First World Conference on Physico-Chemical Methods in Drug ... · HPLC based property measurements of drug discovery compounds to predict in-vivo drug distribution and aid lead optimisation

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

Programme &

Book of Abstracts

Rovinj, Croatia, September 27 - October 01, 2009

First World Conference on Physico-Chemical Methods in Drug Discovery and Development Rovinj, Croatia, September 27 - October 01, 2009

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

The Scientific & Organizing Committee

Alex Avdeef, pIon INC, Woburn, USA

Biserka Cetina-Čižmek, PLIVA, Zagreb, Croatia

Vesna Gabelica Marković, GlaxoSmithKline, Zagreb, Croatia

Marijana Kraljić Roković, University of Zagreb, Croatia

Zoran Mandić, University of Zagreb, Croatia

Ernest Meštrović, PLIVA, Zagreb, Croatia

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CONTENTS

CONFERENCE PROGRAMME ________________________________ i

PLENARY LECTURES ______________________________________ 1

Miniaturized Rotating Disk and Powder Intrinsic Dissolution Measurement in Early Drug Development – Theoretical and Experimental Practice

Alex Avdeef ____________________________________________________________ 3

Physical chemical parameters to predict oral drug absorption Jennifer B. Dressman ____________________________________________________ 4

HPLC based property measurements of drug discovery compounds to predict in-vivo drug distribution and aid lead optimisation

Klára Valkó ____________________________________________________________ 5

The importance of choosing the optimal solid form for the success of a drug Rolf Hilfiker ____________________________________________________________ 6

‘Not Seeing is Just Guessing’ – Applications of Raman and NIR Chemical Imaging in Development of Solid Dosage Formulations

Slobodan Šašić _________________________________________________________ 7

Dissecting Carbohydrate Structure from Cells and Tissues Jasna Peter-Katalinić ____________________________________________________ 8

Solubilizing membrane proteins in nanoparticles and ligand interactions of signaling proteins by NMR

Timothy J. Knowles, Mark Jeeves, Mike Tong, Corinne Smith, Yu-Pin Lin, Tim Dafforn, Michael Overduin ____________________________________________ 9

Mass spectrometry in the elucidation of lead structures for vaccine development Michael Przybylski, Irina Perdivara, Claudia Cozma, Adrian Moise, Gabriela Paraschiv, Stefan Slamnoiu, Marius Iurascu, Mihaela Dragusanu, Alina Petre, Andreas Marquardt, Camelia Vlad, Marilena Manea __________________________ 10

AFM and DFS in the study of protein-ligand interactions Pierre Parot, Jean-Luc Pellequer __________________________________________ 11

Target immobilization provides new opportunities in NMR-based fragment discovery and characterization

Gregg Siegal __________________________________________________________ 12

Rovinj, Croatia, September 27 – October 1, 2009

VI

INVITED LECTURES _____________________________________ 13

pKa, logP, solubility and supersaturation in excipients and biorelevant media. Possibilities to promote a compound’s BCS class

John Comer __________________________________________________________ 15

Cell culture assays to predict oral drug absorption Sibylle Neuhoff _______________________________________________________ 16

Assessing and Improving the Reliability of In Silico Physicochemical Property Predictions by Incorporating In-house Data

P. Japertas, R. Didziapetris, A. Sazonovas, A. Petrauskas _______________________ 17

Solid-state kinetic studies: how to work less and find out more Michaela Horvat ______________________________________________________ 18

Fighting counterfeit drugs by means of XRPD S. Prugovecki, U. Riedel, D. Beckers _______________________________________ 19

Protein instability and stability, a formulation challenge Mario Cindrić, Renata Pavišić ____________________________________________ 20

Identification of complexes in multicomponent systems by IR spectroscopy Dražen Čavužić _______________________________________________________ 21

NMR studies of guanine-rich DNA oligonucleotides: Structures and cation dynamics Primož Šket, Janez Plavec _______________________________________________ 22

Fluorine-19 NMR in drug research Tomislav Biljan _______________________________________________________ 23

Drug Discovery in Academia: Physics based approaches Philip Gribbon ________________________________________________________ 24

Tumor targeting with gonadotropin-releasing hormone derivatives Marilena Manea, Ildikó Szabó, Erika Orbán, Miguel Tejeda, Bence Kapuvári, Žarko Kulić, Peter Öhlschläger, Wilfried Reichardt, Dezső Gaál, Gábor Mező _______ 25

ORAL PRESENTATIONS __________________________________ 27

Hydrogen bonds in the promotive action of bile acids on the kinetics of lidocaine transfer from an aqueous medium to chloroform

Mihalj. M. Poša, Valéria. J. Guzsvány, Janoš. J. Čanadi, Ferenc. F. Gaál ___________ 29

The role of surface free energy and plasticity of the materials in tablet formulation Tamás Sovány, Ilija Ilic, Kitti Papós, Péter Kása jr., Stane Srcic, Klára Pintye-Hódi ___ 30

Improvement of solubility of low soluble drug telmisartan Ivan Simčić, Michaela Horvat, Tijana Dragić, Maja Mihoci, Tina Jelača, Vlatka Džale, Biserka Cetina-Čižmek ______________________________________ 31

1st World Conference on Physico-Chemical Methods in Drug Discovery and Development

VII

Raman analysis of a polymorph within a polymorph Robert Alexander, Stefano Pera ___________________________________________ 32

Study of interactions between API and excipients in solid drug formulation via NIR Zbyněk Oktábec, Josef Jampílek, Anna Zerzaňová , Jiři Dohnal, Vladimír Král _______ 33

Gliclazide cocrystals: In vitro and In vivo evaluations R. K. Averinnei, N. Johan, G. Shavi, K. Armugam, U. Nayak, S. Pandey, N. Udupa ____ 34

Identification of Chlamydia pneumoniae antigens by immuno-proteomics using FTICR-MS and LC-tandem mass spectrometry

Iuliana Susnea, Sebastian Bunk, Corinna Hermann, Michael Przybylski ____________ 35

NMR in drug discovery: Ligand-based NMR spectra demonstrate an additional phytoestrogen binding site for 17β-hydroxysteroid dehydrogenase type 1

Paul J.A. Michiels, Christian Ludwig, Michel Stephan, Christina Fischer Gabriele Möller, Jerzy Adamski, Josef Messinger, Maria van Dongen, Hubert Thole, Ulrich L. Günther ___________________________ 36

Online chemical modeling environment Igor V. Tetko, Iurii Sushko, Sergeii Novotarsky, Robert Körner Anil Kumar Pandey, Matthias Rupp ________________________________________________________ 37

Solvent effects on the structure-activity relationship of 5-substituted-5-phenylhydantoins

Nemanja P. Trišović, Nataša V. Valentić, Tatjana L. Đaković-Sekulić, Gordana S. Ušćumlić ___________________________________________________ 38

Spectroscopic method for the early evaluation of anti-bacterial agents activity Vitaly Erukhimovitch, Mahmoud Huleihel ___________________________________ 39

Echis multisquamatis venom fibrinogenase is a prospective fibrinogen-depletive agent

V. O. Chernyshenko, I. S. Maksymovych ____________________________________ 40

POSTER PRESENTATIONS _________________________________ 41

Determination of dissociation constants of poorly soluble cannabinoid receptor ligands by cosolvent capillary electrophoresis

Carme Calvet, Joan A. Morató, Edmundo Ortega, Adriana Port __________________ 43

Physicochemical Profiling of New Antimalarial Pyridones Ana I. Alvarez, Maria-Teresa Fraile, Ruben Gomez-Sanchez, Angel Santos-Villarejo, J. L. Lavandera _____________________________________ 44

Evaluation of solubility and dissolution of pioglitazone hydrochloride in GI environment

J. Turkalj, I. Erak, D. Raušl, I. Mijakovac, B. Cetina-Čižmek ______________________ 45


VIII

Stability testing of the Spalmotil oral solution during the use (in use test) Biljana Stojanović, Stojanka Vidović, Mirjana Rajić, Ljiljana Petrović, Marija Milovanović ____________________________________________________ 46

Physical methods for in vitro simulation of Sucralfate oral suspension behaviour in vivo

Marina Surjak, Spomenka Milak __________________________________________ 47

Absorption Study of Binary Mixtures Anna Zerzaňová, Zbyněk Oktábec, Josef Jampílek, Jiří Dohnal, Vladimír Král _______ 48

Application of principal component analysis in determination of dehydration behaviour of mesalazine hydroiodide monohydrate

Michaela Horvat, Miljenko Košiček, Ernest Meštrović _________________________ 49

Rheological characterization of semisolid pharmaceutical products Božana Ivković, Petra Golja Gašparović ____________________________________ 50

Particle size characterization of nanosize organometalic complex Petar Tudja, Suzana Šegota, Vesna Svetličić, Biserka Cetina-Čižmek ______________ 51

Optimization of the freeze-drying process by means of thermal methods of analyses Andrea Rožman, Anita Bevetek Moćnik, Michaela Horvat______________________ 52

Electromediated microanalysis for the evaluation of interspecies variation in ester metabolism

Joana Moura, Ana Luísa Simplício ________________________________________ 53

Comparison of original and generic pharmaceutical products safety based on investigation of their influence on membrane structure of blood cells using ESR-spectroscopy

Igor Revelsky, Aleftina Polezneva, Igor Golubev, Evgeniy Gogol _________________ 54

A method for the quantification of L-dopa esters and related metabolites in the blood by LC-electro-spray tandem mass-spectrometry

Igor Mukmenev, Igal Gelboa, Elena Doubijansky, Amir Raichman, Alon Yaar, Eli Heldman __________________________________________________________ 55

Low molecular weight pharmaceutical substances analysis using matrix-assisted laser desorption/ionization (MALDI)

Karine Chamyan, Igor Revelsky, Aleksey Buryak, Valentina Kosenko _____________ 56

FT-IR Spectrometric determination of total ascorbic acid K. Kargosha, S. R Moeinosadat, J. Azad ____________________________________ 57

Anion-induced conformational changes in indole-based receptors Damjan Makuc, Janez Plavec ____________________________________________ 58

Application of HPLC and LC/MS techniques for determination and identification of impurities in Rosiglitazone tablets

S. Biškupić, V. Radić, K. Lacković __________________________________________ 59


IX

Monitoring of the oxidatione stability of sodium picosulfate by a new RP-HPLC method

Ivana Savić, Goran Nikolić,Valentina Marinković, Miroslav Milenković, Ivan Savić, Predrag Sibinović _____________________________________________ 60

Quantification of Erythrocyte s-COMT Activity using a Validated Method Leonel Torrão, Rita Machado, Lyndon Wright, Patrício Soares-da-Silva ____________ 61

Fast pharmaceutical quality control based on elemental analysis Marina Fedoseeva, Elena Kapinus, Valentina Kosenko, Lidia Mitkina, Igor Revelsky __ 62

Computer modelling of possible of binding of calix[4]arenas to the active site of the myosin. A study of influence of calix[4]arenas on the ATP-ase activity of actomyosin and myosin subfragment-1 from the myometrium

Alyona A. Bevza, Alexander V. Bevza _______________________________________ 63

Synthesis, structural and biochemical characterization of oxime bond containing anticancer drug delivery systems

Gábor Mező, Antal Csámpai, Bence Kapuvári, Erika Orbán, Ildikó Szabó, László Radnai, Marilena Manea ___________________________________________ 64

111In and 177Lu radiolabelling and quality control of modified PAMAM dendrimer Veronika Biricová, Alice Lázníčková, Petr Hermann, Ivan Lukeš __________________ 65

Thermodynamic and structural study of pure compounds and mixed crystals in the α,ω-alkanediols family. The C10H22O2-C12H24O2 binary system

Gilgamesh Luis, Màrius Ramírez, Gabriel Luna, María M. Ramos, Martín A. Hernandez ___________________________________________________ 66

Preparation and investigation of bilayer tablets containing theophylline combined titanate nanotubes

Péter Kása jr., Tamás Sovány, Stane Srcic, Endre Horváth, Zoltán Kónya, Imre Kiricsi, Klára Pintye-Hódi ____________________________________________ 67

WORKSHOPS _________________________________________ 69

Challenges of Low Solubility Compounds A. Avdeef, S. M. Stones __________________________________________________ 71

ALPHA Workshop Jiří Šikola _____________________________________________________________ 72

AUTHOR INDEX _______________________________________ 73

Conference Programme


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Sunday, September 27th 09:00 – 11.00 Participant registration

10:00 – 12:00 & 16:00 – 18:00

Course 1, Conference Hall ArupinumPotentials of mass spectrometry in drug discovery Jasna Peter-Katalinić, University of Münster, Germany

10:00 – 12:00 & 16:00 – 18:00

Course 2, Conference Room GrisiaPolymorphs and other solid forms Rolf Hilfiker, Solvias AG, Switzerland

09:30 – 12:00 & 16:00 – 18:30

Course 3, Conference Room CissaNMR methods in Drug Discovery and Development Frederic Girard and Paul Michiels, Spinnovation Analytical Nijmegen, The Netherlands

15:00 – 19.30 Participant registration

20:00 Welcome party, Exhibition Area All Conference Lectures, as well as Oral Presentations will be given in the Conference Hall Arupinum

Monday, September 28th 09:00 Opening Ceremony

Chair: Biserka Cetina Čižmek

09:30 PL 01

Plenary Lecture Alex Avdeef, pION Inc. Woburn USA Miniaturized and Rotating Disk and Powder Intrinsic Dissolution Rate Measurement in Early Drug Development – Theoretical and Experimental Practice

10:15 IL 01

Invited Lecture John Comer, Sirius Analytical, UK pKa, logP, solubility and supersaturation in excipients and biorelevant media. Possibilities to promote a compounds BCS class

11:00 Coffee break

Chair: Klara Valko

11:15 PL 02

Plenary Lecture Jennifer Dressman, J. W. Goethe University, Germany Physical-chemical parameters to predict oral drug absorption


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12:00 IL 02

Invited Lecture Sibylle Neuhoff, Simcyp Ltd. Sheffield, UK Cell culture assays to predict oral drug absorption

12:30 OP 01

Mihalj M. Poša, Valéria J. Guzsvány, Janoš J. Čanadi, Ferenc F. Gaál, University of Novi Sad, Serbia Hydrogen bonds in the promotive action of bile acids on the kinetics of lidocaine transfer from an aqueous medium to chloroform

13:00 Lunch

Chair: Jennifer Dressman

15:00 PL 03

Plenary Lecture Klara Valko, GlaxoSmithKline, Stevenage, UK HPLC based property measurements of drug discovery compounds to predict in-vivo behaviour

15:45 IL 03

Invited Lecture Pranas Japertas, ACD/Labs Inc., Lithuania Assessing and Improving the Reliability of In Silico Physicochemical Property Predictions by Incorporating In-house Data

16:30 OP 02

Tamás Sovány, Ilija Ilic, Kitti Papós, Péter Kása, Stane Srcic, Klára Pintye-Hódi, University of Szeged, Hungary The role of surface free energy and plasticity of the materials in tablet formulation

16:45 OP 03

Ivan Simčić, Michaela Horvat, Tijana Dragić, Maja Mihoci, Tina Jelača, Vlatka Džale, Biserka Cetina-Čižmek, PLIVA Ltd. Zagreb, Croatia Improvement of solubility of low soluble drug telmisartan

17:00 Coffee break

17:15 W 01

17:15 18:00

Workshop, Exhibition Area A. Avdeef, pION Inc. Woburn USA and S.M. Stones, HeathScientific Challenges of Low Solubility Compounds Lecture Live Demonstration

Tuesday, September 29th 08:00 Poster mounting, Conference Room Cissa

Chair: Ernest Meštrović

09:00 PL 04

Plenary Lecture Rolf Hilfiker, Solvias AG, Switzerland The importance of choosing the optimal solid form for the success of a drug

09:45 PL 05

Plenary Lecture Slobodan Šašić, Pfizer, Groton, USA Raman Chemical Imaging in Development of Pharmaceuticals – ‘Not Seeing Is Just Guessing’


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10:30 IL 04

Invited Lecture Michaela Horvat, PLIVA Ltd, Croatia Solid-state kinetic studies: how to work less and find out more

11:00 Coffee break

Chair: Rolf Hilfiker

11:15 IL 05

Invited Lecture S. Prugovečki, U. Riedel, D. Beckers, PANalytical, The Netherlands Fighting counterfeit drugs by means of XRPD

12:00 OP 04

Robert Alexander, Stefano Pera, PerkinElmer, Italy Raman analysis of a polymorph within a polymorph

12:15 OP 05

Zbynek Oktabec, Josef Jampilek, Anna Zerzanova, Jiri Dohnal, Vladimir Kral, Zentiva k.s, Czech Republic Study of interactions between API and excipients in solid drug formulation via NIR

12:30 OP 06

R. K. Averinnei, N. Johan, G. Shavi, K. Armugam, U. Nayak, S. Pandey, N. Udupa, Manipal University, India Gliclazide cocrystals: In vitro and In vivo evaluations

13:00 Lunch

Chairs: Michael Przybylski and Pierre Parot

15:00 PL 06

Plenary Lecture Jasna Peter Katalinić, University of Münster Dissecting Carbohydrate Structure from Cells and Tissues

15:45 PL 07

Plenary Lecture Michael Overduin, University of Birmingham, UK Tools for solubilisation, screening and structural analysis of membrane proteins

16:30 IL 06

Invited Lecture Mario Cindrić, Ruđer Bošković Intitute, Croatia Protein instability and stability, a formulation challenge

17:15 OP 07

Iuliana Susnea, Sebastian Bunk, Corinna Hermann and Michael Przybylski, University of Konstanz, Germany Identification of Chlamydia pneumoniae antigens by immuno-proteomics using FTICR-MS and LC-tandem mass spectrometry

17:30 Poster Session, Conference Room Cissa

17:30 W 02

Workshop, Exhibition Area Jiří Šikola, Bruker Optics, Czech Republic Alpha workshop


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Wednesday, September 30th Chair: Jasna Peter Katalinić

09:00 PL 08

Plenary Lecture Michael Przybylski, University of Konstanz, Germany Mass spectrometry in the elucidation of lead structures for vaccine development

09:45 PL 10

Plenary Lecture Pierre Parot, CEA-Marcoule IBEB SBTN/LIRM, France AFM and DFS in the study of protein-ligand interactions

10:30 IL 07

Invited Lecture Dražen Čavužić, PLIVA Ltd. Croatia Identification of complexes in multicomponent systems by IR spectroscopy

11:00 Coffee break

Chair: Michael Overduin

11:15 PL 01

Plenary Lecture Gregg Siegal, University of Leiden, The Netherlands Target immobilization provides new opportunities in NMR-based fragment discovery and characterization

12:00 IL 08

Invited Lecture Primož Šket, Janez Plavec, NIC, Slovenia NMR studies of guanine-rich DNA oligonucleotides: Structures and cation dynamics

12:30 IL 09

Invited Lecture Tomislav Biljan, PLIVA Ltd. Croatia Fluorine-19 NMR in drug research

13:00 OP 08

Paul J. A. Michiels, Christian Ludwig, Michel Stephan, Christina Fischer, Gabriele Möller, Jerzy Adamski, Josef Messinger, Maria van Dongen, Hubert Thole, Ulrich L. Günther, Solvay Pharmaceuticals Research Laboratories, Weesp, The Netherlands NMR in drug discovery: Ligand-based NMR spectra demonstrate an additional phytoestrogen binding site for 17β-hydroxysteroid dehydrogenase type 1

13:15 Lunch

15:00 Excursion

20:00 Conference Dinner


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Thursday, October 1st Chair: Mario Cindrić

09:00 IL 10

Invited Lecture Philip Gribbon, European ScreeningPort GmbH, Germany Drug Discovery in academia: physics based approaches

09:45 OP 09

Igor V. Tetko, Iurii Sushko, Sergeii Novotarsky, Robert Körner, Anil Kumar Pandey, Matthias Rupp, Helmholtz Center Munich, Neuherberg, Germany Online chemical modeling environment

10:15 OP 10

Nemanja P. Trišović, Nataša V. Valentić, Tatjana L. Đaković-Sekulić, Gordana S. Ušćumlić, University of Belgrade, Serbia Solvent effects on the structure-activity relationship of 5-substituted-5-phenylhydantoins

10:45 Coffee break

11:00 IL 11

Invited Lecture Marilena Manea, Ildikó Szabó, Erika Orbán, Miguel Tejeda, Bence Kapuvári, Žarko Kulić, Peter Öhlschläger, Wilfried Reichardt, Dezső Gaál and Gábor Mező, University of Konstanz, Germany Tumor targeting with gonadotropin-releasing hormone derivatives

11:45 OP 11

Vitaly Erukhimovitch, Mahmoud Huleihel, Ben-Gurion University, Israel Spectroscopic method for the early evaluation of anti-bacterial agents activity

12:15 OP 12

Volodymyr O. Chernyshenko, I. S Maksymovych, Palladin Institute of Biochemistry, Kyiv, Ukraine Echis multisquamatis venom fibrinogenase is a prospective fibrinogen-depletive agent

12:30 Conference ending

Plenary Lectures


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Miniaturized Rotating Disk and Powder Intrinsic Dissolution Measurement in Early Drug Development – Theoretical and Experimental Practice

Alex Avdeef

pION INC, 5 Constitution Way, Woburn, MA 01801 USA [email protected]

Measurement of intrinsic dissolution rate (IDR) and equilibrium solubility profiles of practically insoluble compounds will be described, including comparisons between the literature values of IDR[1] obtained by traditional equipment, and values obtained with a new miniaturized system based on UV detection[2-4]. Dissolution data were collected in 1-10 mL of buffered as well as simulated intestinal fluid media at different pH values for low soluble compounds (e.g., mefenamic acid, glibenclamide, indomethacin, 2-naphthoic acid, phenazopyridine, and griseofulvin - as 50 µg powders and as 5 mg compressed miniaturized pellets). Also described will be the initial results from dissolution measurements using a 96-well microtitre plate prototype instrument. The study demonstrated that in situ UV monitoring is highly versatile technique allowing to measure intrinsic dissolution profiles and equilibrium solubility of compounds while monitoring all kinetic peculiarities of dissolution/precipitation solution, early in preclinical drug development, when only minute quantities of drug candidate compounds are available for study[3]. [1] Yu LX, Carlin AS, Amidon GL, Hussain AS. Feasibility studies of utilizing disk intrinsic

dissolution rate to classify drugs. Int. J. Pharm. 270 (2004) 221-227. [2] Avdeef A, Tsinman O. Miniaturized rotating disk intrinsic dissolution rate measurement: effects

of buffer capacity in comparison to traditional Wood’s apparatus. Pharm. Res. 25 (2008) 2613-2627.

[3] Tsinman K, Avdeef A, Tsinman O, Voloboy D. Powder dissolution method for estimating rotating disk intrinsic dissolution rates of low solubility drugs. Pharm. Res. 2009. in press.

[4] Avdeef A, Tsinman K, Tsinman O, Sun N, Voloboy D. Miniaturization of powder dissolution measurement and estimation of particle size. Chem. Biodiv. 2009. In press.

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Physical chemical parameters to predict oral drug absorption

Jennifer B. Dressman

J.W. Goethe University, Frankfurt, Germany

With the advent of high throughput screening and combinatorial chemistry techniques for the identification and synthesis of potential new drugs, a need has arisen to quickly and simply identify the biopharmaceutical properties of the drug candidates. Since the oral route of administration is the most convenient route of administration, an early assessment of a candidate’s potential bioavailability when administered orally is a particularly important piece of information for assessing “developability” as a pharmaceutical.

Drugs can be absorbed across the gastrointestinal mucosa by several different mechanisms, including transcellular diffusion, paracellular diffusion and active transport. There are also transported in the gut wall that can direct the drug back in to the gut lumen – the so-called exo-transporters. The probability of participation in the paracellular pathway is based on the molecular size and shape of the drug, whereas for active transport, the drug must be recognized as a substrate for the transporter. Thus, access to these pathways is limited and can be assessed through analysis of the structure. Transcellular diffusion represents the most important pathway and it is the transport by this route that we usually wish to predict. The use of physical chemical parameters for predicting transcelluar diffusion is an especially desirable option since these parameters can often be estimated in silico or assessed with simple laboratory experiments.

Current physicochemical methods include LogP, polar surface area, the Lipinsky’s Rules of Five, surface activity profiling as well as assessment of transport across membranes (PAMPA and/or cell cultures). In addition, the extent to which poor solubility can limit the availability of a drug for uptake fro the gut lumen should be assessed. All of these methods will be described in the presentation.

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HPLC based property measurements of drug discovery compounds to predict in-vivo drug distribution and aid lead optimisation

Klára Valkó

Analytical Chemistry, Molecular Discovery Research GlaxoSmithKline Gunnels Wood Road, Stevenage, Herts SG1 2NY United Kingdom

HPLC methodology offers great potential to determine distribution coefficients of biologically active compounds between an aqueous mobile phase and various non polar and bio-mimetic stationary phases via the measurement of retention times[1]. Thus, it provides an easily automated high throughput technique to determine important physico-chemical characteristics of drug discovery compounds.

Fully automated fast gradient HPLC methods have been developed to measure compound’s lipophilicity (using C-18 stationary phase[2]), plasma protein binding (using Human Serum Albumin[3] and Alpha-1-acid-glycoprotein stationary phases) and membrane partition (using Immobilised Artificial Membrane[4] stationary phase). Modern HPLC technology enables parallel measurements to be performed reducing total analysis time, additionally miniaturization helps to further reduce cost and time of analysis; and the application of mass spectrometry enables rigorous peak identification and thus increased data integrity. At GSK we have performed HPLC based physico-chemical property measurements on hundreds of thousands compounds providing lead optimization support for many research programs.

HPLC based bio-mimetic binding data have been successfully used to build models for in vivo drug disposition[5], such as volume of distribution, tissue partition and plasma protein binding. Additionally by measuring binding to proteins and membranes, we can estimate the non-specific/off target binding of compounds. Structure – bio-mimetic binding property relationships can be readily revealed and can be used to guide structural modification and compound selection early in the drug discovery process.

[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] K. Valko, C. M. Du, C. D. Bevan, D. P. Reynolds, M. H. Abraham, J. Pharm. Sci. 89 (2000) 1085 [5] F. Hollosy, K. Valko, A. Hersey, S. Nunhuck, Gy. Keri, C. Bevan, J. Med Chem. 49 (2006) 6958

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The importance of choosing the optimal solid form for the success of a drug

Rolf Hilfiker

Department Solid-State Development, Solvias AG, Basel, Switzerland [email protected]

Solid-state properties of active substances and excipients are of major practical and commercial importance all the way from research to manufacture of the final product. The search for, and more importantly the selection of the optimal solid form (salt, co-crystal, polymorph) is closely linked to the intended application of the final drug product. Good timing of solid-state development activities ranging from first screenings to crystallization optimization is becoming more and more important considering that CMC failures are one of the most important reasons for delays in drug development. An integrated approach to solid-state development is essential for successful development of a drug substance. General concepts like bioavailability of solids will be discussed, followed by examples of solid-state development procedures.

The following issues will be addressed: • How and when in the development cycle should solid state issues be dealt with? • An integrated approach to solid-state development • Discussion of efficient strategies for salt selection. • How to perform effective polymorphism screening, including HTS methods? • How to design a suitable crystallization process?

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‘Not Seeing is Just Guessing’ – Applications of Raman and NIR Chemical Imaging in Development of Solid Dosage Formulations

Slobodan Šašić

Pfizer, Analytical Research and Development, Groton 06344 CT, USA

Vibrational spectroscopic techniques such as Raman and near-infrared (NIR) are steadily gaining in importance in routine applications in the pharmaceutical industry. One of most sophisticated methods based on these two spectroscopies is chemical imaging in which chemical identity and spatial distribution of various components of a pharmaceutical product is determined in the samples of ordinary appearance. This is achieved by tagging micro-Raman or micro-NIR spectra with the spatial coordinates on the surface of a sample from where those spectra are obtained. The vibrational spectra of the components present in the tablets, blends, beads etc are normally quite distinguishable (in particular Raman) and in cases where the signal of satisfactory strength is obtained those differences allow for straightforward identification. This review covers various industrial applications of Raman and NIR chemical imaging exclusively in the field of development of solid dosage formulations in which, currently, those techniques can be used most purposefully. Particular attention is paid to cases in which small quantities of active pharmaceutical ingredients (API) are to be located. APIs are usually excellent Raman scatterers so the combination of efficient Raman scattering and use of microscope (i.e. high power density) allows for relatively simple detection of APIs in the concentration of 1% and less. Similarly, some elusive excipients such as Mg stearate can in most cases be spotted in Raman chemical images. Finally, an address is made to the use of relatively advanced mathematical tools for recovering and distinguishing vibrational spectra. While the spectra per se certainly tend to be distinct, there normally is significant overlap between them that significantly complicates the analysis. The overlap is unavoidable (in particular for the NIR spectra) due to the axial and radial distribution of the impinging excitation light. A few broadly known linear-algebra-based tools (such as principal component analysis or partial least square regression) are mentioned and examples of their applications are listed.

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Dissecting Carbohydrate Structure from Cells and Tissues

Jasna Peter-Katalinić

University of Muenster, Germany and University of Rijeka, Croatia

Cells and tissues produce a variety of glycoproteins and glycolipids that contribute to cell morphology and function in cell adhesion, promoting growth and differentiation, and the induction of apoptosis. The glycodynamics of cells can be studied using primary cell cultures or tissue biopsies obtained from surgery, following molecular changes of glycopatterns by mass spectrometry. Oligomeric glycoconjugates were shown to undergo specific biological interactions in receptor-ligand recognition, cell transport and cell-cell interactions. A major feature of protein glycosylation is the carbohydrate microheterogeneity on the same glycol-sylation site. It is caused by (i) the presence of different structural classes, like high-mannose, hybrid and complex type in N-glycans and of eight possible core structures in O-glycans (ii) different stages of the chain elongation and branching, due to biosynthetic dynamics concerning anabolic or catabolic processes (iii) different terminal epitopes on the same sugar chain, depending on different activity of glycosyl transferases in an organ or an organism and (iv) different modifications of sugars by non-carbohydrate functional groups[1]. Besides, dynamic epigenetic regulation of the glycan precursor chain in N-glycosylation or the N-acetylgalacto-samine precursor in O-glycosylation is influenced by protein structure and its acceptor affinity[2]. Since different glycosylation motifs exert different functions, it is of high relevance to dissect the glycosylation patterns on single N- and O-glycosylation site[3]. Few efficient methods developed to study the distribution of single N- and O-glycosylation sites in annotated proteoms are based on identification of structural changes at the attachment site upon detachment of the glycan chain by enzymatic or chemical methods[4]. However, to determine de-novo glycopatterns in native glycol-proteins proteomics approach seems to be the most efficient one, if the nascent glycopeptides generated by proteolysis can be identified by mass spectrometric (MS) sequencing. Adequate fragmentation could be elaborated using the low-energy collisional activation (CID) under well defined conditions; however, more robust methods were required for high-throughput procedures. Electron Capture Dissociation (ECD) performed within the ion cyclotron resonance (ICR) cell was shown to provide non-ergodic conditions, favourable for retaining the glycan attachment, and for generation of diagnostic ions relevant for the identification of the glycosylation site and the type of the glycan attached[5]. Sequencing of glycopeptides with long chain sugars were most efficiently performed by infrared multiphoton dissociation (IRMPD)[6]. In the set of the presently available methods for standardization of glycosylation analysis[7] novel tools for assessment of glycol-sylation microheterogeneity, like chip-MS and software platform[8,9] will be discussed. [1] J. Peter-Katalinić, Mass Spectrom. Rev. 13 (1994) 77-98; J. Peter-Katalinić, Methods Enzymology 405

(2005) 139-171. [2] F.-G. Hanisch, S. Müller, H. Hassan, H. Clausen, N. Zachara, A. A. Gooley, H. Paulsen, K. Alving, J. Peter-

Katalinić, J. Biol. Chem. 274 (1999) 9946-9954. [3] J. Hofsteenge, K. Huwiler, B. Maček, D. Hess, J. Lawler, D. Mosher, J. Peter-Katalinić, J. Biol. Chem., 276

(2001) 6485-6498; A. Gonzalez de Peredo, D. Klein, B. Maček, D. Hess, J. Peter-Katalinić, J. Hofsteenge, Molecular & Cellular Proteomics 1 (2002) 11-18.

[4] F.-G. Hanisch, M. Jovanović, J. Peter-Katalinić, Anal. Biochem. 290 (2001) 47-59. [5] M. Mormann, B. Maček, A. Gonzalez de Peredo, J. Hofsteenge, J. Peter-Katalinić, Int. J. Mass Spectrom.

234 (2004) 11-21; M. Mormann, H. Paulsen, J. Peter-Katalinić, Eur. J. Mass Spectrom. 11 (2005) 497-511. [6] L. Bindila, K. Steiner, M. Mormann, C. Schäffer, P. Messner, J. Peter-Katalinić, Anal. Chem. 79 (2007)

3271-3279 [7] Y. Wada, P. Azadi, C. E. Costello, et al. Glycobiology 17 (2007) 411-422. [8] L. Bindila, J. Peter- Katalinić, Mass Spectrom. Rev. 28 (2009) 223-253. [9] S. Y. Vakhrushev, D. Dadimov, J. Peter-Katalinić, Anal. Chem. 81 (2009) 3252-3260.

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Page 9

Solubilizing membrane proteins in nanoparticles and ligand interactions of signaling proteins by NMR

Timothy J. Knowles, Mark Jeeves, Mike Tong, Corinne Smith*, Yu-Pin Lin, Tim Dafforn, Michael Overduin

University of Birmingham, Edgbaston, Birmingham, U.K., *University of Warwick, Coventry, U.K.

Membrane proteins are both the most valuable and technically challenging targets for drug discovery. Finding a gentle solution that preserves their structure and activity yet is robust enough for experimental interrogation has represented a major difficulty. A new nanoparticle system has been developed that solubilises helical and barrel membrane proteins using an unusual amphipathic polymer. Proteins maintain their folded structure and binding and enzymatic activities in the polymer-lipid nanoparticles based on NMR and biophysical assays. Solution NMR and thermal shift methods are being used to identify novel interactions of signaling proteins including human kinases and phosphatases and regulatory domains, providing insights into their biological mechanisms and new opportunities for drug discovery.

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Mass spectrometry in the elucidation of lead structures for vaccine development

Michael Przybylski, Irina Perdivara, Claudia Cozma, Adrian Moise, Gabriela Paraschiv, Stefan Slamnoiu, Marius Iurascu, Mihaela Dragusanu, Alina Petre, Andreas Marquardt,

Camelia Vlad, Marilena Manea

Department of Chemistry, Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, University of Konstanz, 78457 Konstanz, Germany

“Soft-ionisation” methods of mass spectrometry (MS), particularly electrospray (ESI-MS) and matrix assisted laser desorption-ionisation (MALDI-MS), have enabled the molecular analysis of peptide and protein chemical structures and their modifications, e.g. in proteome analysis. Affinity-mass spectrometry, using selective proteolytic excision of immobilised protein-ligand complexes in combination with mass spectrometry, has recently emerged as a new tool for the elucidation of molecular recognition structures[1]. Thus, the identification of antigen-epitopes in immune complexes using epitope excision-mass spectrometry has been shown as a cornerstone in the elucidation of lead structures for vaccine development. This is illustrated by studies towards the development of immuno-therapeutic approaches against Alzheimer’s disease (AD), using specific antibodies against ß-amyloid (Aß), the key neurotoxic peptide fragment for the formation of Aß-plaques. The differential epitope structure identification by affinity-MS of (i), therapeutic antibodies upon immunisation with Aß, that disaggregate Aß-plaques, and (ii), physiological Aß-autoantibodies in serum capable of eliciting a protective effect to inhibit the formation of Aß-plaques, provides a breakthrough in the development of (i), AD immuno-therapeutic approaches by immunisation with Aß-specific antibodies, and (ii), the development of molecular AD diagnostic tools with absolute specificity[2]. Most recently, ion mobility mass spectrometry (IM-MS) has been introduced as a new molecular tool to analyse conformation-dependant protein assemblies, and “misfolded” protein aggregates such as Aß-plaques. First applications of IM-MS to identify oligomeric intermediates for aggregates of Aß and other neurodegenerative target proteins, and their implications for vaccine development will be discussed[3]. These studies indicate affinity-MS and ion mobility-MS as powerful new tools for the elucidation and molecular structure characterisation of lead structures for vaccine development against neurodegenerative diseases. [1] McLaurin, J., et al. Nature Med. 8 (2002) 1263-1269; Macht, M., et al. Anal. Bioanal. Chem. 378

(2004) 1102-1111. [2] Stefanescu, R., et al. Eur. J. Mass Spectrom. 13 (2007) 69-75; Przybylski, M. et al., Univ.

Konstanz & Budapest (2008) Eur. & US Patent Applications. [3] Przybylski, M., et al. (2009) Nature Meth., submitted; Dragusanu, M., et al. (2009) Angew.

Chem., submitted.

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Page 11

AFM and DFS in the study of protein-ligand interactions

Pierre Parot, Jean-Luc Pellequer

CEA-Marcoule, IBEB, SBTN/LIRM, F-30207 Bagnols sur Cèze, France

High-resolution imaging of single molecules and their complexes as well as mechanisms involved in protein-ligand recognition, respectively obtained by Atomic Force Microscopy (AFM) and Dynamic Force Spectroscopy (DFS), are very useful to suggest new strategies in molecular biochemistry and potentially for the design of drugs and research reagents for life sciences and medicine. The presentation will focus on DFS measurements of unbinding forces and characterization of the energy landscape of receptor-ligand interactions. We will then introduce molecular recognition kinetics based on the transition state theory and emphasize the treatment of multiple parallel bonds encountered in many biological systems, including a revision of the well-known avidin-biotin system. Detailed analysis of the energy landscape of monoclonal antibodies and dicarboxy-phenanthroline-uranyl interactions will illustrate this new approach of ligand-protein interactions at the single molecule level.

YIELDFINDER II program used in our lab for

Analyzing force-distance curves obtained in DFS

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Target immobilization provides new opportunities in NMR-based fragment discovery and characterization

Gregg Siegal

ZoBio, Leiden, NL and Leiden University, The Netherlands

We have developed a method called TINS, for Target Immobilized NMR Screening, that enables Fragment screening of a greater range of targets including those that are not possible to obtain in large quantities and/or are insoluble such as integral membrane proteins[1]. In TINS, the target (as little as 5 nmol) and a reference are immobilized on a solid support and a collection 1,500 fragments is screened in an automated manner using proprietary hardware[2]. To date soluble proteins, including kinases, GTPases, viral targets and proteases, as well as membrane proteins, have been successfully screened against our collection of commercially available fragments. In addition to far more efficient use of the target, immobilization yields a variety of advantages that are important for the drug discovery process. For one, target specificity can be greatly enhanced by appropriately selecting the reference and avoiding off-target hits. In addition, the sensitivity to weak interactions is extended to the 10 mM range. This sensitivity is critical when targeting Protein-Protein Interactions where typical binding affinties are reduced by a factor of 5-10 with respect to targets with well-defined small molecule binding sites. Here I will present the technology and studies comparing the sensitivity and false negative rates of TINS to other commonly used fragment screening approaches such as solution NMR and SPR. I will also discuss applications of TINS in drug discovery projects and recent developments including quantitating ligand binding and the use of TINS for ligand discovery using membrane proteins. [1] S. Vanwetswinkel, R. J. Heetebrij, J. van Duynhoven, J. G. Hollander,D. V. Filippov, P. J.

Hajduk and G. Siegal,. Chem. Biol. 12 (2005) 207-216. [2] T. Marquardsen, M. Hofmann, J. G. Hollander, C. M. P. Loch, S. R. Kiihne, F. Engelke and G.

Siegal, J. Magn. Reson. 182 (2006) 55-65.

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Invited Lectures


Page 15

pKa, logP, solubility and supersaturation in excipients and biorelevant media.

Possibilities to promote a compound’s BCS class

John Comer

Sirius Analytical, UK

Currently, there is a great deal of interest in supersaturating dosage forms as a means of improving the bioavailability of drugs with poor aqueous solubility. This presentation will describe methods for assessing the amount of supersaturation present in an in-vitro system in early phase drug discovery by studying dissolution and precipitation events in real-time.

Solubility enhancement in the presence of formulation excipients will be presented, showing how this can be used as an opportunity for promoting a molecule’s class in the Biopharmaceutics Classification System. The effects of bio-relevant media (e.g., simulated intestinal fluids) will also be explored. Understanding pKa, logP and solubility enhancement are critical to designing optimal supersaturating dosage forms as well as improving predictive models for absorption by capitalizing upon supersaturation and precipitation events in-vivo.

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Cell culture assays to predict oral drug absorption

Sibylle Neuhoff

Bioinformatics, Simcyp Limited, Sheffield, UK

Transport into and across the cells of the human body is a prerequisite for the pharmacological action of drugs. Orally administered drugs are required to cross the intestinal epithelial cell layer prior to entering the systemic circulation. During this process, intestinal metabolism may occur. Thereafter, transfer of the drugs across the cellular membranes of hepatocytes determines access to intracellular drug-metabolizing enzymes in the liver. For a long time, passive diffusion was considered to be the dominating drug transport process across cell membranes and in vitro-in vivo correlations (IVIVC) leading to qualitative and sometimes quantitative categorization of compounds permeation in the discovery phase of drug development, were established. During the last decade, the complexity of membrane transport has become increasingly apparent. The characterization of the human genome has revealed an excess of proteins facilitating the transmembrane transport of a variety of solutes and over 400 transporters are known today. Many of these proteins have the capacity to transport drug molecules and contribute significantly to the pharmacological and toxic activity of drug molecules. The process involved in transfer of compounds across the canalicular membrane and in renal secretion is nowadays assigned predominantly to active transporters, while oral drug absorption is still viewed as a mixture of passive and active transcellular and paracellular transport.

Cell culture assays are relatively easy to perform and potentially provide a lot of information on the ADME properties of a compound. It is possible to culture some immortalized epithelial cell lines on porous filter supports to form confluent monolayers with reproducible (relative) protein expression and polarity (e.g., Caco-2, MDCK II, LLC-PK1, 2/4/A1). These cells can then be used for automated screening assays as well as manual and mechanistic studies. Although mechanistic studies are more demanding, they may provide valuable information that can be used to explain any unexpected or ambiguous results from high-throughput screening (HTS) or indeed, lead to overall improvement of the experimental design of the HTS assay. Depending on the cell line and the assay conditions, the focus can be on the paracellular pathway or on the transcellular pathway, with and without consideration of active transport and/or metabolism.

This presentation aims to raise an awareness of the cell models and underlying assumptions used for predicting the permeability of drugs, when it is considered to be the rate-limiting step for entering the systemic circulation.

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Assessing and Improving the Reliability of In Silico Physicochemical Property Predictions by Incorporating In-house Data

P. Japertas*, R. Didziapetris*, A. Sazonovas*,**, A. Petrauskas*

*ACD/Labs, Inc., A.Mickeviciaus g. 29, LT-08117 Vilnius, Lithuania; **Faculty of Chemistry, Vilnius University, Naugarduko g. 24,

LT-03225 Vilnius, Lithuania

A number of problems have been long known to prevent the effective use of third-party predictive algorithms in the pharmaceutical industry. Among them is the training set not covering the specific part of the chemical space occupied by the compounds that a certain company is working with or a specific experimental protocol used to measure the property of interest that yields the results contrasting with the experimental values for the same compounds in the training set. Therefore the need arises for a method that would allow any company to tailor a third-party predictive algorithm to its specific needs using proprietary in-house data. Here we present a novel similarity based methodology that provides a possibility for a user to expand the Applicability Domain of the existing Pharma Algorithms models with the help of a custom database of experimental values for the property of interest. A Reliability Index (RI) is also calculated as a measure of the quality of the particular prediction. The use of the method is illustrated with examples of its application in predicting logP, logD and solubility of the compounds. It is shown that a relatively small amount (5 to 10) of similar compounds has to be added to substantially improve the prediction for a group of problematic compounds that is not represented in the original training set. The Reliability Index is shown to be closely related to the overall quality of any given prediction that is represented by a clear correlation of the RI and RMSE values. Given that the improvement of any Pharma Algorithms model in this way is instant as it occurs that very moment when new compounds with experimental values are added to the similarity database and there is no need to retrain the model, this method opens completely new possibilities for their use in the industry.

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Solid-state kinetic studies: how to work less and find out more

Michaela Horvat

Pliva Croatia Ltd., Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia

Solid-state kinetic studies are of great interest to pharmaceutical industry, as the majority of final dosage forms are solid multi-component systems in which components may interact in the course of time. Furthermore, active pharmaceutical ingredients may be present in various polymorphic/pseudo-polymorphic forms with a latent capability to convert from one form to another. In this complex world, understanding the rate, path and conditions under which such transitions occur is essential. The focus is in investigations which include conversions in the solid state; polymorphic transitions, crystallizations, degradation and dehydration reactions. Despite having developed various experimental and computational methods which support kinetic studies in the solid-state, there has been an ongoing discussion and controversy regarding the theoretical framework, approximations and assumptions which have been used. Nevertheless, attention needs to be drawn to another issue: an inconsistency in experimental setup, data analysis and drawing of conclusions, which might often lead to contradicting results and incomplete information about investigated phenomena. A new approach by which these issues can be overcome will be presented. In particular, the kinetics of dehydration of two structurally similar compounds will be shown. The compounds were analyzed by means of isothermal thermogravimetric analysis. It is known that the results of thermal methods of analysis are highly dependant on experimental conditions used. Therefore, all relevant experimental parameters which might have an effect on dehydration behaviour such as particle size, gas flow, sample mass and temperature have been taken into consideration. However, this approach led to numerous data in which much information could have remained hidden, if had it not analyzed by adequate mathematical methods. The obtained data have been subjected to regression analysis and analysis of variance (ANOVA) in order to determine which model best describes the dehydration behaviour. A further step has been taken in data analysis: principal component analysis (PCA) was employed to determine which experimental parameters play the major role in the change of the dehydration mechanism. All results were explained in the light of crystal structure and microscopic observations.

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Page 19

Fighting counterfeit drugs by means of XRPD

S. Prugovecki, U. Riedel, D. Beckers

PANalytical B.V., Almelo, The Netherlands

X-ray powder diffractometry (XRPD) allows to distinguish original medicaments from counterfeit products. The X-ray diffraction patterns are characteristic „fingerprints“ of tablets, capsules or powders and can be measured without a loss of information through the closed blister package. Hence a fast and easy analysis can be performed even by nonqualified personnel without the risk of contact with possibly hazardous drugs like hormones or anticancer drugs. Required instrumentation, experimental setup and analysis steps will be discussed. Examples of identification of counterfeit products and their comparison with originals will be shown.

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Protein instability and stability, a formulation challenge

Mario Cindrić, Renata Pavišić*

Ruđer Bošković Institute., Division of Molecular Medicine, Bijenička cesta 54, Zagreb, Croatia,

*Pliva Croatia Ltd., Bioanalytics, Prilaz baruna Filipovića, 29, Zagreb, Croatia

Development of stable protein or peptide formulation is one of the critical steps in pharmaceutical protein formulation development, towards to the final therapeutic product. A major challenge is to maintain the integrity of a protein drug during pharmaceutical processing, storage, handling and administration, knowing that proteins typically raise more stability issues than “small molecules” as a result of macromolecular instability and complexity (numerous reactive chemical centers and degradable elements). Stability issues start with preservation of delicate 3-D structure stabilized by noncovalent interactions such as hydrogen bonding, ionic interactions, Van der Waals’ forces and hydrophobic packing and end with protection of linear amino acid sequence and potential post-translational modifications.

By determining stabilizing effect of a given stabilizer under the specific physico-chemical conditions, selection and/or comparison of the stabilizers used followed by statistical evaluation bring out general rules that can be applied to prevent unwanted protein/peptide degradation processes. As the science of formulating protein drug today is very much an interdisciplinary problem, together with new advances in the fields such as medicinal research, analytical and pharmaceutical development, manufacturing (process chemistry and engineering), as well as quality and regulatory requirements, new topics in the area of protein formulation emerge.

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Page 21

Identification of complexes in multicomponent systems by IR spectroscopy

Dražen Čavužić

TEVA Pharmaceutical Industries, Pliva, Croatia TAPI R&D, Physical R&D

Classical approach to the multicomponent system analysis is to use techniques which involves separation of the components, like chromatography, extraction, precipitation etc.

Another approach is based on the usage of methods, which selectively detect only one component of the mixture. Common technicques used in this approach are spectroscopy and electrochemistry, often in combination with derivatization or „masking“ of interfering components.

Development of multivariate data analysis – chemometrics, enabled nonselective spectroscopic methods to be used in multicomponent system analysis, which is especially useful in situations when component separation or derivation are not appropriate, or even possible. With no selectivity, its biggest flaw removed, spectroscopy, being simple, fast, robust and inexpensive method, providing plenty of structural information, became an invaluable tool for the characterization of multicomponent systems.

For that reason, spectroscopy methods in conjunction with multivariate calibration (PCR & PLS) are often used for component quantification in many complex systems, like blood, whole tablets, whole grains etc, and in various process control applications.

Beside mentioned quantitative methods, chemometrics encompass many qualitative methods, like Evolving factor analysis, Window factor analysis, Alternate least squares and other „self-modelling“, multivariate curve resolution methods. This methods can reveal number, “pure” spectra and concentration profiles of spectroscopic active components during chemical or physical changes of the system, without the need for any a priory information or even assumptions, about the system.

Application of IR spectroscopy combined with Principal component analysis, Evolving factor analysis and Alternate least squares regression is demonstrated on the example of eutectic paracetamol-propyphenazone mixture, which, after melting forms two different, transient, hydrogen–bonded complexes before final crystallization.

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22

NMR studies of guanine-rich DNA oligonucleotides: Structures and cation dynamics

Primož Šket, Janez Plavec

Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia

DNA molecules can adopt besides the well-known B-type double helix several higher-order structures, including G-quadruplex structures. G-quadruplexes are stable structures adopted by DNA guanine-rich sequences that can be found in telomeres, immunoglobulin switch regions, gene promoter regions and have also been implicated in association with human diseases, as therapeutic targets in drug design and in potential technical applications as nanomolecular devices. In the past years G-quadruplex DNA structures are a subject of great interest since their formation has been suggested to play a role in variety of important biological processes as well as due to their potential therapeutic applications. The main building blocks of G-quadruplex structures are stacks of square-planar arrays of G-quartets, consisting of four guanines that are linked together by eight hydrogen bonds. The major requirement for the formation of such structures is the presence of cations. Development of small molecules that can bind and help in formation and stabilization of G-quadruplex structures has been the focus of search for anti-cancer and anti-viral drugs. By the use of NMR spectroscopy we have shown that the folding topologies of G-quadruplex structures critically depend on the sequence details as well as nature of cations present in solution. Small change in G-rich sequence can cause a dramatic change in topology of G-quadruplex structures. d(G3T4G4) sequence folds into dimeric fold-back G-quadruplex structure, which consists of three G-quartet planes, two overhanging guanine residues and diagonal as well as edge-type loops in the presence of K+, 15NH4

+ or Na+ ions. Furthermore, NMR experiments confirm existence of a mixed mono-K+-mono-15NH4

+ form that represents intermediate in the conversion of di-15NH4

+ into di-K+ form of d(G3T4G4)2 G-quadruplex. Recently, 15N-labeled ammonium ion has been utilized as a non-metallic substitute in combination with 2D NMR spectroscopy to localize cations inside the interior of G-quadruplex structures as well as provide insight into kinetics of their movement. Using this approach we have analyzed cation movement inside a number of G-quadruplex systems of different stoichiometries. Our findings so far give insight into how structural details of G-quadruplexes play a role in controlling cation transport and their kinetics along the central axis as well as into bulk solution. [1] Crnugelj, M.; Sket, P.; Plavec, J., J. Am. Chem. Soc., 125 (2003) 7866-7871. [2] Sket, P.; Crnugelj, M.; Kozminski, W.; Plavec, J., Org. Biomol. Chem. 2 (2004) 1970-1973. [3] Sket, P.; Crnugelj, M.; Plavec, J., Bioorg. Med. Chem. 12 (2004) 5735-5744. [4] Sket, P.; Crnugelj, M.; Plavec, J., Nucleic Acids Res. 33 (2005) 3691-3697. [5] Sket, P.; Plavec, J., J. Am. Chem. Soc. 129 (2007) 8794-8800. [6] Podbevsek, P.; Hud, N. V.; Plavec, J., Nucleic Acids Res. 35 (2007) 2554-2563. [7] Podbevsek, P.; Sket, P.; Plavec, J., J. Am. Chem. Soc. 130 (2008) 14287-14293.

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Page 23

Fluorine-19 NMR in drug research

Tomislav Biljan

PLIVA CROATIA Ltd., European Research and Development, Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia

Among the numerous marketed pharmaceuticals more than 150 drugs are fluorinated compounds[1,2]. The occurrences of a fluorine substituent in commercial drugs continuously increase and today it can be estimated that globally 20-25% of drugs contain at least one fluorine atom. This is a huge frequency when comparing with other halogen containing drugs and especially when one considers the fact that there are virtually no natural products that contain fluorine. Incorporation of fluorine atoms in drugs are driven by the special properties of fluorine atom such as strong electronegativity, small size and the low polarisability of the C-F bond that can significantly impact the behavior of drug molecule in biological environment[3].

NMR spectroscopy is widely used technique in pharmaceutical industry in all phases of drug development. In the context of fluorine containing drugs 19F NMR is very powerful method for studying such molecules. Fluorine-19 nucleus is very attractive for NMR studies due to the 100% natural abundance, high intrinsic sensitivity and a magnetogyric ratio only slightly smaller than that of proton. The fluorine-19 chemical shifts are also highly sensitive to even subtle changes in the magnetic environment, the property that can be used as a probe for distinguishing closely related molecules. The analysis of complex reaction mixtures, formulated drug products and drug metabolites play an important role in pharmaceutical research. However, the analysis of such samples is often very difficult by severe resonance overlap in proton spectrum. Fluorine-19 NMR is a good alternative in such cases if the molecule contains fluorine atoms. Normally excipients, solvents and biological matrices do not contain fluorine atoms and identification of target molecule is much easier. Solid state fluorine-19 NMR is also very useful for identification and quantification of fluorine containing drug molecules and its polymorphs. The use of NMR based screening in drug discovery is very well known. In recent years it has been demonstrated that ligand and substrate based fluorine-19 NMR screening is a powerful tool for identification of novel active compounds[4].

The overview of fluorine-19 NMR spectroscopy use in pharmaceutical research will be presented. [1] S. Purser, P. R. Moore, S. Swallow and V. Gouverneur, Chem. Soc. Rev. 37 (2008) 237. [2] C. Isanbor and D. Hagan, J. Fluorine Chem. 127 (2006) 303. [3] J. P. Bégué and D. Bonnet-Delpon, J. Fluorine Chem. 127 (2006) 992. [4] C. Dalvit, Prog. NMR. Spectrosc. 51 (2007) 243.

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Drug Discovery in Academia: Physics based approaches

Philip Gribbon

European ScreeningPort GmbH, Hamburg, Germany [email protected]

Small molecule Drug discovery within Academia has become firmly established in the past 5 years. New technologies, many of which employ physical based techniques are being adopted more widely and offer advantages over traditional screening and compound profiling techniques, marking a departure from the “one gene, one protein, one target, one compound” model. One critical consideration in small molecule screening is the unequivocal selection of compounds in fluorescence-based assays based on biological activity. Three approaches will be highlighted which have been devised and implemented to identify such interfering compounds (single molecule methods, fluorescence lifetime determination and mathematical modeling of large-scale cellular kinetic responses following receptor agonism). New label free screening have been developed for analyzing kinase activity and which offer sensitivity at the nanomolar level whilst also eliminating artifacts arising from optical interference effects. These surface enhanced Raman approaches will be discussed in detail and their further application on ultra sensitive detection of biomarkers/analytes with applications in Phase 0 (microdosing) and Drug Delivery using nanoparticle systems will be outlined.

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Page 25

Tumor targeting with gonadotropin-releasing hormone derivatives

Marilena Manea1,2, Ildikó Szabó3, Erika Orbán3, Miguel Tejeda4, Bence Kapuvári4 Žarko Kulić1,5, Peter Öhlschläger6, Wilfried Reichardt7, Dezső Gaál4, Gábor Mező3

1Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of

Chemistry, University of Konstanz, Germany 2Zukunftskolleg, University of Konstanz, Germany

3Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös L. University, Budapest, Hungary

4National Institute of Oncology, Budapest, Hungary 5NMR Spectroscopy, Department of Chemistry, University of Konstanz, Germany

6Laboratory of Immunology, Department of Biology, University of Konstanz, Germany 7Department of Diagnostic Radiology/Medical Physics,

University Hospital Freiburg, Germany

Targeted cancer chemotherapy has been developed in order to overcome the drawbacks associated with the application of free anticancer drugs (e.g. lack of selectivity, toxic side effects, multidrug resistance of cancer cells). The attachment of anticancer drugs to a targeting moiety that recognizes tumor-specific or overexpressed receptors on cancer cells might provide efficient chemotherapeutic agents with minimal systemic toxicity. It was found that receptors for peptide hormones, such as gonadotropin-releasing hormone (GnRH), are overexpressed on cancer cells. Consequently, GnRH and its derivatives can be used as targeting moieties to deliver cytotoxic agents directly to tumor cells, thereby increasing the concentration of the drugs in the tumor tissue and sparing normal cells from unnecessary exposure. One of the most promising natural GnRH analogs isolated from sea lamprey is GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2) which specifically binds to GnRH receptors on cancer cells and has lower hormonal effect in mammals than the human GnRH. Daunorubicin (Dau) and doxorubicin (Dox), anthracycline antibiotics widely used in cancer chemotherapy, were attached to the ε-amino group of 8Lys of monomeric GnRH-III via ester, hydrazone, oxime or amide bonds either directly or by insertion of a GFLG tetrapeptide spacer. The drug release from the bioconjugates was determined in the presence of Cathepsin B, chymotrypsin, as well as in human, rat and mouse sera and in rat liver lysosomal homogenates by liquid chromatography in combination with mass spectrometry. The in vitro long term antitumor effect of the compounds was studied on various cancer cell lines such as MCF-7 human breast, C26 murine colon, HT-29 human colon cancer cell lines. The in vivo antitumor activity of free daunorubicin, oxime bond linked GnRH-III(Dau=Aoa-GFLG) conjugate, GnRH-III or GnRH-III(GFLG) hormone analog was determined on C26 colon cancer bearing mice and on TRAMP model (transgenic adenocarcinoma of the mouse prostate). The conjugate had lower toxic effect than the free daunorubicin and its administration resulted in ~ 50% tumor growth inhibition and longer survival time of C26 colon cancer bearing mice and TRAMP mice. Acknowledgement: This work was supported by grants from the Hungarian National Science Fund (OTKA T049814, NK 77485), the Ministry of Health (ETT 202/2006), GVOP-3.2.1.-2004-04-0005/3 and Zukunftskolleg and AFF (Project 01/09), University of Konstanz.

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Oral Presentations


Page 29

Hydrogen bonds in the promotive action of bile acids on the kinetics of lidocaine transfer from an aqueous medium to chloroform

Mihalj. M. Poša, Valéria. J. Guzsvány*, Janoš. J. Čanadi*, Ferenc. F. Gaál*

Laboratory of Physical Pharmacy, Department of Pharmacy, Medical Faculty, University of Novi sad, Hajduk Veljkova 3, Novi Sad, Serbia,

*Department of Chemistry, Faculty of Science, University of Novi Sad, Trg D. Obradovića 3, Novi Sad, Serbia

The equilibrium constant K of the formation of a complex between lidocaine and a number of bile acids was determined by monitoring the shift of the signal of the amide proton of lidocaine in the 1H NMR spectrum as a function of bile acid concentration[1]. Such signal shift of the amide proton of a chloroform solution of lidocaine does not take place in the presence of acetone or methanol. This observation can be explained by the assumption that bile acids in their binding to lidocaine use more than one functional group, involving thus the effect of neighboring groups, as well as the entropic effect due to mutual proximity of the binding groups. The equilibrium constants obtained for the complex formed between lidocaine and bile acids are as follows: 7-monoketocholic acid, K = 14.22 dm3mol-1; 12-mo-noketodeoxycholic acid, K = 9.45 dm3 mol-1; methyl ester of 3α-acetoxy-7-monoketocholic acid, K = 9.36 dm3 mol-1; 3,7,12-triketocholic acid, K = 8.15 dm3 mol-1; 7-monoketocheno-deoxycholic acid, 6.50 dm3 mol-1; 3,12-diketodeoxycholic acid, K = 6.33 dm3 mol-1; 7,12-diketocholic acid, K = 4.44 dm3 mol-1; chenodeoxycholic acid, K = 3.77 dm3 mol-1; 3α,12α-diacetoxy-7-monoketocholic acid, K = 2.04 dm3 mol-1, and methyl ester of 3,7,12-triketocholic acid, K = 0.00 dm3mol-1. A prerequisite for the formation of a complex with lidocaine is that the bile acid has two OH groups, or one keto and one OH group at the corresponding mutual distance. The equilibrium constant of the complex between lidocaine and bile acid decreases with the decrease in the number of possible 1:1 complexes that may be formed with each of the investigated bile acids.

The other part of the study is concerned with the effect of the hydrogen-bonded complex formed between lidocaine and bile acid in chloroform on the kinetics of concentration decrease of lidocaine in the aqueous phase (model for pretreatment with bile acid). The decrease of the lidocaine concentration with and without presence of bile acids in the aqueous phase (pH 5.5; 6.5 and 7.4) can be described by a monoexponential function. Values of the rate constant of lidocaine concentration decrease in the aqueous phase depend on the structure and concentration of the bile acid in chloroform. Structural influence of bile acids on the rate constant may be expressed via the equilibrium constant K of the complex between lidocaine and bile acid in chloroform. The positive correlation between the complex equilibrium constant K and the rate constant of lidocaine depletion in the aqueous phase, k, is stronger if the the bile acid concentaration in chloroform is higher. Bile acids at pH 5.5 and 6.5, apart from increasing the value of the rate constant of lidocaine depeletion in the aqueous phase, decrease at the same time its equilibrium concentration, whereas at pH 7.4 it influences only the rate constant of lidocaine concentration decrease in the aqueous phase. [1] M. Poša, V. Guzsvany, J. Csanadi, S. Kevrešan, K. Kuhajda, Formation of hydrogen-bonded

complexes between bile acids and lidocaine in the lidocaine transfer from an aqueous phase to chloroform, Eur. J. of Pharm. Sciences 34 (2008) 281-292.

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The role of surface free energy and plasticity of the materials in tablet formulation

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

University of Szeged, Department of Pharmaceutical Technology,Szeged, Hungary, *University of Ljubljana, Department of Pharmaceutical Technology, Ljubljana, Slovenia

Tablet is the most common dosage form. To the assurance of their appropriate bioavailability and applicability, strong property requirements need to be fulfilled. The physico-chemical property of the applied materials significantly influences this behaviour, and determines the method of the tablet production. Direct compression, which is the most simple and economic method of tablet preparation requires drugs with good flow properties and compressibility. Flow properties are influenced by the crystal habit, or size distribution and shape of the particles. Small, anisometric particles with great surface free energy have a big potential to electrostatic charging and adhesion. This results poor flow properties, and affect to the compression process, where the unfavourable rearrangement and friction or adhesion to the punches and the die walls cause important problems. The plastic-elastic behaviour of the materials during compression also have influence to the tablet properties; for direct compression materials with good plasticity are necessary. The poorer properties of the drugs can be improved with the use of different excipients. However, to the selection of the suitable excipients, the knowledge of the interactions with the drug is necessary. Determinations of the adhesion work and spreading coefficient have capital importance for the knowledge, how the excipient will change the surface properties of the drug. In a previous study [1], the mechanical properties of tablets prepared from binary excipient mixtures were studied. In present study the effect of the addition of drotaverine HCl (an API with poor flow properties and compressibility) to binary excipient mixtures was investigated. The surface free energy of the materials and mixtures from the results of the optical contact angle measurements. The adhesion work and spreading coefficient of the binary mixtures was also calculated for the determination of the interactions between the materials. These data were then applied to modelling of the surface properties of the compressed systems. As discussed above from compressional point of view the structural properties like plasticity of the materials have also capital importance. The plastic-elastic behaviour of the materials and mixtures was determined based on force measurements during compression. We also tried to model how the plasticity of the mixtures changes with the application of different composition. The results showed that the addition of API greatly changed the postcompressional properties of the original tablets, but with full knowledge of the surface and structural properties, the change can be predicted and taken into account during formulation. Acknowledgement: The work was supported by a Sanofi-Aventis scholarship. [1] T. Sovány, P. Kása jr., K. Pintye-Hódi: Comparison of the halving of tablets prepared with

eccentric and rotary tablet press AAPS PharmSciTech, doi: 10.1208/s12249-009-9225-2

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Improvement of solubility of low soluble drug telmisartan

Ivan Simčić, Michaela Horvat, Tijana Dragić, Maja Mihoci, Tina Jelača* Vlatka Džale, Biserka Cetina-Čižmek

Pliva Croatia Ltd. Generic Research and Development, Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia

*Gradska ljekarna Zagreb, Zdeslava Turića 1, 10000 Zagreb, Croatia

Poorly soluble drugs represent a great challenge in pharmaceutical development. There are many ways how we can improve drug solubility; by particle size reduction, solubilization, amorphization, complexation, formation of salts or choosing other polymorphic forms[1]. The challenge for scientists is a selection of the optimal process which leads to solubility improvement and proper characterization of the drug molecule.

The aim of this study was to select a simple process for solubility improvement of telmisartan, low soluble drug and its characterization.

Telmisartan is practically insoluble in water or in aqueous solution in the pH range from 3 to 9, sparingly soluble in a strong acid and soluble only in a strong base[2]. Crystalline telmisartan exists in two polymorphic forms with different melting points and both of these forms are very poorly soluble in aqueous systems at the physiological pH range (1-7.5) in the gastro-intestinal tract.

In order to improve solubility, telmisartan was dissolved in aqueous solution of NaOH at pH above 10 and then spray-dried. The obtained product showed much higher solubility at pH 7.5. However, when organic basic excipient – meglumine was introduced into spray dry solution even more solubility improvement was observed at pH 7.5.

Characterization of spray-dried products was performed by means of near-infrared spectroscopy (NIR), modulated differential scanning calorimetry (MDSC), X-ray powder diffraction (XRPD) and scanning electron microscopy (SEM). Results of XRPD, MDSC and SEM show that obtained products are spherical particles in amorphous state. NIR analysis confirms interaction of telmisartan and meglumine in basic medium. Based on the results of this study it can be concluded that telmisartan solubility is improved by salt formation with meglumine and by reduction of particle size. [1] Pinnamaneni S., Das N. G., Das S. K., Formulation approaches for orally administered poorly

soluble drugs, Pharmazie, 57 (2002) 291 – 300. [2] W. Wienen, Cardiovascular Drug Review, 18 (2000) 127-154.

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Raman analysis of a polymorph within a polymorph

Robert Alexander, Stefano Pera*

PerkinElmer Limited, Chalfont Road-Seer Green – Beaconsfield, Buckinghamshire HP9 2FX, United Kingdom, email: [email protected]

*PerkinElmer Italia SpA, via Tiepolo 24, 20052 Monza (Milano), Italy, email:[email protected]

The differences in the crystalline structure of polymorph compounds can affect the physicochemical parameters of the substance such as solubility, dissolution rate, density, hardness. This in turn can impact on important pharmaceutical properties as bioavailability, stability and formulation technology of dosage form. The formation of different polymorphs is a controllable process within the formulation and the commercial value of the final product is related to the concentration of the requested polymorphic form, so it is fundamental to determine the polymorphic forms. Raman is recognized as being one of the most powerful and simple techniques for polymorphic analysis. This paper is describing the analysis of Ranatidine hydrochloride which exist in Form I (patented in 1978) and Form II (Zantac, patented in 1984) and now, being both patents expired there are many generic tablets. Raman spectra of Zantac and of a generic tablet were taken with PerkinElmer RamanStation 400, using 1 minute of acquisition time, spectral range 3200-100 cm-1 and resolution 4 cm-1. Raman spectra show that Zantac tablet contains mainly Form II while the generic tablet contains Form I. It is also possible to select the depth of spectra collection in order to acquire the spectra of coatings and subsequently the spectra of coating plus bulk. By spectral subtraction, we can obtain spectrum bulk material without interferences by the coating. After that, subtracting the authentic spectrum of bulk material from the spectrum obtained by “looking through” coating we obtain a spectrum, which was identified as Anatase, a polymorph of titanium dioxide. In summary, this study confirms that Raman spectroscopy is an ideal method for the analysis and identification of both organic and inorganic polymorphs.

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Study of interactions between API and excipients in solid drug formulation via NIR

Zbyněk Oktábec, Josef Jampílek, Anna Zerzaňová , Jiři Dohnal, Vladimír Král

Zentiva k.s., U Kabelovny 130, 102 37 Prague, Czech Republic

A drug formulation usually consists of an active pharmaceutical ingredient(s) – API(s) – combined with excipients that have been added to the formulation to facilitate its preparation, to ensure the required stability of the drug in the formulation during the manufacturing process and storage and to function as a drug delivery system. The interactions in formulations can influence pharmaceutical and biopharmaceutical properties, such as physical stability, dissolution rate, and bioavailability of the finished dosage form. The method of study of these interactions is based on preparation of binary mixtures of API and excipients and their analyses. The preparation of binary mixtures was in correlation with formulation process. The following analyses proceeds by near-infrared spectroscopy (NIR). NIR is a fast and non-destructive analytical technique that offers many advantages for a broad range of applications[1,2]. Main topic was to observe the possible interaction of API and excipients and/or to describe the polymorphism of the API, respectively its change. The analyses confirmed interactions between API and more than one excipient. It also confirmed that even the dissolution of the small ratio of the API, e.g. during the wet granulation process, can result in a change of polymorphs. These facts affect the absorption of an API as PAMPA experiments have shown. [1] G. Reich. Adv. Drug Deliv. Rev. 57 (2005) 1109. [2] J. Luypaert, D. L. Massart, Y. Vander-Heyden, Talanta, 72 (2007) 865.

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Gliclazide cocrystals: In vitro and In vivo evaluations

R. K. Averinnei, N. Johan, G. Shavi, K. Armugam, U. Nayak, S. Pandey, N. Udupa

Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences Manipal University, Manipal, 576 104, India

As per the BCS classification gliclazide comes under the category of Class II drugs, the drugs with low solubility and good permeability. So the present study is aimed at the preparation of cocrystals with the various carriers such as succinic acid, glutaric acid, nicotinic acid and tartaric acid with different ratios. Here the cocrystals are prepared by the slow solvent evaporation method and cogrinding method. The fornmulated cocrystals were evaluated for the vaious in vitro studies like micromeritic properties (carr’s index, hausners ratio, angle of repose) drug content, saturation solubility and varoius evaluations using the differential scanning calorimeter, infrared spectroscopy, scanning electron microscopy and powder X-ray diffraction. Optimized cocrystals were evaluated for the preclinical pharmacokinetic and pharmacodynamic studies in the plain and diabetes induced male wistar rats. Finally the optinmized formulations were compressed into tablets using the direct compression method. From the results it was observed that there is no incompatibility between the selected carries and the gliclazide. Compared to the pure drug of gliclazide the micromeritic properties were greatly improved. The saturation solubility of the gliclazide is increased about 2 folds compared to the plain drug. From the reuslts of the solubility it was observed that the slow solvent evaporation method was found to optimum compared to the cogriding method. The in vitro dissolution studies shows that there is faster release of the gliclazide in form of cocrystals compared to that of the plain drug. The DSC, IR, SEM and PXRD studies indicated that there is alteration in the crystal form of the structure and hence it showed improved solubility and dissoluton. The pharmacokinetic studies shows improved systemic exposure of the gliclazide in the form of cocrystals compared to the plain drug. The pharmacodynamic studies shows the percentage reduction of blood glucose levels in the plain animals are 30.56, 34.03, 53.16 and 61.55 for the cocrystals of gliclazide prepared with the tartaric acid, succinic acid, glutaric acid and nicotinic acid respectively at the end of one hour and the normal blood glucose levels were reached at the end of four hours. Similarly the percentage reduction of blood glucose levels in the diabetes induced animals are 17.81, 18.40, 22.53 and 23.17 respectively for the cocrystals prepared with tartaric acid, succinic acid, glutaric acid and nicotinic acid. Based on the in vitro and in vivo evaluations and considering the stability studies it was concluded that nicotinic acid was a suitable carrier for the formulation of the gliclazide cocrystals. [1] P. Vishweshwar, J.A. McMahon, J.A. Bis, M.J. Zaworotko, Pharmaceutical co-crystals. J Pharm

Sci. 95 (2006) 499-516.

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Identification of Chlamydia pneumoniae antigens by immuno-proteomics using FTICR-MS and LC-tandem mass spectrometry

Iuliana Susnea, Sebastian Bunk*, Corinna Hermann*, Michael Przybylski

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

Laboratory of Biochemical Pharmacology, Department of Biology, University of Konstanz

Chlamydia pneumoniae is an important respiratory pathogen that causes approximately 5 % of all cases of bronchitis and it is believed to be responsible for about 10 % of the cases of community-aquired pneumonia. C. pneumoniae is an obligate intracellular bacteria with an unique life cycle alternating between an infectious but non-replicating elementary body (EB) and a non-infectious but metabolically active reticulate body (RB). More than 50 % of the adult European population has antibodies against C. pneumoniae. There is evidence that C. pneumoniae can persist in the host after primary infection, which may be associated with atherosclerosis leading to cardiovascular diseases. Although this association has been investigated by seroepidemiological studies for more than 15 years, the role of C. pneumoniae infection still remains controversial and is presently unresolved. One major problem of current studies is the lack of reproducibility of serological tests. The aim of the present study is to identify the C. pneumoniae antigen structures that may be suitable biomarkers for serodiagnosis. The combination of 2D-gel-electrophoresis with immunoblotting and mass spectrometry was found to be an excellent tool for the identification of antigens. Detailed studies were carried out on the optimization of the C. pneumoniae culture and the 2D-gel sample preparation. Sera from 27 seropositive and 11 seronegative donors were used for immune-blotting. Approximately 90 antigenic spots were found, the most characteristic of which were subjected to further analysis. Antigenic spots were excised from gels, digested with trypsin and analysed by high resolution FTICR-mass spectrometry or LC-MS/MS. From the twenty two antigenic proteins identified in the present study[1], seven proteins showed a high reactivity (>10%) among the tested sera. These immunodominant proteins may represent potential biomarkers for serodiagnosis. Acknowledgement This work has been supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (Forschergruppe “DNA- and Oligosaccharide Chips – Analyse sekundärer Genprodukte”). [1] S. Bunk, I. Susnea, J. Rupp, J. T. Summersgill, M. Maass, W. Stegmann, A. Schrattenholz, A.

Wendel, M. Przybylski, C. Hermann, Immunoproteomic Identification and Serological Responses to Novel Chlamydia pneumoniae Antigens That Are Associated with Persistent C. pneumoniae Infections, J. Immunol. 180 (2008) 5490-5498

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NMR in drug discovery: Ligand-based NMR spectra demonstrate an additional phytoestrogen

binding site for 17β-hydroxysteroid dehydrogenase type 1

Paul J.A. Michiels, Christian Ludwig*, Michel Stephan**, Christina Fischer Gabriele Möller***, Jerzy Adamski***, Josef Messinger****

Maria van Dongen, Hubert Thole****, Ulrich L. Günther*

Solvay Pharmaceuticals Research Laboratories, Weesp, The Netherlands *HWB-NMR, CR UK Institute of Cancer Sciences,

University of Birmingham, Birmingham, UK **Phosphoenix SARL, Paris, France

***Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Experimental Genetics, Genome Analysis Center, Neuherberg, Germany

****Solvay Pharmaceuticals Research Laboratories, Hannover, Germany NMR spectroscopy is a commonly used technique in the drug discovery process in pharmaceutical industry. It is not only used by the chemist to identify their newly synthesized compound, it can also be used in the screening process for new drugs, hit validation and lead optimization. In this presentation a couple of NMR methods will be discussed that were used for studying the characteristics of the interaction of certain compounds with a protein target, 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1).

The enzyme 17β-HSD1 is an important drug target for breast cancer because it catalyzes the interconversion of estrone to the biologically more potent estradiol which also plays a crucial role in the etiology of breast cancer. Inhibitors for 17β-HSD1 are therefore included in therapy development. Here we have studied binding of 17β-HSD1 to substrates and a number of inhibitors using NMR spectroscopy. Ligand observed NMR spectra show pH dependence for the phytoestrogens luteolin and apigenin but not for the natural ligands estradiol and estrone. Moreover, NMR competition experiments show that the phytoestrogens do not replace the estrogens despite their similar inhibition levels in the in vitro assay. These results support an additional 17β-HSD1 binding site for phytoestrogens which is neither the substrate nor the co-factor binding site. Docking experiments suggest the dimer interface as a possible location. An additional binding site for the phytoestrogens may open new opportunities for the design of inhibitors, not only for 17β-HSD1, but also for other family members of the short chain dehydrogenases.

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Online chemical modeling environment

Igor V. Tetko, Iurii Sushko, Sergeii Novotarsky, Robert Körner Anil Kumar Pandey, Matthias Rupp

Helmholtz Center Munich, Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany

We develop the online chemical modeling environment (http://qspr.eu), a publicly accessi-ble, community-driven database for chemical compound data and predictive models. It facilitates the collection and organization of compounds, together with data on their physico-chemical and biological properties, as well as the development, application, and distribution of predictive models. It is unique in its combination of compound data and predictive models. The main characteristics of the system are: • Open and community-driven. Users are encouraged to provide, curate, and use the data. • High quality data. Compound data includes physico-chemical and biological

properties, measurement conditions, and references to the literature from which the data were obtained. This allows consideration of experimental conditions, easy creation of data sets by properties, and simple access to published data sets.

• Friendly user interface. The web-based user interface supports easy conversion between measurement units, filtering of compounds, as well as application and comparison of published models.

• Descriptor computation. Chemical descriptors can be computed as input for the predictive models, e.g., fragments, E-state indices [1], and quantum chemical descriptors [2].

• Exploratory modeling. Easy model development supports exploratory analysis of compound data by creating, comparing, and sharing different predictive models.

• Publication of models. Predictive models can be stored in the database. This allows other users to use (and cite) the model, to apply it to new data, to improve it, to modify it, and to reproduce results.

Combining compound data and predictive models in one database facilitates not only the efficient creation of such models, but also their comparison and reuse. In this way, the online chemical database-modeling environment might contribute to virtual screening and quantitative structure activity / property relationship modeling, as well as to implementing REACH (registration, evaluation, authorisation and restriction of chemical substances) legislation.

Acknowledgement This study was partially supported by the BMBF GO-Bio project 0313883 and by the FP7 Project CADASTER 212668. [1] L. Hall, L. Kier, Electrotopological State Indices for Atom Types: A Novel Combination of

Electronic, Topological, and Valence State Information, J. Chem. Inform. Comput. Sci., 35 (1995) 1039-1045.

[2] M. Dewar, E. Zoebisch, E. Healy, J. Stewart: AM1: A New General Purpose Quantum Mechanical Molecular Model, J. Am. Chem. Soc., 107 (1985) 3902-3909.

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Solvent effects on the structure-activity relationship of 5-substituted-5-phenylhydantoins

Nemanja P. Trišović, Nataša V. Valentić, Tatjana L. Đaković-Sekulić*, Gordana S. Ušćumlić

Faculty of Technology and Metallurgy, Univerity of Belgrade, Karnegijeva 4,

P.O. Box 3503, 11120 Belgrade, Serbia *Faculty of Science, University of Novi Sad, Trg Dositeja Obradovića 3

21000 Novi Sad, Serbia

Phenytoin (5,5-diphenylhydantoin) is the most widely used anticonvulsant. It targets the neuronal voltage-gated sodium channels (NVSC) to reproduce the normal ion potential. The ability to form hydrogen bonds as well as motional freedom at the level of the phenyl groups are important SAR features in antiepileptic phenytoin-like compounds. The focus of our research has been determination of the structural and chemical behavior of compounds in different solvents using UV/Vis spectroscopic methods[1,2]. In this work, eleven 5-substituted-5-phenylhydantoins (Figure 1) were synthesized and the UV absorption spectra were recorded in the region of 200 to 400 nm in selected solvents of different polarity. The effects of solvent dipolarity/polarizability and solvent/solute hydrogen bonding interactions were analyzed by means of the linear solvation energy relationship (LSER) concept proposed by Kamlet and Taft. The quantitative relationship between the contribution of specific solvent interactions and the corresponding lipophilicity parameter is discussed.

NH NH

O

O

R

Figure 1. Structure of the investigated 5-substituted-5-phenylhydantoins

(R = CH3, C2H5, nC3H7, iC3H7, nC4H9, iC4H9, tC4H9, cycC5H9, cycC6H11, C6H5, CH2C6H5 ) The correlation equations were combined with the corresponding ED50 values and different physicochemical parameters to generate new equations that demonstrate the exact relationships between solvent/solute interactions and the anticonvulsant activity of the studied compounds. [1] N. Trišović, N. Banjac, N. Valentić, G. Ušćumlić, J. Solution Chem. 38 (2009) 199-208. [2] N. Banjac, G. Ušćumlić, N. Valentić, D. Mijin, J. Solution Chem. 36 (2007) 869- 878.

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Spectroscopic method for the early evaluation of anti-bacterial agents activity

Vitaly Erukhimovitch, Mahmoud Huleihel*

Analytical Equipment Unit, Ben-Gurion University of the Negev, Beer-Sheva, Israel *Department of Virology and Developmental Genetics, Faculty of Health Sciences

Ben-Gurion University, Beer-Sheva, 84105, Israel Spectroscopic techniques provide a wealth of qualitative and quantitative information about a biological samples [1,2]. In this study we examined the potential of FTIR microspectroscopy as analytical instrument for early evaluation of anti-bacterial therapy efficiency. For this purpose, the effect of caffeic acid phenethyl ester (CAPE) on the development of bacterial infection in cell culture was examined. CAPE is one of the most active components of propolis which is a natural honeybee product with a potent anti-bacterial activity. Different gram (-) and gram (+) bacteria were grown in the presence and absence of various concentrations of CAPE In general, used control and treated bacteria show similar spectra with some specific consistent differences in all tested bacterial strains Our results show early (2h post treatment) unique and significant spectral indicators for successful treatment with CAPE although some of these biomarkers showed different trends in gram (-) compared to gram (+) bacteria. It seems that FTIR spectroscopy can be used as an effective analytical tool for an early evaluation of the efficiency of the anti-bacterial effect of CAPE and probably other used drugs. [1] V. Erukhimovitch, M. Talyshinsky, Y. Souprun and M. Huleihel, Spectroscopic characterization

of human and mouse primary cells, cell lines and malignant cells, Photochem. Photobiol., 76 (2002) 446-451.

[2] V. Erukhimovitch, V. Pavlov, M. Talyshinsky, Y.Souprun and M.Huleihel, FTIR Microscopy as a method for identification of bacterial and fungal infections, J. Pharmaceut. Biomed. Analysis, 37(5) (2005) 1105-1108.

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Echis multisquamatis venom fibrinogenase is a prospective fibrinogen-depletive agent

V. O. Chernyshenko*,**, I. S. Maksymovych**

*Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine Kyiv, Ukraine

**National Shevchenko University, Kyiv, Ukraine [email protected]

Hyperfibrinogenemia is not an independent thrombosis risk factor, but simultaneously with deep imbalance of haemostasis can cause intravascular coagulation occurrence [1]. There are three main strategies of fibrinogen depletion: usage of thrombin-like enzymes, fibrinolytics and fibrinogenases from snake venoms. Thrombin-like enzymes promote fibrinogen polymerization with further destruction of polymerized fibrin through fibrinolytic system. It is potentially dangerous in consideration of fibrinolysis hyperactivation and bleeding [2]. The development of fibrinolytics is slowed down because of hemorrhagic risk, joined with metalloproteinases intravascular injecton [3]. The prospective fibrinogen-depletion agents are fibrinogenases. Thay do not induce fibrin polymerization, make no influence on fibrinolysis and anticoagulation systems but efficiently decrease fibrinogen clottability. Serine proteinases (some of fibrinogenases belong to this class of enzymes) do not have hemorrhagic activity [4]. We have purified a new fibrinogenase from the venom of Echis multisquamatis [5]. Inhibitory assay showed that it is serine proteinase. Molecular weight is 35 kDa, it possesses strong arginine-amidase and arginine-esterase activity. The target of initial fibrinogenolysis is N-terminus of β-chain, which includes polymerization knobe B, lateral association and platelet-interaction sites. Fibrinogen treated with fibrinogenase is less clottable on 40%; speed of collagen-induced platelets aggregation in the presence of fibrinogenase-treated fibrinogen is lower too. Fibrinogenase from the venom of Echis multisquamatis has no hemorrhagic and haemolytic activity, no influence on platelets. These facts together with determined characteristics of fibrinogenase give us a possibility to say about the potent prospective of its usage as an therapeutic agent. [1] B. Kerlin, B. Cooley, B.H. Isermann, et al, Cause-effect relation between hypefibrinogenemia and

vascular disease, Blood. 103 (2004) 1728-1734. [2] C. R. Prentice, K.K. Hampton, P.J. Grant The fibrinolytic response to ancrod therapy:

characterization of fibrinogen and fibrin degradation products, Br. J. Haematol. 83 (1993) 276-281.

[3] Q. Q. Wang, J. S. Chen, X. X. Liang, et al, Hemorrhagic activity and mechanism of FIIa, a fibrinolytic enzyme from Agkistrodon acutus venom, Acta Pharmacol. Sin. 25 (2004) 514-521.

[4] E. E. Gardiner, R. K. Andrews, The cut of the clot(h): snake venom fibrinogenases as therapeutic agents, J. Thromb. Haemost. 6 (2008) 1360-1362.

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Determination of dissociation constants of poorly soluble cannabinoid receptor ligands by cosolvent capillary electrophoresis

Carme Calvet, Joan A. Morató, Edmundo Ortega, Adriana Port

Laboratorios Dr. Esteve, S.A., Barcelona, Spain

The acidic dissociation constant (pKa) is one of the key physicochemical parameters for characterizing early pharmaceutical candidates. Among other traditionally methods, capillary electrophoresis (CE) has become a powerful technique for performing pKa analysis where large amount of sample, high purity and high aqueous solubility of the test compound are not more a requirement. Several approaches have appeared describing the applications of CE to determine pKa values: medium-throughput methods by pressure-assisted capillary electrophoresis[1-3] combined with short-end injection[4], using co-solvent buffers for insoluble compounds [5-7] or high-throughput multiplexed capillary electrophoresis[7]. Here we describe a methodology for determining aqueous dissociation constants for poorly soluble cannabinoid receptor ligands based on a single capillary instrument integrated with photodiode array detector and short-end injection. The use of increasing methanol concentrations and extrapolation to 0% cosolvent allows resolving pKa values for these lipophilic compounds. Accordingly twenty-four methanol/water cosolvent buffers (pH 1.72 - 11.2) with the same ionic strength covering the range of 30-60% methanol were employed. The

s wpKa values were determined by plotting the effective mobility versus the

pH values of the running buffers and fitting data to the appropriate model equation[3] using SigmaPlot software. The aqueous

w w pKa values were obtained by Yasuda-Shedlovsky

extrapolation method[7]. The application of pressure during the separation combined with short-end injection [1-4] provided the electrophoretic separation to be completed within 3 min, even at lower pH. In addition to the aforementioned compounds, pKa of some other commercial drugs were determined in order to check the goodness of the method. Results agreed well with previous studies found in literature data[8,9]. This methodology enables to measure pKa values at the low pH extreme for very insoluble weak bases (pKa 2-3) even when analysis must be performed with high methanol content in the cosolvent buffer. The low-throughput associated to single CE apparatus is compensated with the use of short-end injection and pressure assisted. This method was confirmed to be very suitable for fast and accurate pKa measurement of very insoluble cannabinoid receptor ligands in early phase of development. [1] Z-Jia, T.Ramstad, M.Zhong, Electrophoresis 22 (2001) 1112-1118. [2] Y. Ishihama, M. Nakamura, T. Miwa, T. Kajima, N. Asakawa. J. Pharm. Sci. 91(4) (2002) 933-942. [3] J. M.Miller, A. C. Blackburn, Y. Shi, A. J. Melzak, H. Y. Ando, Electrophoresis 23 (2002) 2833-2841. [4] H. Wan, A. Holmén, M. Nagard, W. Lindberg, J. Chromatogr. A 979 (2002) 369-377. [5] S. M. C. Buckenmaier, D. V. Mccalley, M. R. Euerby, J. Chromatogr. A 1026 (2004) 251-259. [6] R. Ruiz, C. Ràfols, M. Rosés, E. Bosch, J. Pharm.Sciences 92(7) (2003) 1473-1481. [7] M. Shalaeva, J. Kenseth, F. Lombardo, A. Bastin, J. Pharm. Sci. 97(7) (2008) 2581-2606. [8] J. Kenseth, A. Bastin, D. Tallman, Poster presented at AAPS 2004. [9] A. Llinàs, R. C. Glen, J. M. Goodman, J. Chem. Inf. Model. 48 (2008) 1289-1303.

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Physicochemical Profiling of New Antimalarial Pyridones

Ana I. Alvarez, Maria-Teresa Fraile, Ruben Gomez-Sanchez, Angel Santos-Villarejo, J. L. Lavandera

Computacional, Analytical and Structural Chemistry

DDW Drug Discovery Center, GlaxoSmithKline R&D, Tres Cantos, Spain

Malaria remains the most important health problem in sub-Saharan Africa where it is the causative agent for 90% of deaths related with this disease. It is also considered a burden in some parts of South America and Asia. It is spread by mosquitoes from person to person and caused by protozoan parasites of the genus Plasmodium. Plasmodium falciparum causes cerebral malaria, the most severe form of the infection, and it is responsible for most of the 1 million deaths attributed to malaria annually, mainly pregnant women and young children. In addition to that, the rapid emergence and spread of multidrug-resistance Plasmodium falciparum and Plasmodium vivax diminish the usefulness of current antimalarial treatments. Therefore, there is an urgent need for new drugs acting on new mechanisms or underexploited targets.

Plasmodium mitochondrial electron transport chain is one the most interesting pathways being pursuit for new antimalarial compounds. Atovaquone, one of the most currently used drugs, inhibits Bc1 (complex III).

4(1H)PYRIDONES are potent and selective inhibitors of Plasmodium falciparium cytochrome Bc1 (complex III) that are being developed as a new class of potent antimalarials. However, their low solubility in aqueous media and bioavailability are the main issues to address.

In order to understand those physicochemical factors that influence their PKPD properties, measurements of Hydrophobicity, Plasma Protein Binding and Solubility in bio-relevant media, i.e. Simulated Gastric Fluid (SGF), Fasted Simulated Intestinal Fluid (FaSSIF), Fed Simulated Intestinal Fluid (FeSSIF) and Phosphate Buffer Saline (PBS) were performed. Two different approaches were followed to assess their solubility profile, i.e. thermodynamic equilibrium solubility determinations and dissolution rates experiments.

Compounds GW844520X, GSK325968X and GSK932121A have been studied in this work and the obtained results are presented.

NH

OCl

CH3 CH3

O

O

FF

FNH

OCl

CH3 CH3

N

OF

FF

NH

O

CH3OH

Cl

O

O F

F F

GW844520X GSK325968X GSK932121X

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Page 45

Evaluation of solubility and dissolution of pioglitazone hydrochloride

in GI environment

J. Turkalj, I. Erak*, D. Raušl, I. Mijakovac, B. Cetina-Čižmek

PLIVA CROATIA LTD., Generic Research and development, Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia

*Gradska ljekarna Zagreb, Ilica 301, 10000 Zagreb, Croatia

Pioglitazone hydrochloride is an oral antidiabetic agent that acts primarily by decreasing insulin resistance. Chemically, it is [(±)-5-[[4-[2-(5-ethyl-2-pyridinyl)ethoxy]phenyl]me-thyl]-2,4-] thiazolidinedione monohydrochloride, with pKa values 5.8 and 6.4. It is soluble in N,N-dimethylformamide, slightly soluble in anhydrous ethanol, very slightly soluble in acetone and acetonitrile, practically insoluble in water, and insoluble in ether[1].

The aim of this study was to evaluate the influence of pH and different surfactants on solubility of pioglitazone in order to establish dissolution methods relevant for GI environment.

The solubility of the pioglitazone hydrochloride substance was determined at 37°C by saturated solution method in physiological pH range (aqueous solutions at pH 1.2-7.5). Furthermore, an influence of different surfactants on solubility was evaluated in media pH 4.5 and pH 6.8. Types of surfactants were chosen based on their ionization properties: sodium lauryl sulphate (SLS), Tween 20 and poloxamer.

As a weak base, pioglitazone shows pH-dependant solubility and it is highly soluble in acidic media, while in other tested media solubility decreases by increasing pH.

Results of testing show that at the same conditions non-ionic surfactant Tween 20 and poloxamer do not affect solubility while SLS increases solubility up to 200 times in medium pH 4.5 and 45 times in medium pH 6.8. Furthermore, minimal concentration of SLS sufficient to fulfill sink conditions requirement in medium pH 4.5 was determined to be 0.5% SLS, while in medium pH 6.8 is 2% SLS.

Finally, dissolution methods used to mimic GI tract were set to 900 mL media and paddle apparatus: pH 1.2 with rotational speed of 50 rpm, pH 4.5 by addition of 0.5% SLS and pH 6.8 by addition of 2% SLS as dissolution media with rotational speed of 100 rpm. [1] Martindale - The Complete Drug Reference, Micromedex (R) Healthcare Series 2009

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Stability testing of the Spalmotil oral solution during the use (in use test)

Biljana Stojanović, Stojanka Vidović, Mirjana Rajić, Ljiljana Petrović, Marija Milovanović

Galenika a.d., Institut, Batajnički drum b.b., 11080 Zemun, Serbia

Spalmotil oral solution is used for the relief of bronchospasm in conditions such as asthma and chronic obstructive pulmonary disease.

The goal has been the evaluation of the product stability during the use, in order to define storage conditions and the expiry date after the first opening.

The use of the product has been simulated by pouring off 7,5 ml of oral solution out of the bottle periodically, according to the protocol for the in use test. At the determined time points, the samples stored on 20-25°C (conditions of controlled room temperature) have been analyzed parallely with the samples stored on 5±3°C (in the refrigerator). The duration of the in use test has been 60 days.

According to the proposed specification, some of the parameters have been determined daily ( the appearance - color, clarity, and odor), while the others (relative density, pH, microbial limits and the content of salbutamol, sodium benzoate and salbutamol impurity C) have been determined 15, 30, 45 and 60 days after the first opening. The content of salbutamol and sodium benzoate has been determined by the same HPLC method, and the content of salbutamol impurity C by another appropriate HPLC method. Both HPLC methods were validated and stability-indicating.

In conclusion, the analysis of the data confirms that the product meets all the acceptance criteria of the proposed shelf-life specification, and that it is stable 60 days after the first opening. It is not necessary to store the product in the refrigerator.

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Page 47

Physical methods for in vitro simulation of Sucralfate oral suspension behaviour in vivo

Marina Surjak, Spomenka Milak*

PLIVA Croatia Ltd., Prilaz Baruna Filipovića 29, Zagreb, Croatia *University of Graz, Universitätsplatz 3, Graz, Austria

Sucralfate is basic aluminum salt of sucrose octasulfate. Drugs behavior in vivo is described as adherent to gastric, especially injured mucosa. Sucralfate in its viscous adhesive state in gastroduodenal environment is partially dissociated and consists of polyvalent anions that are composed of the sucrose sulfate moiety with residual aluminum groups[1,2].

Physical methods for characterization of this behavior in vitro were developed. Adhesion to gastric mucosa was monitored as a rheological synergism i.e. viscosity difference between Sucralfate suspension mixed with porcine mucine and Sucralfate suspension only. Viscosity profiles were monitored at shear rates 10 to 300 s-1 and at constant shear rate of 50 s-1, at 37°C.

Sucralfate’s behavior in acidic media was monitored through acid neutralization curves (pH change versus time after addition of 0.1 M HCl), and by visual inspection.

Correlation between reaction in acidic media and adhesivity to mucine was confirmed. [1] Rossi, S.; Bonferoni, M. C.; Caramella, C.; Colombo, P.; Conte, U., Rheological study of

sucralfate humid gel: A contribution to the comprehension of its stability properties, Acta Toxicologica et Therapeutica, 16(1-2) (1995) 70-80.

[2] El-Samaligy, M. S.; Amin, S. Y.; Gindy, H. S., Formulation and evaluation of sucralfate suspension , Egyptian Journal of Pharmaceutical Sciences, 35(1-6) (1994) 365-81.

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48

Absorption Study of Binary Mixtures

Anna Zerzaňová, Zbyněk Oktábec, Josef Jampílek, Jiří Dohnal, Vladimír Král

Zentiva, k.s., U Kabelovny 130, 102 37 Prague, Czech Republic

A pharmaceutical dosage form generally consists of a drug combined with a varying number of excipients that have been added to the formulation to facilitate its preparation and function as a drug delivery system. Although excipients are considered to be inert in therapeutic or biological actions, they should hinder unwanted phase transitions and ensure the required stability of the drug in the formulation during the manufacturing process and storage. On the other hand, the presence of badly chosen excipients could negatively influence behaviour of the formulation. A lot of important parameters of API and formulation, e. g. stability, impurity profile, dissolution or bioavailability could be influenced by excipients. Even more, some excipients are well known as enhancers or inhibitors of the absorption. Thus, a study of binary mixtures should be one of the first steps in a development of a new formulation. Parallel artificial membrane permeability assays (PAMPA) have become a very useful and quite cheap tool for predicting in vivo drug permeability and are well suited as a ranking tool for the assessment of compounds with passive transport mechanisms [1]. An absorption study of binary mixtures or final formulations is also possible on PAMPA plates. Thirty binary mixtures from ten commonly used excipients and three batches of one API were prepared and tested on PAMPA plates. Interesting differences in the absorption were observed. [1] http://www.bdbiosciences.com/discovery_labware/products/display_product.php?keyID=757#

(April 2009)

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Page 49

Application of principal component analysis in determination of dehydration behaviour of mesalazine hydroiodide monohydrate

Michaela Horvat, Miljenko Košiček, Ernest Meštrović

Pliva Croatia Ltd., Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia

Thermal methods of analysis are widely used in solid-state kinetic studies[1]. In particular, thermogravimetric analysis is irreplaceable in investigation of dehydration reactions. It is known that the results of thermal methods of analysis are highly dependant on experimental parameters used and that the results of analysis can be compared only if obtained using the same experimental conditions such as gas flow, pan type, sample mass and heating rate. Moreover, dehydration is a process which can depend on particle size, sample mass and the temperature which the sample is subjected to[2].

If one wants to include all relevant parameters which could have an impact on dehydration behaviour using thermal methods of analysis, one is faced with many variables and numerous data in which much information could remain hidden, if not analyzed by an adequate mathematical approach.

Principal component analysis (PCA) involves a mathematical procedure that transforms a number of possibly correlated variables into a smaller number of uncorrelated variables called principal components with the aim of reducing the dimensionality of the data set and to identify new meaningful underlying variables[3].

An application of principal component analysis in the analysis of thermogravimetric data will be presented. Kinetic of dehydration of mesalazine hydroiodide monohydrate was investigated by means of isothermal thermogravimetric analysis. Dehydration behaviour is monitored in relation to sample mass, gas flow, temperature and particle size. The obtained data were subjected to regression analysis and analysis of variance (ANOVA) in order to determine which model best describes the dehydration behaviour. Principal component analysis (PCA) was employed to determine which experimental parameters play the major role in the change of dehydration mechanism. All results are supported by crystal structure data and hot-stage microscopy observations. [1] A. Khawam, D. R. Flanagan, Basics and applications of solid-state kinetics: a pharmaceutical

perspective, J. Pharm. Sci. 95 (2006) 472-498. [2] A. K. Galwey, Structure and order in thermal dehydrations of crystalline solids, Thermochim.

Acta 355 (2000) 181-238. [3] J. Gabrielsson, N. Lindberg, T. Lundstedt, Multivariate methods in pharmaceutical applications,

J. Chemometrics 16 (2002) 141-160.

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Rheological characterization of semisolid pharmaceutical products

Božana Ivković, Petra Golja Gašparović

PLIVA Croatia Ltd., Prilaz baruna Filipovića 29, 10 000 Zagreb, Croatia The aim of this study was to perform rheological characterization of gel formulation during product development. It is very important to use appropriate methods to investigate influence of critical formulation properties and process parameters (e.g. speed and mixing time, cooling/heating temperature) on rheological behavior. Rheological properties of gel formulation were measured using rotational and oscillatory tests on rheometer Physica MCR 301 (Anton Paar), with 50 mm parallel-plate measuring system.

The influence of pH, temperature and excipient content on rheological properties of gel formulation will be presented. Rotational test was used for measuring viscosity at shear rate values from 0.01 to 500 s-1. The results showed decrease of viscosity with increasing shear rate (shear-thinning behavior), giving information on structural strength and spreadabi-lity[1]. Amplitude sweep test was performed at constant angular frequency with preset shear strain from 0.01% to 500%. Storage (G') and loss modulus (G'') were monitored in order to determine the limit of the linear viscoelastic (LVE) range and crossover point value[2]. The results of the amplitude sweep test showed that in the LVE range the elastic modulus G’ was over G’’ with a significant distance between them. By increasing the shear stress the product started to break down, with G’ and G’’ crossing each other. It is a desirable feature considering the fact that the gel has to spread well on the skin surface before absorption of drug could occur. To investigate structural regeneration, oscillatory test with three intervals, each under constant dynamic – mechanical condition was performed[1]. First and third intervals were rest phase under low-shear conditions (within LVE range) and second interval was load phase under high-shear conditions (outside LVE range). From the obtained results, it can be concluded that tested formulations display thixotropic behavior with almost complete structural regeneration under very short time. Test methods used in this study are proven to be suitable for extensive characterization of gel formulations. [1] T. G. Mezger., The Rheology Handbook, Vincentz Verlag, Hannover, 2002. [2] H. A. Barnes., A Handbook of Elementary Rheology, University of Wales, Aberystwyth, 2000.

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Page 51

Particle size characterization of nanosize organometalic complex

Petar Tudja, Suzana Šegota*, Vesna Svetličić*, Biserka Cetina-Čižmek

Pliva Croatia Ltd. Generic Research and Development, Prilaz baruna Filipovića 29 10000 Zagreb Croatia

*Ruđer Bošković Institute, Division for Marine and Enviromental Research, Bijenička Cesta 54, 10000 Zagreb

Some of organometallic complexes show pharmacological activity, especially those when metal ion is complexed to monosaccharide or disaccharide. They are currently used for treatment of anemia[1, 2].

Metal ion hydroxide carbonate complex is colloidal solution with nanosize particles. Therefore, special techniques should be used for nanoparticles characterization.

The aim of this study is determination and characterization of particle morphology and size using atomic force microscope (AFM) and dynamic light scattering (DLS) methods.

AFM is a very high-resolution type of scanning probe microscopy, with demonstrated resolution of fractions of a nanometer, more than 1000 times better than the optical diffraction. The AFM is one of the foremost tools for imaging, measuring and manipulating matter at the nanoscale. The information is gathered by "feeling" the surface with a mechanical probe. Piezoelectric elements that facilitate tiny but accurate and precise movements on (electronic) command enable the very precise scanning.

DLS is a technique for measuring the size of particles typically in the sub micron region. DLS measures Brownian motion and relates this to the size of the particles. Brownian motion is the random movement of particles due to the bombardment by the solvent molecules that surround them. Normally, DLS is concerned with measurement of particles suspended within a liquid.

Samples of metal complex were measured diluted in AFM analysis to achieve optimum particle concentration while DLS analysis was performed on undiluted samples. Particle size determined by AFM analysis is within range from 3 nm to 12 nm. Samples measured by DLS show particle size distribution in range from 3 nm to 20 nm.

Results obtained by both methods are similar although the methods function on different principles. [1] B.G. Danielson, J. Am. Soc. Nephrol. 15 (2004) .93-98. [2] M. K. Cowman et all, J. Inorg. Biochem. 98 (2004) 1757-1769.

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Optimization of the freeze-drying process by means of thermal methods of analyses

Andrea Rožman, Anita Bevetek Moćnik, Michaela Horvat

Pliva Croatia Ltd. Generic Research and Development, Prilaz baruna Filipovića 29 10000 Zagreb Croatia

Freeze-drying is commonly used process in pharmaceutical industry. The process of freeze-drying consists of three stages: freezing, primary drying, and secondary drying[1]. Based on characteristics of the material, and its behavior during freezing and drying, different experimental conditions are used. Modulated temperature differential scanning calorimetry (MDSC) and freeze-stage microscopy are techniques often employed in optimization of freeze-drying cycle[2]. Many of biopharmaceuticals such as proteins and polypeptides cannot be freeze-dried without cryoprotectants. Cryoprotectants are typically polyhidroxy compounds as sugars or polyalcohols[1].

The aim of this study was to determine glass transition temperature (Tg) and collapse temperature using MDSC and freeze-stage microscopy, in order to optimize freeze-drying cycle. In addition, set of experiments was conducted in order to examine impact of cooling rate on content of amorphous/crystalline phase in the final product.

In this experiment, aqueous solution of polypeptide/mannitol solution was used. Preservation of amorphous state of mannitol was crucial for preservation of secondary structure of polypeptide. The experiment was designed to associate cooling rate of samples with crystallization of mannitol. After cooling at various cooling rates (2°C/min, 1°C/min, 0.5°C/min and 0.3°C/min), all samples were heated at 1°C/min, in order to determine the amount of amorphous content in the material.

Obtained results suggest that samples at slower cooling rates show decreased step change corresponding to glass transition, which can be attributed to smaller amount of amorphous phase and higher crystalline phase present. It has been shown that change of heat capacity Cp calculated at glass transition temperature Tg depends on cooling rate. Higher value of Cp corresponds to higher amount of amorphous mannitol. [1] H. R. Constantino, M J. Pikal; Lyophilization of biopharmaceuticals, American Association of

Pharmaceutical Scientists (2004) AAPS Press. [2] W. Wang; Lyophilization and development of solid protein pharmaceuticals, Int. J. Pharm. 203

(2000) 1–60.

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Page 53

Electromediated microanalysis for the evaluation of interspecies variation in ester metabolism

Joana Moura, Ana Luísa Simplício

ITQB-UNL/IBET, Apartado 12, P-2781-901 Oeiras, Portugal

Electrophoretically mediated microanalysis (EMMA) is a branch of capillary electro-phoresis that presents several advantages in the study of enzyme activity. The method conjugates the potential of a separation technique with the convenience of on-line reaction, thus allowing simultaneous, unattended reaction and detection. EMMA methods also have the advantages of the reduced amount of sample needed and avoiding interference by sample components that might absorb at the same wavelength as the substrate or product.

In this work we present a new high throughput application of EMMA for the evaluation of the esterase activity of the serum of different animals. Dog, cat, cow, horse, sheep, rat and human serum were used after dilution in the appropriate buffer and compared in terms of the ability to hydrolyse common acetylcholinesterase and butirylcholinesterase substrates. The relative metabolic responses for the different esterases and species (see figure) were generally consistent with the observations presented in the literature and obtained with traditional methods.

This approach will allow the establishment on in vivo-in vitro correlations, for interspecies metabolic evaluation, that can subsequently be applied pre-clinically to the study of metabolism and bioavailability of ester containing drugs and pollutants.

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Comparison of original and generic pharmaceutical products safety based on

investigation of their influence on membrane structure of blood cells using ESR-spectroscopy

Igor Revelsky, Aleftina Polezneva, Igor Golubev, Evgeniy Gogol

Lomonosov Moscow State University, Chemistry Department, Moscow, Russia

Very often side effects for generics are much more pronounced then for original pharmaceutical products but comparative testing of both products are carried out usually very rarely using subjects. Some harmful properties of both products may not be reflected during such tests and even during clinical tests for original products. We have developed the new method for comparison of original and generic pharmaceutical products safety based on use of ESR spectroscopy with spin marker for investigation of these products influence on membrane structure of influence of blood cells (erythrocytes, platelets and lymphocytes). It has been shown that this influence has been in most cases quite different for original and generic pharmaceutical products. In case of the former ones it was less pronounced and was reversible in time in case of erythrocytes. As to latter ones the influence was more pronounced and irreversible in most cases for the same cells. For other cells it was quite different. Respective data for different pharmaceuticals will be presented. The method was patented [1]. [1] Igor Revelsky, RF Patent № 2329491, 20.07.2008. Method for comparative estimation of

physiological activity of pharmaceutical substances and related products.

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Page 55

A method for the quantification of L-dopa esters and related metabolites in the blood by LC-electro-spray tandem mass-spectrometry

Igor Mukmenev, Igal Gelboa, Elena Doubijansky, Amir Raichman, Alon Yaar, Eli Heldman

Ben-Gurion University of the Negev P.O.B.653 Beer-Sheva 84105 Israel

Esters of L-3,4-dihydroxyphenylalanine (L-DOPA) may be used as prodrugs for the treatment of Parkinson's disease. In the body, these prodrugs are hydrolyzed and release the active drug, L-DOPA, which is then further metabolized. To prevent the conversion of L-DOPA in the periphery to dopamine, drugs such as carbidopa are used to inhibit peripheral DOPA decarboxylase. For pharmacokinetic studies of these prodrugs and related compounds, an appropriate quantitative assay is needed. However, measurements of L-DOPA and its major metabolites, including dopamine and 3-O-methyldopa have been limited by cumbersome methods and poor sensitivity. We have developed an assay for the quantitative estimation of L-DOPA esters, L-DOPA, 3-O-methyl DOPA, dopamine and carbidopa in human blood. This method is based on separating the compounds to be determined by HPLC Agilent 1100 with column Kromasil C-18 250X2.0 100A (5μm) using 2H-DOPA as an internal standard. Detection was done with a detector Ion Trap MS Esquire 3000 Plus (Bruker Daltonics) containing an Electro-Spray Source. Samples were fed into the HPLC column by an Auto-sampler ALS 1200.

Determination of the concentrations of each compound was based on the MS2 fragments quantification of Positive Ions (M+H) in Ion Trap Mass Analizer. This method allows determining the concentrations of various L-DOPA compounds in the blood with the following detection limits: L-DOPA esters - 25 ng/ml; L-DOPA - 40 ng/ml; 3-O- methyl DOPA - 50 ng/ml; dopamine - 5 ng/ml and carbidopa - 2.5 ng/ml. The determination of these compounds is done in a single injection with a high degree of reliability and accuracy.

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Low molecular weight pharmaceutical substances analysis using matrix-assisted laser desorption/ionization (MALDI)

Karine Chamyan, Igor Revelsky, Aleksey Buryak*, Valentina Kosenko

Lomonosov Moscow State University, Moscow, Russian Federation

*A. N. Frumkin Institute of physical chemistry, Moscow, Russian Federation Matrix-assisted laser desorption/ionization (MALDI) is used mainly for the analysis of macromolecules, such as proteins, oligosaccharides, oligonucleotides and synthetic polymers, combined with time-of-flight (TOF) mass analyzers. There are some publications devoted to MALDI use for the analysis of low molecular weight compounds (<500 Da), in particular for pharmaceutical compounds, however application of MALDI is complicated by matrix ions interferences in the low m/z values range. The application of MALDI for the pharmaceutical substances and related products is very interesting and actual due to as high sensitivity and high productivity of analysis. Our research is devoted to investigation of MALDI possibilities for direct analysis of different pharmaceutical substances with low molecular weight (130-300 Da). It was carried out using Bruker model “Ultraflex” MALDI/TOF mass spectrometer. Mass-spectra of pharmaceutical substances were investigated depending on matrix, analyte quantity, matrix/analyte ratio, method of their mixture preparation. Several matrixes were investigated. The opportunity of fast pharmaceutical substance recognition on trace level has been shown. Respective data will be presented.

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Page 57

FT-IR Spectrometric determination of total ascorbic acid

K. Kargosha, S. R Moeinosadat, J. Azad*

Chemistry and Chemical Engineering Research Center of IRAN, P.O.Box 14355-146, Tehran, Iran

*Chemistry Department, Alzahra University, VANAK, Tehran, Iran

Vitamin C, a water soluble vitamin, is an important micronutrient and plays many physiological roles. Fruit and vegetables are the principal source of vitamin C in most human diets, where it occurs as ascorbic acid (AA), its oxidized form, Dehydroascorbic acid (DHAA) and 2,3-diketogulonate (DKG) which is a degradation product of DHAA.

Both AA and DHAA are unstable in solution at neutral PH with measurable oxidation and degradation, respectively, occurring within minute to hours[1].

This paper reports the application of a new established on-line system of vapour-phase generation (VPG) and FT-IR Spectrometric detection[2] as a direct and highly selective analytical technique for the assay of ascorbic acid. Sodium periodate solution was injected into a reactor, heated at 60°c containing sample solution. The CO gas generated under these conditions of oxidation was transformed by means of N2 carrier gas to an infrared gas cell and appropriate FTIR spectra were recorded in a continuous mode. The maximum absorbance of CO band at 2170 cm-1 was selected as a measurement criterion. Initially, the effect of different chemical and physical parameters, such as concentration and volume of oxidant, PH, equilibrium time, reactor temperature and N2 gas flow rate on the analytical signals were evaluated. Under the optimum experimental conditions, the method provided a relatively broad linear dynamic range, a sampling frequency of 12h-1 and a relative standard deviation of 2.0%. The proposed method dose not have matrix interferences nor dose it require any sample preparation. The present methodology was applied to the determination of total ascorbic acid, (AA, DHAA, and DKG) in pharmaceutical formulations, fruit Juices and soft drinks and result compared well with those obtained by reference methods. [1] I. Koshishi, T.Imanari, Measurment of ascorbate and dehydroascorbate contents in biological

Fluids, Anal, Chem.. 69 (1997) 216-220. [2] K.Kargosha, S.H. Ahmadi, M.Zeeb, S.R Moeinosadat, Vapour phase fourier transform infrared

spectrometric determination of L-cysteine and L-cystine, Talanta 74 (2008) 753-759.

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Anion-induced conformational changes in indole-based receptors

Damjan Makuc, Janez Plavec

Slovenian NMR Centre, National Institute of Chemistry, Ljubljana, Slovenia

Anion receptor chemistry continues to be an area of intense research activity[1-5]. The vivid growth in this area of supramolecular chemistry has been stimulated by many roles played by anions. Remarkably, indole derivatives are found in several enzymes in the sulfate binding proteins and in active site of haloalkane dehalogenase. NH protons may act as hydrogen bond donors and their ability to participate in hydrogen bonds is tuned by the choice of substituents on the heterocyclic scaffold. In the present study we focused on bis-amido and mono-amido-mono-urea 2,7-functionalized indoles which showed interesting new binding properties upon functionalization[6].

6

54

7

3

2

N 1

O

N2β

N 7α

HRH H

H

H

H H

NH

N

S

H

1 2 3 4

O

7γ

OOR:

The conformational analysis of indole receptors 1-4 was performed by a combination of NMR spectroscopy and quantum chemical calculations showing excellent agreement[7]. All four receptors exhibit conformational preorganization in acetone solutions, where anti-anti orientation across C2–C2α and C7-N7α bonds is highly predominant. Anion-receptor interactions have been evaluated through 1H and 15N chemical shift changes. Anion properties affect the anion-receptor interactions and different ways of bindings have been observed. NOE enhancements in the presence of anions revealed that anion-receptor complexes favor the syn-syn conformation of the C2 and C7 substituents.

Acknowledgements We would like to thank Professor Philip A. Gale and Gareth Bates from School of Chemistry, University of Southampton for providing the anion receptors.

[1] A. Bianchi; K. Bowman-James; E. Garcia-España, Eds. Supramolecular Chemistry of Anions; Wiley-VCH: New York, 1997.

[2] P. D. Beer.; P. A. Gale Angew. Chem. Int. Ed. 40 (2001) 486-516. [3] K. Bowman-James, Acc. Chem. Res. 38 (2005) 671-678. [4] J. L. Sessler; P. A. Gale; W. S. Cho, Anion Receptor Chemistry; Royal Society of Chemistry:

Cambridge, 2006. [5] Vilar, R. Eur. J. Inorg. Chem. (2008) 357-367. [6] G. W. Bates, M. E. Triyanti, Light; M. Albrecht; P.A.J. Gale Org. Chem., 72 (2007) 8921–8927. [7] D. Makuc; M. Lenarčič; G. W. Bates; P. A. Gale; J. Plavec Org. Biomol. Chem. (2009) doi:

10.1039/B908947K

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Application of HPLC and LC/MS techniques for determination and identification of impurities in Rosiglitazone tablets

S. Biškupić, V. Radić, K. Lacković

PLIVA CROATIA LTD., Generics R&D, Prilaz baruna Filipovića 29 10000 Zagreb Croatia

Rosiglitazone is an antidiabetic drug in the thiazolidinedione class of drugs. Rosiglitazone is indicated as an adjunct therapy to diet and exercise to improve glycemic control in patients with type 2 diabetes mellitus[1].

During accelerated stability testing of Rosiglitazone tablets, performed at 40°C/75%RH, it was observed that a content of unknown impurity at RRT ~ 0.54 has increased above identification threshold (0.25%). According to international guidelines (ICH Q3B(R2) Impurities in New Drug Product) any degradation product observed in stability studies should be identified when present at level higher than the identification threshold[2].

The aim of this study was to identify observed degradation impurity using HPLC and LC/MS techniques.

Results of experimental study demonstrated that the unknown peak consisted of two peaks and existing HPLC method for determination of impurities was modified by changing the column, content of mobile phase, gradient conditions and sample preparation in order to separate peaks. After HPLC separation, molecular mass of unknown peaks was successfully determined by using Waters Q-TOF Micro MS system. Structural characterization was based on results obtained by LC-MS/MS system. The unknown impurity at RRT ~ 0.54 was identified as (2-(5-{4-[2-(Methyl-pyridin-2-yl-amino)-ethoxy]-benzyl}-2,4-dioxo-thiazolidin-3-yl)-succinic acid), while coeluting peak at RRT ~ 0.56 was identified as diastereoisomer of above mentioned impurity.

The modified method for determination of impurities in Rosiglitazone tablets was validated as stability indicating method to be used for evaluation of the product shelf life. The adequate stability of product has been proved during 24 months of stability study. [1] Martindale - The Complete Drug Reference, Micromedex (R) Healthcare Series 2009. [2] ICH Guideline, Q3B(R2) Impurities in New Drug Product

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Monitoring of the oxidatione stability of sodium picosulfate by a new RP-HPLC method

Ivana Savić, Goran Nikolić,Valentina Marinković*, Miroslav Milenković* Ivan Savić, Predrag Sibinović*

Faculty of Technology, University of Nis, Bulevar oslobodjenja 124, Leskovac, Serbia, *Pharmaceutical and Chemical Industry Zdravlje-Actavis, Vlajkova 199, Leskovac, Serbia.

A sensitive, selective, precise and stability-indicating, new high performance liquid chromatographic method for the analysis of sodium picosulfate both as a bulk drug and in formulations was developed and validated. As the method could effectively separate the drug from its degradation product, it can be employed as a stability-indicating one. The chromatographic separation was achieved on a ZORBAX Eclipse XDB C-18 analytical column. The mobile phase consisted of phosphate buffer (pH 7) : acetonitrile (85:15 v/v). The absorbance was monitored with a DAD detector at 263 nm. The flow rate was 1.5 mL/min. Statistical analysis proved the method is repeatable, selective, and accurate for estimation of sodium picosulfate in the presence its degradation product. Forced degradation study was performed on bulk sample of sodium picosulfate using oxidation (0.1, 0.5 and 1 % v/v hydrogen peroxide, at room temperature). Degradation product formed under these conditions was found to be impurity A.

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Page 61

Quantification of Erythrocyte s-COMT Activity using a Validated Method

Leonel Torrão, Rita Machado, Lyndon Wright, Patrício Soares-da-Silva

Laboratory of Pharmacological Research, Department of Research and Development, BIAL - Portela & Cª S.A., S. Mamede do Coronado, Portugal.

Catechol-O-methyltransferase (COMT) is one of the main enzymes responsible for the metabolism of levodopa, dopamine, other catecholamines and their metabolites. Here we report the validation of an analytical method for quantification of human erythrocyte s-COMT activity. The evaluation of the erythrocyte s-COMT activity was based on measuring the rate of formation of DL-metanephrine (MN) using epinephrine (AD) as substrate, expressed as pmol MN/mg haemoglobin/h, utilising a high performance liquid chromatography coupled with electrochemical detection (HPLC-ED) and a spectrometric assay specific for haemoglobin (Hb) quantification, previously optimized to support clinical studies with COMT inhibitors.

The method provides a linear response from 20 to 400 ng/mL for MN using 250 µl of washed erythrocytes sample. The intra-assay accuracy, calculated as percent error, ranged from –6.6 % to –0.8 % and the precision (% CV) was less than 7.4 %. The inter-assay precision assessment, over 3 days, was less than 6.7 % and the accuracy ranged from −5.2 % to –2.4 %. For the validated range, MN was considered to be stable in erythrocyte extract sample following storage for approximately 24 hours at room temperature and after three freeze-thaw cycles over 5 days below –80 °C. The lower limit of quantification (LLOQ) for Hb was at the nominal concentration of 30 mg/mL and the acceptable upper limit of quantification (ULOQ) was at the nominal concentration of 180 mg/mL. The intra-assay percent error ranged from –1.2 % to 2.0 % and the precision between 0.2 % and 0.8 %. The inter-assay precision assessment was less than 1.5 % and the accuracy ranges from –0.3 % to 2.7 %.

This validated method enables the evaluation of the COMT activity in human erythrocytes[1]. [1] P. Silveira, M. Vaz-da-Silva, L. Almeida, J. Maia, A. Falcao, A. Loureiro, L. Torrao, R.

Machado, L. Wright and P. Soares-da-Silva, Pharmacokinetic-pharmacodynamic interaction between BIA 3-202, a novel COMT inhibitor, and levodopa/benserazida, Eur. J. Clin. Pharmacol. 59 (2003) 603-609.

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62

Fast pharmaceutical quality control based on elemental analysis

Marina Fedoseeva, Elena Kapinus, Valentina Kosenko*, Lidia Mitkina, Igor Revelsky

Lomonosov Moscow State University, Chemistry Department, Moscow, Russia *Roszdravnadzor State Control Department, Moscow, Russia

In our days, one of the actual problems of pharmaceutical analysis is development of fast, not expensive and universal methods of active component content determination in pharmaceuticals. Such methods are need for improving quality control of pharmaceuticals and for fast detection of counterfeits.

We have proposed new approach to fast quality control of pharmaceuticals based on use of elemental analysis[1]. Therefore, the goal of present work was further development of fast and universal method for active component content determination in different pharmaceuticals, based on determination of nitrogen content in pharmaceutical substances and tablets.

Investigation was carried out using elemental analyzer “Dumatherm” (“Gerhard”, Germany). Such analysis of 5 pharmaceutical substances and 9 original and generic pharmaceutical tablet forms products has been carried out. High accuracy of the active component determination has been demonstrated.

The advantage of the proposed method that it is universal, fast and doesn’t require use of the analytes’s standards.

[1] I.A. Revelsky, New approach to fast determination of active component content in pharmaceutical dosage forms based on elemental analysis, 1st BBBB Conference on Pharmaceutical sciences, September 26-30, 2005, Siofok, Budapest, Hungary

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Page 63

Computer modelling of possible of binding of calix[4]arenas to the active site of the myosin. A study of influence of calix[4]arenas on the ATP-ase activity of

actomyosin and myosin subfragment-1 from the myometrium

Alyona A. Bevza, Alexander V. Bevza

O.V. Palladin Institute of Biochemistry, NAS of Ukraine, Kyiv-30 Leontovycha str., 9, Ukraine, e-mail: [email protected]

Calixarenes are emerging as a new class of synthetic perspective scaffolds in the design of the sensors, catalysts and bioactive compounds due to their unique capability to selectively bind ions and neutral molecules selectively[1,2]. As it has been remarkably shown, these macrocycles have a wide spectrum of biological activity [3,4]. We have investigated the inhibition activity of actomyosin ATP-ase and ATP-ase of subfrag-ment-1 (head) myometrium by calix[4]arenes C-97 and C-99 functionalysed at the macrocyclic upper rim by α-hydroxy-phosphonic and α-amino-phosphonic acid fragments, respectively. Actin-myosin (actomyosin complex) is the basic structural and contractile component of smooth muscles cells. The interaction of these two proteins is fueled by the hydrolysis of ATP by ATP-ase that occurs in the catalytic domain of the myosin head [5,6]. It has been shown that calix[4]arene C-99 at a concentration of 100 μM, inhibited enzymatic activity of actomyosin ATP-ase by 50% (the value of the apparent constants of inhibition being 98,8±1,3 μM). At the same time, 100 μM calix[4]arenes C-97 inhibited activity by 70% (I0,5=84,0±2,0 μM). In experiments carried out with subfragment-1 of myosin ATP-ase, it was shown that 100 μM calix[4]arene C-99 inhibited activity by 77+4% (I50 =43+8 μM). In the search for a possible mechanism of co-operation between calix[4]arene C-99 and C-97 (as ligand) with the functionally active part of the myosin subfragment-1 molecule (as receptor), a three-dimensional structure of calix[4]arenes was modeled. We used the spatial structure of myosin, ID 1B7T in the Protein Data Bank. In modeling experiments in the enzyme active center Mg2+ and (or) molecule ATP (ADP), that is necessary for work АТР-ase of myosin was placed. A search for the most powerful and advantageous mutual docking location of ligand (C-99) was conducted in the spacious molecule of myosin. It is possible to assert that the calixarenes co-operate with the functionally active part of the myosin molecule (the area of binding of ATP) was supported by the result of docking. Foreseen in silico binding of calixarene in the enzyme active center (АТР-ase) is confirmed by the results in relation to the influence of calixarenes on the catalytic activity of myosin (subfragment-1) in vitro. The data presented here demonstrates that the calixarenes which we have studied, can influence uterus smooth muscle at the level of the contractile proteins, namely on the ATP-ase of the catalytic domain of the myosin head. The obtained results can be useful in further researches, directed at the use of calixarenes as pharmaceutical substance able normalization of contractile function of uterus in some pathologies of pregnancy and sustain healthy condition of the female reproductive system. The authors are grateful to Prof. V.I. Kalchenko for his donations of extended calix[4]arenes.

[1] G. J. Lumetta, R. D. Rogers, A. S. Gopalan, Calixarenes for Separations, American Chemical

Society: Washington (2000). [2] G. G. Talanova., H.-S. Hwang., V. S Talanov, R. A. Bartsch, Chem. Commun. 9 (1998) 419-420. [3] M. Kopaczynska, T. Wang, A. Schulz, Langmuir. 21 (2005) 8460-8465. [4] B. T. Zhao, M. J. Blesa, N. Mercier, F. Le Derf , M. Salle, J Org Chem. 70 (2005) 6254-6257 [5] S. Highsmith, Biochemistry. 38 (1999) 9791-9797. [6] A. S. Khromov, M .R. Webb., M. A. Ferenczi, D. R. Trentham, Biophys. J. 86 (2004) 2318-2328

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64

Synthesis, structural and biochemical characterization of oxime bond containing anticancer drug delivery systems

Gábor Mező1, Antal Csámpai2, Bence Kapuvári3, Erika Orbán1, Ildikó Szabó1, László Radnai4, Marilena Manea5

1Research Group of Peptide Chemistry, Hungarian Academy of Sciences

Eötvös L. University, Budapest, Hungary; 2Institute of Chemistry, Eötvös L. University, Budapest, Hungary

3National Institute of Oncology, Budapest, Hungary 4Department of Biochemistry, Eötvös L. University, Budapest, Hungary

5Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry and Zukunftskolleg, University of Konstanz, Konstanz, Germany

Chemoselective ligation approaches are widely used in the synthesis of cyclic peptides and peptide conjugates. Oxime bond formation is one of the most commonly used, due to its chemical stability and easy synthesis. The oxime linkage is formed between an oxo group (ketone or aldehyde) and a hydroxylamine derivative such as aminooxyacetic acid. Chemoselective ligation procedures are also employed in the preparation of drug delivery systems in which drugs are attached to peptides or to other types of targeting moieties. The advantages of drug delivery systems is their increased selectivity and lower toxicity resulting in higher therapeutic efficiency. However, many drugs have no functional groups that can be used for conjugation or the modification of the functional groups lead to the loss of the biological activity. Drugs containing oxo groups can be attached via oxime bond to various carriers. In our study, daunorubicin (anthracycline antibiotic) and exemestane (steroidal aromatase inhibitor) as antineoplastic agents were modified with aminooxiacetic acid (Aoa). The isomer composition of the oxime bond of the compounds was determined by NMR spectroscopy. The drugs were attached to the gonadotropin-releasing hormone (GnRH) derivatives. These hormone peptides have own antitumor activity and recognize the GnRH receptors that are overexpressed on tumor cells providing increased selectivity to the conjugates. Daunorubicin (Dau) was coupled to GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2), a weak agonist peptide, while exemestane was incorporated bet-ween two peptide chains of a GnRH antagonist (Ac-D-Trp-D-Cpa-D-Trp-Ser-D-Lys-Asp(Leu-Gln-Pro-D-Ala-NH2)-DEA) resulting in a dimer. Dau has antitumor activity due to its ability to intercalate into the DNA chains. Therefore, the interaction with DNA of Dau, Dau=Aoa-OH and its conjugated form was studied by fluorescence spectroscopy. It was found that Dau and Dau-GnRH-III conjugate bound to the DNA, while the interaction of Dau=Aoa-OH with DNA was less pronounced. The cellular uptake studies of Dau and Dau-GnRH-III conjugate, determined by flow cytometry, showed that Dau was taken up in a higher amount by MCF-7 human breast and C26 murine colon cancer cells than Dau-GnRH-III conjugate. However, the in vitro cytostatic effect of Dau was only one order of magnitude higher than it was obtained in the case of Dau=Aoa-OH and Dau-GnRH-III conjugate. Acknowledgement This work was supported by grants from the Hungarian National Science Fund (OTKA T049814, NK 77485), the Ministry of Health (ETT 202/2006), GVOP-3.2.1.-2004-04-0005/3 and Zukunftskolleg and AFF (Project 01/09), University of Konstanz.

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Page 65

111In and 177Lu radiolabelling and quality control of modified

PAMAM dendrimer

Veronika Biricová, Alice Lázníčková, Petr Hermann*, Ivan Lukeš*

Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Heyrovského 1203 500 05 Hradec Králové, Czech Republic, [email protected]

*Charles University in Prague, Faculty of Science, Albertov 6, Praha 2, Czech Republic

Dendritic highly branched polymeric materials seem to be potential antitumour drug carriers. Their huge surface areas as well as an ability to encapsulate guest molecules in the macromolecule interior make them useful for cancer diagnosis and treatment. The new con-jugate of pyridine-N-oxide derivative of DOTA, 10-[(4-carboxy-1-oxidopyridin-2-yl)me-thyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, with ethylenediamine-core polyamidoamine (PAMAM) dendrimer of a fourth generation (64 amino groups on its surface) was prepared (D-G-4h) and radiolabelled with lutetium-177 and indium-111. The non-radioactive isotopes of the metals were concurrently used with radioactive isotopes to fully saturate the free ligand groups on the surface of the dendrimer. The reaction mixture was incubated at 40°C overnight in purpose to obtain a higher saturation degree of radiometal. Diethylenetriaminepentaacetic acid was added to the mixtures before analysis to scavenge excess of metals possibly non-specifically bound to the dendrimer. Radiolabelled products were analysed using gel permeation chromatography (GPC), polyacrylamide gel electrophoresis (PAGE), instant thin-layer chromatography on silica gel (iTLC-SG) and agarose gel electrophoresis. We have produced and separated the products of a sufficient quality. Radioactivity of the samples was measured on gamma spectrometer. PAGE analysis of purified 177Lu-D-G-4h indicates that radiolabelled conjugate with dendrimer has a negative total charge. Radiolabelled product 111In-D-G-4h also migrates slowly toward the positive electrode during electrophoresis in agarose gel. This study clearly shows that gel permeation chromatography is an effective technique for separation and consecutive quality control of the PAMAM dendrimer conjugate. For the quick quality control of the radiolabelled product iTLC-SG seems to be a convenient method in case of no need to obtain purified product. Polyacrylamide gel electrophoresis and agarose gel electrophoresis are suitable methods for partial characterisation of the conjugate.

Acknowledgement: The study was supported by the Grant Agency of Charles University, Grant No. 111008. This work was also carried out in the framework of COST BM0608 Action.

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Thermodynamic and structural study of pure compounds and mixed crystals in the α,ω-alkanediols family. The C10H22O2-C12H24O2 binary system

Gilgamesh Luis, Màrius Ramírez, Gabriel Luna*, María M. Ramos**, Martín A. Hernandez*

Centro de investigaciones en Ciencias de la Tierra y Materiales,Universidad Autónoma del Estado de Hidalgo, Carretera Pachuca-Tulancingo km 4.5,

Col. Carboneras, 42184, Mineral de la Reforma, Hgo., México. *Centro de Investigaciones y Estudios Avanzados del Instituto Politécnico Nacional Libr. Norponiente 2000 Fracc. Real de Juriquilla, 76230, Querétaro, Qro., México.

**Departamento de Tecnología Química y Ambiental, Universidad Rey Juan Carlos, Tulipán s/n E 28933, Móstoles, Madrid, España.

This work is focused on α,ω-alkanediols with two particular chain lenghts, 1,10-decanediol and 1,12-dodecanediol. Molecular alloys between both molecules have been analyzed by means of powder X-ray diffraction (PXRD) on variable temperature, polarized light microscopy (PLM), and differencial scanning calorimetry (DSC). Experiments proved a solid to solid transition close to melting point, while the pure compounds do not show any transitional effect before fusion. The broadening of the diffraction peaks can be related to defects on crystals. Crystal structures of pure compounds have been solved and refined by the Rietveld method using a rigid body scheme. Cell parameters of molecular alloys, fusion and solid-solid transitions temperatures and enthalpies are reported. The significance of the data is supported by a thermodynamic T-Xi phase-diagram.

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Preparation and investigation of bilayer tablets containing theophylline combined titanate nanotubes

Péter Kása jr., Tamás Sovány, Stane Srcic*, Endre Horváth**, Zoltán Kónya**,

Imre Kiricsi**, Klára Pintye-Hódi

University of Szeged, Department of Pharmaceutical Technology, Szeged, Hungary *University of Ljubljana, Department of Pharmaceutical Technology, Ljubljana, Slovenia

**University of Szeged, Department of Applied and Environmental Chemistry, Szeged, Hungary

The traditional pharmaceutical dosage forms such as tablets can contain different active ingredients. In general the formulation of this preparation mainly depends on the morphological properties of the raw and active materials. The most economic preparation process is the direct tabletting where the materials can compressed immediatelly after the homogenisation. For this tabletting procedure the materials needs to be a good flow properties and a good compressibility and compactibility. For preparing tablets in this way, the flow properties, and the shape of the materials is the most important factor. The long, needle shaped crystals can not process to tablet form in direct compression because their flow properties are very poor. The investigated theophylline crystals was used in native and in incorporated to titanate nanotube form. After the tablet preparation the efficacy of the active materials depends on their solubility in the GI tract after the disintegration. In many cases the aim of the preparation is the slow and constant dissolution rate. The empty titanate nanotubes was prepared with a typical synthesis. The TiO2 powders was dispersed in aqueous NaOH solution under continuous stirring. The prepared suspension was investigated into an autoclave and subjected to alkali treatment. A modified impregnation process was used to prepare titanate nanotube (TiONT) supported theophylline (TP) drugs. Transmission electron microscopy showed that the empty needle-shaped products have a tube structure with an inner diameter of approximately 5 nm and an outer diameter of approximately 8 nm. Before tablet making the properties of the starting materials and the powder mixtures was investigated such as flow properties, loose, tapped and true density, compactibility and compressibility, and morphology. The bilayer tablets were prepared by Kilian SP300 hydraulic tablet press with two different compression forces 4 and 8 kN with manual feeding. After the tablet preparation, the commonly investigated parameters were controlled in all samples such as mechanical properties (friability, breaking hardness). The texture of the two layered preparation and the disintegration and dissolution properties were investigated also.

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Challenges of Low Solubility Compounds

A. Avdeef, S. M. Stones*

pION INC, 5 Constitution Way, Woburn, MA 01801 USA; [email protected] *Heath Scientific, 1 North House, Bond Avenue, Bletchley, MK1 1SW;

[email protected] Solubility measurement is critically important across several stages of pharmaceutical research, from where leads are first selected and optimized to identify promising clinical candidate molecules, to where pre-formulation characterization is done, in preparation for the development of suitable oral formulations. At the later stages, solubility and dissolution become practically inseparable. At the earlier stages, 'solubility' may mean no more than a yes/no answer the question: 'Is my compound soluble enough?’ The lecture part of the workshop will touch on some of the following questions:

• What solubility should be measured in order to gain clinically relevant information?

• Should 'thermodynamic' data ever be used in clinically-relevant investigation? • Why is it so hard to get a straight answer to the questions: 'what's the intrinsic

solubility of terfenadine? amiodarone? itraconazole? miconazole?' • Why are published results on these and similar molecules so inconsistent? • What is the 'sdiff 3-4' approximation? • Can measurement of dissolution be done in 1 mL volumes? In 96-well microtitre

plates? • Can one measure intrinsic dissolution rates with powders of unknown surface

area? • Is the measurement of dissolution ever relevant in drug discovery?

The live demonstration during the workshop will feature micro-dissolution & micro-solubility, using the low soluble model weak base HALOPERIDOL, and the µDISS ProfilerPLUSTM analyzer:

• Demonstration of the instrument features • Constructing a standard curve • Determination of haloperidol intrinsic dissolution rate at pH 6.8 • Determination of haloperidol solubility at pH 6.8 • Use of 2nd derivative data analysis

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ALPHA Workshop

Jiří Šikola

Bruker Optics, Czech Republic

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Author Index

A Adamski, J. .................................................. 36 Alexander, R. ............................................... 32 Alvarez, A.I. ................................................. 44 Armugam, K. ............................................... 34 Avdeef, A. ................................................ 3, 71 Averinnei, R.K. ............................................. 34 Azad, J. ........................................................ 57

B Beckers, D. .................................................. 19 Bevetek Moćnik, A. ...................................... 52 Bevza, A.A. .................................................. 63 Bevza, A.V. .................................................. 63 Biljan, T. ...................................................... 23 Biricová, V. .................................................. 65 Biškupić, S. .................................................. 59 Bunk, S. ....................................................... 35 Buryak, A. .................................................... 56

C Calvet, C. ..................................................... 43 Cetina-Čižmek, B. ............................ 31, 45, 51 Chamyan, K. ................................................ 56 Chernyshenko, V.O. ..................................... 40 Cindrić, M. ................................................... 20 Comer, J. ..................................................... 15 Cozma, K. .................................................... 10 Csámpai, A. ................................................. 64

Č Čanadi, J. ..................................................... 29 Čavužić, D. ................................................... 21

D Dafforn, T. .................................................... 9 Didziapetris, R. ........................................... 17 Dohnal, J. ...............................................33, 48 Doubijansky, E. ........................................... 55 Dragić, T. .................................................... 31 Dragusanu, M. ............................................ 10 Dressman, J.B. .............................................. 4

Dž Džale, V. ..................................................... 31

Đ Đaković-Sekulić, T.L. ................................... 38

E Erak, I. ........................................................ 45 Erukhimovitch, V. ....................................... 39

F Fedoseeva, M. ............................................ 62 Fischer, C. ................................................... 36 Fraile, M-T. ................................................. 44

G Gaál, D. ....................................................... 25 Gaál, F.F. .................................................... 29 Gelboa, I. .................................................... 55 Gogol, E. ..................................................... 54 Golja Gašparović, P. ................................... 50 Golubev, I. .................................................. 54 Gomez-Sanchez, R. ..................................... 44


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74

Gribbon, P. .................................................. 24 Günther, U.L. .............................................. 36 Guzsvány, V.J. ............................................. 29

H Heldman, E. ................................................ 55 Hermann, C. ................................................ 35 Hermann, P. ................................................ 65 Hernandez, M.A. ......................................... 66 Hilfiker, R. ..................................................... 6 Horvat, M. ................................. 18, 31, 49, 52 Horváth, E. .................................................. 67 Huleihel, M. ................................................ 39

I Ilic, I. ........................................................... 30 Iurascu, M. .................................................. 10 Ivković, B. .................................................... 50

J Jampílek, J. ........................................... 33, 48 Japertas, P. ................................................. 17 Jeeves, M. ..................................................... 9 Jelača, T. ..................................................... 31 Johan, N. ..................................................... 34

K Kapinus, E. .................................................. 62 Kapuvári, B. .......................................... 25, 64 Kargosha, K................................................. 57 Kása, P. jr .............................................. 30, 67 Kiricsi, I. ...................................................... 67 Knowles, T.J. ................................................. 9 Kónya, Z. ..................................................... 67 Körner, R. .................................................... 37 Kosenko, V. ........................................... 56, 62 Košiček, M. ................................................. 49 Král, V. .................................................. 33, 48 Kulić, Ž. ....................................................... 25

L Lacković, K. ................................................. 59 Lavandera, J.L. ............................................ 44 Lázníčková, A. ............................................. 65

Lin, Y-P .......................................................... 9 Ludwig, C. ................................................... 36 Luis, G. ........................................................ 66 Lukeš, I. ....................................................... 65 Luna, G. ....................................................... 66

M Machado, R. ................................................ 61 Maksymovych, I.S. ...................................... 40 Makuc, D. .................................................... 58 Manea, M. ...................................... 10, 25, 64 Marinković, V. ............................................. 60 Marquardt, A. ............................................. 10 Messinger, J. ............................................... 36 Meštrović, E. ............................................... 49 Mező, G. ................................................ 25, 64 Michiels, P.J.A. ............................................ 36 Mihoci, M. ................................................... 31 Mijakovac, I. ............................................... 45 Milak, S. ...................................................... 47 Milenković, M. ............................................ 60 Milovanović, M. .......................................... 46 Mitkina, L. ................................................... 62 Moeinosadat, S.R. ....................................... 57 Moise, A. ..................................................... 10 Möller, G. .................................................... 36 Morató, J.A. ................................................ 43 Moura, J. ..................................................... 53 Mukmenev, I. .............................................. 55

N Nayak, U. .................................................... 34 Neuhoff, S. .................................................. 16 Nikolić, G. .................................................... 60 Novotarsky, S. ............................................. 37

O Öhlschläger, P. ............................................ 25 Oktábec, Z. ............................................ 33, 48 Orbán, E. ............................................... 25, 64 Ortega, E. .................................................... 43 Overduin, M. ................................................. 9

P Pandey, A.K. ................................................ 37 Pandey, S. ................................................... 34


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Papós, K. ..................................................... 30 Paraschiv, G. ............................................... 10 Parot. P. ...................................................... 11 Pavišić, R. .................................................... 20 Pellequer, J-L. .............................................. 11 Pera, S. ........................................................ 32 Perdivara, I. ................................................. 10 Peter-Katalinić, J. .......................................... 8 Petrauskas, A. ............................................. 17 Petre, A. ...................................................... 10 Petrović, Lj. .................................................. 46 Pintye-Hódi, K. ....................................... 30, 67 Plavec, J. ................................................ 22, 58 Polezneva, A. ............................................... 54 Port, A. ........................................................ 43 Prugovecki, S. .............................................. 19 Przybylski, M. ........................................ 10, 35

R Radić, V. ...................................................... 59 Radnai, L. .................................................... 64 Raichman, A. ............................................... 55 Rajić, M. ...................................................... 46 Ramírez, M. ................................................. 66 Ramos, M.M. ............................................... 66 Raušl, D. ...................................................... 45 Reichardt, W. .............................................. 25 Revelsky, I. ...................................... 54, 56, 62 Riedel, U. ..................................................... 19 Rožman, A. .................................................. 52 Rupp, M. ...................................................... 37

S Santos-Villarej, A. ........................................ 44 Savić, I. ........................................................ 60 Sazonovas, A. .............................................. 17 Shavi, G. ...................................................... 34 Sibinović, P. ................................................. 60 Siegal, G. ..................................................... 12 Simčić, I. ...................................................... 31 Simplício, A.L. .............................................. 53 Slamnoiu, S. ................................................. 10 Smith, C. ........................................................ 9 Soares-da-Silva, P. ....................................... 61 Sovány, T. .............................................. 30, 67 Srcic, S. .................................................. 30, 67 Stephan, M. ................................................. 36 Stojanović, B. ............................................... 46

Stones, S.M. ................................................ 71 Surjak, M. ................................................... 47 Sushko, I. .................................................... 37 Susnea, I. .................................................... 35 Svetličić, V. ................................................. 51 Szabó, I. .................................................25, 64

Š Šašić, S. ......................................................... 7 Šegota, S. .................................................... 51 Šikola, J. ...................................................... 72 Šket, P. ........................................................ 22

T Tejeda, M. .................................................. 25 Tetko, I.V. ................................................... 37 Thole, H. ..................................................... 36 Tong, M. ....................................................... 9 Torrão, L. .................................................... 61 Trišović, N.P. ............................................... 38 Tudja, P. ..................................................... 51 Turkalj, J. .................................................... 45

U Udupa, N. ................................................... 34 Ušćumlić, G.S. ............................................. 38

V Valentić, N.V. .............................................. 38 Valkó, K. ....................................................... 5 van Dongen, M. .......................................... 36 Vidović, S. ................................................... 46 Vlad, C. ....................................................... 10

W Wright, L. .................................................... 61

Y Yaar, A. ....................................................... 55

Z Zerzaňová, A. .........................................33, 48


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NOTES


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NOTES

First World Conference on Physico-Chemical Methods in Drug ... · HPLC based property measurements of drug discovery compounds to predict in-vivo drug distribution and aid lead optimisation

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