crystals. The adsorbed layer of Entacapone i.e. SALE template in the acetone solution has shown the ability of altering the morphology of the crystals toward more desirable crystal shapes. While in the acetone/water solution SALE acted like an array for assembling the nucleus within the whole bulk solution toward specific crystal polymorph of the form A [1]. Quiescent crystallisation from the acetone/water solution in the presence of bare gold surface immersed vertically, concomitantly yielded single crystals of form A at the gold and form D in the bulk. When gold was electrically polarised, form α crystallised at the gold surface. The binding of Entacapone onto gold was revealed through the study of surface functionality of Entacapone single crystals that have been epitaxially grown at gold surface. The results afford an opportunity for future advancement of the tailor-made crystallisation at the surface and in the bulk solution using self-assembled molecular templates as heterogeneous nucleation catalyst. Reference: [1] A. Kwokal, T.T.H, Nyguen, K.J. Roberts, Polymorph-directing seeding of Entacapone

crystallisation in aqueous/acetone solution using a self-assembled molecular layer on Au (100), Cryst. Growth Des.4 (2009) 4324-4334.

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Physical insight into tautomerization reaction in some pharmaceutically important molecules

Żaneta Wojnarowska, Marian Paluch

Institute of Physics, UniVersity of Silesia, ul. Uniwersytecka 4, 40-007 Katowice, Poland

There are a number of chemical compounds that readily convert to other isomers when their crystalline structure is lost (e.g., during melting or dissolution). This phenomenon is known as tautomerization reaction [1]. The tautomerization process becomes an important problem especially in pharmaceutical industry because a still growing interest in amorphous drugs, characterized by better solubility and bioavailability, is observed. Recently, it has been shown that preparation of the amorphous pharmaceutical, in which tautomerization reaction takes place, leads to the state which is chemically different than its crystalline counterpart. This phenomenon can have positive as well as negative impacts on the quality of the amorphous drug. On the one hand, one can expect that presence of the tautomerization products may affect the drug solubility, and consequently it may enhance the bioavailability of the active substance. Moreover, in many cases the occurrence of two or more tautomers in the sample may strongly reduce the tendency to the drug re-crystallization [2]. On the other hand, tautomerization of drugs might lead to serious medical consequences, since one cannot exclude that biological activity of the given tautomers may be completely different than that expected [3]. Consequently, inactive tautomers may cause a number of drug side effects. In this presentation we will show that broadband dielectric spectroscopy (BDS) can be successfully used for investigation of tautomerization process of various pharmaceutically important molecules. These kinds of measurements give us a possibility to monitor the kinetic of tautomerization process as well as to determine the activation energy of this reaction. The obtained experimental data will be compared with computational simulations.

Acnowledgment: The authors are deeply thankful for the financial support of their research within the framework of the project entitled “From Study of Molecular Dynamics in Amorphous Medicines at Ambient and Elevated Pressure to Novel Applications in Pharmacy” (Contract No. TEAM/2008-1/6), which is operated within the Foundation for Polish Science Team Programme cofinanced by the EU European Regional Development Fund.

References: [1] M. B. Smith and J. March, Advanced Organic Chemistry, 5th ed. (Wiley Interscience, New York,

2001), pp. 1218–1223 [2] Z. Wojnarowska, P. Wlodarczyk, K. Kaminski, L. Hawelek, M. Paluch, M. J. Chem. Phys. 133

(2010) 094507. [3] R.J. Iuliucci, J. Clawson, J.Z. Hu, M.S. Solum, D. Barich, D.M. Grant, C.M. Taylor, Ring-chain

tautomerism in solid-phase erythromycin A: evidence by solid-state NMR Solid State Nucl Magn Reson. 24(1) (2003) 23-38.

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The influence of glycosylation to biological activity of proteins

Gottfried Pohlentz*, Kristina Neue*, Anne Zeck**, Jörg Thomas Regula**, Jasna Peter-Katalinić*,***

*Institute for Medical Physics and Biophysics, University of Muenster, Germany **Proteinanalytics, Biologics Research (pRED), Roche Diagnostics GmbH,

Penzberg, Germany ***Department of Biotechnology, University of Rijeka, Croatia

The carbohydrate part of glycoproteins is involved in a variety of functions including the proper folding and the quality control of newly synthesized glycoproteins in the endoplasmic reticulum and control the serum lifetime of glycoproteins. Glycosylation as a highly complex and dynamic process is usually reflected in the biosynthesis of multiple glycoforms of the same protein by either incomplete glycosylation or processing of the oligosaccarides. An improved protocol for identification of protein glycosylation by a direct nanoESI MS and MS/MS analysis of ZIC-HILIC-enriched proteolytic glycopeptides without additional separation or purification is here presented, in which the proteolytic process is performed by unspecific proteases like thermolysin, elastase, or a trypsin/chymotrypsin mixture [1]. Glycopeptides produced contain short peptide backbones, thus improving the ZIC-HILIC-based separation. The combination of unspecific proteolysis, glycopeptide separation, and their direct MS analysis is highly suitable fast, facile, and inexpensive procedure for monitoring glycosylation patterns at single glycosites of natural and recombinant glycoproteins. A number of therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification which may lead to the generation of products with low efficacy. The naturally present glycosylation repertoire of mammalian host cells promotes the generation of a number of different terminal structures and thus leads to heterogeneous glycosylation. To monitor the functionally relevant glycosylation at Asn-162 site of the recombinant human leukocyte receptor IIIa (hFcγRIIIa) which plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies, glycosylation analysis has been applied to a site directed comparative carbohydrate analysis of hFcγRIIIa derived from human embryonic kidney (HEK) and Chinese hamster ovary (CHO) cells [2]. It could be shown that the difference in the activity of hFcγRIIIa expressed by HEK and CHO cells, respectively, could be assigned to distinct patterns of fucosylation, sialylation and antennarity at the Asn162-glycosite, although the protein sequence and the site of glycosylation remained unchanged for both types of cells.

References: [1] S. Henning, M. Mormann, J. Peter-Katalinić, G. Pohlentz, Identification of single glycosites in

glycoproteins by unspecific protease digestions and HILIC enrichment of glycopeptides, J. Proteome Res. (2011) - submitted

[2] A. Zeck, G. Pohlentz, T. Schlothauer, J. Peter-Katalinić, J. T. Regula: Cell Type-Specific N-Glycosylation Pattern of FcγRIIIa Asn, J. Proteome Res. 10(7) (2011) 3031-3039

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Mass spectrometric approaches for structural elucidation of “misfolding”: aggregating neurodegenerative proteins: Ion mobility- MS and affinity- MS

Camelia Vlad, Claudia Cozma, Marilena Manea, Marius Iurascu, Kathrin Lindner, Jieun

Jung, Martin Scheffner, Marcel Leist, Michael Przybylski

Departments of Chemistry and Biology, and Graduate School Chemical Biology, University of Konstanz, 78457 Konstanz, Germany

A large variety of cellular processes are based on the formation and dynamics of multi/supramolecular protein assemblies, and several diseases, previously thought to be unrelated, such as cancer and neurodegenerative diseases, are characterised by “misfolded” protein aggregates. Chemical structures and reaction pathways of pathophysiological aggregates are only poorly characterised and understood at present. “Soft-ionisation” mass spectrometry, particularly electrospray-MS, have led to substantial progress in the characterisation of protein structures and their modifications; however, HPLC-ESI-MS is often unsuitable to direct analysis of reaction pathways and intermediates in protein assembly. Ion mobility mass spectrometry has recently emerged as a new molecular tool to probe protein assemblies from solution structures, due to its potential for analyzing distinct mixture components by conformation state and topology. Recent applications of IM-MS to in vitro oligomeric products of alpha-synuclein and ß-amyloid (Aß), key proteins for Parkinson’s disease and Alzheimer’s disease, enabled hitherto unknown aggregation, truncation and modified products to be separated and their structures identified. The structures obtained provide a basis for (i), the detailed study of aggregation pathways; (ii), the design of peptides capable to inhibit or modify aggregation; and (iii), the evaluation of immuno-therapeutic lead structures. Recently, the development and application of affinity-mass spectrometry enabled the identification of epitope- specific Aß-antibodies that either disaggregates Aß-plaques, or physiological, neuroprotective Aßautoantibodies that inhibit the formation of Aß- aggregates. These results indicate ion mobility- MS and affinity- MS as powerful complementary tools for the molecular elucidation of structures and intermediates of polypeptide aggregates. Applications to hitherto uncharacterized aggregating proteins, such as key enzymes for lysosomal storage diseases, seem promising in future studies. [1] McLaurin, J., et al. Nature Med. 8 (2002) 1263-1269; [2] Macht, M., et al. Anal. Bioanal. Chem. 378 (2004) 1102-1111; [3] Stefanescu, R., et al. Eur. J. Mass Spectrom. 13 (2007) 69-75; [4] Przybylski, M. et al./Univ. Konstanz & Budapest (2008) Eur. & US Patent Applications.



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Biophysical methods for identifying and characterizing protein-ligand interactions

Geoff Holdgate

AstraZeneca, Discovery, DECS, Macclesfield, Cheshire, UK Over the past few years biophysical methods have been used increasingly to identify and characterize protein-ligand interactions of importance in drug discovery. Several biophysical methods have been used as primary screening approaches, especially in relation to fragment-based lead generation (FBLG). Biophysical fragment binding has also been used to complement traditional high-throughput screening (HTS) methods, in so called fragment assisted lead generation (FALG), where the aim is to identify optimal initial chemistry start points. Some of these same methods have also been applied post HTS as orthogonal screens in order to evaluate the HTS output, so that wasted effort on following up false positives may be reduced. Additional to this role in identifying test compounds which bind specifically to the target of interest, biophysical methods also have been utilized in measuring the affinity, thermodynamic and kinetic properties of binding ligands. Thus, biophysical methods, coupled to structural information, have been utilized to provide a thorough understanding of binding interaction and to facilitate the chemistry driven optimization process. This presentation will describe the importance of protein-ligand interactions in drug discovery and will illustrate how biophysical methods have been employed during the hit to lead process.

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Dynamic Force Spectroscopy for studying high affinity ligand receptor complexes

Pierre Parot, Jean-Luc Pellequer Service de Biochimie et Toxicologie Nucléaire, Laboratoire Interactions et Reconnaissance

Moléculaires (LIRM), CEA Marcoule, BP 17171 30207 Bagnols sur Cèze Cedex, France Dynamic Force Spectroscopy (DFS), based on Atomic Force Microscopy (AFM), and software development are very powerful methods for analyzing long lifetime ligand–receptor interactions. We have chosen the well-known system, (strept)avidin–biotin complex, as an experimental model due to the lack of consensus on interpretations of the rupture force spectrum. We present new measurements of force–displacement curves for the (strept)avidin–biotin complex. These data were analyzed using the YieldFinder software based on the Bell-Evans formalism. In addition, the Williams model was adopted to interpret the bonding state of the system. Our results indicate the presence of at least two energy barriers in two loading rate regimes. Combining with structural analysis, the energy barriers can be interpreted in a novel physico-chemical context as one inner barrier for H-bond ruptures (γ <1Å), and one outer barrier for escaping from the binding pocket which is blocked by the side chain of a symmetry-related Trp120 in the streptavidin tetramer. In each loading rate regime, the presence of multiple parallel bonds was implied by the Williams model. Interestingly, we found that in literature different terms created for addressing the apparent discrepancies in the results of avidin–biotin interactions can be reconciled by taking into account multiple parallel bonds. This study brings new perspectives to design high affinity ligand receptor complexes.



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TRANSIL High Sensitivity Binding Assay: A novel tool for assessing plasma binding of hardly soluble and sticky drugs

Hinnerk Boriss, Kathleen Boehme

Sovicell GmbH, Deutscher Platz 5b, 04103 Leipzig, Germany, [email protected]

Partitioning approaches, like erythrocyte partitioning, have been used over several decades to assess plasma binding of drugs that prove difficult to handle in dialysis or ultrafiltration devices. However, these methods have never gained great popularity since they are cumbersome as well as time and resource demanding. Here we present validation data for an assay that is easy to use, scalable to various needs of automation, and generates results in less time than dialysis. The assay is based on a binding equilibrium shift method to determine the affinity of drugs to plasma. By providing pre-determined ratios of membrane surface areas and plasma concentrations, the partitioning between membrane and plasma results in a series of different equilibria, which in turn define the affinity to plasma. In a standard configuration the assay allows accurate measurement of unbound fractions of at least 0.0001%. At the same time recovery typically remains above 70%, because all assay compartments contain plasma which stabilizes highly lipophilic and barely soluble drugs in solution. For a set of ten less lipophilic and well soluble drugs we demonstrate that the assay yields identical unbound fractions of drug in plasma as dialysis. While for drugs like cyclosporine, the assay yields results comparable to the ultracentrifugation method. We therefore conclude that the assay yields more reliable results for tightly bound and hardly soluble drugs than dialysis or ultrafiltration.

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Chromatographic and spectroscopic studies on the chiral recognition of sulfated β-cyclodextrin as chiral mobile phase additive:

Enantiomeric separation of a chiral amine

Shengli Ma, Sherry Shen, Nizar Haddad,Wenjun Tang, Jing Wang, Heewon Lee, Nathan Yee, Chris Senanayake, Nelu Grinberg

Chemical Development Department, Boehringer Ingelheim Pharmaceuticals, Inc., 900

Ridgebury Road, Ridgefield, CT 06877-0368, USA A fast enantiomeric separation of a chiral aromatic amine was achieved, using ultra high pressure liquid chromatography and highly sulfated beta-cyclodextrin (S-β-CD) as a chiral additive in the mobile phase. The stationary phase consisted of a core–shell support with a particle size of 2.7 μm. Under these conditions the base-line separation was obtained within 2.5 min. The influence of the concentration of the additive, along with the thermodynamics of the separation, was studied. Vibrational circular dichroism (VCD) spectroscopy was applied to assess the absolute configuration of the two enantiomeric analytes, as well as the interaction of these enantiomers with the S-β-CD. The VCD results revealed that S-β-CD undergoes a temperature-induced conformational change. Further, VCD experiments indicate that the interactions of the two enantiomers with the S-β-CD occur through an inclusion of the aromatic part of the analyte, as well as through electrostatic interaction between the protonated amine and the sulfate groups located at the narrow part of the S-β-CD. Molecular mechanics calculations performed according to the VCD results are consistent with experimental data, providing further evidence of these interactions.

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Automated Patch Clamp in Drug Discovery – from the bench to the midfield

Morten Sunesen

Sophion Bioscience A/S, Denmark.

Bringing compounds from research to drug stores is a cumbersome, extremely slow and highly costly process and in recent years much attention has given the various bottlenecks existing in the cascade of bringing new compounds from R&D to clinical phases. For ion channel drug discovery, patch clamp provides a direct and very reliable recording of ionic currents over the cellular membrane – as such a very valuable tool for understanding the effect exerted by compounds directed against ion channels. However, patch clamp was traditionally very slow with throughputs in the range of 2-5 compounds tested per working day. With the advent of automated patch clamp (APC) in early 2000s throughput was increased dramatically. Today hundreds to thousands compounds can be tested on a daily basis – virtually removing a significant bottleneck in ion channel drug discovery. During this talk, a backward looking approach will be taken to describe some of the biological and technical hurdles faced by the field of APC during the early years. This should provide a positive platform to discuss trends and challenges for the future of APC in drug discovery.

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Synergistic and antagonistic effects between selected phenolic

and polyphenolic antioxidants

Ewelina Kowalewska, Grzegorz Litwinienko

University of Warsaw, Faculty of Chemistry, Pasteura 1, Warsaw, Poland

Oxidative stress is involved in most of human diseases [1]. Antioxidants can prevent oxidative damage and alleviate its effects, thus, a progress of a number of diseases including atherosclerosis, chronic inflammatory diseases, Parkinson’s, Alzheimer’s, cancer is claimed to be delayed in the presence of antioxidants. Patients often use antioxidant supplements, many of which contain polyphenolic chain-breaking antioxidants as active constituents. A pair of antioxidants can demonstrate significantly greater or smaller activity than could be expected from the sum of activities for compounds used separately and the effects are described as synergy or antagonism of antioxidants, respectively. Mechanism of this phenomenon for various pairs of phenolic compounds still remains unclear and requires detailed investigation. As we reported previously, mechanism of antioxidant action can be dependent on polarity of a solvent because of partial ionization of phenols [2-4]. Hence, in this work we provide the results considering the pH influence on antioxidant synergy and antagonism between selected flavonoids and flavonolignans with PMHC (analogue of α-tocopherol). The antioxidant activity was evaluated on the basis of oxygen depletion during the autoxidation of methyl linoleate emulsions initiated by water-soluble 2,2’-azobis(2-amidinopropane) dihydrochloride (ABAP) at pH range from 4 to 10. The process was monitored by Clark-type oxygen electrode. We observed a significant dependence of synergistic and antagonistic effects on pH, which will be discussed in detail together with possible mechanisms. References: [1] H. Sies, Oxidative stress, Academic Press, London, 1985 [2] M. Musialik, G. Litwinienko, Scavenging of dpph• radicals by vitamin E is accelerated by its

partial ionization: the role of sequential proton loss electron transfer, Org. Lett. 7 (2005) 4951-4954.

[3] G. Litwinienko, K. U. Ingold, Solvent effects on the rates and mechanisms of reaction of phenols with free radicals, Acc. Chem. Res. 40 (2007) 222-230.

[4] M. Musialik, R. Kuzmicz, T. S. Pawlowski, G. Litwinienko, Acidity of hydroxyl groups: an overlooked influence on antiradical properties of flavonoids, J. Org. Chem. 74 (2009) 2699-2709.

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Compressibility investigation of chewing gum bases used for API containing direct compressed chewing gum tablet preparation

Peter Kása jr., Imre Jojart, Klára Pintye-Hódi

University of Szeged, Department of Pharmaceutical Technology, Hungary

The chewing gum has a more than two thousand years history, but after the first medicated chewing gum (Aspergum, 1928) this pharmaceutical dosage form was approved into Pharmacopoeia in 1991 only. The preparation of chewing gums can be different from the general melting method to the direct compression. The benefit of direct compression method is well known in the case of tablets. The preparation time is shorter but it needs a free flowing and well compressible materials. Nowadays there are different gum bases are available on the market which is directly compressible. With these products it is very easy to develop medicated chewing gum tablets. In this work the authors investigated different gum bases such as Pharmagum C, M, and S. These materials are suggested to use for direct compressed preparation of chewing gum tablets. It was investigated all the physical properties of the bases, focused on the flowing, compactibility, and compressibility properties. For the compressibility test a Korsch EK0 instrumented eccentric tabletting machine was used. There were prepared different gum base mixtures with an Ascorbic acid as a model drug. It was found that the flowing and compressibility properties of Pharmagum C and M not good enough for preparing direct compressed preparations. The Pharmagum S increases all the investigated properties. The authors investigated the breaking process of the preparations too. Comparise with the conventional tablets these preparations shows completely different breaking process, because after reaching the maximum breaking force the tablets did not break into two parts, but just crash down, and than shows a plastic properties. With the increase of the compression force, the properties of the prepared tablets did not make significant changes neither in the physical properties nor the API dissolution. The difference was found only in the breaking time. As conclusion, it was found that the gum bases are good for preparing direct compressed chewing gum tablets. The compressibility properties shows that it is necessary to combine the gum bases for increasing some properties, but it is not necessary to use a high compression forces during preparation.

Acknowledgement: The Project named „TÁMOP-4.2.1/B-09/1/KONV-2010-0005 – Creating the Center of Excellence at the University of Szeged” is supported by the European Union and co-financed by the European Regional Development Fund.

www.nfu.hu www.esza.hu

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Dynamics of enzymes-inhibitor complexes from protein crystallography

J. Hašek, K. Zimmerman*, T. Skálová, J. Dušková, J. Dohnálek

Institute of Macromolecular Chemistry, AS CR, 16206 Praha 6, Czech Republic *MIG INRA, 78352 Jouy en Josas cedex, France

Results of protein crystallography (PX) are usually interpreted by enthalpy terms only (the numbers and/or strengths of intermolecular interactions). In spite of the fact that the PX experiment contains full information on the dynamics in the binding site, a straight interpretation of the PX results in terms of entropy is rare. It is a serious obstacle when trying to predict potential efficiency of newly designed drugs “ab initio”. To cope with the problem, new procedures including the new type of the “water perturbated multisolution refinement of protein structure”, and new concepts of the „shock energy absorbing molecular fragments” and the “energy dissipation elements” were used to deduce both the enthalphy and entropy terms directly from PX. Systematic X-ray analysis of complexes of HIV protease and its mutants (A71V, V82T, I84V) and (L63P, A71V, V82T, I84V) with a series of chemically very similar inhibitors (Ki in range from 10 pM to 400 μM) lead to 12 structures deposited in the PDB. Also, several dynamical experiments with forced withdrawal of inhibitors gave some qualitative insight into the process of inhibitor release from the active site [1-6]. The observed changes of Ki by several orders of magnitude [3] induced by the enzyme mutations and the inhibitor tuning were newly explained by: 1. enthalpy terms related to interaction energy of protein-inhibitor complex, i.e.

strengths of H-bonds, π-π interactions, water removal during inhibition, etc. 2. entropy related terms connected with dynamics of the system: • conformational flexibility and mobility of bulky side chains within the inhibitor region

increase stability of the complex, • the shock energy absorbing blocks built in the inhibitor molecule decrease the chance

of inhibitor to escape from binding site, • mutations along the binding site lowering a number of low-energy metastable states

decrease the entropy of the protein-inhibitor complex, • the distant mutations (here, stiffeness of the β-sheet induced by A71V) can modulate

low frequency vibration modes changing thus dissipation of energy, • the multi-step mechanism of the inhibitor binding and releasing found by molecular

dynamics simulations increases stability of the system.

The research was supported by 305/07/1073 and IAA 500500701.

References: [1] Dohnálek J. et al. (2002) J. Med. Chem. 45, 1432-1438. [2] Skálová T. et al. (2003) J. Med. Chem. 46, 1636-1644. [3] Petroková H. et al. (2004) Eur. J .Biochem. 271, 4451-4461. [4] Hašek J. et al. (2005) Acta Crystallogr. A61, C242-243. [5] Skálová T. et al. (2006) J. Med .Chem. 49, 5777-5784. [6] Dušková J. et al. (2006) Acta Crystallogr. D62, 489-497.

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38

Online SAW-Bioaffinity- Mass Spectrometry: New Bioanalytical Application in

Detection, Structure Determination and Quantification of Protein-Ligand Interactions from Biological Material

Michael Przybylski

Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, University of Konstanz, Germany

In recent years, bioaffinity analysis using biosensors such as surface plasmon resonance have become established techniques for the detection and quantification of biomolecular interactions; however, a severe limitation of biosensors is their lack of providing structure information of affinity-bound biopolymer ligands. Bioaffinity- mass spectrometry is a new, combined analytical approach for the detection, quantification, and structure determination of affinity-bound ligands. We have developed an online combination of a surface acoustic wave (SAW) biosensor with electrospray ionization mass spectrometry (SAW-ESI-MS) that enables the direct structure determination and quantification of affinity-bound ligands dissociated from a protein-ligand complex on a gold chip [1]. An interface for the coupling of SAW-biosensor chip and ESI- MS provides a sample concentration and in-situ desalting step for the MS analysis of the ligand eluate solution. First applications of the online SAW-MS combination with chip- immobilized Ig-antibody and polypeptide ligands show broad bioanalytical applications to the simultaneous, label-free structure determination and quantification of biopolymer- ligand interactions as diverse as antigen-antibody and lectin- carbohydrate complexes. Dissociation constants (KD) are determined in the milli- to nano- molar range directly from biological material, e.g. cell lysate and brain homogenate. Identification of molecular ligand recognition structures and antibody-epitopes are feasible for a broad range of biopolymer interactions, as diverse as antigen-antibody and lectin- carbohydrate complexes. Reference: [1] Dragusanu M. et al. (2010) J. Am. Soc. Mass Spectrom. 21, 1643-1647.

S 1

– Se

min

ar

Poster Presentations


Page 41

Characterization of topical delivery systems by in vitro release

and permeability testing

Maja Radić, Marjana Durrigl, Biserka Cetina-Čižmek

PLIVA CROATIA Ltd., Generics Research and Development Zagreb, Zagreb, Croatia Saturated dicarboxylic acid, our chosen active substance, has been traditionally formulated as a cream, but has been recently formulated as water-soluble gel used for skin therapy. In order to determine drug release and permeability characteristics in vitro release method was developed. During drug development in vitro release test is often used for optimization of topical delivery system which has to provide maximum therapeutic effect, to control product during development and production, and its performance over the time. Drug could penetrate into skin layers after topical application. Therefore, once the final formulation has been selected and optimized it has to be evaluated for efficacy. It is important to know where topically applied drug penetrates; which quantity remains on the skin surface or penetrates into the stratum corneum and viable skin, and which quantity is finally absorbed into the bloodstream. [1] A Franz diffusion cell apparatus was used for in vitro release method development as well as for permeability testing. Permeability studies were performed using pig skin mounted on Franz diffusion cell and additionally, using EpiSkin model (SKINETHIC) obtained by culturing human keratinocytes with terminal differentiation and the reconstruction of an epidermis. In vitro release method parameters were defined based on the release behavior of active substance from gel formulations, with special attention on membrane selection as well as the receptor medium composition. Permeability experiments conducted on EpiSkin model showed comparable drug concentration in the receptor compartment obtained from cream and different gel formulations. Comparable amounts of the drug in the receptor compartment released from gel and cream formulation were also found using pig skin. When this model was used, it was found that significant amount of the drug from gel remained in the viable skin. It could be due to the ability of the gel vehicle to more effectively deliver the drug into the skin. Similar permeability values were obtained for different gel formulations. In vitro release and permeability testing of topical delivery systems combining different skin models gave us better understanding of drug delivery based on formulation characteristics. References: [1] Z.D. Draelos, Cosmetic Dermatology, 16(11) (2003) 56-58

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Investigating alkyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxoazepan-1-yl)hexanoates as potential transdermal penetration enhancers

Kateřina Brychtová, Josef Jampílek, Lenka Dvořáková, Radka Opatřilová,

Jiři Dohnal

Department ofChemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 61242 Brno, Czech Republic,

E-mail: [email protected] Transdermal penetration enhancers are special pharmaceutical excipients that interact with skin components to increase penetration of drugs from the topical dosage forms to blood circulation. Numerous compounds have been evaluated as penetration enhancers and many potential sites and modes of action have been identified for them [1]. This is a follow-up contribution to our previous articles; synthesis, physico-chemical properties, 3D structures and in vitro transdermal penetration-enhancing activity of the below mentioned compounds derived from 6-aminohexanoic acid [2,3] are considered herein. Modification of the ester part of the molecule with linear alkyl chains is aimed at increasing penetration/carrier activities.

NO

R

O

O

N

O

R = -C6H13, -C7H15, -C8H17, -C9H19, -C10H21, -C11H23, -C12H25

O

This study was supported by the Czech Science Foundation (GACR P304/11/2246) and by IGA VFU Brno 20/2010/FaF. References: [1] J. Jampilek, K. Brychtova, Azone analogues: Classification, design, and transdermal penetration

principles, Med. Res. Rev. (2011) in press, 31, DOI 10.1002/med.20227. [2] K. Brychtova, J. Jampilek, R. Opatrilova, I. Raich, O. Farsa, J. Csollei, Synthesis, physico-chemical

properties and penetration activity of alkyl-6-(2,5-dioxopyrrolidin-1-yl)-2-(2-oxopyrrolidin-1-yl)hexanoates as potential transdermal penetration enhancers, Bioorg. Med. Chem. 18 (2010) 73-79.

[3] K. Brychtova, R. Opatrilova, I. Raich, D.S. Kalinowski, L. Dvorakova, L. Placek, J. Csollei, D.S. Richardson, J. Jampilek, Investigating the activity of 2-substituted alkyl-6-(2,5-dioxopyrrolidin-1-yl)hexanoates as skin penetration enhancers, Bioorg. Med. Chem. 18 (2010) 8556-8565.

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A Chromatographic Method to determine the

1-Octanol/Water Partition Coefficient (log Po/w) of Drugs

J.M. Pallicer*, M. Rosés*, C. Ràfols*, E. Bosch*, R. Pascual**, C. Calvet**, A. Port**

*Departament de Química Analítica and Institut de Biomedicina (IBUB), Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain **ESTEVE, Av. Mare de Déu de Montserrat, 221, 08041 Barcelona, Spain

1-octanol/water partition coefficient has been widely used to express the lipophilicity of organic compounds, which is a key parameter in the pharmacokinetic behavior of drugs. In previous studies a chromatographic method to determine the 1-octanol/water partition coefficient (logPo/w) was reported. This method allows the determination of the logPo/w from a single chromatographic run combined with several descriptors derived from the structure of the drug. The chromatographic measurements can be performed in conventional HPLC but also in UHPLC conditions without any significant difference in the final results. A set of 34 drugs with different structures and functionalities have been selected in order to validate the new logPo/w determination method by comparison with logPo/w values obtained by the RefinementPro pHmetric method and also by the shake-flask reference method. References: [1] J. Sangster, Octanol-Water Partition Coefficients: Fundamentals and Physical

Chemistry, Wiley, Chichester, 1997. [2] E. Bosch, P.Bou, M. Rosés, Anal. Chim. Acta 229 (1994) 219 [3] R. Bosque, J. Sales, E. Bosch, M. Rosés, M.C. García-Álvarez-Coque, J.R. Torres-Lapasió,

J. Chem. Inf. Comput. Sci. 43 (2003) 1240 [4] J. M. Pallicer, S. Pous-Torres, J. Sales, M. Rosés, C. Ràfols, E. Bosch. J. Chromatogr. A

1217 (2010) 3026. [5] J. M. Pallicer, J. Sales, M. Rosés, C. Ràfols, E. Bosch, submitted for publication.

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Setup and validation of a shake-flask procedure for the determination of the

partition coefficient log D at pH 7.4 using less than 2 mg of drug

Axel Andrés*, Martí Rosés*, Clara Ràfols*, Elisabeth Bosch*, Sonia Espinosa**, Víctor Segarra**, Josep Mª Huerta**

*Departament de Química Analítica and Institut de Biomedicina (IBUB), Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona (Spain)

**Computational and Structural Drug Discovery Department, R&D Centre, Almirall S.A., Laureà Miró 408-410, 08980 Sant Feliu de Llobregat, Barcelona (Spain)

The distribution coefficient between octanol and water, log Po/w, is the most common parameter to express the lipophilicity of drugs. log D7.4 stands for this distribution at pH 7.4, that is at the physiological pH, and it is a key value in the interpretation of pharmacokinetic behavior of drugs. In this work a procedure, based on the shake-flask technique, to determine the log D7.4 of a wide variety of compounds is established and optimized to use a minimum amount of drug. The experimental partition is carried out using aqueous phases buffered with phosphate. Different partition ratios to be used according to the estimated lipophilicity of the compound have been proposed. The analysis of both, aqueous or organic phases, is performed by HPLC and UV detection. Every measurement was carried out using 2 mg of substance at most. A 28-substance set, with a lipophilicity range that went from -2.0 to 4.5 (log D at pH 7.4 values), was chosen to establish and validate the procedure. Experimental values are compared with the reference ones, showing a nice agreement between both series of values. The standard deviation, obtained from different partition ratios, is lower than 0.25 for each compound. References: [1] Sangster J. Octanol-Water Partition Coefficients : Fundamentals and Physical Chemistry. Wiley.

1997. Chichester. [2] BioLoom online database: http://www.davidhoekman.com/bio-loom/bin/details. [3] Stopher D., McClean S., J. Pharm. Pharmacol., 42 (1990) 144

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Physico-chemical profiling of antibacterial fluoroquinolones

Gábor Vizserálek, Gergely Völgyi, Kin Y. Tam*, Krisztina Takács-Novák

Semmelweis University, Department of Pharmaceutical Chemistry, H-1092 Budapest, Hőgyes E. u. 9., Hungary,

*AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK104TG, United Kingdom

Drug absorption from the gastrointestinal tract is infuenced by several biological and physico-chemical factors. Permeation through the membranes of amphoteric compounds containing at least one acidic and one basic functional group is largely determined by the protonation state of the molecule at the given pH and the lipophilicity of individual microspecies. Our aim was to investigate the effect of these physico-chemical properties on the intestinal absorption. Amphoteric fluoroquinolone drug molecules were selected as model compounds. Today, fluroquinolones are one of the most commonly prescribed antibacterial agents. Due to their broad-spectrum they are often used in the treatment of urinary tract and upper respiratory tract infections. Pharmacokinetics of orally administered fluoroquinolones are favorable, and they are well absorbed from the intestine. In this work, the protonation macro- (logK1 and logK2) and microconstants (logkC) of 10 fluoroquinolone molecules used in the therapy (I-IV. generation) were determined by potentiometry and UV/pH titrations. The protonation scheme of norfloxacin is presented here as an example:

N

COO-

O

CH3

N

N

F

H

N

COO-

O

CH3

N

N+

F

HH

N

COOH

O

CH3

N

N

F

H

N

COOH

O

CH3

N

N+

F

HH

kC

kNkC

N

kNC

X-

XH XH2+

K1 K2

Using the determined protonation macroconstants and logkC value, the other microconstants can be calculated as follows:

K1 = kC + kN and K1 K2 = kC kNC = kN kC

N

Their octanol/water partition coefficients at the isoelectric point (logDiep) were also measured. From these data, the logarithm of the true (micro) partition coefficients of nonionized, neutral species (logp) was calculated using the following formula:

logp = logD + log(1+1/kC [H+] + kNC /kC

N + kNC [H+])

The true (micro) partition coefficients will be used to rationalized the passive membrane permeability and intestinal absorption of the selected fluroquinolones.

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Correlation between the lipophilicity and the antioxidant activity of the parabens

Trifon M. Angelov, Elena I. Korotkova*, Ana Vlasenko**

Unipharm JSCo, QC Department, 3. Trajko Stanoev str. 1797Sofia, Bulgaria [email protected]

*Tomsk Polytechnic University, Departmentof Physical Chemistry, 30Lenin Blvd., 634021 Tomsk, Russia

**V.N. Karzin Kharkiv National University, 4. Svobodysq., Kharkiv 61077, Ukrain

Investigations of the lipophilicity and the antioxidant activity of the parabens are very important for their preservative action. The lipophilicty was expressed as log P determinate by liquid/liquid extraction [2], and as log k’ determinate by HPLC [3]. The correlation between lipophilic parameters obtained with both methods was investigated. The antioxidant activity was determinate with differential impulse voltammetry and was expressed as K (Total antioxidant activity coefficients) [1]. Practicall calculations of the lipophilicity of the parabens, and determination of their total antioxidant activity coefficients as K, were performed indicating that relationship between their lipophilicity and antioxidant activity exists. The dependency is linear with excellent correlation, 0.999 for the relationship K/log P, and 0.998 for the relationship K/log k’. At almost twice increasing of the lipophilicity the antioxidant activity increases from methyl to butyl paraben with about 15%.

References: [1] C. Hansch, T. Fujita, A Method for the Correlation of Biological Activity and Chemical Structure,

Journal of the American Chemical Society. 86, (1964), 1616-1626. [2] M. Ramos-Nino, M. Clifford, M. Adams, Quantitative structure activity relationship for the

effect of benzoic acids, cinnamic acids and benzaldehydes on Listeria monocytogenes, Journal of Applied Bacteriology. 80, (1996), 303-310.

[3] E. I. Korotkova, O. A. Avramchik, T. M. Angelov, Y. A. Karbainiov, Investigation of antioxidant activity and lipophilicity parameters of some preservatives, Electrochim. Acta. 51, (2005), 324-332.

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Zwitterionic characteristics of piroxicam and a novel carboxy methylated pyridoindole derivative as expressed in the retention/pH behavior on reversed

phase and ion interaction HPLC

Nikos Triantos*, Milan Stefek**, Anna Tsantili-Kakoulidou*

*Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimiopolis, Zografou, Athens 15771, Greece

**Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Dubravska cesta 9, 841 04 Bratislava, Slovakia

Ampholytes, forming zwitterions, represent a particular type of solutes with intra- and inter-molecular interactions, which influence their physicochemical characteristics, among them lipophilicity. In fact, their partitioning behavior changes as a function of pH resulting in most cases in a bell shaped profile, while a U-shaped profile may also be obtained, depending to a large extent by the tautomeric constant Kz. On the other hand charged molecules can form ion pairs with hydrophobic counter ions. This association may have a dramatic impact in the zwitterionic species formation altering their partitioning profile. Reversed phase HPLC has been widely used to assess lipophilicity, as an alternative of the time consuming direct partitioning experiments. The effect of ion reagents on retention factors in ion interaction chromatography has also been established. In the present study we applied both chromatographic techniques in order to investigate the zwitterionic characteristics of piroxicam and a novel carboxy methylated pyridoindole derivative, designed and synthesized as aldose reductase inhibitor. Retention measurements have been performed over a wide pH range. The mobile phase consisted of different methanol fractions and appropriate buffer. For ion interaction chromatography 0.1 M sodium hexane-sulfonate was added in the mobile phase. Isocratic logk and extrapolated logkw retention factors were plotted versus pH. In the case of piroxicam a bell shaped profile was obtained. Upon addition of hexane-sulfonate, lower retention was observed, as a result of partial disruption of the zwitterionic structure, ion pair formation of the protonated basic center and thereupon exposure of the anion to the environment. In the case of carboxy methylated pyridoindole derivative an irregular logkw/pH profile was obtained, while plots of isocratic logk values versus pH revealed a U-shaped profile. Addition of hexane sulfonate did not produce any significant effect at pH around the isoelectric point. The different behavior of the carboxy methylated pyridoindole derivative in regard to piroxicam, indicates a weaker interaction between the oppositely charged centers in the molecule, which is affected by the interference of the ionized silanol sites on the stationary phase. This interference is more evident in mobile phases rich in methanol, since the water may act as a masking agent. More to the point, in contrast to piroxicam, the carboxy methylated pyridoindole derivative, according to the acidic / basic pKa difference, should be considered to exhibit a rather large Kz, which suggests that a U-shaped profile is also possible.

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Study of pH-dependent drugs solubility in water

Aneta Pobudkowska, Urszula Domańska, Aleksandra Pelczarska

Warszaw University of Technology, Faculty of Chemistry, Department of Physical Chemistry, Poland

Thermodynamic behaviour, including pKa, pH and the solubility of solid drugs in liquid solvents, plays a pivotal role in the design of drug compounds, as well as in the development and optimization of the drug manufacturing processes The rate at which a drug goes into solution, when it is dissolved in a medium is proportional to the solubility of the drug in the medium. Many drugs have different solubilities at different pHs. These pH-dependent solubility differences lead to pH-dependent dissolution profiles. In general, pH-dependent dissolution is an undesirable product characteristic [1, 2]. Solubility and pKa have a profound influence on the transport properties of drug molecules.The pKa values are useful for physicochemical measurements describing the extent of ionization of functional groups with respect to pH. The traditional pH-metric titration method of determining the pKa value is less employed because of the drug’s poor aqueous solubility. The basic thermodynamic properties of pure substances, such as melting point and enthalpy of fusion and of solid−solid phase transition determined by the differential scanning calorimetry (DSC) technique can be used for the correct estimation of the ideal solubility of drug compounds in water and organic solvents [3]. The solubilities of drug-compounds ( for example: Atropine, Bifonazole, Cimetidine, Estrone, Fenbufen, Indomethacin, Nadolol, Nitrofurantoin, Pentoxifylline, Phenylbutazone, Triamterene) have been measured at constant temperature (298.15 K) by the classical saturation shake flask method. The experimental conditions that affect equilibrium solubility values as composition of aqueous buffer, technique and time of separation of two phases and stirring time at certain pH have been examined. The best conditions have been chosen and are recommended for getting the repeatable results. The Henderson-Hasselbalch (HH) relationship has been used to predict the pH-dependent solubility profiles of drugs. References: [1] B. Faller, P. Erlt, Computational approaches to determine drug solubility, Adv. Drug Deliv. Revs.

59 (2007) 533-545. [2] L. Du-Cuny, J. Huwyler, M., Wiese, M. Kansy, Computational aqueos solubility prediction for

drug-like compounds in congeneric series, Eur. J. Med. Chem. 43 (2008) 501-512. [3] U. Domańska, A. Pobudkowska, A. Pelczarska, Ł. Żukowski, Modelling, Solubility and pKa of five

sparingly soluble drugs, Int. J. Pharmaceut. 403 (2011) 115-122.

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Solubility – pH Profiles of Ionizable Compounds

Elham Shoghi, Clara Ràfols, Elisabet Fuguet, Elisabeth Bosch

Departament de Química Analítica and Institut de Biomedicina (IBUB), Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain

Solubility is a key physicochemical property of molecules. An accurate measurement of this parameter is important for many processes, but it is of special relevance in the pharmaceutical industry. In fact, solubility plays a main role in the absorption and other pharmaceutical properties of new drugs, so it must be addressed during the early stages of drug discovery programs in order to avoid the development of molecules with inadequate solubility under physiological conditions [1]. As most drugs are ionizable in aqueous solution, their solubility is pH dependent. Therefore, solubility – pH profiles provide valuable information about molecules which are likely to have problematic pharmacokinetic profiles. For a given compound, the relationship between solubility and pH is provided by the Henderson Hasselbach (HH) equation. To obtain the profiles two parameters of the compound are needed: the acidity constant(s) and its intrinsic solubility (S0). In this work we show the solubility – pH profiles obtained through the respective HH equations of compounds of different nature. Two different methods have been used in order determine the S0 values: the classical shake flask method [2] and the potentiometric CheqSol method [3]. A comparison of the profiles is provided, and good agreement is generally observed. In addition, the effect of the counter-ion of the buffer electrolytes on solubility has also been studied. To this end, two different series of buffers were used to determine solubility at different pH values. Whereas the buffers of the first series were made exclusively of water, HCl and NaOH, so a real interaction was not expected, the buffers of the second series were electrolytes (phosphate, citrate, formiate, acetate, etc. at a given ionic strength) whose counter-ion may affect the solubility of the compounds. Several deviations were observed when the experiemental solubility values obtained with the buffers of the second series were compared with the theoretical profiles. References: [1] H. van de Waterbeemd, H. Lennernäs, P. Artursson (Eds.), Drug Bioavailability, Wiley-VCH,

Weinheim, 2003. [2] EPA, Product Properties Test Guidelines, OPPTS 830.7840, Water Solubility: Column Elution

Method; Shake Flask Method, 1998. [3] M. Stuart, K. Box, Chasing Equilibrium: Measuring the Intrinsic Solubility of Weak Acids and

Bases, Anal. Chem. 77 (2005) 983-990.

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Increasing of drugs water solubility using pectines and their derivatives

Zbyněk Oktabec*,**, Josef Jampílek*, Jiři Dohnal*, Martin Čulen*, Vladimir Král***

*Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and

Pharmaceutical Sciences, Palackeho 1/3, 61242 Brno, Czech Republic e-mail: [email protected]

**Department of Toxicology, Institute of Forensic Medicine and Toxicology, General Teaching Hospital in Prague, U nemocnice 2, 12808 Prague 2, Czech Republic

***Department of Analytical Chemistry, Faculty of Chemical Engineering, Institute of Chemical Technology, Technicka 5, 16628 Prague 6, Czech Republic

It is estimated that about 50% marketed active pharmaceutical ingredients (APIs) and 40% of active new chemical entities (NCEs) identified in R&D screening programs employed by many pharmaceutical companies are poorly water soluble. These poorly soluble molecules are distinguished by incomplete absorption and low, erratic bioavailability. An effective strategy for overcoming low aqueous solubility is to convert the water-insoluble parent drug into a soluble complex with complexation agents or solubilizing excipients. An approach discussed in this paper deals with complexation/encasement of poorly soluble molecules of native pectine salts and various esterified or amidated pectines. Pectin is a structural heteropolysaccharide (complex polysaccharides that contain 1,4-linked α-D-galactosyluronic acid residues) contained in the primary cell walls of terrestrial plants. It is produced commercially as a white to light brown powder, mainly extracted from citrus fruits, and is used in food industry as a gelling agent particularly in jams and jellies. It is also used in fillings, sweets, as a stabilizer in fruit juices and milk drinks and as a source of dietary fibre. In nature, around 80% of carboxyl groups of galacturonic acid are esterified with methanol. The ratio of esterified to non-esterified galacturonic acid determines the behaviour of pectin in applications. The non-esterified galacturonic acid units can be either free acids or salts. The complexation capability of native pectine salts and various esterified or amidated pectines were investigated. Solid adducts were evaluated by spectroscopic methods compared with the pure starting materials that were treated under the same conditions. Generally, it can be concluded that amidated pectine derivatives showed low or medium activity. Native and esterified pectines possessed complexing activity. Some pectine esters with a particular degree of esterification and specific alcohol used for esterification showed a very strong complexing activity for insoluble or poorly soluble drugs.

Patents applications WO/2011/063774 and WO/2011/063775

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Development of biorelevant dissolution method for sparingly water soluble drug product with different polymorphs

Jasmina Turkalj, Anamarija Tomljenović Gluhak, Biserka Cetina-Čižmek

PLIVA CROATIA Ltd., Generics Research and Development Zagreb, Zagreb, Croatia Development of biorelevant dissolution method for drug product with limited solubility has been challange during product development1. Polymorphism of drug substance can have significant influence on solubility and dissolution rate, consequently altering drug absorption form a solid dosage form after oral administration and bioavailability2. The aim of this study was to develop dissolution method relevant for prediction of in vivo drug product behaviour, based on pharmacokinetic properties and physicochemical characteristics of selected drug substance which shows polymorphism. The solubility of both polymorphs of the drug substance, the most stable and metastable, was determined at 37°C by solvent saturated method in physiological pH range (aqueous solutions at pH 1.0 - 6.8), as well as in Simulated Gastric Fluid (SGF). Since the drug substance showed low solubility in aqueous media, an influence of different concentration of surfactant sodium lauryl sulphate (SLS) on solubility has been evaluated. Additionaly, solubility rate of different polymorphs has been determined by intrinsic dissolution at medium pH 1.0. Dissolution testing was performed using paddle apparatus, at rotational speed of 50, 75 and 100 rpm, in 900 ml volume of the following media: 0.1N HCl + 1% SLS, 0.01N HCl, SGF, SGF + 0.5% SLS. Results show pH-dependant solubility. There is no difference in solubility of both polymorphs over a tested range with maximum solubility reached at pH 2.0. Polymorph characterized as the most stable has lower intrinsic solubility rate than polymorph characterized as metastable. Results indicate that dissolution method considered as the most biorelevant for in vivo prediction is using paddle apparatus, with rotational speed of 100 rpm in medium Simulated Gastric Fluid. References: [1] C. Noory, N. Tran, L. Ouderkirk, and N. Shah, Dissolution Technology, February 2000, 16-18. [2] Guidance for Industry - ANDAs: Pharmaceutical Solid Polymorphism (2007)

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Crystal structures and dissolution behaviour of co-crystals of meloxicam with

carboxylic acids

Svetlana A. Myz, Tatyana P. Shakhtshneider, Nikolay A. Tumanov, Elena V. Boldyreva*

Institute of Solid State Chemistry & Mechanochemistry SB RAS, Novosibirsk, Russia

*REC-008 “Molecular Design and Ecologically Safe Technologies” Novosibirsk State University, Novosibirsk, Russia;

[email protected]; [email protected]; [email protected]; [email protected]

Co-crystallization with a suitable co-former is one of the methods of improving the bioavailability of poorly water-soluble drugs. 17 cocrystals of meloxicam with different carboxylic acids were obtained using solvent-drop co-grinding, crystallization from solution, heating of preliminary mechanically activated mixtures of components [1,2]. We argue, that not the meloxicam dimers, as was claimed in [3], but a fragment containing two molecules of meloxicam linked via a carboxylic acid molecule is the primary structure-forming element in all the 2:1 meloxicam: carboxylic acid co-crystals known up to now [4]. All co-crystals of meloxicam with carboxylic acids show an increase in the dissolution rate in comparison with meloxicam [1,2,4]. The supersaturated solution is formed at the initial stage of dissolution of co-crystals, and the supersaturation is preserved for hours. This effect is the most pronounced for the meloxicam-terephtalic acid, for which it significantly exceeds the increase in the dissolution rate and apparent solubility, as compared not only with commercial meloxicam, but also with the more soluble meloxicam samples subjected to mechanical treatment [4]. The improved dissolution of meloxicam co-crystals as compa-red to poorly soluble meloxicam is supposed to be related to the presence of H-bonded meloxicam dimers in the crystals of meloxicam and to the absence of these dimers in co-crystals.

Acknowledgements: This work was supported by grants from CRDF (RUX0-008-NO-06), RF Ministry of Education and Science (2.2.2.2/10470), RFBR (10-03-00252, 11-03-00684), Projects 5.10, 21.44 and 5.6.4 of the RAS.

References: [1] S.A. Myz, T.P. Shakhtshneider, K. Fucke, A.P. Fedotov, E.V. Boldyreva, V.V. Boldyrev, N.I.

Kuleshova, Synthesis of co-crystals of meloxicam with carboxylic acids by grinding, Mend. Commun. 19 (2009) 272-274.

[2] S.A. Myz, N.A. Tumanov, T.P. Shakhtshneider, E.V. Boldyreva, Co-crystals of meloxicam with carboxylic acids, Russian Chem. Bull. (in the press).

[1] M.L. Cheney, D.R. Weyna, N. Shan, M. Hanna, L. Wojtas, M.J. Zaworotko, Supramolecular architectures of meloxicam carboxylic acid cocrystals, a crystal engineering case study, Crystal Growth & Design. 10 (2010) 4401-4413.

[2] N.A. Tumanov, S.A. Myz, T.P. Shakhtshneider, E.V. Boldyreva, Are meloxicam dimers really the structure-forming units in the 'meloxicam - carboxylic acid' co-crystals family? Relation between crystal structures and dissolution behavior, CrystEngComm, under review.

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Development and validation of dissolution tests and analysis methods for Cetraxate HCl capsules and Sodium picosulfate in Korean Pharamceutical Codex

Juyoung Jung*, Chungsik Min*, Minjung Kim*, Sheenhee Kim*, Soonhan Kim*, Dongsup Kim**,Youngja Lee*, Rackseon Seong*

*Center for Food & Drug Analysis, Busan Regional Korea Food and Drug Administration, **Pharmaceuticals and Medical Divices Research Department, National Institute of Food

and Drug Safety Evaluation, Chungcheongbuk-do 363-951, Korea

Dissolution testing is an essential requirement for the development, establishment of in vitro dissolution and in vivo performance and quality control of solid oral dosage forms such as tablets and capsules. High level and intensive analytical method is required to ensure the drug's quality and safety. The most important analytical technique used during the various steps of drug development and manufacturing is high performance liquid chromatography (HPLC). The objective of this study was to set up dissolution specifications of drugs preparation which have no existing dissolution specifications and the analysis method using HPLC system instead of UV measurement method in Korean Pharmaceutical Codex (KPC). Therefore, we selected Cetraxate hydrochloride (HCl) capsules and sodium picosulfate tablets. Cetraxate HCl capsule is an anti-ulcer drug with a mucosal protective effect. Sodium picosulfate tablets is a medicine known as a stimulant laxative. To set up optimal conditions and dissolution standards to carry out the dissolution test, the dissolution profiles of these drugs were executed at the various condition (pH 1.2, 4.0, 6.8 buffer and distilled water). The in vitro dissolution samples were analyzed using validated HPLC methods in this study. Using a C18 column, the best result of cetraxate HCl was obtained at 220 nm with a mobile phase consisting of distilled water: methanol: 0.5M ammonium acetate 50:35:15 (pH 6.0) v/v. And as for sodium picosulfate, the best result was gained at 220 nm with a mobile phase consisting of phosphate buffer (pH 7): acetonitrile 80:20 v/v. The methods showed accuracy, precision, linearity and specificity within the acceptable range. Taken together, the outcome of this study is going to be applied to next edition of Korean Pharmaceutical Codex.

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Validation of Analysis Method to Study the Dissolution Behavior of Acetylcysteine Capsules and Sodium Loxoprofen Tablets

Raeseok Jung, Sanghyeok Sohn, Oksoon Heo, Miae Na, Minchul Kim, Wooyoung Jung, Soyoung Wang, InKyu Kim*

Daejeon Regional Food and Drug Administration, Hazardous Substance Analysis Division, 120 Sunsa-ro, Seo-gu, Daejeon, 302-828, Korea

*Pharmaceutical Standardization Research and Testing Division, National Institute of Food and Drug Safety Evaluation, Chungcheongbuk-do 363-951, Korea

Corresponding Author(s): Raeseok Jung ([email protected]) Dissolution tests are used for the development, establishment of in vitro and in vivo performance, registration and quality control of solid oral dosage forms. It has been used for testing the bioavailability of drugs instead of bioequivalence measurements. The dissolution test methods by HPLC were developed and validated for evaluation of the dissolution behavior of drugs containing acetylcysteine and sodium loxoprofen respectively. The analytical method of HPLC was validated in specificity, linearity, precision and accuracy. And inter-laboratory comparison assays were performed in two labs. The dissolution test described here could be proposed as a pharmacopeial standard to assess the performance of formulations about acetylcysteine capsules and sodium loxoprofen tablets.

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Development of Dissolution testing method for Sobrerol capsules in Korean pharmaceutical codex

E. J. Kim, J. H. Lee , Y.M. Jung, K. H. Sohn*, L. Choi*, I. K. Kim*, D. S. Kim**

Hazardous Substances Analysis Division, Gwangju regional Food & Drug Administration, Korea

*Pharmaceutical Standardization Research and Testing Division, National Institute of Food and Drug Safety Evaluation, Chungcheongbuk-do 363-951, Korea

**Pharmaceuticals and Medical Divices Research Department, National Institute of Food and Drug Safety Evaluation, Chungcheongbuk-do 363-951, Korea

Although the dissolution test can serve as an effective tool of quality control and predictor in vivo performance, there are a number of drugs with no established dissolution specifications in Korean Pharmaceutical Codex (KPC). So, we tried to develop the dissolution testing method of sobrerol capsules among commercially available drug preparations which have no dissolution specifications. To determine the dissolution medium, we studied the dissolution profile observed with various types of dissolution media, pH 1.2, pH 4.0, pH 6.8 buffer solution and distilled water, based on the "Guidelines on Specifications of Dissolution tests for Oral dosage forms" of Korea Food & Drug Administration(KFDA) [1]. The dissolution test was carried out in 900 ml media at 37°C using KP apparatus II(paddle) at 50rpm. The analysis method was studied using HPLC. Chromatography was performed using Capcellpak C18 (5 μm, 4.6×250 mm) as column and monitored at UV 210 nm. The mobile pahse used for elution of sobrerol in distilled water was 45% methanol [2] and that in each buffer solution was acetonitrile/metha-nol/dilstilled water (49/21/170). The developed method was linear (R2>0.999) in the range of 10~130ug/mL, precise (RSD%<2%) and accurate. At pH 1.2, a change of sobrerol was observed, resulting in increased impurity peak in HPLC chromatogram with the passage of time. The dissolution degrees of sobrerol capsules in pH 4.0 and distilled water were approximately 85% and 70% in 2 h, respectively. However, this was increased up to 90% within 90 min in pH 6.8 medium. Similar dissolution profiles were investigated in all manufacturers’ sobrerol capsules on the market. These results suggest that pH 6.8 as the dissolution medium could be suitable to develop the dissolution specification of sobrerol capsules in the revised version of KPC. Moreover, the analysis method was demonstrated to be adequate for quality control of that. References: [1] KFDA, The Korean Pharmacopoeia, ninth ed., Seoul, Korea, 2007. [2] Tao, M.A. Pereira, Quantification of carvone, cineole, perillaldehyde, perillyl alcohol abd

sobrerol by isocratic high-performance liquid chromatography, J. Chr. A. 793 (1998) 71-76.

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Particle characterization in the pharmaceutical industry

Iva Tunjić, Biserka Cetina-Čižmek

PLIVA CROATIA Ltd., Generics Research and Development Zagreb, Zagreb, Croatia

The particle size distribution and morphology of drug substance and excipients are important physical characteristics of the material used to create pharmaceutical products. The size, distribution and shape of the particles can affect bulk properties, dissolution, absorption rates, product performance, processability, stability and appearance of the end product. Techniques used for particle characterization are based on several principles including sieving, electrical sensing zone, laser diffraction, dynamic light scattering, acoustic attenuation and image analysis [1]. Different techniques will measure different properties of the particle and will thus generate different results. Particle-size results obtained through various methodologies are all correct and equally accurate but each method simply might be expressing the results in different terms. Particle-sizing techniques usually measure some one-dimensional property of the particle and relate this to a spherical particle of the same property as that of our irregular particle. Therefore, for irregularly shaped particles, characterization of particle size must include information on particle shape. The aim of this work is to present a variety of methodologies that can be used to deduce information about the shape and size distribution of particles used in drug product development. Methodology of low angle laser light scattering, dynamic light scattering, scanning electron microscopy, optical microscopy, static image analysis and dynamic image analysis is described. Properties measured and information gathered by each technique are explained by experimental results. It is shown that different particle sizing techniques and image analyses give complement results and in order to fully characterize particle size distribution and morphology of pharmaceutical material information of different particle sizing and image analysis should be gathered. References: [1] A.F. Rawle, Analytical tools for suspension characterization, in: A.K. Kulshreshtha, O.N. Singh,

G.M. Wall (Eds.), Pharmaceutical suspensions, Springer, New York, 2010, pp. 177-230.

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Effect of the physico-chemical properties of materials on the properties of direct compressed bi-layer tablets

Tamás Sovány, Kitti Papós, Péter Kása jr., Ilija Ilič*, Stane Srčič*, Klára Pintye-Hódi

Department of Pharamceutical Technology, University of Szeged, Eötvös u. 6. H-6720, Szeged, Hungary

*Department of Pharmceutical Technology, University of Ljubljana, Aškerčeva cesta7 1000 Ljubljana, Slovenia

The use of combination preparations has a lot of advantage in the medication. The patient compliance can be significantly improved with the reducing of the number of necessary preparations, and the fix combinations will provide enhanced therapeutic effect. However a number of problems such as chemical incompatibly, or the need of different dissolution profile for the different drugs can occur. These problems can be solved by the formulation of bi-layer tablets. The small contact surface between the two layers reduces the possibility of chemical interactions, and different dissolution profiles can be provided. Nevertheless the formulation of this dosage form has considerable challenges, such as prohibition of lamination. This necessitates the better understanding, how the physicochemical properties of materials, and their interactions influence the mechanical behaviour of the compressed tablets, which can show considerable differences from single layer tablets. Present study was focused how the differences in the physico-chemical properties of the compressed materials influence the mechanical properties of the compressed tablets. The surface characteristics of materials were determined with optical contact angle measurement and the ratio of the plastic-elastic behaviour of materials was calculated according to the Stamm-Mathis equation based of force-displacement measurements on an instrumented tablet press. The results showed that beside the ratio of the cohesive forces inside a layer and the adhesive force between layers, the presence of shape-locking bindings also plays an essential role in the lamination of tablets. It can be concluded based on these results that with appropriate selection of the excipients and composition an optimal quality and bioavailability of tablets can be assured. Acknowledgements: The work was suported by the SI-17/2009 Hungarian-Slovenian bilateral Science and Technology Transfer project and by the Project named „TÁMOP-4.2.1/B-09/1/KONV-2010-0005 – Creating the Center of Excellence at the University of Szeged” is supported by the European Union and co-financed by the European Regional Fund.

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An investigation on the polymorphism of racemic betaxolol

Teresa M. R. Maria, Ricardo A. E. Castro*, António O. L. Évora, Suse S. Bebiano, M. Ramos Silva**, Hugh D. Burrows, M. Luísa Ramos, Licínia L. G. Justino,

João Canotilho*, M. Ermelinda S. Eusébio

Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal *Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal **Physics Department, University of Coimbra, 3004-516 Coimbra, Portugal

Polymorphism, the ability to originate different crystalline phases with different molecular arrangements and/or conformations is a common phenomenon among active pharmace-utical ingredients, APIs[1]. Different polymorphs are prone to have distinct properties and, actually, the recognition of the impact of polymorphism at the different stages of a drug product life cycle makes the evaluation and characterization of polymorphic forms of drugs an essential and mandatory task[2]. Betaxolol, Figure 1, is a selective β1-adrenergic anta-gonist drug which finds large application in medical practice, for instance in the treatment of arterial and intraocular hypertension. As many β-blockers the betaxolol molecule comprises an isopropyl-amino-2-propanol group attached to an aromatic ring. In the betaxolol molecule the phenyl group is also para-substituted by a 2-(cyclopropylmethoxy)ethyl group. Therefore, in a supramolecular assembly, different kind of intermolecular hydrogen bond interactions involving the hydroxyl, the amine and the ethers groups may be envisaged. Moreover, the betaxolol molecule displays considerable flexibility. Both effects point out the existence of different polymorphic forms, either packing or conformational ones, as expectable. In the present work research is carried out on the polymorphism of racemic betaxolol, using melt crystallization as the means to generate solid specimens. In melt cooling/heating experiments investigatd using a multidisciplinary approach, by differential scanning calorimetry, polarized light thermomicroscopy, infrared spectroscopy, X-ray powder diffraction and magic angle 1H NMR, two metastable phases could be identified: a low ordered, low birrefrigent phase, III, which gives rise on heating to a less symmetrical, less disordered phase, II which melts at T ~ 39 oC. The crystallization of the liquid just obtained originates the stable polymorph, I, Tfus = 69 oC[3]. References: [1] G. P. Stahly, Diversity in Single-and Multiple-Component Crystals. The Search for and

Prevalence of Polymorphs and Cocrystals, Cryst. Growth Des. 7 (2007) 1007-1026. [2] R. Hilfiker, ed., Polymorphism in the Pharmaceutical Industry; Wiley VCH: Weinheim, 2006. [3] J. Canotilho, R. A. E. Castro, M. T. S. Rosado, M. R. Silva, A. M. Beja, J. A. Paixão, J. S. Redinha,

The structure of betaxolol from single crystal X-ray diffraction and natural bond orbital analysis, J. Mol. Struct. 891 (2008) 437-442.

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Figure 1. Molecular structure of betaxololFigure 1. Molecular structure of betaxolol


Page 59

Deciphering Interactions of Macrolide Antibiotics with Membrane-Mimetics by NMR Spectroscopy

Evelyne Schrank, Simone Kosol, Katarina Čuljak*, Christoph Göbl, Klaus Zangger, Predrag Novak*

Institute of Chemistry, University of Graz, Austria *Department of Chemistry, University of Zagreb, Croatia

Macrolide antibiotics, such as erythromycin and azythromycin, have been in widespread clinical use for over 50 years and are effective against Gram-positive and certain Gram-negative microorganisms [1]. They inhibit bacterial protein synthesis by binding to the bacterial ribosome, blocking the exit tunnel through which new polypeptides leave. Peptide synthesis is halted at, typically, the tetrapeptide or pentapeptide stage. As a side-effect macrolides accumulate in lysosomal membranes and induce phospholipidosis by inhibiting phospholipase activity [2]. While there is currently no report that could show that phospholipidosis is detrimental to the effected organism, the onset of phospholipidosis in preclinical testing in animals can delay or even prevent the drug development process. Here we report on interactions of selected macrolide compounds with membrane mimetics using a series of solution NMR spectroscopy techniques. The orientation and location of the drugs in the membrane-mimetic was determined using relaxation enhancements after the addition of the freely soluble and inert paramagnetic agent Gd(DTPA-BMA) [3]. Furthermore, possible interactions of macrolides with phospholipase A1 and phosphatidylserine were investigated. The combined data show that the tested macrolide antibiotics bind close to surface of phosphocholine membrane-mimetics and protect it from lipolytic degradation rather than direct inhibition of phospholipase A1. [1] Macrolide Antibiotics; W. Schönfeld, S. Mutak, Eds.; Birkhäuser Verlag: Basel, 2002. [2] M.J. Reasor, S. Kacew, Exp.Biol.Med. 226 (2001) 825-830. [3] M. Respondek, T. Madl, C. Göbl, R. Golser, K. Zangger, J.Am.Chem.Soc. 129 (2007) 228-5234.

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Highly sensitive NMR system for discovery and structure elucidation of natural

drug leads

Masashi Tsuda, Keiko Kumagai

Science Research Center, Kochi University, Japan *Center for Advanced Marine Core Research, Kochi University, Japan

Marine dinoflagellates of the genus Amphidinium are well-known as a producer of unique cytotoxic metabolites. We have isolated a number of potently cytotoxic macrolide [1,2] and polyketides against cancer cells, from the benthic dinoflagellate Amphidinium species collected off Iriomote Island, Japan. In our research for anticancer drug leads from marine dinoflagellates, we planned to apply the highly sensitive NMR equipped with cold probes for discovery and structure elucidation for new metabolites. HMQC measurement of the extracts is the most convenience for the detection of macrolides. Although the extract contained a large amount of unsaturated fatty acid and their glycerides, the macrolide-maker signals were clearly observed. On the other hand, structure elucidations of submicrogram of samples are possible by using inverse and CH cold probes. In this symposium, we will describe the application of highly sensitive NMR for discovery and elucidation of new cytotoxic macrolides. References: [1] Tsuda, M., Oguchi, K., Iwamoto, R., Okamoto, Y., Kobayashi, J., Fukushi, E., Kawabata, J., Ozawa,

T., Masuda, A., Kitaya, Y., Omasa, K. Iriomoteolide-1a, a potent cytotoxic 20-membered macrolide from a benthic dinoflagellate Amphidinium species. J. Org. Chem. 72 (2007) 4469-4474.

[2] Oguchi, K., Tsuda, M., Iwamoto, R., Okamoto, Y,; Kobayashi, J., Fukushi, E., Kawabata, J., Ozawa, T., Masuda, A., Kitaya, Y., Omasa, K. Iriomoteolide-3a, a cytotoxic 15-membered macrolide from a marine dinoflagellate Amphidinium species. J. Org. Chem. 73 (2008) 1567-1570.

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Investigation of the Entericidins EcnA/EcnB – a membrane-bound toxin-

antitoxin system

Gabriel Wagner, Simone Kosol, Klaus Zangger

Institute of Chemistry/Organic and Bioorganic Chemistry, 8010 Graz, Austria

Toxin-Antitoxin (TA) systems have been found on various plasmids but also on bacterial chromosomes. As the first ones are important for plasmid survival and in turn for antibiotic resistance, structural studies on TA systems hold promise for the design of new antibiotics and might open a new route towards fighting antibiotic resistance. The role of chromosomal TA systems, on the other hand, is still under debate. However, it has been hypothesized that they play a central role in stress response and programmed cell death, amongst others. TA systems consist of a short-lived antitoxin and a stable toxin. An interesting fact about them is that neither sequences nor folds are conserved among toxin or antitoxins, which - combined with the great variability in the combination of different toxin and antitoxin folds – makes them an interesting target for structural studies. TA systems are classified into type I and type II systems, depending wether the antitoxin is an antisense RNA or a protein. Here we investigate a chromosomal TA system from E. Coli consisting of the antidote Entericidin A (EcnA) and Entericidin B (EcnB) acting as the toxin. This TA system is the only one know which can not be chategorized into type I or II as both the toxin and antitoxin are small membrane-bound peptides. Both are small amphipathic lipoproteins, which are activated under high osmolarity conditions in the stationary phase and simultaneously repressed by the osmoregulatory signal transduction pathway. The two peptides are proposed to adopt a α-helical conformation and to bind parallel to the surface of the membrane. As for many membrane-bound peptides structural studies with crystallization techniques are not possible, due to the problem of producing diffracting crystals. Therefore, solution state NMR Spectroscopy is the method of choice for the investigation of small hydrophobic peptides. Knowledge, not only of the structure but also about the interaction with membranes or membrane-mimetic systems, is important to understand the biological function. In order to circumvent line broadening caused by big molecular assemblies, small membrane-mimetic systems like micelles have to be used. As the exact targets of these toxins are not known - though they are supposed to affect bacterial membranes– the determination of the topology of these helical peptides in a micelle would further increase the understanding of the mechanism of this peptidic TA system. Towards this end we have developed a method that uses paramagnetic relaxation enhancements (PREs) caused by the freely-soluble agent Gd(DTPA-BMA), allowing us to determine surface distances for different protons. Additionally, we want to address the important question, how Ecn A counteracts the toxic cell-lytic effects of Ecn B and if this happens via direct interaction between them or via a more complex mechanism.

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The role of cation - π interactions in stabilizing charged peptide residues

inside of biological membranes

Walter Hohlweg, Christoph Göbl, Klaus Zangger

Institute of Chemistry, Department of Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria.

The cation - π interaction in proteins and peptides is a noncovalent binding mode characterised by the interaction of a cation with the polarisable pi electron cloud of an aromatic ring such as that of amino acids Phe, Trp and Tyr. Cations involved include the sidechains of Lys and Arg, cationic ligands or metal ions. Cation - π interactions have been found to occur very frequently in structures found in the Protein Data Bank, with a typical protein displaying several such interactions, and they have been argued to be considered as an important factor in stabilizing protein structure alongside the more conventional hydrogen bonds, salt bridges and hydrophobic effects [1]. Theoretical and experimental studies have shown that cation - π interactions are comparatively strong, ranging from approximately 6 to 12 kcal/mol [2]. Apart from defining protein structure, cation - π interactions have been found to play a role in ligand recognition, for instance in the binding of acetylcholine to the nicotinic acetylcholine receptor, or the binding of serotonin to the serotonin channel 5-HT3AR, both achieving binding through the ammonium moiety of the respective ligand [3]. We specifically investigated the potential role of cation - π interactions in stabilizing charged residues buried inside of biological membranes. We used a variety of model peptides, including peptides from the WALP peptide family, and TM7, a transmembrane helix of the vacuolar V-ATPase in yeast. Employing both “solvent PREs” and conventional NMR spectroscopy, we have identified an arginine residue in V-ATPase TM7, which is buried inside of the biological membrane and seems to interact with two adjacent aromatic residues, probably through cation - π interactions. This finding could aid in unravelling the still elusive mechanism of proton transport in V-ATPases. Our pH titration studies suggest that the arginine residue remains protonated inside of the lipid membrane, stabilized by the cation - π interaction; therefore our research also ad-dresses the open question of the protonation state of arginine residues in a hydrophobic environment. Our results point to an important role of these interaction in the structure and function of membrane-bound peptides, and we think that cation- π interactions should thus be considered e.g. in designing membrane-bound antimicrobial peptides.

References: [1] J.P. Gallivan, D.A. Dougherty, Cation - π interactions in structural biology, PNAS 96 (1999) 9459-

9464 [2] Y. Mo, G. Subramian, J. Gao, DM. Ferguson, Cation−π Interactions: An Energy Decomposition

Analysis and Its Implication in δ-Opioid Receptor−Ligand Binding, JACS 124 (2002) 4832-4837 [3] D.L. Beene, G.S. Brandt,W. Zhong, N.M. Zacharias, H.A. Lester, D.A. Dougherty, Cation−π

Interactions in Ligand Recognition by Serotonergic (5-HT3A) and Nicotinic Acetylcholine Receptors: The Anomalous Binding Properties of Nicotine, Biochemistry 41 (2002), 10262-10269

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Influence of Risperidone on Pharmacokinetics of Caffeine in Rabbits

Nino Lominadze, Manuchar Gvaramia, Manoni Kurtanidze, Maka Alexishvili, Marina Rukhadze

Faculty of Exact and Natural Sciences, Tbilisi State University, 3 I.Chavchavadze ave, Tbilisi, 0179, Georgia, e-mail: [email protected]

Risperidone (RP) is most important among atypical or the second-generation antipsychotic drugs. It is very effective in treatment of both the positive and the negative symptoms of schizophrenia. RP is a benzisoxazole derivative and belongs to a chemically new class of drugs [1]. From the point of view of neuropharmacology RP represents antagonist of serotonin (5HT2) and dopamine (D2), also α1-, α2- adrenergic and H1-histaminergic receptors [2]. Caffeine (Caff) is a strong stimulator of central nervous system. Stimulation of metabolic processes in different organs and tissues, as well as secretory action of stomach proceeds under the influence of Caff [3]. It enters the human organism during consumption of beverages like tea, coffee, Coca-cola and so on. Therefore study of influence of Caff on pharmacokinetics of other drugs is important.

Pharmacokinetics of Caff is strongly affected by RP: the additional multiple peaks are appeared on the “C-t”curves, Cmax is increased and tmax is reduced on the first peaks (Figure). Besides AUC is increased, whereas Cl and Vd remain constant. The obtained results and consideration of pharmacokinetic curves of Caff in different mixtures of drugs, also rates of absorption and elimination of Caff indicate that the induction of metabolism of Caff takes place under the influence of RP. Besides its rate of absorption is practically constant.

References: [1] M. Aravagiri, S. Marder. Brain plasma and tissue pharmacokinetics of risperidone and 9-

hydroxyrisperidone after separate oral administration to rats. Psychopharmacology, 159 (2002) 424–431.

[2] D. Caetano. Risperidone: New atypical antipsychotic - survey on its pharmacokinetics and pharmacodynamics. Journal Brasileiro de Psiquiatria, 44 (1995) 305-310.

[3] M. Kot, W.A. Daniel. Caffeine as a marker substrate for testing cytochrome P 450 activity in human and rat. Pharmacological Reports, 60 (2008) 789-797.

0

5000

10000

15000

20000

0 10 20 30 40

C , ng/ml

t, hInfluence of risperidone on pharmacokinetics of caffeine in rabbits(1) 80 mg caffeine

(2) 10 mg risperidone + 80 mg …

1 2

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Interaction structure of the complex between neuroprotective factor humanin and Alzheimer’s β-amyloid peptide revealed by affinity-mass spectrometry and

molecular modelling

Madalina Maftei*, Xiaodan Tian*,**, Marilena Manea*,***, Thomas E. Exner**** Daniel Schwanzar*****, Christine A.F. von Arnim*****, Michael Przybylski*

*Laboratory of Analytical Chemistry and Biopolymer Structure Analysis,

Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany **Agilent Technologies, 12312 Waldbronn, Karlsruhe, Germany

***Zukunftskolleg, University of Konstanz, 78457 Konstanz, Germany ****Laboratory of Computational and Theoretical Chemistry,

Department of Chemistry, University of Konstanz *****Department of Experimental Neurology, IZKF Ulm, University of Ulm,

89081 Ulm, Germany Humanin (HN) is a linear 24 aa peptide recently detected in a human AD brain. HN specifically inhibits neuronal cell death in vitro induced by ß-amyloid (Aß) peptides, amyloid precursor protein and presenilin gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism(s) is only partially understood. We report here the identification and characterization of the interaction structure between HN and Aβ(1-40). Wild-type HN and HN-sequence mutations were synthesized by SPPS and the HPLC-purified peptides characterized by MALDI-mass spectrometry. The interaction epitopes between HN and Aß(1-40) were identified by affinity-mass spectrometry using proteolytic epitope- excision and -extraction, followed by elution of the affinity-bound peptides. The affinity-mass spectrometry analyses revealed HN(5-15) as the interaction epitope of HN, while Aß(17-28) was identified as the interaction epitope of Aβ(1-40). The interactions sites were ascertained by ELISA of the complex of HN-mutant peptides with immobilized Aβ(1-40) and by ELISA with Aβ(1-40) and Aß-partial sequences as ligands to HN. Specificity and affinity of the HN-Aß(1-40) interaction were characterized by direct electrospray ionization mass spectrometry of the HN-Aß(1-40) complex, and by bioaffinity analysis using a surface acoustic wave (SAW) biosensor, providing a KD of 610 nM. A molecular dynamics simulation of the HN-Aß(1-40) complex was fully consistent with binding specificity and shielding effect of the interaction epitopes. These results indicate a specific, strong association between HN and Aß(1-40), and provide a molecular basis for understanding the neuroprotective function of HN and the design of AD-specific therapeutic peptides.

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Binding parameters involved in the interactions between some acidic drugs with human and bovine serum albumin

Sílvia Zarza, Clara Ràfols, Elisabeth Bosch

Departament de Química Analítica and Institut de Biomedicina (IBUB), Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain

Three well known non-steroidal anti-inflammatory drugs (NSAIDs), naproxen, flurbiprofen and ibuprofen, and also the warfarin have been selected to study their interactions with albumin. This is a serum transport protein, which play a very significant role in the pharmacokinetics of drugs. Albumin from two different origins, human (HAS) and bovine (BSA) serum has been tested. Isothermal titration calorimetry (ITC) and the front analysis capillary electrophoresis (CE/FA) are used to evaluate the binding constants (Kb) and the stochiometry (n) of the interactions of drugs with BSA. Measurements have been carried out at physiological pH (7.4) with HEPES buffer and ionic strength 50 mM. The results achieved by means of both experimental approaches, as well as the literature values; allow us to test the suitability of ITC and CE/FA to evaluate the protein-drug affinity for the studied systems. ITC has also been used to measure Kb, n, and the variations of enthalpy (ΔH) and entropy (TΔS) of the selected drugs with HAS and with BSA. The same experimental conditions mentioned above have been used. Results confirm a significant difference between the interaction parameters of two kind of albumin binding the selected drugs References: [1] J. E. Ladbury, M. L. Doyle Biocalorimetry 2. Applications of Calorimetry in the Biological

Sciences, Wiley, Chichester, 2004. [2] E. Freire, A. Schön, A. Velazquez-Campoy, Methods in Enzymology: Biothermodynamics , Vol.

455, Chapter 5, Elsevier Inc., 2009. [3] K. Vuignier, J. Schappler, J.L. Veuthey, P-A. Carrupt, S. Martel, J. Pharm. Biomed. Anal. 53,

(2010), 1288

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Brain Tissue Binding of Drugs: evaluation and validation of solid supported porcine brain membrane vesicles (TRANSIL) as a novel high -throughput method

Hinnerk Boriss, Kathleen Boehme

Sovicell GmbH, Deutscher Platz 5b, 04103 Leipzig, Germany, [email protected]

Estimating the unbound fraction of drugs in brain has become essential for the evaluation and interpretation of the pharmacokinetics and pharmacodynamics of new central nervous system drug candidates. Dialysis-based methods are considered to be accurate for estimating the fraction unbound in brain, however these techniques are hampered by a low throughput. Here we present a novel matrix-free high -throughput method for estimating the unbound fraction, based on a sample pooling approach combining the TRANSIL brain absorption assay with liquid chromatography mass spectrometry. The base measurement of the TRANSIL approach is the affinity to brain membranes and this is used directly to predict the free fraction in brain. The method was evaluated by comparing free fractions fu,brain obtained using the TRANSIL brain absorption assay and equilibrium dialysis methods for a test set of 65 drugs (27 marketed and 38 GlaxoSmithKline proprietary drugs). A good correlation (r2 >0.93) of fu,brain between TRANSIL brain absorption assay and equilibrium dialysis was observed. Moreover, we compared the lipid composition of rat and porcine brain and analyzed the influence of the brain albumin content on brain tissue binding measurement. The comparison of the lipid composition indicated only minor differences between rat and porcine brain and albumin appears to have a low impact on brain tissue binding measurements. The TRANSIL brain absorption assay with sample pooling methodology not only significantly reduces the biological matrix required but also increases the throughput as compared to the conventional dialysis methods.

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Page 67

Effect of chicken egg white cystatin on proliferation of cancer cells

Renata Brykner, Łucja Cwynar-Zając, Katarzyna Juszczyńska*, Agnieszka Sosnowska**, Krzysztof Gołąb*, Jakub Gburek*, Piotr Dzięgiel, Antoni Polanowski**, Tadeusz Trziszka**

Department of Histology and Embryology, Wroclaw Medical University, Wroclaw, Poland *Department of Pharmaceutical Biochemistry, Wroclaw Medical University,

Wroclaw, Poland ** Department of Animal Products Technology and Quality Management,

University of Environmental and Life Sciences, Wroclaw, Poland

Cysteine peptidases play a crucial role in pathogenesis of cancer including invasion through degradation of extracellular matrix, metastasis by enhancing cell motility and angiogenesis due to yet unknown mechanisms. In cancer cells peptidases such as cathepsin B and L are shuttled to the plasma membrane where they can activate receptor-bound prourokinase-type plasminogen activator. The peptidase participate extracellular matrix degradation directly by its intrinsic activity and indirectly by activation of matrix metalloproteinases. It is well known that activity of cysteine cathepsins are controlled by endogenous inhibitors belonging to cystatin superfamily. Recently it has been reported that cystatins can also inhibit metalloproteineses. Chicken egg white cystatin is one of the best characterized members of those inhibitors and has been used in many studies as a model protein in pathophysiological studies. In this study we have investigated the influence of the inhibitor on cancer and normal cell proliferation. Three cell lines have been examined: normal human dermal fibroblasts (NHDF), human Beidegröm melanoma cells (BM) and breast cancer cells Michigan Cancer Foundation – 7 (MCF-7). Cells were grown on microtiter plates in medium containing cystatin or medium alone for 72 h. Cell proliferation rate was determined by cell protein content using sulforhodamine B colorimetric assay (SRB). We observed dose dependent inhibition of NHDF cells proliferation reaching 25 % at the 20 µM cystatin concentration. Growth of the MCF-7 cells was also inhibited but to the lesser extends (16%). We did not observed significant impact of cystatin on proliferation of BN cells. Our results indicate different susceptibility of distinct cell lines to cystatin treatment. Further studies on phenotype level are needed to clarify these differences. Inhibition of MCF-7 cell proliferation by cystatin suggests that cysteine peptidase inhibitors may of value in anticancer therapy. Surprisingly, also the growth of normal fibroblasts was significantly retarded. This should be taken into consideration in future research of cystatin-based anticancer drugs.

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Polyethylenimine-functionalized single-walled carbon nanotube for efficient delivery of plasmid DNA

Mohammad Ramezani, Behzad Behnam*, Khalil Abnous*

*Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, P.O. Box 91775-1365, Mashhad, Iran

**Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, P.O. Box 91775-1365, Mashhad, Iran;

The high priority of finding safe and efficient vectors for gene delivery has led to diverse studies for developing novel delivery systems. One promising strategy has been to structurally modify polyethylenimine (PEI) by imparting more hydrophobic nature to its structure as it has been reported by our group [1]. On the other hand, there has been great attention to carbon nanotubes (CNTs) as cellular cargos for different molecules. CNT has a very hydrophobic surface make it insoluble in biological media [2]. In this study, PEI has been attached to the single-walled CNT (SWNT) through a pegylated phospholipid in order to both solubilize SWNT and impart hydrophobic characteristics to PEI structure. Distearoyl phosphatidylethanolamine-PEG 2000-COOH (DSPE-PEG 2000) molecules were attached to SWNTs hydropobically during bath sonication. Undissolved CNTs and excess of DSPE-PEG were removed by several centrifugation (22,000 g) and filtration through 100 kDa filters, respectively. PEI was attached to the DSPE-PEG 2000 through amide linkage using EDC/BtOH coupling reagents. The ability of the vector to condenseDNA was evaluated by ethidium bromide exclusion assay. For transfection studies, murine neuroblastoma cells (Neuro2A) were used for expression of luciferase. Stable solutions of CNTs were made by functionalization of the SWNT using DSPE-PEG. The conjugation of PEI to SWNT-DSPE-PEG was assessed by appearance of a new peak at 1643 cm-1. Vectors conjugated with either PEI 10 or 25 kDa were strongly condense plasmid (C/P = 1.2 for SWNT-PEI 1800, 0.2 for SWNT-PEI 10 KDa and 0.1 for 25 kDa). The great potential of these vectors for condensing plasmid could be due to the fact that each SWNT-PEI molecule might have several PEI molecules attached to it where it could provide more positive ions to easily interact with DNA. Transfection data indicated that the new reported vectors had nearly 9.5 fold less concentrations of PEI mollecules compared to control group (PEI itself) but with the same transfection potency while their toxicity are low at C/Ps where the transfections were the highest. The novel vectors based on SWNT and PEI obtained in this study (i.e. SWNT-PEI 10 and 25 kDa) exhibited significant condensation ability with high transfection activity compared to the parent molecules while their cell toxicity remains intact making them good candidate for in vivo studies.

References: [1] R.K. Oskuee, A. Dehshahri, W.T. Shier, M. Ramezani. Alkylcarboxylate grafting to

polyethylenimine: asimple approach to producing a DNA nanocarrier with low toxicity J. Gene Med. 11 (2009) 921–932.

[2] Z. Liu, S. Tabakman, K. Welsher, H. Dai. Carbon Nanotubes in Biology and Medicine: In vitro and in vivo Detection, Imaging and Drug Delivery. Nano Res. 2 (2009) 85-120.

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Supercritical CO2 extraction and large-scale separation of natural drug leads

Keiko Kumagai, Masashi Tsuda*

Science Research Center, Kochi University, Japan *Center for Advanced Marine Core Research, Kochi University, Japan

The supercritical fluid is any compound at a temperature and pressure above the critical values. The supercritical CO2 extraction has focused on advocating on reduction in the use of organic solvents. Besides ecological benefits, one of the most interesting properties of supercritical CO2 is the high diffusion coefficients lipid in supercritical fluid, far greater than in organic solvents. Marine dinoflagellates of the genus Amphidinium are well-known as a producer of unique cytotoxic metabolites. We have isolated a number of potently cytotoxic macrolide [1,2] and polyketides against cancer cells, from the benthic dinoflagellate Amphidinium species collected off Iriomote Island, Japan. Usually, our separation scheme of these polyketides is follows. The harvested algal cells obtained by mass-cultivation were extracted with organic solvents (MeOH/toluene). The extract was partitioned between toluene and water. The toluene-soluble materials were subjected to three-step separation with column chromatographies (SiO2 gel, C18, and then NH2-SiO2 columns) and C18 HPLC. Contents of these metabolites in the dried cells were less than 1%, while major components in the cells are lipids such as glycerolipids, fatty acids, and steroids. In our investigation on bioactive substances from natural products, we developed supercritical CO2 extraction for separation of these macrolides in the lipid-rich cells of cultured dinoflagellates. So, we set up the sequential system of supercritical CO2 extraction and trap HPLC. Furthermore, an automatic HPLC separation system with 50 mm internal diameter column was set up. Evaporation of obtained fractions was performed by the high-speed vacuum evapotator and/or centrifuged vacuum evaporator systems. In this presentation we will report on systematic large-scale separation system for natural drug leads. References: [1] Tsuda, M., Oguchi, K., Iwamoto, R., Okamoto, Y., Kobayashi, J., Fukushi, E., Kawabata, J., Ozawa,

T., Masuda, A., Kitaya, Y., Omasa, K. Iriomoteolide-1a, a potent cytotoxic 20-membered macrolide from a benthic dinoflagellate Amphidinium species. J. Org. Chem. 72 (2007) 4469-4474.

[2] Oguchi, K., Tsuda, M., Iwamoto, R., Okamoto, Y,; Kobayashi, J., Fukushi, E., Kawabata, J., Ozawa, T., Masuda, A., Kitaya, Y., Omasa, K. Iriomoteolide-3a, a cytotoxic 15-membered macrolide from a marine dinoflagellate Amphidinium species. J. Org. Chem. 73 (2008) 1567-1570.

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Development of Analytical Method of Compounds in Drugs Using HPLC instead of UV Spectrophotometer Method

JaeYong Song, SeungEun Jang, SunHoi Kim, GilBong Lee, JeaMan Lee, YongHee Kim

Pharmaceuticals Analysis Devision, Incheon Metropolitan City Institute of Health and Environment, Korea

There are some analytical methods behind the times in The Korean Pharmaceutical codex so far. Many research institutes, especially Korean Food and Drug Administration(KFDA), have enforced to improve the efficient and accurate qualification. This study is necessary to make better standard criterion of analytical method which was developed long time ago and to unify each analytical method for maintaining its quality to match the criterion effectively. Also, to develop new research methods to certify drug qualification result of accuracy and precision The analytical method by HPLC was validated. The analytical best conditions are as follows; the separation of Benproperine Phospate, Fenoterol Hydrobromide and Midodrine hydrochloride was achieved on Capcell Pak C18 UG120 (4.6mm×250mm, Particle size 5µm) column using a 0.1M Ammonium acetate(pH 3.3)·Methanol(250:750), Buffer solution(pH3.2): ACN (1000:270) and Methanol: 0.1M NaH2PO4 (300:700) mobile phase. These compounds were eluted isocratically at flow rate 1.0 mL/min, analyzed with UV detection at 270, 276, 290nm, respectively. The RSD (%) of new method using HPLC analyzing for Benproperine Phosphate, Fenoterol Hydrobromide, Midodrine hydrochloride is 0.96, 0.74, 0.57 %, that of existing method using UV Spectrophotometer showed 3.46, 1.76, 2.05 %. Therefore, this new method showed specificity, linearity, precision and accuracy as well as reproducibility within acceptable range. This alternative analytical method was adequate for its purpose and could be helpful for improving qualification methods in Pharmacopeia .

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Chromatographic characteristics of inorganic analytes in nonionic micellar liquid

chromatography with ion-pair additives

Nino Lominadze , Levan Akhalkatsi, Manuchar Gvaramia , Marina Rukhadze,

Faculty of Exact and Natural Sciences, Tbilisi State University, 3, I.Chavchavadze ave, Tbilisi, 0179, Georgia - e-mail: [email protected]

Separation and determination of inorganic ions by ion interaction or ion-pair chromatography substantially has limited wide application of ion-exchange and ion chromatographic methods[1]. Modification of reversed stationary phase surface with mixed nonionic and cationic surfactants resulted in the improvement of efficiency is proposed recently[2,3]. Chromatographic behavior of some inorganic ions by using of micellar mobile phase on the basis of Brij-35 was investigated in the presented work. Different ion-pair reagents (surfactants and surface inactive compounds as well) e.g. sodium dodecyl sulfate, cetylpyridinium chloride, hexadecyltrimethylammonium bromide, cholic acid and ornithine chloride were introduced in the mobile phase in order to increase an electrostatic interaction. Iodide, bromide and chromium (III and VI) ions were selected as model compounds. Influence of concentration of Brij-35 and ion-pair additives, also pH of mobile phase on retention and separation factors of sample ions was studied. Results have shown that separation Cr3+ and Cr04

2- ions is improved at the presence of ornithine chloride in mobile phase together with Brij-35 in comparison with ion-pair additives sodium dodecyl sulfate, cetylpyridinium chloride, hexadecyltrimethylammonium bromide and cholic acid below their micelle critical concentration. The optimum concentration of ornithine chloride and Brij-35 were selected which provides to avoid elution of Cr3+ ions with void volume and simultaneously to reach sufficient resolution (Rs=1.1) for Cr3+ and chromate ions. At the same time Rs=0.8 is observed for bromide and chloride ions. A suppression action of buffer ions on retention of solutes was revealed at the preparation of mobile phase on the basis of phosphate buffer, therefore Brij-35 and ion-pair additives were added to pure water. Micellar liquid chromatography has been successfully employed for separation of some inorganic ions. Importantly, that the conventional HPLC instrumentation was used in this work. Proposed technique was flexible because concentration of nonionic surfactant and ion-pair additives could be readily varied to obtained good separation characteristics. References: [1] T. Cecchi. Application of Ion Pairing Chromatography to the Analysis of Inorganic

Analytes:Review. Journal of liquid Chromatography & Related Technologies. 30 (2007) 1205-1225.

[2] Zh. Yan, P.R. Haddad, J.S.Fritz. Ion chromatography on reversed-phase materials coated with mixed cationic and nonionic surfactants. Journal of Chromatography A. 985 (2003) 359-365.

[3] J.S.Fritz; Zh. Yan, P.R. Haddad. Modification of ion chromatographic separations by ionic and nonionic surfactants. J.Chromalography A. 997(1-2) (2003) 21-31.

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DFT Calculations on pregabalin as a pharmaceutical compound for the treatment of neuropathic pain

E. Vessally, M. Rezaei*, M. Saber*

Miyaneh Branch, Islamic Azad University, Miyaneh, Iran *Payame Noor University, Department of Science, P. O. Box: 19395-4697 Tehran, Iran

**Tabriz Branch, Islamic Azad University, Miyaneh, Iran

Pregabalin (PGN); 3-(aminomethyl)-5-methylhexanoic acid (Fig. 1), is structurally related to the inhibitory neurotransmitter aminobutyric acid (GABA) [1]. It is a white crystalline solid and soluble in water as well as in both basic and acidic aqueous solution [2]. The PGN is a new anticonvulsant and analgesic medication that has been recently approved for adjunctive treatment of partial seizures in adults [3–6] and for the treatment of neuropathic pain from postherpetic neuralgia to diabetic neuropathy. The PGN was structurally and pharmacologically related to the anticonvulsant and analgesic medication gabapentin [7]. In this work, the optimization calculations were carried out on Pregabalin and its ground state conformations were compared with its obtained X-Ray data. These calculations were carried out by using B3LYP/6-311++G(d,p) and HF/6-311++G(d,p) levels of theory. The geometrical parameters, natural bonding orbital (NBO) charge at atoms HOMO and LUMO, the chemical hardness (η), chemical potential (μ), electrophilicity (ω) and the maximum amount of electronic charge, ΔNmax, for PGN and its analogues were obtained. The results and discussions should be presented.

References: [1] M. Van Ameringen, M. A. Rynn, T. K. Murphy and F. Mandel, Europ Psych., 23 (2008) S221-

S222. [2] Rajinder Singh Gujral, Sk Manirul Haque and Sanjeev Kumar, Afr. J. Pharm. Pharmacol., 3 (2009)

327-334. [3] D. M. Tassone, E. Boyce, J. Guyer, D. Nuzum, Clin. Ther., 29 (2007) 26-48 [4] K. Hamandi, J. W. Sander. Seizure.,15 (2006) 73-78. [5] R. Freynhagen, K. Strojek, T. Griesing, E. Whalen, et al., Pain. 115 (2005) 254-263. [6] U. Sharma, J. Young, L. LaMoreaux, Pain. 6 (2005) S29. [7] D. Wesche, H. Bockbrader., Pain. 6 (2005) 29.

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Ab initio Calculations on Galantamine as a pharmaceutical compound for the treatment of Alzheimer’s Disease

Maryam Motallebzadeh, Mehdi Rezaei*, Esmail Vessally*

Young Researchers club, Tabriz Branch, Islamic Azad University, Tabriz, Iran *Payame Noor University, Department of Science, P. O. Box: 19395-4697 Tehran, Iran

Pregabalin (PGN(-)-Galantamine (1) {(4aS,6R,8aS)-4a,5,9,10,11,12-hexahydro-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-ef][2]-benzazepin-6-ol} (Scheme 1) is an alkaloid which isolated from the Caucasian snow-drop (Galanthus woronowii) and from the bulbs of different species of the Amaryllidaceae family [1]. From the pharmacological point of view, (-)-Galantamine is a centrally acting, selective, reversible, and competitive acetylcholinesterase (AChE) inhibitor [2-3]. In this work, the optimization calculations were carried out for different conformations of galantamine. The ground state conformations were compared with its obtained X-Ray data. These calculations were carried out by using DFT/6-31G(d) and HF/6-31G(p) levels of theory. The geometrical parameters, natural bonding orbital (NBO) charge at atoms HOMO and LUMO, the chemical hardness (η), chemical potential (μ), electrophilicity (ω) and the maximum amount of electronic charge, ΔNmax, for PGN and its analogues were obtained. The results and discussions should be presented.

References: [1] Proskurnina, N. F.; Yakoleva, A. P. J. Gen. Chem. 22 (1952) 1899. [2] Bores, G. M.; Kosley, R. W. Drugs Future 21 (1996,) 621. [3] Rainer, M. Drugs Today 33 (1997) 273.

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A new method of producing monoclinic paracetamol suitable for direct compression

Andrey G. Ogienko, Elena V. Boldyreva, Andrey Yu. Manakov, Vladimir V. Boldyrev,

Alexander S. Yunoshev, Anna A. Ogienko, Svetlana A. Myz, Alexei I. Ancharov, Andrey F. Achkasov, Tatyana N. Drebushchak

Research and Education Centre “Molecular Design and Ecologically Safe Technologies”,

Novosibirsk State University, Novosibirsk, Russia; Nikolaev Institute of Inorganic Chemistry, SB RAS, Novosibirsk, Russia;

Institute of Solid State Chemistry and Mechanochemistry, SB RAS, Novosibirsk, Russia; Lavrentiev Institute of Hydrodynamics, SB RAS, Novosibirsk, Russia;

Institute of Cytology and Genetics, SB RAS, Novosibirsk, Russia; [email protected]; [email protected]

In the contribution we discuss the method of preparing paracetamol form I in a new form suitable for direct compression by spray freeze-drying of acetone-water solutions and the results of characterizing the properties of the samples by scanning electron microscopy, X-ray powder diffraction, dissolution dynamics experiments, as well as the results of evaluating compaction properties [1]. The tensile strength of the tablets from the paracetamol sample prepared by freeze-drying in this work (1.27±0,27 МPа) was somewhat higher than that for the tablets prepared from the orthorhombic paracetamol form II (~ 0.95 МPа for the tablets prepared with the compression pressure 335 МPа) [2], and considerably larger than the strength required to crush the tablets compressed from the commercial monoclinic paracetamol form I. Thus, the new form is comparable to polymorph II in compactability, but is stable. The same method based on freeze-drying of solutions in mixed aqueous-organic solvents can be used to prepare new improved forms of other molecular solids for pharmaceutical applications Acknowledgements: This work was supported by grants from CRDF (RUX0-008-NO-06), RF Ministry of Education and Science (2.2.2.2/10470), RFBR (10-03-00252, 11-03-00684), the Integration Project № 109 of SB RAS, Russian Ministry of Education and Science (State Contract of RF № 14.740.11.1023), Projects 5.10, 21.44 and 5.6.4 of the Russian Academy of Sciences. References: [1] A.G. Ogienko, E.V. Boldyreva, A.Yu. Manakov, V.V. Boldyrev, A.S. Yunoshev, A.A. Ogienko, S.A.

Myz, A.I. Ancharov, A.F. Achkasov, T.N. Drebushchak, A new method of producing monoclinic paracetamol suitable for direct compression, Pharm. Res. (2011) DOI 10.1007/s11095-011-0502-x.

[2] E. Joiris, P. Di Martino, C. Berneron, A.-M. Guyot-Hermann, J.-C. Guyot, Compression behavior of orthorhombic paracetamol, Pharm. Res. 15 (1998) 1122-1130.

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DFT Calculations on Quetiapine Hemifumarate as a pharmaceutical compound

for the treatment of schizophrenia

E. Vessally, A. A. Jafari, M. Rezaei

Payame Noor University, Department of Science, P. O. Box: 19395-4697 Tehran, Iran Quetiapine, is described 2-(2-(4-dibenzo(b,f)(1,4)thiazepine-11-yl-1-piperazinyl)ethoxy) ethanol with molecular formula C21H25N3O2S and molecular weight 383.51 (Fig. 1). Quetiapine is used for the treatment of schizophrenia and recently has Food and Drug Administration (FDA) approval for treatment of manic depression [1,2]. It is also to treat other disorders, such as post-traumatic stress disorder, alcoholism, obsessive compulsive disorder, anxiety disorders, and hallucinations in Parkinson's disease patients using ropinirole and as a sedative for those with sleep disorders. Quetiapine is the most commonly prescribed antipsychotic drug in America and has been used by more than 19 million patients worldwide in 1997. The mechanism of action of Quetiapine, as with other drugs having efficacy in the treatment of schizophrenia and acute manic episodes associated with bipolar disorder, is unknown. In this work, the optimization calculations were carried out on Quetiapine Hemifumarat and its ground state conformations were compared with its obtained X-Ray data. These calculations were carried out by using B3LYP/6-31G(d) level of theory. The DFT calculations clarified a boat structure for dibenzothiaazepine part of the molecule which piperazine section was occurred in a chair conformation. The results will be presented and discussed.

Acknowledgment: Payame Noor University is gratefully acknowledged for their financial support. References [3] L.A. Arvanitis, B.G. Miller, Biol. Psychiatry 42 (1997) 233-246. [4] D.E. Casey, J. Clin. Psychiatry, 57 (1996) 40-52.

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Author Index A Abnous, K .................................................... 68 Achkasov, AF ............................................... 74 Akhalkatsi, L ................................................ 71 Alexishvili, M ............................................... 63 Ancharov, AI ............................................... 74 Andrés, A .................................................... 44 Angelov, TM ................................................ 46

B Bebiano, SS ................................................. 58 Behnam, B .................................................. 68 Boehme, K ............................................ 32, 66 Boldyrev, VV ............................................... 74 Boldyreva, EV .................................. 24, 52, 74 Bolger, MB .................................................. 12 Boriss, H ................................................ 32, 66 Bosch, E .................................... 43, 44, 49, 65 Brychtová, K ................................................ 42 Brykner, R ................................................... 67 Burrows, HD ................................................ 58

C Cabot, JM .................................................... 14 Calvet, C ...................................................... 43 Canotilho, J ................................................. 58 Carlomagno, T ............................................. 16 Castro, RAE ................................................. 58 Cetina-Čižmek, B ............................. 41, 51, 56 Chen, H ....................................................... 13 Choi, L ......................................................... 55 Comer, J ........................................................ 9 Cozma, C ..................................................... 29 Cwynar-Zając, Ł ........................................... 67

Č Čulen, M ................................................. 7, 50 Čuljak, K ................................................ 17, 59

D Dohnálek, J .................................................. 37 Dohnal, J .................................................. 7, 50 Domańska, U ............................................... 48 Dove, S ........................................................ 11 Drebushchak, TN ......................................... 74 Durrigl, M .................................................... 41 Dvořáková, L ................................................ 42 Dušková, J ................................................... 37 Dzięgiel, P .................................................... 67

E Elz, S ............................................................ 11 Espinosa, S .................................................. 44 Eusébio, MES ............................................... 58 Évora, AOL ................................................... 58 Exner, TE ..................................................... 64

F Fraczkiewcz, R ............................................. 12 Fuguet, E ............................................... 14, 49

G Gburek, J ..................................................... 67 Giron, D ....................................................... 21 Göbl, C ............................................. 17, 59, 62 Gołąb, K ....................................................... 67 Grinberg, N .................................................. 33 Grzybowska, K ............................................. 25 Gvaramia, M .......................................... 63, 71

H Haddad, N ................................................... 33 Hašek, J ....................................................... 37 Heim, R ....................................................... 11 Heo, O ......................................................... 54 Hilfiker, R ..................................................... 22


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Hohlweg, W .......................................... 17, 62 Holdgate, G ................................................. 30 Huerta, JMª ................................................. 44

I Ilič, I ............................................................ 57 Iurascu, M ................................................... 29

J Jafari, AA ..................................................... 75 Jampílek, J ......................................... 7, 42, 50 Jang, SE ....................................................... 70 Jojart, I ........................................................ 36 Jung, J ................................................... 29, 53 Jung, R ........................................................ 54 Jung, W ....................................................... 54 Jung, YM ..................................................... 55 Justino, LLG ................................................. 58 Juszczyńska, K ............................................. 67

K Kása jr., P .............................................. 36, 57 Kessler, H .................................................... 15 Kim IK .......................................................... 54 Kim, D ......................................................... 53 Kim, DS ........................................................ 55 Kim, EJ ......................................................... 55 Kim, IK ......................................................... 55 Kim, M .................................................. 53, 54 Kim, Sh ........................................................ 53 Kim, SH........................................................ 70 Kim, Soo ...................................................... 53 Kim, YH ....................................................... 70 Korotkova, EI............................................... 46 Kosol, S ........................................... 17, 59, 61 Kowalewska, E ............................................ 35 Král, V ......................................................... 50 Kumagai, K ............................................ 60, 69 Kurtanidze, M ............................................. 63 Kwokal, A .................................................... 26

L Lavrič, Z ....................................................... 20 Lee, GB ........................................................ 70 Lee, H .......................................................... 33 Lee, JH ......................................................... 55 Lee, JM ........................................................ 70

Lee, Y ........................................................... 53 Leist, M ........................................................ 29 Li, B .............................................................. 13 Lindner, K .................................................... 29 Litwinienko, G ............................................. 35 Lominadze, N ........................................ 63, 71 Louit, G ........................................................ 10 Luísa Ramos, M ........................................... 58 Lužnik, J ....................................................... 20

M Ma, S ........................................................... 33 Mafte, M ..................................................... 64 Manakov, AYu ............................................. 74 Manea, M .............................................. 29, 64 Maria, TMR ................................................. 58 Mehrens, S .................................................. 19 Meštrović, E ................................................ 23 Min, C .......................................................... 53 Motallebzadeh, M ....................................... 73 Myz, SA.................................................. 52, 74

N Na, M........................................................... 54 Neue, K ........................................................ 28 Ngai, KL ....................................................... 25 Novak, P ................................................ 17, 59

O Ogienko, AA ................................................ 74 Ogienko, AG ................................................ 74 Oktabec, Z ................................................... 50 Opatřilová, R ............................................... 42

P Pallicer, JM .................................................. 43 Paluch, M .............................................. 25, 27 Papós, K ....................................................... 57 Parot, P ........................................................ 31 Pascual, R .................................................... 43 Pelczarska, A ............................................... 48 Pellequer, J-L ............................................... 31 Pepin, X ....................................................... 10 Peter-Katalinić, J .......................................... 28 Pintye-Hódi, K ....................................... 36, 57 Pirnat, J ....................................................... 20 Pobudkowska, A .......................................... 48


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Pohlentz, G ................................................. 28 Polanowski, A ............................................. 67 Port, A ......................................................... 43 Przybylski, M ................................... 29, 38, 64

R Radić, M ...................................................... 41 Ràfols, C ............................... 14, 43, 44, 49, 65 Ramezani, M ............................................... 68 Ramos Silva, M............................................ 58 Regula, JT .................................................... 28 Řezáčová, A ................................................... 7 Rezaei, M ........................................ 72, 73, 75 Roberts, KJ .................................................. 26 Rosés, M ......................................... 14, 43, 44 Rukhadze, M ......................................... 63, 71

S Saber, M ..................................................... 72 Scheffner, M ............................................... 29 Schrank, E ................................................... 59 Schwanzar, D .............................................. 64 Segarra, V ................................................... 44 Senanayake, C ............................................. 33 Seong, R ...................................................... 53 Shakhtshneider, TP ..................................... 52 Shen, S ........................................................ 33 Shoghi, E ..................................................... 49 Silva, ME ..................................................... 11 Skálová, T .................................................... 37 Sohn, KH ..................................................... 55 Sohn, S ........................................................ 54 Song, JY ....................................................... 70 Sosnowska, A .............................................. 67 Sovány, T ..................................................... 57 Srčič, S ................................................... 20, 57 Stefek, M .................................................... 47 Strasser, A ................................................... 11 Sugano, K ...................................................... 4 Sunesen, M ................................................. 34

Š Šašić, S ........................................................ 19

T Takács-Novák, K ...................................... 8, 45 Tam, KY ................................................... 3, 45

Tang, W ....................................................... 33 Tian, X ......................................................... 64 Tomljenović Gluhak, A ................................ 51 Triantos, N ................................................... 47 Trontelj, Z .................................................... 20 Trziszka, T .................................................... 67 Tsantili-Kakoulidou, A ................................. 47 Tsuda, M ............................................... 60, 69 Tudja, P ....................................................... 23 Tumanov, NA............................................... 52 Tunjić, I ........................................................ 56 Turkalj, J ...................................................... 51

V Vacher, M .................................................... 10 Valkó, K ......................................................... 5 Vessally, E ........................................ 72, 73, 75 Vizserálek, G ................................................ 45 Vlad, C ......................................................... 29 Vlasenko, A .................................................. 46 Völgyi, G ...................................................... 45 von Arnim, CAF ........................................... 64

W Wagner, G ................................................... 61 Waldman, M ............................................... 12 Wang, J ........................................................ 33 Wang, JF ...................................................... 13 Wang, S ....................................................... 54 Watts, A ...................................................... 18 Whitlock, M ................................................. 19 Wojnarowska, Ż .................................... 25, 27 Woltosz, WS ................................................ 12

Y Yang, HB ...................................................... 13 Yee, N .......................................................... 33 Yunoshev, AS ............................................... 74

Z Zangger, K ................................. 17, 59, 61, 62 Zarza, S ........................................................ 65 Zeck, A ......................................................... 28 Zhuang, D .................................................... 12 Zimmerman, K ............................................. 37


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