DDR_2007

INTERNATIONAL SYMPOSIUM on

DRUG RESEARCH and DEVELOPMENT

“From Chemistry to Medicine”

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYEMay 17-20, 2007

Hacettepe University Medicinal Chemistry Research, Development and Application Center

(MAGUM)

iiiKervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE

“From Chemistry to Medicine” DRD 2007

A big THANK YOUSupporting Organizations

Sponsoring Organizations

ABBOTT LABORATUVARLARI İTHALAT İHRACAT ve TİC.LTD.ŞTİ.

ABDİ İBRAHİM İLAÇ SANAYİ ve TİC. A.Ş.

ADEKA İLAÇ SAN. A.Ş.

BİEM TIBBİ CİHAZ ve İLAÇ SANAYİ LTD. ŞTİ.

THE SCIENTIFIC & TECHNOLOGICAL RESEARCH COUNCIL OF TÜRKİYE

Pharmaceutical Companies

ASSOCIATION OF RESEARCH - BASED PHARMACEUTICAL COMPANIES

TURKISH PHARMACEUTICAL INDUSTRIES ASSOCIATION

TURKISH PHARMACEUTICAL MANUFACTURERS ASSOCIATION

THE SOCIETY OF PHARMACEUTICAL SCIENCES OF ANKARA

TURKISH PHARMACEUTICAL TECHNOLOGY SCIENTISTS ASSOCIATION

TURKISH ASSOCIATION OF PHARMACEUTICAL AND MEDICINAL CHEMISTRY

iv May 17-20, 2007

International Symposium on Drug Research and Development

BİLİM İLAÇ SANAYİ ve TİC. A.Ş.

FAKO İLAÇLARI A.Ş.

GLAXOSMITHKLINE İLAÇLARI A.Ş.

JOHNSON & JOHNSON SIHHİ MALZEME SAN. ve TİC. LTD. ŞTİ.

LİLLY İLAÇ TİCARET LİMİTED ŞTİ.

MUSTAFA NEVZAT İLAÇ SANAYİ A.Ş.

NOVAGENIX BİO ANALİTİK İLAÇ AR-GE MERKEZİ SAN. TİC. A.Ş.

NOVARTIS SAĞLIK GIDA ve TARIM ÜRÜNLERİ SAN. VE TİC. A.Ş.

PFIZER İLAÇLARI LTD. ŞTİ.

ROCHE MÜSTAHZARLARI SAN. A.Ş.

SANOFİ-AVENTİS İLAÇLARI LTD.ŞTİ.

SANOVEL İLAÇ SAN. VE TİC. A.Ş.

SANTA FARMA İLAÇ SANAYİ A.Ş.

SCHERING PLOUGH TIBBİ ÜRÜNLER TİC. A.Ş.

SERVIER İLAÇ ve ARAŞTIRMA A.Ş.

ULKAR KİMYA SAN. VE TİC. A.Ş.

vKervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE


ALBİO KİMYEVİ MADDELER İTHALAT ve TİC. A.Ş.

ANAMED ANALİTİK ve MEDİKAL SİSTEMLER A.Ş.

ANT TEKNİK CİHAZLAR PAZ. ve DIŞ TİC. LTD.ŞTİ.

ATOMİKA MAKİNA TİC. LTD. ŞTİ.

BETA LABORATUVAR CİHAZLARI LTD. ŞTİ.

FARGEM, FARMASÖTİK ARAŞTIRMA GELİŞTİRME MERKEZİ SAN. ve TİC.A.Ş.

HACETTEPE TEKNOKENT A.Ş.

IŞIN TIP ARAŞTIRMA ÜRÜNLERİ TİC. LTD. ŞTİ.

İNCEKARA TEKNİK CİHAZLAR ENDÜSTRİSİ ve TİC. A.Ş.

İNFO KİMYA LABORATUVAR CİH. TİC. LTD. ŞTİ.

İNTERLAB LABORATUVAR ÜRÜNLERİ SANAYİ ve TİCARET A.Ş.

KUTAY LABORATUVAR CİHAZLARI TİCARET A.Ş.

Institute

TURKISH PATENT INSTITUTE

Manufacturers/Distributors of Analysis Products and Equipments

vi May 17-20, 2007


LABOR İLDAM LAB. MALZ. TİC. LTD. ŞTİ

MEDSANTEK LABORATUVAR MALZEMELERİ SANAYİ ve TİCARET LTD.ŞTİ.

NÜVE SANAYİ MALZEMELERİ İMALAT ve TİC. A.Ş.

REDOKS KİMYASAL BİYOLOJİK MAD. ve LAB. CİH. PAZ. İTH. İHR. SAN. ve TİC. LTD. ŞTİ.

REFERANS KİMYA LTD. ŞTİ.

SARTONET SEPERASYON TEKNOLOJİLERİ LTD. ŞTİ.

SESA ELEKTRONİK SAN. TİC. A.Ş.

TETRA ENDÜSTRİYEL ve TEKNİK SİSTEMLER DIŞ TİCARET A.Ş.

BİOSTAR

LEDA PASTAHANESİ

SAYDANÜÇ AS TEKSTİL

SAYDAN ÜÇ AS TEKSTİL

SOYDANLAR KIRTASİYE

ZEKİ KIRTASİYE

Others

viiKervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE


Honorary Chairman Prof. Tunçalp ÖZGEN, MD

President of Hacettepe University

Organizing CommitteesChairman : Prof. Ünsal ÇALIŞ, Ph. D.

Scientific Secretary : Prof. Selma SARAÇ, Ph. D.Treasurer : Assoc. Prof. Kezban ULUBAYRAM, Ph. DMembers : Prof. Sedef KIR, Ph. D.

Prof. Gülberk UÇAR, Ph. D. S. Kutay DEMİRKAN, Pharm. D.

Analytical Chemistry

Sacide ALTINÖZ Hacettepe University, Türkiye

José BARBOSA University of Barcelona , Spain

Nursabah BAŞCI Hacettepe University, Türkiye

Nuran ÖZALTIN Hacettepe University, Türkiye Biochemistry

Hamdi ÖĞÜŞ Hacettepe University, Türkiye

İnci ÖZER Hacettepe University, Türkiye

Nazmi ÖZER Hacettepe University,Türkiye Angelino PARINI Rangueil Institute of Molecular Medicine,

France Kevser PİŞKİN Hacettepe University, Türkiye

Mercedes UNZETA Barcelona Autonoma University, Spain Biology

Nilüfer AKSÖZ Hacettepe University, Türkiye

Vasıf HASIRCI Middle East Technical University, Türkiye

Aşkın TÜMER Hacettepe University, Türkiye

Chemistry and Chemical Engineering

Ayhan S. DEMİR Middle East Technical University, Türkiye

Adil DENİZLİ Hacettepe University, Türkiye

Emir Baki DENKBAŞ Hacettepe University, Türkiye

Metin BALCI Middle East Technical University, Türkiye

Menemşe GÜMÜŞDERELİOĞLU Hacettepe University, Türkiye

Olgun GÜVEN Hacettepe University, Türkiye

Nesrin HASIRCI Middle East Technical University, Türkiye

Yunus KARA Atatürk University, Türkiye

Erhan PİŞKİN Hacettepe University, Türkiye

Bekir SALİH Hacettepe University, Türkiye

Muzaffer TALU Gazi University, Türkiye Pharmaceutical and Medicinal Chemistry

Peter CROOKS University of Kentucky, USA

Sevim DALKARA Hacettepe University, Türkiye

Şeref DEMİRAYAK Anadolu University, Türkiye

Erçin ERCİYES Ege University, Türkiye

Dilek EROL Yeditepe University, Türkiye

Mevlüt ERTAN Hacettepe University, Türkiye

Marianne FILLET University of Liege, Belgium

Alexander V. KABANOV University of Nebraska, USA

Ningur NOYANALPAN Gazi University, Türkiye

Seçkin ÖZDEN Ankara University, Türkiye

Wolfgang SIPPL Martin Luther University, Germany Ljubica SUTURKOVA Ss. Cyril and Methodius University

Skopje, R. Macedonia Fethi ŞAHİN Gazi University, Türkiye Pharmaceutical Technology

Sema ÇALIŞ Hacettepe University, Türkiye

Nevin ÇELEBİ Gazi University, Türkiye

Ruxandra GREF University of Paris-Sud, France

Süeda HEKİMOĞLU Hacettepe University, Türkiye

A. Atilla HINCAL Hacettepe University, Türkiye

Filiz ÖNER Hacettepe University, Türkiye

Levent ÖNER Hacettepe University, Türkiye

A. Yekta ÖZER Hacettepe University, Türkiye

Nilüfer TARIMCI Ankara University, Türkiye

Pharmaceutical Microbiology

Ufuk ABBASOĞLU Gazi University, Türkiye

Sulhiye YILDIZ Ankara University, Türkiye Pharmacognosy and Pharmaceutical Botany

Ahmet BAŞARAN Hacettepe University, Türkiye

Ömür DEMİREZER Hacettepe University, Türkiye

Nurten EZER Hacettepe University, Türkiye

Erdem YEŞİLADA Yeditepe University, Türkiye Pharmacology

İsmail Hakkı AYHAN Ankara University, Türkiye

E. Rüştü ONUR Hacettepe University, Türkiye

İnci ŞAHİN Hacettepe University, Türkiye

Serdar UMA Hacettepe University, Türkiye Toxicology

Sema BURGAZ Gazi University, Türkiye

Filiz HINCAL Hacettepe University, Türkiye

Gönül ŞAHİN Hacettepe University, Türkiye

Scientific Committee

viii May 17-20, 2007


ixKervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE


Contents

• Exhibitor A - Z ....................................................................................................................................................................................... x

• Scientific Program .............................................................................................................................................................................. xi

• Lectures ................................................................................................................................................................................................. 1

SMART MOLECULES IN TARGETED THERAPIES ..........................................................................................................................................................3

PROPARGYLAMINES AS NEUROPROTECTIVE AGENTS IN NEURODEGENERATIVE DISEASES ............................................................................................4

OXIDATIVE STRESS AND MONOAMINE OXIDASES: FROM BASIC STUDIES TO NOVEL THERAPEUTICAL INTERVENTIONS ....................................................7

CHARACTERIZATION OF GLYCOPROTEINS BY CAPILLARY ELECTROPHORESIS ELECTROSPRAY MASS SPECTROMETRY

(CE-ES-MS). APPLICATIONS TO DIAGNOSIS IN BIOMEDICINE .....................................................................................................................................8

BIOMARKER DISCOVERY FOR CHRONIC INFLAMMATORY DISEASES USING PROTEOMIC SERUM PROFILING .................................................................14

IN SILICO MEDICINAL CHEMISTRY –

UNDERSTANDING BIOLOGICAL EFFECTS THROUGH MOLECULAR DOCKING AND MOLECULAR DYNAMICS SIMULATIONS ...............................................17

SURFACE-MODIFIED NANOPARTICLES FOR DRUG ENTRAPMENT AND TARGETING .....................................................................................................18

CHALLENGES IN THE TREATMENT OF GRAM-POSITIVE INFECTIONS ..........................................................................................................................20

CHALLENGES IN TREATMENT OF GRAM-NEGATIVE BACTERIA ...................................................................................................................................22

CHALLENGES IN ANTIFUNGAL THERAPY ................................................................................................................................................................24

NATIONAL AND INTERNATIONAL PATENT PROTECTION, FREE PATENT SEARCH TOOLS (ESPACENET) AND STRATEGIES ..................................................25

PATENTABILITY OF MEDICAL AND PHARMACEUTICAL INVENTIONS ..........................................................................................................................27

IMPORTANCE OF POLYMORPHISM IN DRUG RESEARCH AND DEVELOPMENT .............................................................................................................28

A VAST SOURCE FOR THE DISCOVERY OF NOVEL DRUG LEADS: TRADITIONAL MEDICINES ...........................................................................................29

PARTHENOLIDE ANALOGS AS ANTILEUKEMIC AGENTS WITH CLINICAL POTENTIAL ....................................................................................................35

SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL CAMPTOTHECIN CLASS TOPOISOMERASE I INHIBITORS .............................................................36

NANOMATERIALS IN MEDICINE .............................................................................................................................................................................38

NANOMEDICINE FOR CENTRAL NERVOUS SYSTEM (CNS) DRUG DELIVERY ................................................................................................................39

• Oral Presentations ............................................................................................................................................................................. 41

• Poster Presentations ......................................................................................................................................................................... 49

• Author Index ....................................................................................................................................................................................... 91

x May 17-20, 2007


ABDİ İBRAHİM İLAÇ SANAYİ VE TİC. A.Ş. .......................................................... A – 3ALBİO KİMYEVİ MADDELER İTHALAT VE TİC. A.Ş. ............................................ C – 11ANAMED ANALİTİK VE MEDİKAL SİSTEMLER A.Ş. ........................................... C – 10ANT TEKNİK CİHAZLAR PAZ. VE DIŞ TİC. LTD.ŞTİ. ............................................... A – 4ATOMİKA TEKNİK CIH. TİC. LTD. ŞTİ. ..............................................................A – 10BİEM TIBBİ CİHAZ VE İLAÇ SANAYİ LTD. ŞTİ. ..................................................... B – 9BİLİM İLAÇ SANAYİ VE TİC. A.Ş. ....................................................................... B – 6FABAD .......................................................................................................... A – 8FARGEM, FARMASÖTİK ARAŞTIRMA GELİŞTİRME MERKEZİ SAN. VE TİC.A.Ş. ....... B – 5HACETTEPE TEKNOKENT A. Ş. ......................................................................... A – 7IŞIN TIP ARAŞTIRMA ÜRÜNLERİ TİC. LTD. ŞTİ. .................................................. B – 7İNCEKARA TEKNİK CİHAZLAR ENDÜSTRİSİ VE TİC. A.Ş. .................................... C – 12İNFO KİMYA LABORATUVAR CİH. TİC. LTD. ŞTİ. ...............................................A – 12İNTERLAB LABORATUVAR ÜRÜNLERİ SANAYİ VE TİCARET A.Ş. .........................A – 18LABOR İLDAM LAB. MALZ. TİC. LTD. ŞTİ. .......................................................... B – 8

MEDSANTEK LABORATUVAR MALZEMELERİ SANAYİ VE TİCARET LTD.ŞTİ. .........A – 19NOVAGENIX BİO ANALİTİK İLAÇ AR-GE MERKEZİ SAN. TİC. A.Ş. ........................A – 17NÜVE SANAYİ MALZEMELERİ İMALAT VE TİC. A.Ş. ............................................. C – 9ONDÖRT MAYIS DERGİSİ ............................................................................. A – 14PATENT ENSTİTÜSÜ ............................................................................... A – 15,16PFIZER İLAÇLARI LTD. ŞTİ. .............................................................................. A – 5REDOKS KİMYASAL BİYOLOJİK MAD. VE LAB. CİH. PAZ. İTH. İHR. SAN. VE TİC.LTD.ŞTİ ...........................................A – 1REFERANS KİMYA LTD. ŞTİ. ............................................................................. C – 7SESA ELEKTRONİK SAN. TİC. A.Ş. ...................................................................A – 20TERRALAB LABORATUVAR MALZEMELERİ SANAYİ VE TİCARET A:Ş. ................... C – 8TUBİTAK ....................................................................................................... A – 6TÜFTAD ........................................................................................................ A – 9TÜRK KİMYA DERNEĞİ ................................................................................. A – 13ULKAR KİMYA SAN. VE TİC. A.Ş. ...................................................................... B – 4

Exhibitor A - Z

xiKervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE


www.drd2007.org

PROGRAMMEMAY 17, 2007 – THURSDAY

16.16-16.0 Registration

Hall A 16.00-17.30 OPENING CEREMONY Plenary Lecture Smart molecules in targeted therapies Emin Kansu (Hacettepe University, Türkiye)

19.19-19.0 WELCOME COCKTAIL

MAY 18, 2007 - FRIDAY

Hall A09.00-10.20 SESSION I Simultaneous Turkish translation will be provided. (New Approaches to Neurodegenerative Diseases) Chairpersons: Peter Crooks (University of Kentucky, USA) Emin Kansu (Hacettepe University, Türkiye)09.00-09.40 Propargylamines as neuroprotective agents in neurodegenerative diseases

Mercedes Unzeta (Universitat Autonoma de Barcelona, Spain)09.40-10.20 Oxidative stress and monoamine oxidases: From basic studies to novel therapeutical

interventions Angelo Parini (Rangueil Institute of Molecular Medicine, France)

10.20-10.50 Coffee Break

Hall A10.50-12.10 SESSION II Simultaneous Turkish translation will be provided. (Analytical Methods in Medicine) Chairpersons: Wolfgang Sippl (Martin Luther University, Germany) Nursabah Başçı (Hacettepe University, Türkiye)10.50-11.30 Characterization of glycoproteins by capillary electrophoresis electrospray mass spectrometry

(CE-ES-MS). Applications to diagnosis in biomedicine José Barbosa (University of Barcelona, Spain)

11.30–12.10 Biomarker discovery for chronic inflammatory diseases using proteomic serum profiling Marianne Fillet (University of Liege, Belgium)

12.10–12.35 Investigation of noncovalent protein-protein interactions by matrix- assisted llaser desorption/ionization mass spectrometry

Bekir Salih (Hacettepe University, Türkiye)

12.35-13.30 LUNCH

xii May 17-20, 2007


Poster Hall13.30-14.30 Poster Session

Industry Hall All presentations will be in Turkish. (Mission and Vision of the Pharmaceutical Companies) Chairperson: Nilüfer Tarımcı (Ankara University, Türkiye)13.30-14.00 Bilim İlaç Sanayii ve Ticaret A.Ş. (Türkiye) 14.00-14.30 Farmasötik Araştırma Geliştirme Merkezi Sanayii ve Ticaret A.Ş.- FARGEM (Türkiye) Satellite Hall All presentations will be in Turkish.Chairperson: Seçkin Özden (Ankara University, Türkiye)13.30-14.00 New Technology: Near Infrared (NIR) Analysis Systems

İncekara Holding/FOSS Analytical A.Ş. (Türkiye) 14.00-14.30 XRD Applications in Pharmaceutical Industry: Determination of Crystal Structure

Ant Teknik Ltd. Şti./Shimadzu Corp. (Türkiye)

Hall A14.30-16.00 SESSION III Simultaneous Turkish translation will be provided (Molecular Modeling) Chairpersons: José Barbosa (University of Barcelona, Spain) Sevim Dalkara (Hacettepe University, Türkiye)14.30-15.10 In silico medicinal chemistry – Understanding biological effects through molecular docking and

molecular dynamics simulations Wolfgang Sippl (Martin Luther University, Germany) 15.10-15.35 Molecular dynamics simulation of thrombin: Target for anticoagulant drugs Özge Kül (Hacettepe University, Türkiye) 15.35-16.00 Molecular dynamics simulatıon of hirudins: Specific thrombin inhibitor isolated from medicinal leech Fulya Çağlar, (Hacettepe University, Türkiye)

Hall B14.30-16.00 SESSION IV (Current Approaches for Development of Novel Drug Delivery Systems)Chairpersons: Alexander V. Kabanov (University of Nebraska, USA) Nevin Çelebi (Gazi University, Türkiye)14.30-15.10 Surface-modified nanoparticles for drug entrapment

Ruxandra Gref (University of Paris-Sud, France)15.10-15.35 Dendrimers as drug delivery agents to bone Rana Sanyal (Boğaziçi University, Türkiye)15.35-16.00 Recent advances in brain drug delivery Yılmaz Çapan (Hacettepe University, Türkiye)


Anamed Hall Presentations will be in Turkish.16.00-16.30 Coffee Break SessionChairperson: Ayhan S. Demir (Middle East Technical University, Türkiye) See the Future:”Microwave Synthesis from µL’s to L’s” Nejla Kılıç (ANAMED & ANALİTİK Grup, Türkiye)

Hall A16.30-18.15 SESSION V Simultaneous Turkish translation will be provided (Design and Synthesis of New Anticancer Drugs)Chairpersons: Fethi Şahin (Gazi University, Türkiye) Vildan Adar (Hacettepe University, Türkiye)16.30-16.55 Rational design of novel photosensitizers as potential photodynamic therapy reagents Engin Umut Akkaya (Middle East Technical University, Türkiye)16.55-17.20 A novel potential antitumor active drug: Platinum blue complex containing sulfur-donor ligand Şeniz Özalp-Yaman (Atılım University, Türkiye) 17.20-17.45 Synthesis of acetophenone and substituted acetophenone derived Mannich bases and their

biological activities H. İnci Gül (Atatürk University, Türkiye)

xiiiKervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE


MAY 19, 2007 - SATURDAY

Hall A09.00-10.30 SESSION VI Simultaneous translation will be provided (Experiences of Clinicians: New Horizons and Expectations From Research &

Development in Antimicrobial Agents)Chairpersons: Angelo Parini (Rangueil Institute of Molecular Medicine, France) Ahmet Başaran (Hacettepe University, Türkiye)09.00-09.30 Challenges in treatment of gram positive bacteria

Serhat Ünal (Hacettepe University, Türkiye)09.30-10.00 Challenges in treatment of gram negative bacteria

Recep Öztürk (İstanbul University, Türkiye)10.00-10.30 Challenges in antifungal agents

Ömrüm Uzun (Hacettepe University, Türkiye)


Info Kimya Hall Presentations will be in Turkish.10.30-11.00 Coffee Break Session Chairperson: Filiz Öner (Hacettepe University, Türkiye) Technological Applications in Pharmaceutical QA/QC and R&D Oğuzhan Ay (Info Kimya Laboratuar Cihazları Tic. Ltd. Şti, Türkiye)

Hall A11.00-12.10 SESSION VII Simultaneous translation will be provided (Patenting in Health and Pharmaceutics)Chairperson: Levent Öner (Hacettepe University, Türkiye)11.00-11.40 National and international patent protection, free patent search tools (espacenet) and strategies

Hakan Bayram (Turkish Patent Institute, Türkiye)11.40-12.10 Patentability of health and pharmaceutical inventions

Serkan Özkan (Turkish Patent Institute, Türkiye)

12.10-13.30 LUNCH

Poster Hall13.30-14.30 Poster Session

Industry Hall All presentations will be in Turkish. (Mission and Vision of the Pharmaceutical Companies)Chairperson: H. İnci Gül (Atatürk University, Türkiye)13.30-14.00 Novartis (Türkiye)14.00-14.30 Abdi İbrahim İlaç Sanayii ve Ticaret A.Ş. (Türkiye)

Satellite Hall All presentations will be in Turkish.Chairperson: Yılmaz Çapan (Hacettepe University, Türkiye)13.30-14.00 New Horizons in Drug Industry: Technological Platforms

Ahu Yücesoy (The Scientific & Technological Research Council of Türkiye-TÜBİTAK)14.00-14.30 Bioavailability and Bioequivalency Studies in Türkiye

Tuncel Özden (Novagenics, Türkiye)

xiv May 17-20, 2007


Hall A14.30-16.25 SESSION VIII Simultaneous Turkish translation will be provided (Pharmacology and Molecular Pharmacology)Chairpersons: Mercedes Unzeta (Universitat Autonoma de Barcelona, Spain) İnci Erdemli (Hacettepe University, Türkiye)14.30-15.10 Importance of polymorphism in drug research and development M. Fethi Şahin (Gazi University, Türkiye)15.10-15.35 Studies on histone deacetylase inhibitory activity of some carboxylic acid derivatives and their

structure-activity relationships Gamze Bora (Hacettepe University, Türkiye)15.35-16.00 Free-radical scavenger activities of newly synthesized 2-benzoxazolinone derivatives containing

thiosemicarbazide, triazole, thiadiazole and hydrazone Samiye Yabanoğlu (Hacettepe University, Türkiye)16.00-16.25 UVB-Induced apoptotic effect of 11 P53 actinic keratosis mutations Ayşe Ercan (Hacettepe University, Türkiye)

Hall B14.30-16.00 SESSION IX (Natural Compounds as Drug Leads)Chairpersons: Engin Umut Akkaya (Middle East Technical University, Türkiye) Bekir Salih (Hacettepe University, Türkiye)14.30-15.10 A vast source for the discovery of novel drug leads: Traditional medicines

Erdem Yeşilada (Yeditepe University, Türkiye)15.10-15.50 Parthenolide analogs as antileukemic agents with clinical potential

Peter Crooks (University of Kentucky, USA)15.50-16.30 Synthesis and biological evaluation of novel camptothecin class topo-isomerase inhibitors

Ayhan S. Demir (Middle East Technical University, Türkiye)


Atomika Hall Presentations will be in Turkish.16.30-17.00 Coffee Break SessionChairperson: Füsun Acartürk (Gazi University, Türkiye) Particle Characterization in the Pharmaceutical Industry: New Developments and Techniques for

Size, Shape and Chemical Analysis in Laboratory and Process Environments Stuart Wakefield ( Malvern Instruments Ltd. England) Naci Saraçoğlu (Atomika Teknik Ltd., Türkiye)

Hall A17.00-18.30 PANEL Simultaneous English translation will be provided. (Expectations of Pharmaceutical Industry from Drug Research & Development Studies) Panelists: Altan Demirdere (Novartis, Türkiye) (Moderator)

Erdal Akalın (Pfizer, Türkiye) Vedat Eğilmez (Abdi İbrahim İlaç Sanayi ve Ticaret A.Ş., Türkiye)

20.00-24.00 GALA DINNER

xvKervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE


MAY 20, 2007 - SUNDAY

Hall A10.10-10.10 SESSION (Nanomedicine and Nanomaterials)Chairpersons: Ruxandra Gref (University of Paris-Sud, France) Sema Çalış (Hacettepe University, Türkiye)09.30-10.20 Nanomaterials in medicine

Erhan Pişkin (Hacettepe University, Türkiye)10.20-11.00 Nanomedicine for central nervous system (CNS) drug delivery Alexander V. Kabanov (University of Nebraska, USA)

11.00-11.20 CLOSING REMARKS

MAY 18, 2007 - FRIDAY

TÜFTAD SATELLITE SYMPOSIUMorganized by

Turkish Pharmaceutical Technology Scientists’ Association (TÜFTAD)

Projects and Support Sources in the Development of Active Pharmaceutical Ingredients and Drug Formulations in University and Industry Cooperation

TÜFTAD Hall All presentations will be in Turkish. Simultaneous English translation will not be provided.

30.30-30.30 İlaç Etkin Maddesi ve Formulasyon Geliştirmede Üniversite Sanayi İşbirliği Projeleri ve Destek Kaynakları

TÜBİTAK Sanayi AR-GE Projeleri DestekleriDr. Bülent İçgen (Türkiye Bilimsel ve Teknolojik Araştırma Kurumu -TEYDEB)

Eczacıbaşı Özgün Kimya’da Faydalanılan AR-GE TeşvikleriDr. Mustafa Adıyaman (Eczacıbaşı Özgün Kimya)

Sanayide AR-GE’nin RolüDoç.Dr. Tuncer Aslan (Ulkar İlaç Sanayi ve Tic. A.Ş.)

TÜBİTAK Sanayi AR-GE Destekleri ve Etkilerinin DeğerlendirilmesiUzm. Ecz. Ece Kut (Abdi İbrahim İlaç Sanayi)

TÜBİTAK-TEYDEB Projelerinde Hakemlik ve İzleyicilik DeneyimleriProf. Dr. A.Atilla Hıncal (H.Ü. Eczacılık Fakültesi)Prof. Dr. Nilüfer Tarımcı (A.Ü. Eczacılık Fakültesi)


Dear Participants,

You can access to the “DRD 2007 Symposium Abstract e-Book” via internet using the following address:

http://www.magum.hacettepe.edu.tr/drd/drd2007.htm

DRD 2007

Organizing Committee

The authors are responsible for their presentations published in this Abstract Book.

Lectures

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Kervansaray Convention Center & Hotel, LARA, ANTALYA / TÜRKİYE


In recent years there has been a significant progress in the field of cancer cell biology and molecular oncology. Many researchers outlined very impor-

tant processes of cancer genesis, growth, invasion and metastasis. As a result of this progress a large number of molecular abnormalities that are “specific” to can-cer cells and that are a critical feature of cancer phe-notype have been discovered. These advances in the field of moleculer oncology over the past decade have led to a new era in cancer therapeutics and several strategies directed to selective molecular targets. This new class of anticancer agents has been named “tar-geted therapies”, because these structures target spe-cific cellular molecular and/or abnormalities.

Tumour cells acquire the ability to proliferate uncon-trollably, resist apoptosis, sustain angiogenesis and evade immune surveillance. Different from conventi-onal chemotherapy agents, which mainly kill prolife-rating cells by interacting with general cellular pro-cesses, “targeted agents” are expected to affect only cells in which the specific molecular alteration is pre-sent, induce predominantly antiproliferative effects, and be specific for cancer cells versus normal tissu-es. In this context, there have been very significant progress in the anti-cancer “targeted molecules” inc-luding tyrosine kinase inhibitors, angiogenesis inhi-bitors, modulators of cell matrix interactions, agents that interact with the cell cycle and cell death (apop-tosis) and protein trafficking regulators.

Several of these new therapeutic agents are showing promise in the clinic and many more are being de-veloped. The RAS proteins control signalling path-ways that are key regulators of several aspects of nor-mal cell growth and malignant transformation. They are aberrant in most human tumours due to activat-ing mutations in the RAS genes themselves or to al-terations in upstream or downstream signalling components. Rational therapies that target the RAS pathways might inhibit tumour growth, survival and

spread. The p53 is an attractive therapeutic target in oncology because its tumour-suppressor activity can be stimulated to eradicate tumour cells. Inhibiting the p53–MDM2 interaction is a promising approach for activating p53, because this association is well charac-terized at the structural and biological levels. MDM2 inhibits p53 transcriptional activity, favours its nucle-ar export and stimulates its degradation, so inhibiting the p53–MDM2 interaction with synthetic molecules should lead to p53-mediated cell-cycle arrest or apop-tosis in p53-positive stressed cells.

Angiogenesis inhibitors are a new class of drugs, for which the general rules involving conventional chem-otherapy might not apply. The successful translation of angiogenesis inhibitors to clinical application de-pends partly on the transfer of expertise from scien-tists who are familiar with the biology of angiogenesis to clinicians.

STAT proteins — especially STAT3 and STAT5 — reg-ulate all of these processes and are persistently activat-ed in a surprisingly large number of human cancers. Consequently, STAT proteins are emerging — unex-pectedly — as ideal targets for cancer therapy.

Monoclonal antibodies have become the most rapidly expanding class of pharmaceuticals for treating a wide variety of human diseases, in-cluding cancer. Six antibodies including Trastu-zumab (Herceptin), Rituximab (Mabthera), Ale-mtuzumab (Campath), Cetuximab, Gefinitib and Bevacizumab are now approved for cancer therapy. Coupled antibodies to toxins or radionuclides is the most widely investigated means for increasing their antitumour activity. Two anti-CD20 radioimmu-noconjugates, Bexxar (tositumomab; 131iodine) and Zevalin (ibritumomab tituxetan; 90yttrium), and My-lotarg (anti-CD33-calicheamicin conjugate) are ap-proved for cancer therapy and currently in clinical practice.

SMART MOLECULES IN TARGETED THERAPIES

Emin KANSUHacettepe University, Institute of Oncology, Ankara, Turkey

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Parkinson´s disease is a neurodegenerative dis-order characterized by the progressive loss of the dopaminergic neurons in the nigrostriatal

system. Different factors have been suggested to in-duce Parkinsonism: environmental toxins, alteration of the intracellular calcium homeostasis, mitochon-drial dysfunction, genetic factors, oxidative stress etc., [1]. All them are able by themselves to induce apop-totic cell death [2].

Oxidative stress, arising from an imbalance between the production of reactive oxygen-containing free-radical species (ROS) and antioxidant protective mechanisms, has been shown to induce apoptosis in dopaminergic cells. Dopaminergic neurones in the substantia nigra (SN) are particularly vulnerable to oxidative stress because of relatively low antioxidant levels. Dopamine itself can be readily autooxidised at physiological pH, with generation of ROS and, fur-thermore, it is metabolised by monoamine oxidase in a reaction that forms H2O2. Furthermore, elevat-ed levels of MAO-B activity have been reported in the SN of patients with Parkinson´s disease [3]. Thus, the increase of the oxidation of dopamine by MAO-B, might generate sufficient ROS to trigger the death of nigrostriatal neurones [4].

Besides the symptomatic therapeutically treatments based on L-Dopa administration, a precursor of do-pamine, at present there is a great interest on the use of neuroprotective agents such as the antiapoptotic factors [5]. Apoptosis has been reported to be present in post-mortem human brain from Parkinson´s pa-tients [6, 7]. Apoptosis (Programmed cell death), is a highly regulated physiological process that oc-curs in all vertebrates and controls the cellular turn-over from fetal development to aging [8]. Apoptosis is morphologically characterized by chromatin con-densation, DNA fragmentation, cell shrinkage and plasma membrane blebing. The apoptotic pathway is induced by a cascade of events in which a family of cysteine proteases named caspases, leads to the cleav-age and activation of different cellular substrates.The apoptotic death is under genetic control and is char-acterized by the expression of some genes that en-hance the apoptosis (bax, bad ) and others that in-

hibits it (bcl-2, bcl-xL, bcL-w, mcl-1). Defects in the physiological apoptotic pathway, leading to inappro-priate cell death underlies in some neurodegenerative diseases.

The programmed cell death may contribute to human neurodegeneration. It has been reported that apopto-sis is involved in the death of the dopaminergic neu-rons in Parkinson´s disease [6, 7, 9].

In humans, MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tet-rahydropyridine) produces a behavioural syndrome similar to the neuropathological features of idiopathic Parkinson´s disease [10] and involves a selective de-struction of nigrostriatal dopaminergic neurons. Al-though MPTP itself displays low chemical reactivity, it is biotransformed by glial MAO-B, to form MPP+ (1-methyl-4-phenylpyridinium) as the effective tox-in [11]. MPP+ is able to inhibit the Complex-I of the respiratory chain, inducing the free radical formation, ATP depletion, and cellular death [12].

The programmed cell death may contribute to the hu-man neurodegeneration probably due to the free rad-icals formation as a consequence of the mitochon-drial dysfunction [13]. The mitochondrial process implicated in the toxic and neurodegenerative condi-tions is related with the opening of the mitochondrial pore in the inner membrane, that leads to the mem-brane depolarization, release of small proteins such as Cytochrome c, apoptosome complex formation, acti-vation of the executer caspase 3, poly (ADP-ribose) polymerase (PARP) activation, DNA damage and cell death.

Recent studies have demonstrated that ER stress in-duced by protein missfolding as a consequence of the inhibition of protein glycosylation, trafficking or al-teration of the Ca+ homeostasis in the endoplasmic reticulum (ER), may contribute to the pathogenesis and neurodegeneration in Parkinson and Alzheim-er diseases. It has been reported that ER stress can in-duce apoptotic cell death in mice trough another mo-lecular pathway different to mitochondrial one that involves the activation of an ER-specific Caspase 12 [14]. Furthermore, pharmacological agents able to in-

PROPARGYLAMINES AS NEUROPROTECTIVE AGENTS IN NEURODEGENERATIVE DISEASES

Elisenda SANZ, José Luis MARCO, Mercedes UNZETADepartament Bioquimica y Biologia Molecular, Facultad de Medicina-Instituto de Neurociencias, Universitat Autonoma de Barcelona, 8193 Barcelona, Spain

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hibit the perturbation of the mitochondria and ER function may represent a potential therapeutically approach for the prevention of neurotoxin-induced Parkinson´s disease.

In this context, the design and synthesis of new an-tiapoptotic molecules, able to interfere with different apoptotic molecular pathways, inhibiting the open-ing of the mitochondrial pore, preserving the mem-brane potential and/or inducing the gene expression of some antiapoptotic proteins, is expected to protect neurons from the cell death and to slow progression of chronic neurodegenerative diseases [1, 15-17].

It has been designed and synthesised, a novel series of acetylenic and allenic tryptamine derivatives as po-tential MAO inhibitors [18]. Among them there is a new non-amphetamine molecule, the PF9601N [N-(2-propynyl)-2-(5-benzyloxy-indolyl) ethylamine, with antioxidant properties [19], that resulted to be a MAO B inhibitor, more potent and selective than se-legyline (l-deprenyl), widely used as coadjuvant of l-DOPA in PD therapy [20]. Moreover, PF 9601N did not show the ‘amphetaminergic’ side-effects of l-de-prenyl [21] and was able to inhibit the dopamine up-take in human and rat striatum. PF 9601 N showed a neuroprotective effect in vivo, using several dopamin-ergic toxins in different experimental models such as MPTP, [22] and 6-hydroxydopamine-striatal lesion [23]. Furthermore, PF 9601N also enhanced the du-ration of l-DOPA-induced contralateral turning in 6-hydroxydopamine lesioned rats [24].

A structurally different non-amphetamine com-pound, Rasagiline, has also been reported to be neu-roprotective [25], but this compound also differs from l-deprenyl in not being an inhibitor of presynaptic dopamine uptake [26]. In that respect, it differs from PF9601N and thus comparison of its behaviour with that of Rasagiline should reveal whether this trans-port inhibition is an important factor in the neuro-protective spectrum of activities.

The present study was performed to determine the molecular mechanism involved in the neuroprotective effect of PF 9601N observed in vivo. In this context it has been studied the cytoprotective and antiapop-totic effect of PF 9601N using human neuroblastoma SHSY5Y cells lesioned with MPP+ as a toxin that in-duces the apoptotic mitochondrial pathway, and le-sioned as well with Brefeldin A, Tunicamycin and Thapsigargin, as toxins inducers of the ER stress ap-optotic pathway.

The neuroprotective properties of PF9601N in these different apoptotic cell death models, were assessed using several viability assays (Alamar Blue reduc-tion and Calcein-AM staining). PF9601N pretreat-

ment significantly reduced MPP+ induced cell death and diminished the activation of one of the main ex-ecutioner caspases, Caspase-3 and also inhibited the PARP activation. PF9601N showed the same neu-roprotective behaviour when cells were treated with ER-stress toxins. Further effects of PF9601N in the maintenance of mitochondrial membrane potential or changes in the expression of Bcl-2 family proteins were also examined by RPA analysis.

MPP+ treatment also induced a prominent increase in p53 expression, nuclear translocation of this tran-scription factor and transactivation of p53 response elements. Additionally, p53 inhibitor pifithrin-alpha partially prevented MPP+-induced apoptosis, suggest-ing that activation of p53 contributes to cell death.

PF9601N pre-treatment was able to partially avoid MPP+ induced cell death through preventing an in-creasing p53 expression and thus reducing transcrip-tional activity of p53. PF9601N was also able to show the same degree of protection that pifithrin-alpha supporting that PF9601N would act inhibiting p53 pathway activation as well as Caspase 2 activation.

These results allow us to conclude that the pharma-cological target of PF9601N is the p53 molecular pathway. In this context, this new non-amphetamine MAO B inhibitor, could have a therapeutical potential use for those diseases where neurodegenation is de-pendent upon the p53 induced apoptosis. PF 9601N is a good candidate to be used in the therapy of the neurodegenerative diseases.

References 1. Olanow CW, Tatton WG. Etiology and pathogenesis of

Parkinson´s disease, Ann Rev Neurosci., 22, 123-144, 1999.

2. Simonian NA, Coyle JT. Oxidative stress in neurodegen-erative diseases, Annu Rev Pharmacol Toxicol, 36, 83-106, 1996.

3. Riederer P, Jellinger K. Neurochemical insights into monoamine oxidase inhibitors, with special reference to deprenyl (selegiline), Acta Neurol Scand Suppl , 95, 43-55, 1983.

4. Cohen G. Monoamine oxidase and oxidative stress at dopaminergic synapses, J Neural Transm 32 (Suppl), 229-38, 1990.

5. Kragten E, Lalande I, Zimmermann K, Roggo S, Schin-dler P, Múller D, Oostrum J van, Waldemeir P, Fürst P. Glyceraldehide-3-phosphate deshydrogenase, the puta-tive target of the antiapoptotic compounds CGP3466 and l-deprenyl, J Biol Chem., 273, 5821-5828, 1998.

6. Hartmann A, Hirsch EC. Parkinson’s disease. The apop-tosis hipótesis revisited, Adv Neurol., 86, 143-153, 2001.

7. Mochizuki H, Goto K, Mori H, Mizuno Y. Histochemi-cal detection of apoptosis in Parkinson´s disease, J Neurol Sci., 137, 120-123, 1996.

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8. Oppenheim RW. Cell death during development of the nervous system, Ann Rev Neurosci., 14, 453-501, 1991.

9. Tatton NA, Maclean-Fraser A, Tatton WG, Perl DP, Olanow CW. (1998), A fluorescent double-labeling meth-od to detect and confirm apoptotic nuclei in Parkinson´s disease, Ann Neurol., 44, S142-S148.

10. Langston JW, Ballard PA, Tetrud JW, Irwin I. Chronic parkinsonism in humans due to a product of meperidine analog synthesis, Science, 219, 979-980, 1983.

11. Chiba K, Trevor A, Castagnoli N. Metabolism of the neurotoxic tertiary amine MPTP, by brain monoamine oxidase, J Biochem Biophys Res Commun., 120, 574-578, 1984.

12. Sanchez-Ramos J, Hefti F, Hollinden DE, Jasik T, Rosenthal M. (1988) Mechanisms of neurotoxicity oxygen radicals and mitochondrial inhibition hypothesis in Progress in Parkinson´s Research (Hefti F, eds), Plenum Press New York pp 145-152.

13. Beal MF. Mitochondria, free radicals and neurodegenera-tion, Curr Opin Neurobiol., 6, 661-666, 1996.

14. Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, Yuan J. Caspase-12 mediates endoplasmic-reticu-lum-specific apoptosis and cytotoxicity by amyloid-beta, Nature, 403, 98-103, 2000.

15. Djaldetti R, Melamed E. New drugs in the future treat-ment of Parkinson´s disease, J Neurol., 249 (Suppl), II30-II35, 2002.

16. Wellington CL, Hayden MR. Caspases and neurodegen-eration: on the cutting edge of new therapeutic approach-es, Clin Genet., 57, 1-10, 2000.

17. Tajrena A, Cesari V, Borlongan RLM, Faull CEW, Ros GC, Gluckman PD, Hughes PE. Neuroprotective strate-gies for basal ganglia degeneration: Parkinson´s and Huntington¨s diseases, Prog Neurobiol., 60:5, 409-470, 2000.

18. Cruces MA, Elorriaga C, Fernandez-Alvarez E. Acety-lenic and allenic derivatives of 2-(5-methoxyindolyl) and 2-(5-hidroyindolyl) methylamines: synthesis and invitro evaluation as monoamine oxidase inhibitors, Eur Med Chem., 26, 33-41, 1991.

19. Sanz E, Romrea M, Bellik L, Marco JI, Unzeta M. Indo-lalkylamines derivatives as antioxidant and neuropro-tective agents,in a experimental model of Parkinson´s disease, Med Sci Monit., 10, BR477-484, 2004.

20. Perez V, Marco JL, Fernandez-Alvarez E, Unzeta M. Rel-evance of benzyloxy group in 2-indolyl methylamines in the selective MAO-B inhibition, Br J Pharmacol., 127, 869-76, 1999.

21. Lees AJ. Parkinson´s disease research group of the United Kingdom. Comparison of therapeutic effects and mor-tality data of levpdopa and levodopa combined with l-deprenyl in patienst with early, mild Parkinson´s disease, Br Med J., 311, 1602-1607, 1995.

22. Perez V, Unzeta M. PF 9601N [N-(2-propynyl)-2-(5-ben-zyloxy-indolyl) methylamine] a new MAO- B inhibitor, attenuates MPTP-induced depletion of striatal dopamine levels in C57/Bl 6 mice, Neurochem Int., 42, 221-229, 2003.

23. Cutillas B, Ambrosio S, Unzeta M. Neuroprotective effect of the monoamine oxidase inhibitor PF9601N [N-(2-propynyl)-2-(5-benzyloxy-yndolyl) methylamine ] on rat nigral neurons after 6-hydroxydopamine-striatal lesion, Neuroscience Lett., 329, 165-168, 2002.

24. Prat G, Perez V, Casas ARM, Unzeta M. The novel type B-MAO inhibitor PF 9601N enhances the duration of L-DOPA-induced contralateral turning in 6-hydroxy-dopamine lesioned rats, J Neural Transm., 107, 409-417, 2000.

25. Sterling J, Veinberg A, Lerner D, Goldenberg W, Levy R, Youdim M, Finberg JP. (R)(+)-N-propargyl-1-ami-noindan (rasagiline) and derivatives: highly selective and potent inhibitors of monoamine oxidase B, J Neural Transm (Suppl), 52, 301-305, 1998.

26. Lamensdorf I, Porat S, Simantov R, Finberg JP. Effect of low-dose treatment with selegiline on dopamine trans-porter (DAT) expression and amphetamine-induced dopamine release in vivo, Br J Pharmacol., 126, 997-1002, 1999.

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OXIDATIVE STRESS AND MONOAMINE OXIDASES: FROM BASIC STUDIES TO NOVEL THERAPEUTICAL INTERVENTIONS

Angelo PARINIINSERM U858, Institut de Médecine Moléculaire de Rangueil – I2MR, Toulouse, France

Biogenic amines, including catecholamine and serotonin, regulate a variety of cell functions through the interaction of G-coupled mem-

brane receptors. During the last years, we described a novel mechanism of action of dopamine and serot-onin that occurs independently of membrane recep-tor stimulation and requires hydrogen peroxide gen-eration by monoamine oxidases (MAO).

Using different models of renal and cardiac cells we showed that, in addition to the classical receptor-de-pendent effects, dopamine and serotonin induces cell proliferation and hypertrophy by a mechanism inde-pendent of receptor activation. At higher concentra-tions (up to 10 µM), dopamine and serotonin cause cell apoptosis by sequential i) increase in the ratio of Bax/Bcl2 proteins, ii) mitochondrial cytochrome c release, iii) caspase 3 activation and iv) DNA frag-mentation. Both proliferative and apoptotic effects of dopamine and serotonin were not inhibited by specif-ic receptor antagonists but were prevented by amine transporter inhibitors, the irreversible MAO inhibitor

pargyline and the antioxidant N-acetylcysteine. These data show that dopamine and serotonin induces cell proliferation, hypertrophy and apoptosis by a recep-tor-independent mechanism requiring amine inter-nalisation into the cells, their degradation by MAOs and hydrogen peroxide production.

Based on these findings, we next investigated the po-tential role of hydrogen peroxide generated by MAOs on cell death in vivo. Our results showed that MAO inhibition largely reduced renal and myocardial dam-age induced by ischemia/reperfusion in rat. The pro-tective effects of MAOs inhibitors were associated with the prevention of post-ischemic oxidative stress, ceramide accumulation, neutrophil infiltration and mitochondrial-dependent cell death.

In conclusion, our results show the key role of H2O2 produced by MAOs in mediating cell effects of bio-genic amines and propose these enzymes as a phar-macological target for prevention of organ damag-es.

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CHARACTERIZATION OF GLYCOPROTEINS BY CAPILLARY ELECTROPHORESIS ELECTROSPRAY MASS SPECTROMETRY (CE-ES-MS). APPLICATIONS TO DIAGNOSIS IN BIOMEDICINE

J. BARBOSA, V. SANZ-NEBOT, F. BENAVENTE, E. GIMÉNEZDepartament de Química Analítica, Facultat de Química, C/Martí i Franquès 1, 08028 Barcelona, Spain

Genomics and, even more important, the wide field of proteomic and related clinical appli-cations like biomarkers, dramatically increase

the demand of sensitive and selective analytical tools for the analysis of biological samples. The sugar con-tent of proteins has been demostrated to be critical for its biological activity, and it is influenced during its manufacturing process by the cell line and the in-cubation culture conditions [1]. The polymorphism associated with the amount, the size, and the struc-ture of the carbohydrate chains is known as micro-heterogeneity, and the molecular species generated are termed glycoforms. In this context, investigations of glycoproteins has become increasing important, in particular with respect to the variations in glycosyla-tion patterns observed in sera from healthy individu-als and patiens.

Glycosylation is the most common posttranslation-al modification in proteins carbohydrates partici-pate in many biological processes and encode infor-mation for molecular recognition, protein folding, stability and pharmacokinetics [2]. The number and type of glycoforms for a certain glycoprotein may change as a consequence of pathological process-es [3]. For example, patients with Congenital Dis-orders of Glycosylation (CDG) or chronic alcohol-ism present hypoglycosylation of several plasmatic glycoproteins as transferrin (Tf), analysis of which is used as a model glycoprotein for CDG diagnosis [4]. Tf is one of the twenty high abundance human plasma proteins. Other different analytical problem is analysis of erythropoietin which is found as very low abundance protein. Erythropoietin (EPO) is a glycoprotein hormone, which regulates erythropoi-esis and has been used extensively for the treatment of several anemias associated with acute and chronic diseases [5]. Despite the many benefits of EPO in the clinic, they have been most widely publicized on ac-count of their extensive misuse as performance en-hancing agents in endurance sports [6].

Until recently, isoelectric focuosing electrophore-sis was the reference method for glycoforms analy-sis due to its high selectivity allowing an easy detec-tion of genetic glycoprotein variants. However, this

complex and time consuming procedure favoured the introduction of new alternative methods. Cap-illary electrophoresis (CE), has become one of the most important techniques for glycoform separa-tion, combining high resolution power under non-denaturing conditions [7]. In previous studies Tf sialoforms were resolved by CE-UV (4). Also the two commercial ready-to-use pharmaceuticals of re-combinant human EPO (rHuEPO), epoetin-α and epoetin-β, and the hyperglicosilated EPO derivated NESP were analysed [8, 9]. Separation and charac-terization of the different glycoforms from each glyc-oprotein are presented. However endogenous EPO are generally found at subnanomolar levels such as other endogenous hormones and the poor concen-tration detection sensitivity of CE preclude the di-rect analysis of these hormones at the levels found in biological fluids.

The high values of the concentration limits of de-tection in CE are closely related with the small vol-ume capacity of the capillary columns. Several in-strumental, electrophoretic and chromatographic modifications have been described in order to over-come this limitation. In solid phase extraction cou-pled on-line to capillary electrophoresis (SPE-CE), a microcartridge or analyte concentrator is insert-ed near the inlet of the separation capillary [10]. The analyte concentrator contains a solid phase extrac-tion sorbent which retains the target analyte, ena-bling large volumes of sample to be intruduced. The captured analyte is eluted in a small volume of an ap-propiate solution, resulting in sample clean-up and concentration enhancement, with minimum sam-ple handling. Several researchers have perceived the suitability of SPE-CE to perform selective and sensi-tive analysis of proteins and peptides in complex di-luted samples. In addition, the use of solid-phase ex-traction coupled on-line to capillary electrophoresis electrospray mass spectrometry (SPE-CE-ESI-MS) has demonstrated improved capabilities for charac-terization of compounds found at low concentration in complex matrix [10].

Mass spectrometry (MS) has emerged as a powerful tool for the analysis of large biomolecules. Howev-

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er, direct analysis of intact glycoproteins by MALDI-TOF and conventional electrospray ionization mass spectrometry (ESI-MS) has presented some problems in order to resolve microheterogenous structures. Thus, a previous glycoform separation is mandato-ry to obtain valuable information about the carbohy-drate heterogeneity of glycoproteins. CE-ESI-MS has been successfully used for this purpose [11-13]. How-ever, volatile background electrolytes (BGE) are nec-essary to provide suitable electrospray ionization and therefore, obtain good sensitivity. The non-volatile buffer used in the current CE-UV methods for gly-coform separation preclude the CE-ESI-MS coupling [14-16].

In our work, CE-UV methods for the separation of glycoforms in volatile BGE have been devel-oped. The CE-ESI-MS separation method for in-tact rHuEPO has been improved as a consequence of the use of a novel acrylamide-based coating that provides a stable suppression of EOF and al-lows a succesful glycoform separation [7, 17]. Also a method for the separations of Tf sialofor-ms has been developed, that permits the diagnostic of Congenital Disorders Glycosylation and chron-ic alcoholism. A stable negative modified capillary is performed by a first amino quaternary coating (Polybrene) attached to the capillary wall, followed by a second anionic coating (Dextran) obtaining a negative coated capillary [6, 7]. In order to improve de detection limits of CE-ESI-MS methodologies, the use of solid-phase extractions coupled on-line to capillary electrophoresis using electrospray mass spectrometry detection (SPE-CE-ESI-MS) is stud-ied for the analysis of peptide hormones in dilute solution [10]. This resulted in sample clean-up and concentration enhancement, with minimum sam-ple handling. The CE-ESI-MS developed methods are applied for the characterization of rHuEPO gly-coforms. The achieved separation and the highly mass-resolving time of fligh (TOF) mass detection allows to establish the most probable rHuEPO gly-coforms.

EXPERIMENTAL PARTChemicalsAll chemicals used in the preparation of buffers and solutions are analytical reagent grade. Standard hu-man transferrin (partially saturated, min 98%), insu-lin, sodium dextran sulphate (M=500000), and hex-adimethrin bromide (Ploybrene, PB, M=15000) are purchased from Sigma. Ultra TolDynamic PreCoat LN was provided by Target Discovery (Palo Alto, CA, USA). Trypsin Gold, Mass Spectrometry Grade, was obtained by Promega (Madison, WI, USA). Stand-ard rHuEPO was obtained as BRP from Pharmaco-poeia (EDQM, European Pharmacopoeia, Council of Europe, Strasbourg, France). Epoetin-α (Eprex) 6000

IU from Janssen-Cilag (Neuss, Germany) and epoe-tin- (NeoRecormon) 4000 IU from Roche (Man-nheim, Germany) were obtained as ready-to-use drugs. Deionised and organic-eliminated water was obtained using a Milli-Q water purification system (Millipore, Schwalbach, Germany). All solutions and background electrolytes were degassed by ultrasoni-cation before use.

InstrumentalCE-ESICE was performed on a Hewlett Packard CE (Agi-lent Technologies, Waldbronn, Germany). For CE-MS coupling, a coaxial sheath-liquid sprayer was used (Agilent Tecnologies). For intact EPO glycopro-tein analysis, separation was performed in capillar-ies coated with polybren (PB) or ultra Tof Dynam-ic Pre-Coat LN (LN). For Tf analysis the separation capillary is coated with a polybren-dextran (PB-DE) double layer couting.

MS Mass spectrometric Tf analysis are carried out in a Marimer TOF mass spectrometer (Perseptive Bio-systems, Framingham, MA, USA) coupled to CE sys-tems whereas the hormones spectrometric investiga-tions were performed with the CE system compled to a MSD Ion Trap mass spectrometer (Agilent Tech-nologies) and characterization of glycoprotein were performed using a CE-microTOF (Bruker Daltonik), an orthogonal accelerated TOF mass spectrometer (oaTOF-MS).

RESULTS AND DISCUSSIONTransferring glycoform analysis by CE-UV and CE-ESI-MS Application to CDG chronic alcoholism diagnosisA CE-UV separation method has been developed us-ing a MS compatible buffer with 25 mM NH4Ac at pH 8.5. Best separation conditions have been obtained in a coated capillary based on a Successive Multiple Ion-ic Layer (SMIL) performed by a first layer of PB and a second layer of dextran (DS). This coating provides a constant and positive EOF, and minimizes the inter-actions between Tf and capillary walls improving sep-aration reproducibility.

In order to deplete albumin and the most abundant immunoglobulins from sera, a commercial kit based on dyes and immunoaffinity capture has been used prior to electrophoretic analysis. Figure 1 shows the obtained electropherograms in two different sera, one from a healthy individual and the other from a CDG patient.

A clear difference on the electrophoretic profiles is observed. The CE-UV developed method is now ap-plied in clinical diagnosis.

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Using CE-ESI-MS methodologies the best sensitivi-ty is obtained with a sheath liquid containing 90% of methanol and 0.5 % formic acid. Other experimental parameters as sheath liquid flow rate, nebulizer gas and ionization potentials have been optimized in or-der to achieve good sensitivity and separation .Sepa-ration of different proteins present in serum has been achieved and mass spectra can be deconvoluted. A glycoform of 77387 Da is obtained in a serum from a CDG patient that is not observed for a control se-rum (Figure 2).

Analysis of hormones by on line SPE-CE-ESI-MSEndogenous hormones are generally found at sub-nanomolar levels, e.g. between 100 and 1 ng L de-pending on the hormone, in biological samples. In our studies, solid-phase extraction coupled on-line to capillary electrophoresis electrospray mass spectrom-etry (SPE-CE-ESI-MS) is explored for the preconcen-tration and separation of dilute solutions of peptide hormones. First, a CE-ESI-MS methodology was de-veloped and validated. Limits of detection (LOD) of around 1 g mL were obtained for all the studied hormones. For SPE-CE-ESI-MS experiments, a home-

Figure 1. CE-UV electropherograms obtained for different serum samples. a) Non-treated healthy, b) healthy serum and c) CDG serum both passed through the albumin depletion kit.

Figure 2. a) Total Ion Electropherogram obtained for a serum from a healthy individual in the CE-ESI-MS optimized conditions. b) and c) deconvoluted mass spectra obtained from the beginning and the end of the Tf peak respectively. d) Total Ion Electropherogram obtained for a serum from a CDG patient. e) and f ) deconvoluted mass spectra obtained from the two partial resolved glycoforms of Tf. The most probable glycan composition is displayed below the deconvoluted mass spectra.

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made SPE microcartridge containing a C18 sorbent was constructed near the inlet of the separation capil-lary. (Figure 3). After optimizing the on-line precon-centration methodology, LOD between 10 and 0.1 ng mL-1 were achieved. The preconcentration method-ologies have been applied to rHuEPO analysis using an in-line inmunoaffinity solid phase extraction(IA-CE-ESI-MS).The preliminary results obtained using a custom-made inmunoaffinity sorbent prepared from an anti-human EPO polyclonal antibody and glutar-aldehyde-glass beads show the potential of this nov-el approach.

Characterization of rHuEPO glycoforms by CE-ESI-MSSeveral recombinant human erythropoietins (rHuE-PO) from different origen have been analysed. Coat-ed capillaries are mandatory in order to decrease or suppress the adsorption of glycoproteins to the sil-ica capillary wall. In our works two different cap-illary coatings have been used: (1) polybrene (PB), an amino quaternary polymer with positive charges that reverses the EOF, and (2) UltraTolDynamic Pre-Coat Low Normal (LN), an acrylamide polymer that suppresses the EOF at low pH. The best sensi-tivity has been obtained in sheath liquids contain-ing 1% acetic acid and high resolution TOF-MS has been found to be the most suitable mass analyzer for detect intact glycoproteins differing in few Da. In CE-ESI-MS analyses of intact glycoproteins, nu-merous and complex data are obtained and there-fore, extracting useful and valuable information

from spectra is not as obvious as in more straight-forward samples. Figure 4 shows the separation and mass spectra obtained from rHuEPO in a LN coated capillary, summarizing the procedure performed in the data analysis.

In order to obtain more complete information about the carbohydrate moiety of glycoproteins, a study of the composition and structure of the glycan is nec-essary. In our work a CE-ESI-MS separation meth-od has been developed for the N-glycans obtained by PNGase F release from the glycoproteins Fig-

Figure 4. Schematic view for data processing of intact CE-ESI-MS glycoprotein analysis in a LN coated capillary, BGE: 2M acetic acid, separation voltage + 30kV. Sample: Pharmacopoeia rHuEPO 2.5 µg/µL injected for 15s, at 50 mbar. a) Base Peak Electropherogram (BPE) obtained, b) mass spectrum obtained in 25.0-25.2 min, c) deconvoluted mass spectrum, d) ion identification for 29888.2 Da glycoform, e) Extracted Ion Electropherogram (EIE) obtained for 29888.2 Da glycoform (in purple) and for some of the different sialoforms present in rHuEPO (in grey).

Figure 3. Scheme of the manufacturing procedure for the C18 analyte concentrator (C18AC).

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ure 5. Also the sialic acid content of O-glycosyla-tion site has been characterized based on the glyco-peptides obtained by trypsin digest and CE-ESI-MS analysis. Therefore, the probability of every intact sialoform has been calculated taking into account the number of glycosylation sites, and the percent-ages of the N-glycans and the O-glycans depend-ing on their sialic acid content. The data have been compared with the normalized areas obtained from the EIE of the intact sialoforms of Pharmacopoeia rHuEPO, and therefore the sialic acid assignment has been performed. Once sialic acid number has been assigned, a global carbohydrate composition is deduced in high confidence. Thus, a main molecu-lar mass of 29888.2 Da, consist of the protein back-bone (165 amino acids, 18235.8 Da), 22 hexoses, 19 N-acetylhexosamines, 3 fucoses and 13 sialic acids (Figure 6). Table 1 shows the main observed molec-ular masses and the respective carbohydrate com-position.

References 1. Skibeli, V, Nissen-Lie G, Torjesen P. Blood, 98, 3626-3634,

2001. 2. Mechref Y, Novotny V. Chem. Rev., 102, 321-369,

2002. 3. Balaguer E, Benavente F, NeusüB C, Sanz-Nebot V, Bar-

bosa J. Electhrophoresis, (2006) in press. 4. Sanz-Nebot V, González P, . Toro I, Ribes A, Barbosa J. J.

Chromatogr.B, 798, 1-7, 2003.

Table 1. Main intact rHuEPO glycoforms and the respective carbohydrate composition obtained in a LN coated capillary.

Figure 5. Typical tetra-antennary N-glycan of rHuEPO.

Figure 6. One possible glycan composition for an intact rHuEPO glycoform of 29888.2 Da containing 22 hexoses (), 19 N-acetylhexosamines (), 3 fucoses (◄) and 13 sialic acids ().

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5. Fisher JW. Exp. Biol. Med., 228, 1-14, 2003. 6. Sanz-Nebot V, Benavente F, Vallverdú A, Guzmán NA,

Barbosa J. Anal. Chem., 756, 5220-5229, 2003. 7. Balaguer E, Demelbauer U, Pelzing M, Sanz-Nebot V,

Barbosa J, NeusüB C. Electrophoresis, 27, 2638-2350, 2006.

8. Sanz-Nebot V, Benavente F, Giménez E, Barbosa J. Elec-trophoresis 26, 1451-1456, 2005.

9. Giménez E, Benavente F, Barbosa J, Sanz-Nebot V.(2006) in press.

10. Benavente F, Vescina MC, Hernández E, Sanz-Nebot V, Barbosa J, Guzman N. J. Chromatogr. A, 1140, 205-212, 2007.

11. Kelly JF, Locke SJ, Ramaley L, Thibault P. J. Chromatogr. A, 720, 409-427, 1996.

12. Demelbauer UM, Plematl A, Kremser L, Allmaier G, Josic D, Rizzi A. Electrophoresis 25, 2026-2032, 2001.

13. Neusüss C, Demelbauer U, Pelzing M. Electrophoresis, 26, 1442-1450, 2005.

14. Klampfl CW. Electrophoresis, 27, 3-34, 2006. 15. Stutz H. Electrophoresis, 26, 1254-1290, 2005. 16. Hernández-Borges J, NeusüB C, Cifuentes A, Pelzing M.

Electrophoresis, 25, 2257-2281, 2004. 17. Benavente F, Gimenéz E, Olivieri AC, Barbosa J, Sans-

Nebot V. Electrophoresis, 27, 4008-4015, 2006.

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BIOMARKER DISCOVERY FOR CHRONIC INFLAMMATORY DISEASES USING PROTEOMIC SERUM PROFILING

M. FILLET1, D. de SENY2, M-A. MEUWIS1, P. GEURTS4, L. WEHENKEL4, Ed. LOUIS3, M. MALAISE2, M-P. MERVILLE1

1Laboratory of Clinical Chemistry, 2Rheumatology, 3Gastroenterology, 4Department of Electrical Engineering & Computer Science, GIGA Research, University of Liège, and CHU, 4000 Liège, Belgium

The development of new technologies in the last decade in the field of genomic and proteom-ic analysis has brought novel optimism to the

discovery of new biomarkers. One of the novel strat-egies employed for the discovery of new biomarkers is the analysis of the peptides and proteins in plasma or serum samples by mass spectrometry. Protein dif-ferential display techniques such as two-dimensional gel electrophoresis (2-DE), one- or two dimensional liquid chromatographic approaches (LC-MS) or sur-face-enhanced laser desorption and ionization time of flight (SELDI-TOF) mass spectrometry have be-come increasingly useful to establish fingerprint pro-files of both disease and non-disease states from large number of samples.

These methods are based on the assumption that the pathology will affect the physiology of the organism and cause more or less severe changes in the expres-sion level of proteins. Proteins common to both groups are ignored when up- and downregulated proteins be-come of the main interest and potential biomarkers.

SELDI’s ProteinChip Arrays distinguish this tech-nology from other mass spectrometry-based analyt-ical systems. ProteinChip Arrays provide a variety of surface chemistries that allow researchers to opti-mize protein capture and analysis. The surface chem-istries of the arrays include a series of classic chroma-tographic chemistries and specialized affinity capture surfaces. Classic chromatographic surfaces include normal phase for generic protein binding; hydropho-bic surfaces for reversed-phase capture; cation- and anion-exchange surfaces; and immobilized metal af-finity capture (IMAC) for metal-binding proteins. Specific proteins of interest can be covalently immo-bilized on pre-activated surface arrays, enabling cus-tomized experiments to investigate antibody-antigen, DNA-protein, receptor-ligand, and other molecular interactions. Then, the unbound proteins are washed away, the chips are overlaid with an energy-absorbing matrix and finally spectra are acquired by using laser ionization and TOF separation mass spectrometry.

Blood is an ideal source of markers because it is eas-ily accessible and reflects secondary systemic chang-

es, as it perfuses all the tissues of the body, sothat it carries not only specific blood proteins but also pro-teins or fragments shed by diseased tissue. In order to analyse circulating proteins and peptides, the cellular components of blood are removed, either in the pres-ence of anticoagulants, which yields plasma, or after blood coagulation, which yields serum.

Analysis of plasma or serum is a challenge because of the huge dynamic range of protein abundance and forms. It is well known that the concentration of the proteins present in the blood covers at least 10 orders of magnitude, ranging from albumine (35-50mg/ml in serum) to IL6 (0-5 pg/ml in serum). Ninety-sev-en percent of the proteins found in plasma belong to one of the seven major groups of high-abundant plas-ma proteins: albumin, immunoglobulin, fibrinogen, alpha 1-antitrypsin, alpha 2-macroglobulin, transfer-ring and lipoproteins. The remaining 3% is a com-plex mixture of middle and low abundance proteins, including proteins from the family of complements, hormones, other proteins originated from normal tis-sue secretion or from tissue leakage upon cell death or damage. The dynamic range of protein mount that can be detected in a single mass spectrum (2-3 or-ders of magnitude) is insufficient to cover the range present in blood sample. To overcome this disadvan-tage, two non-exclusive strategies have been devel-oped. One consists of the selective depletion of the most abundant proteins in the plasma or the serum and the second one, on the fractionation of the sam-ple. The use of antibodies for the albumin, immu-noglobulins and other high-abundance proteins is at present the most efficient and specific method. The major drawback is the high cost. These resins are ro-bust, highly specific and yield to reproducible results. However, it is known that albumin and other high-abundance proteins bind some less abundant pep-tides in the blood and act as their carriers. Moreover, the depletion shows a certain degree of cross-reactiv-ity with non-targeted proteins.

In order to prevent the finding of artefactual biomar-kers, it is important to eliminate as many of variables. The all procedure has to be standardized from the me-dical history of the blood donor to the acquisition of

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the spectra. When preparing the plasma, the nature of the anticoagulant added to the blood and the protocol employed to remove the cellular components influen-ces the protein content of the sample. Similarly, when preparing serum, the material of the tube for blood collection (plastic or glass) and the conditions of the clotting affect the peak pattern.

This work describes how SELDI-TOF applied to blood samples may generate complex protein profile for diagnostic and prognostic evaluation and become a valuable tool to reliably predict arthritis and inflam-matory bowel diseases (IBD) outcome. The aim is to accurately distinguish patients with Rheumatoid Ar-thritis or Crohn disease from patients with other in-flammatory diseases and from healthy controls.

Chronic inflammatory diseases like Rheumatoid Ar-thritis (RA) and Crohn disease (CD) are autoimmune diseases of unknown etiology for which diagnosis are based on a pattern of clinical symptoms completed by unspecific biological tests for RA and endoscop-ic tissue histology for CD. Biomarkers for early di-agnosis or prognosis do not exist and clinicians of-ten manage the patients empirically and secondarily adapt the therapeutic strategy according to the clin-ical evolution.

The project requires the establishment of a biobank including serum, tissue samples and epidemiologi-cal data, from large cohort of patients afflicted with RA, CD and related diseases (Psoriatic Arthritis -PsA- and Ulcerative rectocolitis -UC-). The num-ber of samples and types of samples is one of the most important parameters that determine the suc-cess of a project. Usually, we profile at least 30 sam-ples in each classification group (e.g., disease versus healthy or treated versus untreated). This number of samples is usually enough to give us > 90% statisti-cal confidence in single marker with p values < .01 and is also enough to use different forms of multi-variate analysis.

Control sera were selected on the basis of matching for age, sex and Caucasian race. All samples were al-iquoted and frozen at -80°C until thawed specifical-ly for SELDI analysis. We paid a careful attention thoughout all our experiments to avoid as much as possible sources of variation in the procedure. For ex-ample, the sera freeshly collected have been aliquot-ed, stored at –80°C and were only ones unfreezed. Our quality control serum allowed us to detect any unusual features during the process. All those precau-tions allowed us to obtained a very good repetability and a good reproducibity between the results.

A quality control serum sample was taking from a healthy control. It was used to determine reproduc-

ibility and as a control protein profile for each SEL-DI experiment.

Several chip arrays such as strong anion-exchange (SAX), weak cation-exchange (CM10) and hydro-phobic (H4) were tested in order to determine which would provide the optimal profile for serum in terms of number and resolution of peaks. pH (from 4 to 9) and salt concentration (from 30 mM to 1M) in wash-ing buffers were optimized using ion-exchange ar-rays as well as the percentage of acetonitrile (ACN) (from 10 to 60 %) in H4. CM10 and H4 were finally observed to give the best results.

Mass accuracy was calibrated externally using peptide molecular mass standards in order to cover a larger range of mass (0 to 20 000 Da).

Several processing steps are required before data analysis such as baseline subtraction, normaliza-tion or peak detection. Baseline subtraction was achieved by employing a varying-width segment-ed convex hull algorithm that eliminates any base-line signal caused mostly by matrix distortions. Nor-malization was essential to eliminate any systematic effects between samples due to varying amounts of protein or degradation over time in the sample or variation in the instrument detector sensitivity. All data were normalized according to the total ion cur-rent normalization function by following the soft-ware instruction. Peak detection was performed us-ing the ProteinChip Biomarker software version 3.0 (Ciphergen Biosystems, Inc.). The part of the spec-trum m/z values < 1000 was not used for analysis, as the energy absorbing matrix signal generally in-terfered with peak detection in this area. Peaks be-tween 1000 and 20 000 m/z ratio were autodetected with a signal:noise ratio >3 and the peaks clustered using second-pass peak selection with signal:noise ratio >2 and a 0.3% mass window.

Standardization of all experimental conditions was drawn up to minimize the effects of irrelevant sourc-es of fluctuation and coefficient of variations (CVs) were calculated to evaluate the reproducibility of ex-periments using SELDI-TOF-MS approach.

Moreover, we chose to include patients with a vari-ety of very close pathologies to RA (osteoarthritis, PsA) in our control groups to mimic real life diagnos-tic difficulties. We also wanted to include heterogene-ous control groups, because we wanted to differenti-ate markers that are inflammatory in nature from RA specific molecules.

Data were analyzed with an original multivariate sta-tistical method based on multiple decision trees al-gorithms. One of the challenges in the analysis of

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SELDI mass spectrometry-generated data is avoid-ing the false discovery of proteins peaks, of which the discrimatory power is due to random variation. The statistical approach we used to analyse the SEL-DI profiles reduces this problem by ranking all of the detected protein peaks according to their relative con-tribution to the separation of distinct data sets and by using bootstrap cross-validation. As an additional safeguard against the identification of discriminating peaks that are merely artefacts, we analysed all of the samples in duplicate, and only the peaks that exhibit-ed a reproducibly high ranking in both sets of analy-sis were used. For most comparisons, bioinformatics analysis yielded a panel of 10 or more distinguished between patients groups. Once the optimum discrim-inatory decision process is created, blinded samples can then be subjected to analysis to confirm the utili-ty of the algorithm.

In a first approach, we compared RA spectra versus control spectra (inflammatory controls and non-in-flammatory controls). According to the boosting sta-tistical analysis a sensitivity superior to 75% and 85% was obtained on CM10 and H4 arrays respectively.

In the second approach, RA spectra were compared to PsA spectra. PsA is a chronic disease, whose clin-ical manifestations are very close to the ones of RA. Sensitivity reaches 81% on CM10 and 90% on H4 arrays. Correlation between discriminant values obtained by boosting and p-values was also per-formed.

Finally, the multivariate analysis based on multiple decision trees generated several models that could classify samples with good sensitivity and specificity (minimum 80%) discriminating inflammatory bowel diseases versus controls (inflammatory or healthy) or Crohn versus ulcerative colitis.

In conclusion we can say that Surface Enhanced La-ser Desorption Ionization-Time Of Flight-Mass Spec-trometry technology combined with the use of the multiple decision trees method, as robust statistical tool, led to the selection of protein biomarker patterns that may reveal to be helpful for diagnosis and under-standing of chronic inflammatory diseases.

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IN SILICO MEDICINAL CHEMISTRY – UNDERSTANDING BIOLOGICAL EFFECTS THROUGH MOLECULAR DOCKING AND MOLECULAR DYNAMICS SIMULATIONS

Wolfgang SIPPLInstitute of Pharmaceutical Chemistry, Martin-Luther-University of Halle-Wittenberg, 06120 Halle/Saale, Germany

Protein homology models have been used in conjunction with structure-based approach-es to successfully identify novel inhibitors over

the last few years. It is widely accepted that for exam-ple docking to homology models is more challenging and less successful than docking to X-ray structures of proteins. To successfully apply structure-based methods to homology models, in addition to accu-rate docking programs, high quality protein models are needed.

The present talk will highlight the results obtained for two protein targets where we used a combina-tion of comparative protein modelling and struc-ture-based methods in order to find novel leads. For both targets (the nuclear hormone receptor CAR1,2 and the histone modifying enzymes PRMT1 and SIRT2) we were able to identify new ligands based on a structure-based virtual screening. Besides the successful identification of PRMT13 and SIRT24 in-hibitors as well as CAR agonists the validated ho-mology models were used to explain the structural basis for yet not understood biological effects. Our findings suggest that high quality homology mod-

els can be used as structural basis for lead finding of yet not crystallized protein targets and are able to provide important information concerning their bi-ological effects.

References 1. Windshügel B, Jyrkkärinne J, Poso A, Honkakoski P, Sippl

W. Molecular dynamics simulations of the human CAR ligand binding domain: Deciphering the molecular basis for constitutive activity, J. Mol. Mod., 11, 69-79, 2005.

2. Jyrkkärinne J, Windshügel B Mäkinen J, Ylisirniö M, Peräkylä M, Poso A, Sippl W, Honkakoski P. Amino acids important for ligand specificity of the human constitu-tive androstane receptor, J. Biol. Chem., 280, 5960-5971, 2005.

3. Spannhoff A, Heinke R, Bauer I, Trojer P, Metzger E, Gust R, Schüle R, Brosch G, Sippl W, Jung M. Target-based approach to inhibitors of histone arginine methyl-transferases, J. Med. Chem., 2007, in press.

4. Trapp J, Jochum A, Meier R, Saunders L, Marshall B, Kunick C, Verdin E, Goekjian P, Sippl W, Jung M. Adenosine mimetics as inhibitors of NAD+-dependent histone deacetylases - from kinase to sirtuin inhibition, J. Med. Chem., 49, 7307-16, 2006.

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SURFACE-MODIFIED NANOPARTICLES FOR DRUG ENTRAPMENT AND TARGETING

Ruxandra GREFUMR CNRS 8612, Paris-Sud University, Châtenay-Malabry, France

Over the last decades, there has been a grow-ing interest in the development of a drug car-rier that is small enough for intravenous ad-

ministration and has the ability to bypass the normal physiological defense processes of the organism. This type of “stealth” carrier could therefore circulate for prolonged times in the bloodstream, opening in this way new therapeutic opportunities, such as control-led drug release into the vascular compartment, or targeted delivery.

There are numerous potential applications for such a system, for example, the protection of highly sen-sitive active molecules against in vivo degradations, the reduction of the toxic side effects that can occur when highly active drugs such as those used in cancer therapy are administered as a solution, the increase of patient comfort by avoiding repetitive injections or the use of perfusion pumps, and the achievement of more favorable drug pharmacokinetics. The success-ful administration of protein drugs is also often prob-lematic. Hydrolyzed or denaturated in the gastroin-testinal tract, most proteins cannot be administered orally, and usually, they are quickly metabolized if in-travenously injected. Their typical half-lives,ranging from 2 to 30 min, could be improved by encapsulat-ing them in long-circulating drug delivery devices acting as circulating microreservoirs.

Many efforts have concentrated in recent years on the design of submicronic long-circulating drug carri-ers such as liposomes, lipid emulsions, micelles, solid lipid nanoparticles, and nanoparticles. Among all of these systems, we will focus on nanoparticles, which were defined as solid colloidal particles ranging in size from 10 to 1000 nm, consisting of macromo-lecular materials in which the active principle is dis-solved, entrapped, and encapsulated and/or to which the drug is adsorbed or attached.

The strategy of choice in the design of submicron-ic long-circulating drug carriers was to sterically ex-clude the cellular immune system from recognizing and responding to the particles’ artificial surfaces. To achieve this, polymers were covalently bound or ad-sorbed on surfaces. Among these, poly (ethylene gly-

col) (PEG) were the most commonly used polymers for surface modification.

Core-shell (PEG) nanoparticles revealed to be of out-most interest; the biodegradable core is able to entrap and release a variety of active molecules, whereas the hydrophilic PEG surface governs the in vivo interac-tions and determines the fate after intravenous ad-ministration.

Examples of applications will be given, including the entrapment of proteins and lipophilic drugs. The ac-cent will be put on the challenge of busulfan entrap-ment. This small fragile molecule with a strong ten-dency to crystallize is indeed particularly difficult to be encapsulated.

Busulfan is a bifunctional alkylating agent, which is widely used at high dose as a part of myeloablative regimen before both allogenic and autologous bone marrow transplantation for the treatment of haema-tological malignancies and non-malignant disorders such as immunodeficiency. For a long time, busulfan has been available only in oral form and a wide in-tra-patient and inter-patient bioavailability variabili-ty in both adult and children has been reported [1]. Moreover, severe side effects were reported such as the veino-occlusive disease. This pathology has been correlated with a high systemic exposure to busulfan expressed as the area under the plasma concentra-tion-time curve.

In order to overcome these problems, intravenous formulations of busulfan were developed, using co-solvent mixtures [2]. However, these organic solvents have their own well-documented toxicity. Therefore, to avoid the massive use of organic solvents, injecta-ble colloidal carriers, such as conventional liposomes [3] have been elaborated. However, these carriers had encapsulation efficiencies lower than 1% (w/w).

We aimed at designing composite core-shell nanopar-ticles able to combine the ability of the biodegradable polymeric cores to efficiently encapsulate busulfan, with the excellent steric repulsive properties of the di-block copolymer, poly (�-caprolactone)-poly (ethyl-

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ene glycol) (PCL-PEG), which is expected to provide increased blood half lives to the nanoparticles [4].

In a first step, we have chosen the biodegradable poly-mer able to encapsulate the highest amounts of busul-fan. For this, busulfan was entrapment by nanoprecip-itation into polyesters and five different types of poly (alkyl cyanoacrylate) polymers. The polymers leading to the highest busulfan loading efficiencies were poly (isobutyl cyanoacrylate) (PIBCA) and poly (ethyl cy-anoacrylate) (PECA). Molecular modelling along with energy minimization process was employed to identify the nature of the interactions occurring be-tween busulfan and PIBCA. Further, optimization studies enabled to obtain PIBCA nanoparticles dis-playing busulfan loading ratios equal to 5.9 % (w/w) together with nanoparticle yields of 71 % (w/w). Since busulfan is a highly reactive molecule, we per-formed 1H-NMR spectroscopy experiments showing that chemical integrity of the drug was preserved after loading into nanoparticles. The in vitro release stud-ies under ‘sink’ conditions, in water or in rat plasma showed a fast release in the first minutes, followed by a slower one over 6 hours.

The composite busulfan-loaded core-shell nanopar-ticles were obtained by co-precipitation of mixtures of PIBCA and of PCL-PEG, in different mass ratios [5]. The nanoparticle size, morphology and surface charge were assessed. For example, electron micros-copy studies revealed a particular core-shell structure typical of the composite nanoparticles (Fig. 1).

The chemical composition of the top layers was deter-mined by X-ray photo-electron spectroscopy (XPS). 3H-labelled busulfan was used in order to determine the drug loading efficiency and the invitro drug re-lease by liquid scintillation counting. Physico-chem-ical techniques such as Zeta potential determination and XPS analysis provided evidence about a pref-

erential surface distribution of the PCL-PEG poly-mer. Therefore, composite nanoparticles have a ‘core-shell’-type structure, where the “core” is essentially formed by the PIBCA polymer and the “shell” by the PCL-PEG copolymer. The use of PIBCA to form the core of the nanoparticles leads to a 2-4 fold drug load-ing increase, in comparison to the single PCL-PEG nanoparticles. In addition, the complement activa-tion results showed a significant difference between the composite nanoparticles and the single PIBCA nanoparticles, thus demonstrating that PEG at the surface of the nanoparticles reduced the complement consumption, one of the main opsonin responsible for nanoparticle removal from the blood stream.

These data strongly suggest that PEG-coated nano-particles could serve as long-circulating busulfan car-riers.

References 1. Hassan M, Oberg G, Bekassy AN, Aschan J, Ehrsson H,

Ljungman P, Lonnerholm G, Smedmyr B, Taube A, Wal-lin I, et al.. Pharmacokinetics of high-dose busulphan in relation to age and chronopharmacology, Cancer Chem-other. Pharmacol., 28(2), 130-134, 1991

2. Bhagwatwar HP, Phadungpojna S, Chow DSL, Andersson BS. Formulation and stability of Busulfan for intrave-nous administration in high-dose chemotherapy, Cancer Chemother. Pharmacol., 37, 401-408, 1996.

3. Hassan Z, Nilsson C, Hassan M. Liposomal Busulfan: bioavaibility and effect on bone marrow in mice, Bone Marrow Transplant., 22, 913-918, 1998.

4. Layre AM, Gref R, Richard J, Requier D, Chacun H, Appel M, Domb AJ, Couvreur P. Nanoencapsulation of a crys-talline drug, Int. J. Pharm., 298(2), 323-327, 2005.

5. Layre AM, Couvreur P, Chacun H, Richard J, Passirani C, Requier D, Benoit JP, Gref R. Novel composite core-shell nanoparticles as busulfan carriers, J. Contr. Release, 111(3), 271-80, 2006.

Figure 1. Typical transmission electron microscopy studies of PCL-PEG (left) and composite PIBCA/ PCL-PEG (right) nanoparticles.

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CHALLENGES IN THE TREATMENT OF GRAM-POSITIVE INFECTIONS

Serhat ÜNALHacettepe University, Faculty of Medicine, Department of Medicine, Section of Infectious Diseases, Ankara, Turkey

The introduction of benzylpenicillin in the 1940s was the start of an era in the treatment of bacterial infections but is soon became appar-

ent that some strains of Staphylococcus aureus were resistant due to production of β-lactamase. Since the mid-1970s resistance to antimicrobials has become an escalating problem. A striking change over the past quarter-century has been the increasing role of Gram-positive bacteria which might have resulted from the emphasis placed on controlling Gram-negative infec-tions. Today we have to deal with infections caused by multidrug-resistant organisms, particularly methicil-lin resistant staphylococci, penicilin- and erythromy-cin-resistant pneumococci and vancomycin-resistant enterococci. The emergence and rapid spread of me-thicillin- resistant S.aureus (MRSA), which were re-sistant not only to all β-lactams but also to the main antibiotic classes, resulted a an increase use of glyco-peptide antibiotics, namely vancomycin and teicho-planin. Unfortunately, the first glycopeptide-resistant enterococci were described in late 1980s, they rapidly spred in many different countries.

Against this background, there is a definitive need for new antimicrobial agents, as well as for prudent use of existing agents.

Linezolid (Zyvox®) is the first of a new class of anti-microbial agents, the oxazolidinones. It was approved by the U.S. Food and Drug Administration (FDA) in 2000 for the treatment of uncomplicated and compli-cated skin and soft tissue infections, including diabet-ic foot infections without concomitant osteomyelitis, community acquired and nosocomial pneumonia and vancomycin-resistant Enterococcus faecium infec-tions including cases with concurrent bacteremia. Al-though clinical trials have shown linezolid to be as ef-fective as best established therapy, resistance has been reported. In addition, high rates of adverse effects, in-cluding thrombocytopenia and anemia, are seen with prolonged courses of therapy. Serotonin syndrome, potentially irrevesible peripheral neuropathy and lac-tic acidosis are also reported.

Daptomycin (Cubicin®) is the first agent of a new class of cyclic lipopeptides The drug exhibits a calcium–

dependent antimicrobial effect against Gram-posi-tive microorganisms, including MRSA. Its once–dai-ly dosing and and the favourable safety profile (except some concerns about rhabdomyolysis and neuropa-thy) are making daptomycin an attractive option for the treatment of Gram-positive infections. Succesful results had been reported for the treatment of bac-teremia and right-sided infective endocarditis but not for the treatment of community acquired pneumo-nia.

Tigecycline (Tygacil®) is a new, semisynthetic glycyl-cycline that was approved by US FDA for the treat-ment of skin, soft tissue and intra-abdominal infec-tions It appears to be very active not only against MRSA but also against glycopeptide-resistant staphy-lococci and enterococci. Nausea and vomitting are the most common side-effects.

Quinupristin-dalfopristin (Synercid®) is fixed mic-ture of semisynthetic streptogramin derivatives. It is bacteriostatic against Enterococcus faecium and bac-tericidal against methicillin-susceptible and methi-cillin-resistant staphylococci. It is inactive against Enterococcus faecalis. Pain and inflammation at the infusion site, arthralgia, myalgia, drug interactions and liver function abnormalities have occured in pa-tients treated with quinupristin-dalfopristin.

Oritavancin, telavancin, dalbavancin are the new glycopeptides in clinical development which appear as potent molecules wiht favourable pharmacokinetic and pharmacodynamic properties.

Ceftobiprole is an investigational novel cephalosporin that is active in vitro against streptococci and staphy-lococci, including penicilin-resistant strains of pneu-mococci and MRSA, while maintaining the activity of extended-spectrum cephalosporins against Gram-negative organisms. In vivo, screening models predict good activity for ceftobiprole against Gram-positive and Gram-negative bacteria.

Changing patterns of resistance have compound-ed and exacerbated the need for new antimicrobi-al agents. The appropriate indications and cost-effec-

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ctiveness of these molecules will determine our future treatment options. Until then the prudent use of ex-isting antibiotics with strict reinforcement of infec-tion control should be the rules of our practice in the treatment of gram-positive infections.

References 1. Appelbaum PC. MRSA-the tip of the iceberg, Clin Micro-

biol Infec., 12 (Supp 2), 3-10, 2006.

2. Pace JL, Yang G. Glycopeptides: update on an old suc-cessful antibiotic class, Biochem Pharmacol., 71, 968-980, 2006.

3. Eliopoulos GM. Current and new antimicrobial agents, Am Heart J, 147, 588-91, 2004.

4. Torres-Viera C, Dembry LM. Approaches to vancomy-cin-resistant enterococci, Curr Opin İnfec Dis., 17, 541-7, 2004.

5. Bosso JA. The antimicrobial armamentaium: Evaluating current and future treatment options, Pharmacother., 25, 55S-62S, 2005.

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CHALLENGES IN TREATMENT OF GRAM-NEGATIVE BACTERIA

Recep ÖZTÜRKİstanbul University, Cerrahpaşa Medical Faculty, Infectious Diseases and Clinical Microbiology, İstanbul, Turkey

The issue of resistance among nosocomial or even community acquired gram-negative mi-coorganisms including primarily extend-

ed-spectrum beta-lactamase producing Enterobac-teriaceae (Escherichia coli, Klebsiella pneumoniae), multidrug-resistan (MDR) Pseudomonas, and MDR Acinetobacter produce a challenge for their tratement. Recently carbapenem-resistant, ESBL producing en-teric bacilli and panresistant Pseudomonas and Aci-netobacter strains emerged [1].

Developing new antimicrobials have considerable problems since the resistance shortens market life and thus increases its cost. Pharmaceutical pipelines are less encouraged to maintain new agents. Discov-ery of novel antibiotics is not expected to give much alternatives in near future [2, 3]. On the other hand, a clear and urgent need for developing new anti-in-fectives is apparent. However especially develeop-ing drugs for the agents especially for the multidrug- or pan-resistant ones is a social responsibility of the companies [3, 4].

Antibiotics such as colistin that have been available but had limited use due to its toxicity gained a new interest in this challange posed by resistant microor-ganisms. Recently available drugs against gram-nega-tives with ongoing studies are as follows:

TigecyclineIt is the first member of the glycylcycline class antibi-otics, which are the synthetic analogues of classical tet-racyclines. The modification of tetracycline at its D-9 position of the central four-ring carbocyclic skeleton enabled the molecule with much broader spectrum of antimicrobial activity as well as defense against tet-racycline resistance. It exhibits activity against gram-positive bacteria including MRSA and VRE as well as gram-negative bacteria such as ESBL producing en-teric rods and multidrug-resistant Acinetobacter spp. β-lactamase negative and positive strains of Haemo-philus influenzae and Moraxella catarrhalis are within its spectrum. Moreover, it is active against the anaer-obic bacteria and MOTT (Mycobacterium other than tuberculosis) rods. It has limited activity against Pseu-domonas aeruginosa and Proteus spp. It is indicated in

complicated skin, soft tissue and intra-abdominal in-fections. Its activity in the treatment of community- and hospital-acquired pneumonias is under investi-gation. It is approved by the Ministry of Health in our country. Nausea, vomiting and diarrhea are among the side effects [2, 5].

CeftobiproleIt exhibits activity against resistant gram-posi-tive bacteria such as MRSA and penicillin-resistant Streptococcus pneumoniae as well as gram-negative bacteria such as E. coli, K. Pneumoniae and Entero-bacter cloacae. It has oral and parenteral formula-tions. Complicated skin and soft tissue infections, and community- and hospital-acquired pneumonias are among the probable indications for its usage [2, 6].

DoripenemIt is a carbapenem class antibiotic with activity against gram-positive, gram-negative and anaerobic bacte-ria. It exhibits activity against ESBL-producing enter-ic rods similar to imipenem and meropenem. It has partial activity against carbapenem-resistant P. aeru-ginosa. It is under investigation for the treatment of nosocomial- and ventilator-associated pneumonias, complicated urinary tract infections, pyelonephritis and complicated intra-abdominal infections. It has parenteral formulation [7, 8].

FaropenemIt is a carbapenem class antibiotic with oral formula-tion available. It has wide-spectrum of activity similar to other carbapenems [2].

GarenoxacinIt is a new quinolone antibiotic undergoing phase III clinical trials. It exhibits activity against gram-posi-tive, gram-negative (M. catarrhalis and H. influenzae) and anaerobic bacteria [3].

Prulifloxacin This new quinolone is the prodrug of ulifloxacin that exhibits activity against gram-positive, gram-negative (Enterobacteriaceae, H. influenzae and M. catarrhalis) and anaerobic bacteria [3].

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New ribosomal inhibitorsThere is ongoing research on these new class antibiot-ics with wide spectrum (A-72310 and A-692345). This class exhibits activity against gram-positive, gram-neg-ative and respiratory pathogens such as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus and Moraxella catarrhalis [9]. Though similar to quinoles in spectrum of activity, they differ in struc-ture. As they have a different mechanism of action, they are not affected by cross-resistance to other ribos-omal inhibitors such as macrolides, tetracyclines, chlo-ramphenicol, aminoglycosides and oxazolidinones.

Other developmentsThere is ongoing research on compounds and extracts other than the ones mentioned above. In addition, there are studies to counteract resistance development mechanisms using molecular methods [2, 3, 10, 11].

References 1. McGowan JE Jr. Resistance in nonfermenting gram-nega-

tive bacteria: multidrug resistance to the maximum, Am J Infect Control., 34(5 Suppl 1), S29-37, 2006.

2. Bosso JA. The Antimicrobial Armamentarium: Evaluat-ing Current and Future Treatment Options, Pharmaco-therapy, 25(10 Pt 2), 55S–62S, 2005.

3. Khardori N. Antibiotics--past, present, and future, Med Clin North Am., 90(6), 1049-76, 2006.

4. Bush K. Antibacterial drug discovery in the 21st century, Clin Microbiol Infect.., Nov;10 Suppl 4, 10-7, 2004.

5. Stein GE, Craig WA. Tigecycline: a critical analysis, Clin Infect Dis., 2006 Aug 15; 43 (4), 518-24.

6. Ceftobiprole Medocaril: BAL5788, JNJ 30982081, JNJ30982081, RO 65-5788, RO 655788. Drugs R D. 2006;7(5):305-11.

7. Anderson DL. Doripenem, , 42(6), 399-404, 2006. 8. Jones RN, Huynh HK, Biedenbach DJ. Activities of dorip-

enem (S-4661) against drug-resistant clinical pathogens, Antimicrob Agents Chemother., 48, 3136–40, 2004.

9. Dandliker PJ et al. Novel antibacterial class, Antimicrobial Agents and Chemotherapy; 47, 3831-9, 2003.

10. Mahady GB. Medicinal plants for the prevention and treatment of bacterial infections, Curr Pharm Des., 11, 2405-27, 2005.

11. Wiart C, Hannah A, Yusof M, Hamimah H, Sulaiman M. Growth inhibition of foodborne and nosocomial pathogens by aqueous fraction of bearded Argostemma (Argostemma involucratum Hemsl., Rubiaceae), J Herb Pharmacother., 5, 97-102, 2005.

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CHALLENGES IN ANTIFUNGAL THERAPY

Ömrüm UZUNHacettepe University School of Medicine, Department of Medicine, Section of Infectious Diseases, Ankara, Turkey

The incidence of invasive fungal infections has increased, and the spectrum of etiologic agents has widened in recent years. Recent advanc-

es in newer classes of antifungals havepromised bet-ter outcome, especially in difficult-to-treat invasive mould infections; however this has not proven to be true. The empirical use of antifungal therapy in pa-tients with neutropenia who remain febrile in spite of adequate antibacterial therapy has been a standard approach for almost two decades. However, thesta-tistical weakness of data, the relatively low incidence of IFI in patients not undergoing allogeneic hemat-opoietic stem cell transplantation together with the recent progress in the diagnostic accuracy of IFI has made the routine use of empirical antifungal therapy in all patients with fever and neutropenia question-able. Recently, new developments in fungal diagno-

sis have rompted a reappraisal of the concept of em-pirical antifungal therapy. The time period between the biologicalstart of a fungal infection and the ap-pearance of clinicalsigns and symptoms represents a window of opportunity that, if identified through prospective screening, may allow earlier therapeutic intervention and may potentially improve outcome. Such ‘preemptive’ strategy would not be triggered by fever, but would rest on (1) a better identification of those patients who are at the highest risk for fungal infections so that they can be closely monitored;and (2) the availability of sensitive techniques that facil-itate rapid and early diagnosis of invasive fungal in-fections. On the other hand, the concept of combina-tion therapy has become appealing, but there is still a long way to go.

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NATIONAL AND INTERNATIONAL PATENT PROTECTION, FREE PATENT SEARCH TOOLS (ESPACENET) AND STRATEGIES

Hakan BAYRAMPatent Examiner, Turkish Patent Institute, Hipodrom Street 115, Yenimahalle, Ankara, Turkey

About PCTThe PCT was concluded in 1970, amended in 1979, and modified in 1984 and 2001.

It is open to States party to the Paris Convention for the Protection of Industrial Property (1883). Instru-ments of ratification or accession must be deposit-ed with the Director General of WIPO. The Treaty makes it possible to seek patent protection for an in-vention simultaneously in each of a large number of countries by filing an “international” patent applica-tion. Such an application may be filed by anyone who is a national or resident of a Contracting State. The procedure under the PCT has great advantages for the applicant, the patent offices and the general public: (i) the applicant has up to 18 months more than

he has in a procedure outside the PCT to reflect on the desirability of seeking protection in for-eign countries, to appoint local patent agents in each foreign country, to prepare the necessary translations and to pay the national fees; he is assured that, if his international application is in the form prescribed by the PCT, it cannot be rejected on formal grounds by any designated Office during the national phase of the process-ing of the application; on the basis of the inter-national search report or the written opinion, he can evaluate with reasonable probability the chances of his invention being patented; and the applicant has the possibility during the in-ternational preliminary examination to amend the international application to put it in order before processing by the designated Offices;

(ii) the search and examination work of patent of-fices can be considerably reduced or virtually eliminated thanks to the international search report, the written opinion and, where applica-ble, the international preliminary examination report that accompany the international appli-cation;

(iii) since each international application is pub-lished together with an international search re-port, third parties are in a better position to formulate a well-founded opinion about the patentability of the claimed invention.

About EPCThe Convention on the Grant of European Patents of 5 October 1973, commonly known as the Europe-an Patent Convention (EPC), is a multilateral trea-ty instituting the European Patent Organisation and providing an autonomous legal system according to which European patents are granted. Although the term European patent is used to refer to patents grant-ed by the EPO, after grant such a patent is not a uni-tary right, but a group of essentially independent na-tionally-enforceable, nationally-revocable patents, subject to revocation and/or narrowing as a group pursuant a time-limited, unified, post-grant opposi-tion procedure.

The EPC provides a legal framework for the granting of European patents, via a single, harmonized pro-cedure before the European Patent Office. A single patent application in one language, may be filed at the European Patent Office at Munich, at its branch-es at The Hague or Berlin or at a national patent of-fice of a Contracting State, if the national law of the State so permits. This latter provision is important in countries such as the United Kingdom, in which it is an offence for a UK resident to file a patent ap-plication for inventions in certain sensitive areas abroad without

About Patent databases

As any exclusive ownership rights, paatents must be accessible to the public if the owner wants to exert his right. Today, patents are usually published and acces-sible through electronic means.

As any exclusive ownership rights, patents must be accessible to the public if the owner wants to exert his right. Today, patents are usually published and acces-sible through electronic means.

In several technical fields, Patents are the most ef-ficient source of information for the following rea-sons.

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About Coverage and Content Do not expect to cover the whole state-of-the-art by searching in national Patents only.

Databases available on espa@cenet® have also some limitations in the number and the quality of fields ac-cessible that should be considered when drawing con-clusions from the

result of a search. Commercial patent databases usu-ally offer extra value, by the number of fields you can access, and due to the content of the abstract that has been rewritten to facilitate easieraccess to the docu-ment by keyword searches.

To sum up, a patent database user should always be aware of the content of the database he is using and know the limitations of the fields.

About Classification Patents are classified according to various classifica-tion schemes covering all possible technical domains. When conducting a search, it is essential to systemat-ically use this tool that offers an objective criteria to access relevant documents. Keywords are much more subjective as there are many words to describe a same concept.

• Accessibility: All patent documents have a universal format for the bibliographic data. More than 50 different fields, each representing valuable technical or strategic information, are accessible for each Patent. In addition to this, Patents are classified according to internationally agreed systems, which divides technical domains into more than 100,000 subdivisions.

• Content: A patent, to be valid, should enable a person skilled in that particular area to reproduce the invention. This strict requirement explains why 70% of the information contained in Patents is not available elsewhere. When a catalogue or an article describesa product in a few lines, the corresponding Patent often consists of 20 pages. Patentsrepresent about 350 million of A4 pages containing very relevant technical information.

• Concentration: Full patent collections are often present in national Patent Office archives. From a relevant list of patent documents you will need one or two hours in a patent libraryto collect all the data. In comparison, you will usually need several weeks to order andreceive the references quoted in a thesis.

• Up to date: A company is not inclined to make its inventions public. To get a legal protection for the invention, the company generally files the Patent Application at the earliest possible stage. Patent Applications are normally published 18 months after their first filing date and therefore very often represent the first published information available.In other words newly published Patents are the most up-to-date information available ina specific field.

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PATENTABILITY OF MEDICAL AND PHARMACEUTICAL INVENTIONS

Serkan ÖZKANPatent Examiner, Turkish Patent Institute, Hipodrom Street 115, Yenimahalle, Ankara, Turkey

Intellectual property rights are the rights given to persons over the creations of their minds. They usually give the creator an exclusive right over the

use of his/her creation for a certain period of time. In-tellectual property rights are customarily divided into two main areas: (i) Copyright and rights related to copyright; and (ii) Industrial property rights.

Industrial property rights are a result of intellectual activities in the industrial spheres such as trademark laws, representing an indication of a business, or de-sign rights, utility model rights, and patent rights.

Patents are given for any inventions, whether prod-ucts or processes, in all fields of technology without discrimination, subject to the normal tests of novel-ty, inventiveness and industrial applicability. Patents give the right holder exclusive right to make, use and sell the invention.

Inventions, which are new, have an inventive step that is not obvious to someone with knowledge and experience in the subject, and capable of being made or used in some kind of industry, are protected by patents.

Non-Patentable subject matters and inventions are mentioned in the article 6 of the Decree-Law No.551 Pertaining to The Protection of Patent Rights. Ac-cording to subparagraph (e) of this article 6, meth-ods of diagnosis, therapy and surgery applying to hu-man or animal body shall remain outside the scope the patent protection. The provision under subpara-graph (e) of the Article 6, shall apply neither to the products and compositions (per se) used in connec-tion with these methods nor to their process of man-ufacturing. Patent shall not be granted for inventions

in respect of following subject matter: (a) Inventions whose subject matter is contrary to the public order or to morality as is generally accepted and (b) Plant and animal varieties/species or processes for breed-ing/plant or animal varieties/species, based mainly on biological grounds.

In order to develop new drugs, mechanisms will have to be put in place that foster innovation and the de-velopment of new products, while at the same time ensuring that patients have rapid access to the fruits of such research. The pharmaceutical sector is a ma-jor user of the patent system. While only a small - and declining - number of new chemical entities are ap-proved annually, thousands of patents are applied for to protect variants of existing products, process-es of manufacture or, where admitted, second uses of known pharmaceutical products.

The number of international patent applications con-tinues to rise with impressive growth. The great-est number of international applications (PCT) pub-lished in 2006 were in telecommunications (10.5%), pharmaceuticals (10.4%), and information technolo-gy (10.4%). The fastest growing technology areas are semiconductors (28% increase), information technol-ogy (22%) and pharmaceuticals (21%).

There is a remarkable growth rate in the number of pat-ent applications received by the Turkish Patent Insti-tute (TPI) in last years. Number of patent applications increased from 1152 to 5165 between 2003 and 2006. Also, TPI experienced a %50 growth in the number of patent applications in 2006 as compared to 2005. Sim-ilar to the increase in the total number of patent ap-plications, number of pharmaceutical applications in-creased from 320 to 467 between 2005 and 2006.

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IMPORTANCE OF POLYMORPHISM IN DRUG RESEARCH AND DEVELOPMENT

Fethi ŞAHİNGazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey

Polymorphism is the property of a compound found in different crystal forms. Different crystal forms exhibit different dissolution pro-

file and polymorphs of recently discovered drug sub-stances show different lipophilicity. During the de-velopment of an original active principal all the polimorphs should be determined before clinical in-vestigations. The more stable olymorph which is dis-covered after clinical studies may cause repeating of clinical studies or withdrawal of the medicine from the market. (example Ritonavir).

This circumstance cause the more intense studies on the polymorphs and their patent protection.

Importance of polymorphism in pharmaceutical in-dustry can be classified as follows:

• Thermodynamic stability • Chemical stability • Mechanical features • Solubility

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A VAST SOURCE FOR THE DISCOVERY OF NOVEL DRUG LEADS: TRADITIONAL MEDICINES

Erdem YEŞİLADAYeditepe University, Faculty of Pharmacy, Kayışdağı 34755 İstanbul, Turkey

Discovery of new drug leads may be defined as a SACRED ART. In order to create MASTER-PIECES in this field, scientists should have to

embroider each detail obtained through recent com-putational techniques, SAR studies, ethiological stud-ies on the pathogenesis of diseases, etc. Once a viable structure is designed, further process is to obtain the compound in ample quantities by chemical synthesis or biosynthesis for the succeeding procedures.

Alternatively, during the photosynthesis hundreds of primary or secondary metabolites as well as phy-toallexins have been synthesized in each plant speci-men in the nature. Therefore, plants are well-known as an important source of many biologically active compounds. Since ancient times of civilizations, peo-ple have been relying on plants as either prophylac-tic or therapeutically arsenal to restore and maintain health.

However, random exploiting of these rich sources for developing new drug leads is a long, tedious and ex-pensive task and various tools are developed in order to increase success. Traditional medicine is among the most extensively implemented approach for this pur-pose. Recently breeding sciences of Ethnobotany for documentation of traditional wealth of information and Ethnopharmacology for the scientific evaluation of this information to afford biologically active drug molecules are useful tools for discovery of new drug leads. Ethnopharmacology is defined as “the interdisci-plinary scientific exploration of biologically active agents traditionally employed or observed by man“ (Bruhn and Holmstedt, 1981). The bio-rational approaches within the drug discovery process are based on the ethnobo-tanical knowledge. A careful evaluation of ethnobotan-ical inquiries based on field surveys are compulsory to attain an idea about the selection of the correct materi-al, the way of preparation for the administration of the remedy (ethnopharmaceutics), and the symptoms of pathologies which the remedy claimed to be effective by the lay people (ethnomedicine) may yield success-ful results. The latter issue is of special importance for the elaboration of pharmacological studies. The diffus-er the descriptions about the diseases and troubles to be cured with, the blurrier the focused pharmacolog-

ical targets. As the initial process the crude extract(s) are investigated by in vitro and/or in vivo techniques to comb for the potential targets and in turn, to confirm or disapprove the applications from folk medicines. This is the most critical step to select the convenient pharmacological technique for the success of study. For example, during our field survey in Anatolia in a loca-tion was reported that the decoction of hawthorn (Cra-taegus tanacetifolia) roots was used against rheumat-ic pain. Actually such utilization was interesting since was not previously reported for hawthorn. We pre-pared aqueous and methanol extracts from the roots of the plant and tested for their effects on carrageenan-induced hind paw edema model in mice, a well-known technique for the assessment of anti-inflammatory ac-tivity. However, we could not find any activity on this model and considered as possibly false information. In a following attempt, we decided to test the in vitro ac-tivity of the extracts against phospholipase A2 enzyme and the methanol extract revealed a potent activity. Through activity-guided fractionation and isolation procedures we obtained a polyphenolic constituent with a large molecular weight as the active ingredient. As known, carrageenan-induced edema model is a con-venient technique for the investigation of nonsteroidal anti-inflammatory agents which act through cycloox-ygenase pathway in the inflammatory process, while phospholipase A2 is an enzyme catalyze the hydrolysis of membrane phospholipids to arachidonic acid and a convenient model for the corticosteroid type agents. In order to emphasis the importance of “bioassay model selection”, I would like to give another example; the oily extract from the fruits of bitter cucumber (Momordica charantia) has been used in Anatolia to treat wounds and ulcers. In a previous study, targeting to evaluate the antiulcer potential of this extract was employed water immersion-induced stress ulcer model in rats, howev-er was found totally ineffective. Water immersion-in-duced stress ulcer model is known as a representative model mainly for the determination of agents acting through inhibition of acid secretion. However, the oily extract of the plant was also recommended as an effec-tive remedy for wound healing, which suggests that the activity of the extract might possibly, at least partial-ly, through a cytoprotective mechanism. Therefore we tested the effects of the extracts on indomethacin-in-

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duced ulcer model as a representative model for assess-ing cytoprotective activity and a potent antiulcer activ-ity was found (Gürbüz et al., 2000).

I’m always enthusiastic that how the people had dis-covered the biological effects of those remedies! How many of them had sacrificed their life? For example, during our expeditions in south Anatolia (Isparta), Spanish broom (Spartium junceum) flowers were men-tioned to be effective in treating peptic ulcers. Due to the alkaloid content (spartein type), this plant is known as toxic and we deliberately recorded this information. However, in vivo experimental studies had shown that the crude extract was highly effective in rat ulcer mod-els without inducing any apparent toxicity. In further bioassay-guided fractionation procedures, one fraction which accumulates alkaloids induced sudden death of animals, while active fraction yielded a new triterpe-noid saponin with potent antiulcer activity (Yeşilada et al., 2000a). Actually, because of the lowest alkaloid con-tent inhabitants have used the flowers of plant in thera-py without any apparent toxicity. If leaves or other parts of the plant were used, instead of flowers, might possi-bly cause death, due to the higher alkaloid content.

Bioassay-guided fractionation and isolation proceduresThe most effective way for the discovery of drug leads from the natural sources in ethnopharmacology is the “Bioassay-guided Fractionation and Isolation” proce-dures (BGFI). This process asks for a carefully selection of target, respectively bioassay. As soon as an activity is manifested by the preliminary tests, the extract is sub-jected to successive chemical fractionation and isola-tion procedures and each fraction and/or components is then submitted to bioassay model, which eventual-ly yield the active ingredient(s). The chemical struc-ture of the isolated constituent is elucidated through detailed spectroscopic analysis, i.e., mass spectrometry and 1H- and 13C-NMR techniques. Although, this pro-cedure sounds quite simple and logical, in compare to dealing with a collection of pure natural compounds a lot of experience is required for the successful chem-ical and pharmacological processing, in particular to exclude false positive results. One of the most common problems frequently encountered in BGFI procedures is the stated activity may be loss during chemical frac-tionation, or may be spread among many fractions and compounds. This may be, in particular, due to the syn-ergistic interaction where many constituents in the ex-tract/fraction of sometimes even in different chemical classes contributing to a pharmacological effect. An-other problem is the possible decomposition of the active ingredient(s) during the chemical separation processing; due to the loss of co-metabolites responsi-ble for the stability of the active ingredient or wrong ex-traction or chromatographic conditions employed for the isolation of constituents.

In particular, in the last two decades, extensive re-searches on BGFI procedures have been carried out for the identification and isolation of pharmacolog-ically active principles from the plants. In Turkey, we have conducted such studies for the evaluation of Turkish traditional medicines.

Turkish folk medicine for the discovery of drug moleculesTurkey has a rich flora as well as cultural diversi-ty which yielded the accumulation of notable tradi-tion originated remedic information. Through ex-tensive field surveys between 1986 and 1994, we have documented this wealth of knowledge using scientif-ic ethnobotanical techniques. A database of Turkish folk medicine was established (TUHIB) and 8000 folk remedies have been accumulated so far.

Several chemical and pharmacological studies have been conducted in order to evaluate this wealth of in-formation and through application of BGFI proce-dures a number of molecules have been defined with anti-inflammatory, antinociceptive, antidiabetic, anti-Helicobacter, IL-2 inducing, antihepatotoxic, antiul-cerogenic, antioxidant or antiviral activity so far. In order to demonstrate the different strategies for lead discovery from folk remedies, we will focus on several selected ethnopharmacological studies on TFM.

Anti-inflammatory and antinociceptive agents from Turkish folk medicines (TFM)The upper ground part of water speedwell (Veronica anagallis-aquatica), Scrophulariaceae, is used to treat rheumatic pain in the northeast part. Herbs boiled in a cauldron and then the patient takes bath inside while still warm as well as the pulp applied external-ly on affected joints. Through BGFI techniques three catalpol derivative of iridoids were isolated. The anti-inflammatory (carrageeenan-induced hind paw ede-ma) and antinociceptive (p-benzoquinone-induced writing) activities were found to be shared by three compounds but, in particular, catalposide and ver-proside showed higher activity and were safe as re-gard to gastric toxicity (Küpeli et al., 2005).

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An isoflavone derivative, scandenone, was isolat-ed from osage orange (Maclura pomifera) (Morace-ae) fruits by BGFI techniques showed comparable anti-inflammatory (carrageenan-induced hid paw edema, TPA-induced ear edema) and antinocicep-tive (PBQ-induced writhings) activity to indometh-acin, without inducing any gastric lesion (Küpeli et al., 2006a).

Due to widespread distribution of Clematis spe-cies at the northern sphere, have been reported to be used in the traditional medicines worldwide. The aerial parts of various Clematis species are used as analgesic, antipyretic and diuretic, against rheumat-ic pain, eye infections, gonorrhoeal symptoms, bone illnesses, chronic skin disorders, gout and varicosity in particular in the Europe and Eastern Asia. In the field works, we have determined that different Turk-ish species of Clematis are used as remedy among public of Anatolia. The most salient application re-lated to folkloric usage is the one that is applied in the treatment of rheumatic ailments. It is stated that in Northern Anatolia, C. cirrhosa ve C. flammula leaves or the aerial parts are implemented to pro-vide temporary relief in joint pains. Fresh herbs or leaves are applied on inflammatory joints kept for about 15-30 minutes after battering, as it irritates the skin opens a hole for draining the edema. In fact, in particular cases, in order to provide continuous draining of the edema for 20-25 days, the wound is plugged by inserting a grape dreg into the hole,

which is occasionally removed to drain the accumu-lated inflammation out. On the other hand, for cur-ing this open wound a fresh leaf of Plantago major ssp. major L. is applied. Moreover, C. vitalba branch-es are also used to stop toothache by smoking like a cigarette in Northwestern Anatolia.

Recently we have isolated a potent anti-inflamma-tory, antinociceptive and antipyretic principle from the dried herbs of Clematis vitalba, as a new C-gly-cosylflavon, vitalboside (4’-coumaroyl isovitexin). This compound was shown to possess a powerful and broad activity spectrum against a battery of tests; acute inflammation tests: carrageenan-, PGE2-, serot-onin-induced paw edema, inhibitory activity on ace-tic acid-induced vascular permeability, castor oil-in-duced diarrhea; subacute inflammation test: Air-sac test; and chronic inflammation test:, Freund’s com-plete adjuvant (FCA)-induced chronic arthritis; anti-pyretic tests: yeast- and FCA-induced pyrexia; anti-nociceptive test: p-benzoquinone-induced writings without inducing any apparent gastric toxicity (Yes-ilada and Küpeli, 2007).

Another C-glycosylflavon with potent anti-inflam-matory and antinociceptive activity isolated from several plants was isoorientin. Even in a very low dosage, isoorientin provided activity as potent as of indomethacin without inducing any apparent gast-ric toxicity (Küpeli et al., 2004). This compound was also found to possess a potent antihepatotoxic (Kü-peli et al., 2004) and antidiabetic (Sezik et al., 2005) activities.

In Anatolia, oil made from the flowers of mullein (Verbascum species) is used to help soothe earache and can be applied externally for eczema and other types of inflammatory skin conditions, wounds and rheumatic pain. Through BGFI procedures from the flowers of Verbascum lasianthum (Scrophulariaceae) an iridoid glucoside, aucubin, and a triterpenoid sa-ponin, ilwensisaponin A, were isolated as the potent anti-inflammatory and antinociceptive components without inducing any apparent acute toxicity or gas-tric damage (Küpeli et al., 2007).

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Bath prepared with the whole plant (roots and aerial parts) of spruge olive (Daphne oleoides ssp.oleoides) (Thymelaeaceae) has been used to treat rheumatism, lumbago and against fever in TFM. Through activ-ity guided fractionation and isolation techniques 17 active constituents were isolated and their struc-tures were chemically elucidated. Diterpenes [genk-wadaphnin, gnidilatin, gnidicin and 20-palmitates of these compounds and 1,2-dehydrodaphnetoxin], a coumarin derivative [daphnetin], Lignan deriva-tives [eudesmin, wikstromol, matairesinol]. Effects of these molecules were studied against a group of cytokines known to play key role in inflammatory disorders; interleukin-1α and –1β and tumor necro-sis factor-α. Genkwadaphnin, 1,2-dehydrodaphne-toxin and daphnetin were found to possess potent inhibitory activity on these cytokines, but others showed a moderate activity (Yesilada et al., 2001).

The roots of Astragalus membranaceus (Fabaceae), a Chinese species, has a reputation of immunomodula-tory and have been implemented against various dise-ases including cancers. In Turkey, ca.500 species have been recorded, however, almost none of these species are used in Turkish folk medicine. Fourteen triterpen saponins isolated from the roots of three Turkish spe-cies, A.oleifolius, A.cephalotes, A.trojanus, were stu-died using ELISA tests against cytokines induced by bacterial lipopolysaccharide stimulation; IL-1ß, IL-8, TNF- α, and phorbolacetate stimulation; IL-2, IL-4, INF-γ. Among the compounds studied only Astraga-loside VII was isolated from Astragalus oleifolius sho-wed a potent IL-2 stimulatory activity (142%). Since a few local healers claimed that several Astragalus spe-cies (not revealed) from southeast Anatolia to possess healing effect on cancers (personal note), it is suppo-sed that this result may partially support the above as-sertions (Yesilada et al., 2005).

Cistus laurifolius (Cistaceae), rockrose, is a wide-spread and perennial bush with charming flowers. The leaves are used externally as a remedy against rheumatic pain, urinary inflammations, high fever, peptic ulcers and diabetes in TFM. For this purpose, a warm decoction was used as a bath to relieve pain, or wilted leaves were externally applied to joints. The leaves were found to inhibit effectively IL-1α, an in-flammatory cytokine (Yesilada et al., 1997).

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Internal use of flowers for treatment of peptic ul-cer was also reported. In a recent study, to evaluate antirheumatic activity in TFM, we isolated 16 com-pounds mainly flavonoids and lignans through ac-tivity-guided procedures and major component 3-O-methyl quercetin was found to possess potent prostaglandin inhibitory and antioxidant activity; in-hibitory on PGE1 and PGE2-induced contractions in guinea pig ileum and DPPH (Sadhu et al., 2006). This compound was also found to possess potent anti-Heli-cobacter pylori activity (Üstün et al., 2006).

Other flavonoids, kaempferol-3,7-O-α-dirhamnoside and quercetin-3,7-O-α-dirhamnoside from the leaves of Tilia argentea showed antinociceptive (34% and 40% inhibition, respectively) and anti-inflammatory (36% for both) activities without inducing any appa-rent gastric lesions (Toker et al., 2004).

Antiulcerogenic activity

Tea prepared from the flowers of Spanish broom (Spartium junceum L.) (Fabaceae) are used in TFM to treat ulcers. Since Spanish broom is known to pos-sess spartein type alkaloids, utilization of this plant as a folk remedy seemed quite risky. However, aqueous extract and polar fractions showed 100% inhibition against water immersion and restraint-induced stress ulcer in rats. BGFI techniques yielded a new triter-penoid, spartitrioside. This compound showed po-tent inhibitory activity against a wide range of ulcer models; EtOH-ulcer (100%**), pyloric ligation-ulcer (84%*), gastric secretion (49%*), gastric pH (2.3 to 4.5**), peptic activity (61%*), gastric acidity (83%*) (Yesilada et al., 2000a). Several flavonoids with potent antioxidant activity were also isolated from the flow-ers (Yesilada et al., 2000b).

Fresh flowers of yellow starthistle (Centaurea solstitia-lis ssp.solstitialis) (Asteraceae) are swallowed to treat ulcers in TFM. Methanol extract showed 100% in-hibition against EtOH-induced lesions in rats. BGFI

techniques yielded two sesquiterpenes as the active components; Chlorojanerin (90%***), 13-acetyl sol-stitialin A (89%*), while Solstitialin A showed a weak activity (31%) (Yesilada et al., 2004; Gürbüz and Yes-ilada, 2007).

Ethnopharmacological studies have been conducted on TFM so far revealed that flavonoids are the most promising phytochemicals as drug leads. A wide activ-ity spectrum for flavonoids has been reported since at least two or three decades and many studies have been conducted on the semi- or full synthesis of various fla-vonoid type compounds. Among the isolated flavo-noids in the course of ethnopharmacological studies from TFM; 3-O-methyl quercetin (isorhamnetin) de-serves further detailed attention. This compound sho-wed a potent anti-Helicobacter pylori activity (MIC 3.9 µg/ml; AMP 0.06 µg/ml) (Üstün et al., 2006), anti-inf-lammatory and antinociceptive activities (Küpeli and Yesilada, 2007), prostaglandin inhibitory and antioxi-dant activities (Sadhu et al., 2006) and antihepatotoxic activity (Küpeli et al., 2006b). Further studies may yield successful results for the discovery of new drug leads.

References 1. Bruhn, J.G., Holmstedt, B., 1981. Ethnopharmacology:

objectives, principles and perspectives. In: Natural Prod-ucts as Medicinal Agents. J.L. Beal, E. Reinhard (Eds). Hippokrates Verlag: Stuttgart, pp.18-19.

2. Gürbüz, İ., Akyüz, Ç., Yeşilada, E., Şener, B., 2000. Anti-ulcerogenic Effect of Momordica charantia L. fruits on Various Ulcer Models in Rats. Journal of Ethnopharmacol-ogy 71, 77-82.

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3. Gürbüz İ., Yeşilada, E., 2007. Evaluation of anti-ulcero-genic effect of sesquiterpene lactones from Centaurea solstitialis L. ssp.solstitialis by using various in vivo and biochemical techniques. Journal of Ethnopharmacology (in press).

4. Küpeli E., Aslan M., Gürbüz İ., Yeşilada E., 2004. Evalua-tion of the in vivo biological activity profile of isoorientin. Zeitschrift für Naturforshung C 59c, 787-89.

5. Küpeli E., Harput Ş., Varel M., Yeşilada, E., Saraçoğlu, İ.., 2005. Bioassay-guided isolation of iridoid glucosides with antinociceptive and anti-inflammatory activities from Veronica anagallis-aquatica. Journal of Ethnopharmacol-ogy 102, 170-176.

6. Küpeli E., Orhan İ., Toker G., Yesilada E., 2006a. Anti-inflammatory and antinociceptive potential of Maclura pomifera (Rafin.) Schneider fruit extracts and its major isoflavonoids, scandenone and auriculasin. Journal of Ethnopharmacology 107, 169-174.

7. Küpeli E., Orhan Deliorman D., Yeşilada E., 2006b. Effect of Cistus laurifolius L. leaf extracts and isolated flavonoids on acetaminophen-induced hepatotoxicity in mice. Jour-nal of Ethnopharmacology 103, 455-60.

8. Küpeli, E., Tatlı, İ.İ., Akdemir, Z.S., Yeşilada E., 2007. Bioassay-guided isolation of anti-inflammatory and anti-nociceptive glycoproteins from the flowers of Verbascum lasiniathum Boiss. ex Bentham.. Journal of Ethnopharma-cology 110, 444-450.

9. Sadhu S.K., Okuyama E., Fujimoto H., Ishibashi M., Yesi-lada E., 2006. Prostaglandin Inhibitory and Antioxidant Components of Cistus laurifolius, a Turkish Medicinal Plant. Journal of Ethnopharmacology 108, 371-78.

10. Sezik E., Aslan M., Yeşilada E., Ito S., 2005. Hypogly-caemic activity of Gentiana olivieri and isolation of the active constituent through Bioassay-guided fractionation techniques. Life Sciences 76, 1223-38.

11. Toker G., Küpeli E., Temizer Memişoğlu M., Yesilada E., 2004. Flavonoids with antinociceptive and anti-inflam-matory activity from the leaves of Tilia argentea (Linden). Journal of Ethnopharmacology 95, 393-7.

12. Üstün O., Özçelik B., Akyön Y., Abbasoğlu U., Yeşilada E., 2006. Flavonoids with anti-Helicobacter pylori activity from Cistus laurifolius leaves. Journal of Ethnopharmacol-ogy 108, 457-61.

13. Yeşilada, E., Üstün, O., Sezik, E., Takaishi, Y., Ono, Honda, G., 1997. Inhibitory effects of Turkish folk remedies on inflammatory cytokines: interleukin-1a, interleukin-1β and tumor necrosis factor α. Journal of Ethnopharmacol-ogy 58, 59-73.

14. Yeşilada, E., Takaishi, Y., Fujita, T., Sezik, E., 2000a. Anti-ulcerogenic effects of Spartium junceum flowers on in vivo test models in rats. Journal of Ethnopharmacology 70, 219-226.

15. Yeşilada, E., Tsuchiya, K., Takaishi, Y., Kawazoe, K., 2000b. Isolation and characterization of free radical scav-enging flavonoid glycosides from the flowers of Spartium junceum by activity-guided fractionation. Journal of Eth-nopharmacology 73, 471-78.

16. Yeşilada E., Taninaka H., Takaishi Y., Honda G., Sezik E., Momota H., Ohmoto Y., Taki T., 2001. In vitro effects of Daphne oleoides ssp. oleoides on inflammatory cytokines and activity-guided isolation of active constitu-ents. Cytokine 13, 359-64.

17. Yeşilada E., Gürbüz İ., Bedir E., Tatlı İ., Khan I.A., 2004. Isolation of antiulcerogenic sesquiterpene lactones from Centaurea solstitialis L. ssp.solstitialis through bioassay-guided fractionation procedures in rats. Journal of Ethno-pharmacology 95, 213-9.

18. Yeşilada, E., Bedir, E., Çalış, İ., Takaishi, T., Ohmoto, 2005. Effects of triterpene saponins from Astragalus species on in vitro cytokine release. Journal of Ethnopharmacology 96, 71-7.

19. Yeşilada E., Küpeli E., 2007. Clematis vitalba L. aerial part exhibits potent anti-inflammatory, antinociceptive and antipyretic effects. Journal of Ethnopharmacology 110, 504-515.

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PARTHENOLIDE ANALOGS AS ANTILEUKEMIC AGENTS WITH CLINICAL POTENTIAL

Peter A. CROOKSUniversity of Kentucky, College of Pharmacy, 501A Pharmacy Building, Rose Street, Lexington, KY 40536-0082

The sesquiterpene lactone, parthenolide, the principal constituent of the medicinal plant Feverfew (Tanacetum parthenum), has been

shown to be capable of inducing human leukemia stem cell (LSC) death in vitro, but is without effect on hematopoietic stem cells. LSCs are believed to play a central role in the pathogenesis of acute leukemia (AML), and are known to be involved in both initi-ation of AML and relapse. Thus, novel compounds such as parthenolide, that selectively target LSCs, rep-resent potential novel clinical agents for the treat-ment of AML. Unfortunately, parthenolide has poor physicochemical properties that preclude its devel-opment as an effective clinical agent. Utilizing clas-sical solution-phase Michael-addition chemistry, we have designed, synthesized and screened a small li-

brary of chirally defined parthenolide analogs with improved water-solubility and in vivo bioavailability. From these studies we have identified a lead clinical candidate, DMAPT fumarate, which induces selective and rapid death of primary human LSCs from both myeloid and lymphoid leukemias. The mechanism of action of DMAPT fumarate includes NF-кB inhibi-tion, p53 activation, and induction of oxidative stress responses. Pharmacokinetic and metabolic studies in rodents indicate that DMAPT fumarate has ~70% bi-oavailability via the oral route, while studies in mouse xenograft models and in dogs suffering from sponta-neous acute leukemias indicate that DMAPT has in vivo bioactivity. Thus, based upon the above preclin-ical data, DMAPT fumarate appears to be a potential clinical candidate for the treatment of human LSCs.

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SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL CAMPTOTHECIN CLASS TOPOISOMERASE I INHIBITORS

Ayhan S. DEMİRMiddle East Technical University, Department of Chemistry, 06531 Ankara, Turkey

Camptothecin (CPT) is a pentacyclic alkalo-id first isolated from extracts of the Chinese tree Camptotheca acuminata. Camptothecin

is thought to inhibit the proliferation of cancer cells by interfering with the breakage/reunion reaction of the enzyme topoisomerase I, a nuclear enzyme impli-cated in DNA replication and RNA transcription. A camptothecin drug stabilizes and forms a reversible enzyme-camptothecin-DNA ternary complex, des-ignated the cleavage complex. The formation of the cleavable complex specifically prevents the reunion step of the breakage/union cycle of the topoisomer-ase reaction.1

The natural fluorescent properties of CPT and its de-rivatives have been exploited to monitor its concen-tration in living cells. Significant attention has been paid to the equilibrium between the lactone and its ring-opened carboxylate form, which influences its antitumor ability.2

Unfortunately, camptothecin and many structural-ly-related camptothecin analogs are water insolu-ble. This water insolubility makes administration of camptothecin compounds difficult. In an effort to address this problem a number of synthetic efforts have been directed to derivatizing the A-ring and/or B-ring to improve water-solubility while maintaining cytotoxic activity.

To date, some water-soluble camptothecin derivatives have been prepared by derivatizing the A and B rings and by opening the lactone E-ring. Water-soluble pro-drug type camptothecin compounds, in which the 20-position hydroxyl group is esterified. Enzymes present within the body can break the ester bond af-ter injection to form the parent camptothecin com-pound. Camptothecin and its analogs are presently under study worldwide in research laboratories and cancer clinics. In lab tests and in clinical trials, these camptothecin drugs have aroused considerable inter-est as a result their ability to halt the growth of a wide range of human tumors. For example, these drugs ex-hibit unprecedented high levels of antitumor activi-ties against human colon cancer.3 Camptothecin has also been shown to be effective against other experi-mental cancer types such as lung, breast, and malig-nant melanoma.

A need continues to exist for the development of new and better camptothecin compounds having still high-er anti-tumor activity and still more improved water-solubility while exhibiting low levels of toxicity.

The primary object of the present work to provide new camptothecin analogs and derivatives displaying cytotoxic activity and improved water-solubility for ease and efficiency of administration/delivery.

The compounds are prepared by substitution of OH to form camptothecin prodrugs, which can be con-verted to active form by physical methods and sub-

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stitution of the 7-position of camptothecin or ho-mocamptothecin to form water-soluble derivatives containing a 7- aminoalkyl moiety. Generally, the camptothecins serving as starting materials are sus-pended in organic solvents or water. To the mixture is added the following compounds which give equiv-alent structure to Z: halo alcohols, amino alcohols, amino aldehydes, halo aldehydes, halo ketones, azido alcohols, azido aldehydes, sulfuric acid, iron sulfate and oxidizing reagents such as per acids and hydro-gen peroxide. The mixture is then stirred to complete the reaction. Any precipitate which forms is removed by filtration and the product is isolated after remov-al of the solvent.

We have evaluated the abilities of the novel camp-tothecins described here to inhibit the topoisomer-ase I enzyme. In these experiments the oligonu-cleotide was labeled at the 3 ′-end of the upper (scissile) strand with 32P-α-cordycepin and termi-nal transferase.4 Topoisomerase I-mediated cleav-age generates DNA fragments that can be readily detected by electrophoresis and quantitated by phos-phorimager analysis.5 Several CPT analogs such as 7-(1-Guanidinopropyl)-20(S)camptothecin and 7-(1-Guanadinoethyl)-20(S)camptothecin are good inducers of cleavable complex formation and the dis-tribution of cleavage sites are comparable to those

generated by camptothecin or 7-ethyl-10-hydroxyca-mptothecin (SN-38). This slower reversal may corre-spond to more stable cleavable complexes. The gua-nadino amine compounds show cleavable complexes with intermediate stabilities between camptothecin and SN-38.

References 1. Camptothecins: New Anticancer Agents; Potmesil, M.,

Pinedo, H., Eds.; CRC Press: Boca Raton, FL, 1995. (b) Garcia-Carbonero, R.; Supko, J. G. Clin. Cancer Res. 2002, 8, 641-661. (c) Croce, A. C.; Bottiroli, G.; Supino, R.; Favini, E.; Zuco, V.; Zunino, F. Biochem. Pharm. 2004, 67, 1035-1045.

2. Burke, T. G.; Mi, Z. J. Med. Chem. 1994, 37, 40-46. (b) Mi, Z.; Burke, T. G. Biochemistry 1994, 33, 10325-10336. (c) Mi, Z.; Burke, T. G. Biochemistry 1994, 33, 12540-12545. (d) Burke, T. G.; Malak, H.; Gryczynski, I.; Mi, Z.; Lakow-icz, J. R. Anal. Biochem. 1996, 242, 266-270.

3. Giovanella B C; Stehlin J S; Wall M E; Wani M C; Nicholas A W; Liu L F; Silber R; Potmesil M. . Science 1989, 246: 1046-1048.

4. Pommier, Y., Kohlhagen, G., Kohn, F., Leteurtre, F., Wani, M.C., and Wall, M.E. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 8861-8865.

5. Tanizawa, A., Kohn, K.W., Kohlhagen, G., Leteurtre, F., and Pommier, Y. Biochemistry 1995, 34(21), 7200-6.

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NANOMATERIALS IN MEDICINE

Erhan PİŞKİNHacettepe University, Chemical Engineering and Bioengineering; Biyomedtek: Society for Biomedical Technologies, Beytepe, Ankara, Turkey

Nanotechnology deals with materials and sys-tems having at least one dimension of about 1-100 nm and aims to produce materials with

superior electrical, chemical, mechanical and optical properties. Nanotechnology has a great potential for medical applications both in diagnosis and therapy. Nanoparticles made of metallic, ceramic, polymer-ic or their composites have been proposed in many applications in medical diagnosis, both in vitro and in vivo. Nanocarriers (particles, micelles and soluble

polymers) have been utilized in controlled/targeted delivery of several active agents including drugs, plas-mid DNA, etc., for effective therapy of a wide range of diseases. Nanofibers can be electrospun and form po-rous nonwoven films as potential dressings and scaf-folds for tissue engineering. This presentation will cover some diverse medical applications of nano-structured materials including author’s contributions and ongoing studies.

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NANOMEDICINE FOR CENTRAL NERVOUS SYSTEM (CNS) DRUG DELIVERY

A. V. KABANOVDepartment of Pharmaceutical Sciences, and Center for Drug Delivery and Nanomedicine, University of Nebraska Medical Center, 985830 Nebraska Medical Center, Omaha, Nebraska 68198-5830

Neurodegenerative and infectious disorders in-cluding Alzheimer’s and Parkinson’s diseas-es, amyotrophic lateral sclerosis, and stroke

are rapidly increasing as population’s age. Alzheimer’s disease alone currently affects 4.5 million Americans, and more than $100 billion is spent per year on med-ical and institutional care for affected people. Such numbers will double in the ensuing decades. Current-ly disease diagnosis for all disorders is made, in large measure, on clinical grounds as laboratory and neu-roimaging tests confirm what is seen by more routine examination. Achieving early diagnosis would ena-ble improved disease outcomes. Drugs, vaccines or regenerative proteins present “real” possibilities for positively affecting disease outcomes, but are limit-ed in that their entry into the brain is commonly re-stricted across the blood-brain barrier. This presen-tation will review highlights how these obstacles can

be overcome by polymer science and nanotechnolo-gy. Specific examples developed in our work include 1) inhibition of drug efflux transport systems in the blood-brain barrier by amphiphilic block copolymers; 2) chemical modification of proteins with hydropho-bic anchors groups and amphiphilic polymers to en-hance their transport across the blood-brain barrier; 3) development of nanogels capable of carrying anti-sense oligonucleotides and siRNA across the blood-brain barrier; 4) cell-mediated delivery of nanozymes to the brain. Such approaches may improve diagnos-tic and therapeutic outcomes. New developments in polymer science coupled with cell based delivery strategies support the notion that diseases that now have limited therapeutic options can show improved outcomes by advances in nanomedicine. The work is supported by the United States National Institutes of Heath (RO1 NS36229 and RO1 NS051335).

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O 01 Ref: 0053

INVESTIGATION OF NONCOVALENT PROTEIN-PROTEIN INTERACTIONS BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY Bekir SALİH, Basri GÜLBAKAN, Özlem DEMİREL Hacettepe University, Faculty of Science, Department of Chemistry, 06532 Beytepe-Ankara, Turkey

Matrix-Assisted Laser Desorption/Ionization-Mass spectrometry (MALDI-MS) is a very important and rapidly evolving analyti-cal tool that is used in many different areas, including protein in-teraction analysis. The work described herein was devised and implemented using MALDI-MS to investigate different types protein-protein interactions. Experimental conditions have been found in which specific noncovalent interactions in solu-tion are maintained throughout the sample preparation and ionization process. Noncovalent interaction between Aβ 1-42 pep-tide and Aβ 1-42 antibody is studied. Aβ 1-42 peptide and Aβ 1-40 antibody was used as negative control to determine the specificity. Aβ 1-42 peptide was digested with trypsin and digestion prod-ucts were incubated with Aβ 1-42 antibody and Aβ 1-40 antibody respectively. Fibril form of the Aβ 1-40 peptide was studied. The ef-fect of TFA and Aβ 1-40 antibody onto disaggregation of Aβ 1-40 fibril peptide was followed by capillary electrophoresis (CE) and MALDI-MS. Interaction of bovine serum albumin (BSA) with Aβ 1-42 was investigated with varying working concentrations by MALDI-MS and CE. Intensity fading MS was also applied to determine the degree of interaction with varying working concentrations. Protein A-IgG interaction was further studied to show the applicability of intensity fading approach in determination of protein-protein inter-actions. Finally, a very complex and high molecular weight enzyme, cytochrome C oxidase, was studied by using CE and MALDI-MS to explore the capability of MALDI-MS in complex protein-protein interactions.

1. Farmer TB, Caprioli RM. Determination of protein-protein interactions by matrix-assisted laser desorption/ionization mass spectrometry, Jour-nal of Mass Spectrometry, 33(8), 697-704, 1998.

2. Frenkel D, Solomon B, Benhar I. Modulation of Alzheimer’s amyloid neurotoxicity by site-directed single-chain antibody, Journal of Neu-roimmunology, 106, 23–31, 2000.

3. Heck AJ, Van Den Heuvel RH. Investigation of intact protein complexes by mass spectrometry, Mass Spectrometry Reviews, 23(5), 368-89, 2004.

4. Solomon B, Koppel R, Hanan E. Katzav T. Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer β amyloid peptide, Pro-ceedings of National Academy of Science USA, 93, 452–455, 1996.

O 02 Ref: 0010

MOLECULAR DYNAMICS SIMULATION OF THROMBIN: TARGET FOR ANTICOAGULANT DRUGS 1Özge KÜL, 1Fulya ÇAĞLAR, 1Vildan ADAR, 2Timuçin YALÇINKAYA 1Hacettepe University, Faculty of Science, Department of Chemistry, Computer-aided Drug Design Group, Ankara, Turkey2Forte Technology, Simulation Group, Ankara, Turkey

Thrombin is a primary target for the development of novel anti-coagulants, since it plays two important and opposite roles in he-mostasis as procoagulant and anticoagulant. Two allosteric forms, slow (S) and fast (F), are present that recognize natural substrates and inhibitors with significantly different affinities. Two forms are almost equally populated. THROMBIN is an enzyme that causes blood clotting when vascular system is injured, thus preventing the

loss of blood. Thrombin inhibitors are very important in preventing the formation of blood clots inside veins and arteries.

Trombin consists of two polypeptide chains which are chain A and chain B. Chain A consists of 36 aminoacid, Chain B consists of 259 aminoacid. There are 492 X-ray structures which has been dissolved in the Protein databank (http://www.rcsb.org/pdb/home/home.do).

One of the principal tools in the theoretical studies of biological molecules is molecular dynamics simulations (MD). Md gives us dy-namic picture of molecules and their motions.

X-Ray structures of thrombin with bound different substrates of 1qj6, 1qj7, 1qj1, 1qhr ve 1ppb [5] are taken from the protein da-tabank.

Figure 1. X-Ray structure of thrombin (pdb code:1ppb).

Molecular dynamics simulations an energy minimizations were carried out on Sun Fire x4600 x64 server: (8 Core AMD Opteron) using the GROMACS 3.3.1 [1] package of programs. SYBYL 7.3 [2] was used to model of hirudins. For visualization of structures and trajocteries the VMD [3] 1.8 and PyMOL [4] respectively were used. After gained free forms of this structures (without water,ligands) by sybyl programme this structures are calculated by GROMACS 3.3.1.

Some structural properties of the thrombin with different sub-strates including pdb code and number of residues are given in Table 1. All water molecules and ligands from X-Ray structures are remo-ved and proteins were solvated with water. Trajectories were sampled at 2 ps. interval.

Table 1. MD simulation parameters of 1qj6,1qj7,1qhr,1ppb.

Start structure

1qj6 1qj7 1qhr 1ppb

No.of atoms (chain A)

287 287 287 287

No.of atoms (chain B)

2093 2093 2093 2093

No.of solvent molecles

8559 8545 8501 8695

Total charge in the system

3.000e 3.000e 3.000e 3.000e

Box geometry octahedron octahedron octahedron octahedron

Box volume (nm3)

126.84 124.07 126.27 148.15

Temperature 300 K 300 K 300 K 300 K

Simulation time

20 ns. 20 ns. 20 ns. 20 ns.

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MD simulations have been performed for thrombins (1qj6, 1qj7, 1qj1, 1qhr ve 1ppb) to explore conformational transition from fast to slow forms.It was carry out with a time step of 20 ns. at 300K.

Analysis of MD simulations was examined by the root mean square deviation(RMSD), the root mean square fluctation (RMSF). Active sides of thrombin has been determined.

1. Berendsen HJC, Van der Spoel D, van Drunen R. GROMACS: A mes-sage-pssing parallel molecular dynamics implantation, Comp. Phys. Comm., 95, 43-56, 1995. http://www.gromacs.org/

2. http://www.tripos.com, SYBYL 7.0, Tripos Inc., 1699 South Hanley Rd., St. Louis, Missouri, 63144, USA.

3. Humphrey W, Dalke A, Schulten K. VMD - Visual Molecular Dynamics, J. Molec. Graphics, 14, 33-38, 1996.

4. DeLano WL. The PyMOL Molecular Graphics System, DeLano Scien-tific, Palo Alto, CA, USA, 2002.

5. Berman M, Westbrook J, Feng Z, Gilliland G, Bhat TNH, Weissig IN, Shindyalov PE. Bourne: The Protein Data Bank, Nucleic Acids Research, 28, 235-242, 2000. http://www.rcsb.org/

AcknowledgementWe would like to thank Turkish Scientific and Technical Research Asso-

ciation (TUBITAK) for the financial support (Grant no: TBAG-106T088) in this research.

This work was carried out under the HPC-EUROPA project( RH3-CT-2003-50607), with the support of the European Community’s Research Infra-structures action of the European Research Area Programme.

O 03 Ref: 0011

MOLECULAR DYNAMICS SIMULATION OF HIRUDINS: SPECIFIC THROMBIN INHIBITOR ISOLATED FROM MEDICINAL LEECH 1Fulya ÇAĞLAR, 1Özge KÜL, 1Vildan ADAR, 2Uğur BÜYÜKDEMİRCİ 1Hacettepe University, Faculty of Science, Department of Chemistry, Computer-aided Drug Design Group, Ankara, Turkey2Forte Technology, Simulation Group, Ankara, Turkey

Hirudin is a low molecular weight anticoagulant peptide (~7 kDa) excreted naturally from the salivary glands of the medicinal leech, Hirudo medicinalis [1]. Hirudin is the most powerful natural inhibi-tor of thrombin, due to its binding specificity. Native hirudin is not a single, homogeneous protein, but rather includes several isoforms. Three variants, designated HV-1, HV-2, and HV-3, have been iso-lated from Hirudo medicinalis [2-4]. In Figure 1 x-ray structure of HV1 obtained from 1hrt is given.

Figure 1. Structure of HV1 taken from complex, 1hrt.

X-Ray structures of hirudin variant 1 (HV1) taken from the Brook Protein Data Bank [5], with the pdb codes 5hir and 1hrt.5hir

which is free form of HV1, whose first 49 residues’ structure has been determined in solution by nmr spectroscopy, and 1hrt which is the structure of a complex of bovine alpha-thrombin and HV1, whose whole chain (65 residues) structure has been determined by x-ray diffraction. X-Ray structure of hirudin variant 2 (HV2) with the pdb code 4htc, structure of the hirudin-thrombin complex, is modelled based on sequence of HV1, extracted from 1hrt, since there is no coordinates for whole chain of HV2. Also hirudin variant 3 which has no x-ray structure is modelled based on sequence of HV1, extracted from 1hrt.

Molecular dynamics simulations and energy minimizations were carried out on Sun Fire x 4600 x 64 server: (8 Core AMD Opteron) using the GROMACS 3.3.1 [6] package of programs. SYBYL 7.3 [7] was used to model hirudins. For visualization of structures and tra-jocteries the VMD [8]1.8 and PyMOL [9] respectively were used.

MD simulations have been performed to determine time-depend-ent characteristic of hirudin variants (HV1, HV2, HV3). All systems were minimized prior to simulation using the steepest descent meth-od followed by the conjugate gradient method. MD simulations were carried out with a time step of 20 ns temperatures over 300 K.

Some structural properties of the hirudin variants, including pdb code, the number of residues, resolution are given in Table 1.

Table 1. MD simulation parameters.

1hrt (HV1) 1hrt (HV1) 5hir (HV1) 1hrt (HV2) 4htc (HV2)

No. of residues 65 49 49 65 65

No. of atoms 483 347 348 478 479

No. of solvent molecules

10404 2881 3183 10284 10751

Total charge -9 -3 -3 -7 -6

Box geometry octahedron octahedron octahedron octahedron octahedron

Box volume (nm^3)

326,893 93,46 104,041 321,3 341,39

Temperature (K) 300 300 300 300 300

Simulation time (ns)

20 20 20 20 20

Methods X-Ray Diffraction

X-Ray Diffraction

NMR X-Ray Diffraction

X-Ray Diffraction

A detailed comparison of simulations were examined by comput-ing the root mean square deviation (RMSD), the root mean square fluctuation (RMSF), the radius of gyration of a group of atoms and the radii of gyration. They are shown that hirudin is composed of a compact N-terminal region (residues 1-47, cross-linked by three di-sulfide bridges) and a flexible C-terminal tail (residues 48-65), and it has a disordered region made up of residues 31-36.

AcknowledgementWe would like to thank Turkish Scientific and Technical Research Asso-

ciation (TUBITAK) for the financial support (Grant no: TBAG-106T088) in this research.

This work was carried out under the HPC-EUROPA Project (RH3-CT-2003-50607), with the support of the European Community’s Research Infra-structures action of the European Research Area Programme.

1. Markwardt F. Untersuchungen uber Hirudin, Naturwissenschaften, 42, 537–538, 1955.

2. Dodt J, Muller HP, Seemuller U, Chang JY. The complete amino acid sequence of hirudin, a thrombin-specific inhibitor, Applied Microbiology and Biotechnology, 165, 180–184, 1984.

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3. Harvey RP, Degryse E, Stefani L, Schamber F, Cazenave JP,Courtney M, Tolstoshev P, Lecocq JP. Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medici-nalis. Proc Natl Acad Sci USA, 83, 1084–1088, 1986.

4. Tripier D. Hirudin: a family of iso-proteins. Isolation and sequence deter-mination of new hirudins, Folia Haematol (Leipzig), 115, 30–35, 1988.

5. Berman M, J. Westbrook Z, Feng G, Gilliland TN, Bhat H, Weissig IN, Shindyalov PE. Bourne: The Protein Data Bank. Nucleic Acids Research, 28, 235-242, 2000. http://www.rcsb.org/

6. Berendsen HJC, Van der Spoel D, van Drunen R. GROMACS: A mes-sage-pssing parallel molecular dynamics implantation, Comp. Phys. Comm. 95, 43-56, 1995. http://www.gromacs.org/

7. http://www.tripos.com, SYBYL 7.0, Tripos Inc., 1699 South Hanley Rd., St. Louis, Missouri, 63144, USA

8. Humphrey W, Dalke A, Schulten, K. VMD - Visual Molecular Dynamics, J. Molec. Graphics, 14, 33-38, 1996.

9. DeLano, WL. The PyMOL Molecular Graphics System (2002), DeLano Scientific, Palo Alto, CA, USA.

O 04 Ref: 0113

DENDRIMERS AS DRUG DELIVERY AGENTS TO BONE Rana SANYAL Boğaziçi University, Faculty of Arts and Sciences, Department of Chemistry, 34342 Bebek, İstanbul, Turkey

Dendrimers have emerged as promising candidates for targeted drug carriers since they can be tailored to accommodate a high den-sity and wide variety of functional groups on their surface 1]. Along with their well-defined molecular structure, segmented spherical construction of dendrimers offers an interesting architecture. While one of these segments is ornamented with active drug molecules, the other one can be decorated with targeting groups. Since direct appli-cation of drug molecules to the diseased tissue or organ increases the effect of the therapy and decreases the side effects, targeted drug de-livery promises to solve the cytotoxicity issues associated with many drug candidates. Presentation will provide a brief survey of some of the advances in this field as well as introduce some new dendritic carriers developed in our group aimed towards bone targeted deliv-ery [2, 3]. Synthesis of different generations of biodegradable den-dritic compounds containing bone targeting molecules for targeting chemotherapy agents to bone tissue will be presented.

1. Lee CC, MacKay, JA, Frechet JMJ, Szoka FC. Designing dendrimers for biological applications, Nature Biotech., 23, 1517-1526, 2005.

2. Wang D, Miller SC, Kopeckova P, Kopecek, J. Bone-targeting macromo-lecular therapeutics, Adv. Drug Delivery Rev., 57, 1049-1076, 2005.

3. Gittens SA, Bansal G, Zernicke, RF, Uludağ, H. Designing proteins for bone targeting, Adv. Drug Delivery Rev., 57, 1011-1036, 2005.

O 05 Ref: 0111

RECENT ADVANCES IN BRAIN DRUG DELIVERY Yilmaz ÇAPAN Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100 Ankara, Turkey

Drug delivery to the brain possess a major challenge due to the blood brain barrier (BBB). Thus, several strategies have been devel-oped to overcome the BBB and to achieve successful brain drug de-livery. Here, we describe two different approaches for drug delivery to the brain. In the first approach, we describe the formulation of mitox-antrone (MTZ) into poly (lactide-co-glycolide) (PLGA) microspheres for local delivery. In the other approach, we describe the development of a chitosan nanoparticle carrier system, functionalized with PEG-BIO-SA/ OX26 for the delivery of a caspase-3 inhibitor.

Mitoxantrone (MTZ) has potent in vitro activity against malignant glioma cell lines, and MTZ-loaded poly(lactide-co-glycolide) (PLGA) microspheres may be injected into the peritumoral area and into tu-mor tissue to provide effective and sustained local drug concentrations without causing systemic side effects. Thus, in rats treated with MTZ-loaded microspheres, tumor volumes were significantly reduced. No tumor formation was observed when glioma cells and MTZ-load-ed PLGA microspheres were implanted concomitantly. Moreover, no systemic side effects or parenchymal inflammatory infiltration were observed in either group of rats. Brain MTZ concentration was highest at the injection site and declined with time and dis-tance from the injection site. The data demonstrate that MTZ-loaded PLGA microspheres can deliver therapeutic concentrations of the drug to the tumor and prevent glioma growth without caus-ing side effects. Therefore, this treatment method may increase the efficiency of antineoplastic therapy and positively impact survival. The peptide Z-DEVD-FMK is a specific caspase inhibitor, which sig-nificantly reduces vulnerability to the neuronal cell death. The inhi-bition of the caspase-3 enzyme is reported to increase neuronal cell survival following cerebral ischemia. Using the avidin (SA)-biotin (BIO) technology, we have accomplished the design of chitosan (CS) nanoparticles conjugated with poly(ethylene glycol) (PEG) bearing the OX26 monoclonal antibody whose affinity for the transferrin receptor (TfR) may trigger receptor-mediated transport across the BBB. Fluorescently labeled CS-PEG-BIO-SA/ OX26 nanoparticles were administered systemically to mice in order to evaluate their ef-ficacy for brain translocation.

The results showed that an important amount of nanoparticles were located in the brain, outside of the intravascular compartment. These findings, which were also confirmed by electron microscopic examination of the brain tissue, indicate that this novel targeted na-noparticulate drug delivery system was able to translocate into the brain tissue after iv administration. Consequently, these novel nano-particles are promising carriers for the transport of the anticaspase peptide Z-DEVD-FMK into the brain.

O 06 Ref: 0109

RATIONAL DESIGN OF NOVEL PHOTOSENSITIZERS AS POTENTIAL PHOTODYNAMIC THERAPY REAGENTS Engin Umut AKKAYA Middle East Technical University, Department of Chemistry, Ankara, Turkey

Photodynamic therapy (PDT) is a noninvasive method of treating malignant tumors and age-related macular degeneration, and par-ticularly promising in the treatment of multidrug-resistant (MDR) tumors. The PDT strategy is based on the preferential localization of certain photosensitizers in tumor tissues upon systemic adminis-tration. The sensitizer is then excited with red or NIR light, gener-ating reactive oxygen species (ROS) including singlet oxygen (1O2) and thus irreversibly damaging tumor cells. Current practice of PDT is limited to a few functionalized porphyrins, however these com-pounds are not considered to be ideal drugs for use in PDT. Among the limitations, the most prominent is the low extinction coefficient of porphyrins in the body’s therapeutic window (650-800 nm, low absorptivity region in typical mammalian tissues). Therefore, there is a significant impetus to develop novel and better efficiency sensi-tizers for use in PDT. Partially reduced porphyrins are an alternative. As non-porphyrin photosensitizers, texaphyrins, phthalocyanines, squaraines, chalcogenopyrylium dyes, aza-boradiazaindacenes and perylenediimides have been suggested. There is also a recent report of a diiodo-substituted boradiazaindacene (BODIPY) as a sensitizer, but it requires excitation outside of the therapeutic window.

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Boradiazaindacenes with methyl substituents on 3 or 5 positions were previously shown to undergo condensation reactions with aldehydes to yield longer wavelength absorbing dyes (100 nm red shifted) of internal charge transfer (ICT) characteristics. The extend-ed conjugation in these dyes moves the absorption peak to 590-600 nm. Incorporation of a second styryl group would result in further red shifts in the absorption spectrum. We targeted dyes 1-3 in our synthesis work, and demonstrated that these dyes have strong ab-sorptions in the 650-680 nm region. The singlet oxygen generation efficiency was studied using the singlet oxygen trap 1,3-diphenyl-isobenzofuran (DPBF). In order to facilitate comparison to previ-ously reported sensitizers, the activity was studied in isopropanol. Even at very low concentration levels of 9 nM dyes (1-3) and under relatively weak red LED irradiation at 625 nm, remarkable efficiency was observed. Encouraged by these observations, we tested the most promising sensitizer 3 on K562 human erythroleukemia cells. EC50 value less than 200 nM was obtained [1].

Thus, we demonstrated that novel di-styrylborodiazaindacene dyes with bromo system are very efficient singlet oxygen-substitu-ents on the fluorochrome pi- generators. In addition, these water sol-uble photosensitizers were shown to have spectacular photoinduced cytotoxicity at very low concentrations and even under low fluence rate LED irradiation. Dark toxicity was nil at the concentration range studied. Structure-activity fine tuning of the sensitizer with further in vitro and in vivo studies is likely to result in highly promising reagents for use in PDT. We are working on the design and synthesis of further red shifted photosensitizers and novel delivery systems in-cluding functionalized carbon nanotubes.

1. Atılgan S, Ekmekçi Z, Doğan AL, Güç, D, Akkaya EU. Water soluble distyryl-boradiazaindacenes as efficient photosensitizers for photodyna-mic therapy. Chem. Commun., 4398-4400, 2006.

O 07 Ref: 0115

A NOVEL POTENTIAL ANTITUMOR ACTIVE DRUG: PLATINUM BLUE COMPLEX CONTAINING SULFUR-DONOR LIGAND 1Şeniz ÖZALP-YAMAN, 1İsmail ERİLHAN, 1S. Belgin İŞGÖR, 2Hüseyin İŞÇİ1Atılım University, Engineering Faculty Chemistry Group, 06836-İncek, Ankara, Turkey2Middle East Technical University, Chemistry Department, Ankara, Turkey

Cisplatin (cis-diamminedichloroplatinum(II), Pt(NH3)2Cl2 (CDDP) was chemically described in 1845, but its antitumor proper-ties were only found accidentally by Rosenberg in 1965 [1]. Decipite the success of cisplatin in chemotherapy, serious side effects limits

the dose that can be administered to patients. The clinical incon-vencies associated with cisplatin therapy prompted the design and synthesis of more effective and less toxic platinum analogs.

A family of deeply colored platinum compounds, usually called platinum blues, has attracted wide interest for years not only because of their unusual color and intriguing chemistry but also for their high antitumor activities [2]. In contrast to the usual yellow, orange, red, or colorless platinum complexes, platinum blues are unusual for their intense blue or purple colors [3]. The first blue platinum compound was prepared by German chemists in 1908 [4]. ]. This unusual material was prepared by the reaction of Ag2SO4 with yellow cis- PtIICl2(CH3CN)2 and was first proposed to have a mononuclear composition of PtII(CH3CONH)2.H2O. However, the compound was later proposed to be polymeric with bridging acetamidate linkages [5].

Moreover, special attention was paid to the platinum blues produ-ced from the reactions between the hydrolysis product of cis-DDP (i.e., cis-[Pt(NH3)2(OH2)2]

2+) and pyrimidine bases such as uracil, since these so-called “platinum-pyrimidine-blues” were found to have a high index of antitumor activity with a lower associated neph-rotoxicity than cis-DDP [6].

Various platinum blues complexes containing nitrogen and oxy-gen have been synthesized and reported in literature, however, no platinum blues compound containing sulphur and nitrogen has been reported.

The reaction of tetrachloroplatinate (II), [PtCl4]2-, with 2-, and

3-aminothiophenol first time have been investigated. The reaction with 2-aminothiophenol, first yielded a yellow solid, then a very dark blue colored product, which has a very distinct absorption band at 724 nm in acetonitrile. Elemental analysis, UV-Vis, IR, ESR, ESCA, SEM,1H-NMR, 13C-NMR , 195Pt-NMR and electrochemical measure-ments made on this dark blue colored compound suggest that it is a new “platinum blue” type complex.

DNA binding studies of the complex were carried out by voltam-metric and UV titrations supporting the electrostatic binding of ct-DNA with suggesting preferential stabilization of PtIII over PtII on binding to DNA.

The effect of platinum blue complex on GST, one of the most im-portant enzymes involved in drug conjugation and biotransforma-tion reactions ,were also investigated.

1. Rosenberg B, VanCamp L., Nature, 205, 698, 1965. 2. Tejel C, Ciriano MA, Oro LA., Chem. Eur. J., 5, 1131, 1999. 3. Matsumoto K, Sakai, K. Adv. Inorg. Chem., 49, 375, 2000. 4. Hoffmann KA, Bugge G., Berichte, 41, 312, 1908. 5. Gillard RD, Wilkinson G., J. Chem. Soc., 2835, 1964. 6. Davidson JP, Faber PJ, Fisher RG., Cancer Chemother. Rep., 59, 287,

1975.

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O 08 Ref: 0055

SYNTHESIS OF ACETOPHENONE AND SUBSTITUTED ACETOPHENONE DERIVED MANNICH BASES AND THEIR CYTOTOXIC ACTIVITIES H. İnci GÜLAtatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey

Mannich bases are generally formed by the reaction between a com-pound containing a reactive hydrogen atom, formaldehyde and a sec-ondary amine. The process whereby these compounds are formed is known as the Mannich reaction [1]. Their cytotoxic activities mostly depend on the alkylation of cellular thiols. Since available alkylating anticancer drugs in market have resistance problem or low selectivity against cancer cells [1], there is a need to find some new compounds with anticancer activity. Our laboratory has been focused on the syn-thesis of acetophenone derived Mannich bases and investigation of their cytotoxic activities against some cancer cell lines.

In this presentation, the synthesis of mono Mannich bases, 1-aryl-3-amino-1-propanone hydrochlorides (series 1), bis Mannich bases, bis(beta-aroylethyl) methyl/ethylamine hydrochlorides (series 2), 2-benzoyl-1,3-diaminopropan dihydrochlorides (series 3), piperidi-nols, 3-aroyl-4-aryl-1-ethyl-4-piperidinol hydrochlorides (series 4), which are structural non classical isomers of bis Mannich bases of series 2, and azine derivatives of some mono Mannich bases, N, N’-bis(3-amino-1-aryl-propylidene)hydrazine dihydrochlorides (series 5) and evaluation of their cytotoxic activities against some cancer cell lines such as Jurkat, Renca and Androgen-independent Prostate Cancer cell line, PC-3, will be mentioned [2-6].

Aryl part was changed as phenyl, substituted phenyl and 2-thienyl and amine part was methyl or ethylamine, dimethylamine, piperid-ine, morpholine in series depending on our design. Representative quaternary derivatives of series 1, 3 and 4 were also synthesized since the quaternary derivatives of these series were expected to deami-nate faster comparing to their corresponding nonquaternary deriva-tives. Bis Mannich bases were also designed with an expectation that they may produce additional alkylating centers compared to their corresponding mono derivatives. The logic behind the synthesizing piperidinol compounds was to see alterations in cytotoxicity in iso-mers. Azine derivatives were considered as bifunctional alkylating agents which may produce additonal alkylating centers compared to the Mannich bases they are derived from. Cytotoxic activities were evaluated depending on chemical structure. The effects of the repre-sentative compounds on glutathione and related enzymes were test-ed in order to determine the mechanism of thiol alkytion to confirm the stability study of some representative compounds [7, 8]. A pre-liminary study has also been performed to determine the effects of mono-Mannich bases and a cyclic Mannich base having piperidinol sturucture on the expression of cytoprotective heat shock proteins (HSC70 and GRP75) and on the levels of thioredoxin (TRX) and glutaredoxin (GRX) in Jurkat cells [9].

1. Dimmock JR, Kumar P. Current. Med. Chem., 1, 1-22, 1997.2. Gül Hİ, Vepsalainen J, Gül M, Erciyas E, Hanninen O. Pharm. Acta Helv., 4,

393-8, 2000.3. Gül Hİ, Gül M, Erciyas E. Arzneimittel Forschung, 52, 628-35, 2002.4. Gül Hİ, Gül M, Vepsalainen J, Erciyas E, Hanninen O. Biol. Pharm. Bull. 26,

631-7, 2003.5. Gül Hİ, Das U, Pandit B, Li PK. Arzneimittel Forschung, 56, 850-4, 2006.6. Gül M, Gül Hİ, Das U, Hanninen O. Arzneimittel Forschung, 55, 332-7,

2005.7. Gül M, Atalay M, Gül Hİ, Nakao C, Lappalainen J, Hanninen O. Toxicol. In

Vitro, 19, 573-80, 2005.8. Gül Hİ, Gül M, Erciyas E. Arzneimittel Forschung, 52, 628-35, 2002.9. Gül M, Gül Hİ, Hanninen O. Toxicol. In Vitro, 16, 107-12, 2002.

O 09 Ref: 0117

UVB-INDUCED APOPTOTIC EFFECT OF 11 P53 ACTINIC KERATOSIS MUTATIONS 1Ayşe ERCAN, 2İ. Hamdi ÖĞÜŞ, 3Douglas BRASH1Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 06100 Ankara, Turkey2Hacettepe University, Faculty of Medicine, department of Biochemistry, 06100 Ankara, Turkey3Yale University, Faculty of Medicine, Department of Therapeutic Radiology, New Haven, USA

The incidence of skin cancer is increasing every year where 95 % of them are non-melanoma skin cancers. Actinic Keratosis (AK) is a non-melanoma skin cancer which is characterized by aberrant proliferation and cell differentiation. UV contributes to the etiology of skin cancers by creating DNA photoproducts which generates mutations, thus causing damage on the genes. The signature of UVB damage is C to T transitions which have been found on the p53 tu-mor suppresor gene. The role of the P53 protein is to suppress tumor growth by arresting cell cycle and inducing apoptosis. P53 is mutated in more than 50 % of the cancers, where most of these are missen-se mutations. The aim of this work was to determine whether the selected 11 p53 AK mutations have different abilities as a response to UVB-induced apoptosis. For this purpose, after a bioinformatic study 11 mutations were selected and generated on an expression vector by site-directed mutagenesis. The mutated and wild type vectors were transiently transfected into primary mouse fibroblasts and the cells were irradiated with 1000 J/m2 UVB in order to induce apoptosis. The apoptosis percentage was detected by flow cytometry. Taken all the data together, the results show that the physical and chemical change the mutation acquires, being on an evolutionary conserved domain, being close to the DNA binding region confers to the protein different properties on apoptosis.

Keywords: Actinic keratosis, UV, P53, site-directed mutagenesis, apoptosis.

O 10 Ref: 0084

FREE-RADICAL SCAVENGER ACTIVITIES OF NEWLY SYNTHESIZED 2-BENZOXAZOLINONE DERIVATIVES CONTAINING THIOSEMICARBAZIDE, TRIAZOLE, THIADIAZOLE AND HYDRAZONE 1Samiye YABANOĞLU, 2Umut SALGIN-GÖKŞEN, 2Nesrin GÖKHAN-KELEKÇİ, 1Gülberk UÇAR 1Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 06100 Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Ankara, Turkey

It has been reported that some benzoxazolinone, triazole, thiadia-zole and hydrazone derivatives showed significant analgesic and an-tiinflammatory activities [1-3]. It has been speculated that non-ster-oidal antiinflammatory agents (NSAIDs) can act as the free radical scavengers and show antioxidant activity. It is also well documented that oxidative stress can play an important role in the side effects of many xenobiotics including NSAIDs. Therefore, in the present study, it was thought that the combination of different pharmacophores in one frame may lead to compounds with interesting pharmacologi-cal profiles and the effects of thiosemicarbazide, triazole, thiadiazole and hydrazone bearing 2-benzoxazolinone were investigated on free radical scavenging activity.

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Figure 1.

Thiosemicarbazide, triazole, thiadiazole and hydrazone deriva-tives (Figure 1) were synthesized according to a previous method [4]. Antioxidant activities of the novel compounds were evaluated by the determinations of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, superoxide radical scavenging activity, hydrogen peroxide scavenging activity, nitric oxide scavenging activity and re-ducing power. The various antioxidant activities were compared to standard antioxidants such as butylated hydroxytoluen and ascorbic acid and the results were expressed as IC50 values [5,6].

All of the newly synthesized compounds exhibited varying degrees of scavenging capacity on different active radicals; but compounds 13-18 (hydrazone derivatives) proved to be most active derivatives. Among those, compound 17 and 18, having p-methoxy and p-hy-droxyl groups on their phenyl rings, respectively showed the highest scavenging capacity of active radical species. These preliminary in vitro results may contribute to explain the potency of antiinflamma-tory activity of the compounds.

1. Köksal M, Gökhan N, Küpeli E, Yeşilada E, Erdoğan H. Synthesis, an-algesic and antiinflammatory properties of certain 5-/6-acyl-3-(4-sub-stituted-1-piperazinylmethyl)-2-benzoxazolinones derivatives, Arch. Pharm., 338, 117-125, 2005.

2. Labanauskas L, Kalcas E, Udrénaité E, Gaidelis P, Brukštus A, Daukšas, V. Synthesis of 3-(3,4-dimethoxyphenyl)-1H-1,2,4-triazole-5-thiol and 2-amino-5-(3,4-dimethoxyphenyl)-1,3,4-thiadiazole derivatives exhibit-ing anti-inflammatory activity, Pharmazie, 56, 617-619, 2001.

3. Sondhi SM, Dinodia M, Kumar A. Synthesis, anti-inflammatory and an-algesic activity evaluation of some amidine and hydrazone derivatives, Bioorg. Med. Chem., 14, 4657-4663, 2006.

4. Salgın U et al. Unpublished data 5. Ferreira ICFR, Baptista P, Vilas-Boas M, Barros L. Free-radical scaveng-

ing capasity and reducing power of wild edible mushrooms from north-east Portugal: individual cap and stipe activity, Food Chem., 100, 1511-1516, 2007.

6. Kumaran A, Karunakaran RJ. In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India, LWT, 40, 344-352, 2007.

O 11 Ref: 0108

STUDIES ON HISTONE DEACETYLASE INHIBITORY ACTIVITY OF SOME CARBOXYLIC ACID DERIVATIVES AND THEIR STRUCTURE-ACTIVITY RELATIONSHIPS 1Gamze BORA, 1Didem DAYANGAÇ-ERDEN, 2Peruze AYHAN, 3Kemal YELEKÇİ, 4Sevim DALKARA, 2Ayhan DEMİR, 1Hayat ERDEM-YURTER 1Hacettepe University, Faculty of Medicine, Department of Medical Biology, Ankara, Turkey2Middle East Technical University, Department of Chemistry, Ankara, Turkey3Hadir Has University, Faculty of Arts and Sciences, İstanbul, Turkey4Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey

Histone deacetylation is responsible for transcriptional repres-sion whereas histone hyperacetylation facilitates gene expression. Therefore histon deacetylating enzymes (HDACs) are popular tar-gets in drug development and HDAC inhibitors are potential drug candidates for cancer, inflammation and some single gene disorders caused by splicing defects of mRNA.

During the past 15 years, a number of structurally diverse HDAC inhibitors have been identified including short chain carboxylic ac-ids. To date, a limited number of compounds such as butyric acid (BA), phenylbutyric acid (PBA) and valproic acid (VPA) are in phase trails for the treatment.

In this study, we designed eighteen BA, PBA and VPA analo-gous to understand the structural requirements for HDAC in-hibitory activity. To develop structure-activity relationships the modifications listed below are realized in the molecules: . increase or decrease the length of alkyl chain, . branching in the alkyl chain, . introduction of double bond and hydroxyl group into the chain, . replication of carboxylic group, . amino acid analogy and . isosteric replacements.

HDAC inhibition activities of these molecules were investigated in vitro by using HeLa nuclear extract in a fluorimetric assay. Sodium butyrate was used as reference compound to compare the effects of molecular modifications on HDAC inhibitory activities. Activity re-sults were evaluated to establish structure-activity relationship.

AcknowledgementThis project was supported by The Scientific & Technological Research

Council of Turkey, Project No: 105G014.

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P 001 Ref: 0003

PRICEITE DOES NOT INDUCE GENOTOXICITY IN HUMAN LYMPHOCYTES IN VITRO Hasan TÜRKEZ, Fatime GEYİKOĞLUAtatürk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, Turkey

Boron does not exist by itself in nature. This element combines with oxygen and other elements to form boric acid, or inorganic salts called borates. People need borates, too, as an important part of a healthy diet and an essential ingredient in many products necessary for an acceptable standard of living. And borates frequently used in indus-trial, cosmetic, and medical settings. Priceite also (or pandermite = Ca4B10O19.7H2O) is one of the most important commercial boron compounds produced in large. The aim of the present study was to investigate the ability of priceite to induce sister-chromatid exchange (SCE) and micronucleus (MN) in cultured human lymphocytes.

With this aim, whole heparinized blood samples were taken from eight healthy non-smoking donors. Thirteen experimental concen-trations of priceite were used, ranging from 5 to 500 mg/L.

The peripheral blood cultures which were applied 400 and 500 mg/L of priceite was found to be sterile. Treatment with priceite did not cause an increase in the frequency of SCE per cell at all other concentrations. Moreover, there were no significant aneugenic and/or clastogenic effects observed in the micronucleus assay.

Our results firstly indicated that priceite is not a genotoxic agent in human blood cultures and safe for use in medical and cosmetic applications.

P 002 Ref: 0004

THE EFFECTS OF SOME LICHEN SPECIES AGAINST SISTER-CHROMATID EXCHANGE FREQUENCY INDUCED BY TITANIUM DIOXIDE IN HUMAN LYMPHOCYTES Fatime GEYİKOĞLU, Hasan TÜRKEZAtatürk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, Turkey

Despite the increasing use of factory-made synthetic drugs, natu-ral healing materials have persisted as the treatment of choice for a multitude of health problems in populations throughout the world. Investigations of genotoxicity and anti-genotoxicity can help evalu-ate the safety and effectiveness of herbal health products. Titanium dioxide (TiO2) is widely used in the pharmaceutical and cosmetic industries. It is also used for sterilization of waste water. The pur-pose of this work was to evaluate the effects of four lichen species (Dermotocopon intestiniformis, Pseudevernia furfuracea, Rhizoplaca melanophthalma and Xanthoparmelia pulla) against the genotoxicity induced by titanium dioxide (TiO2) for the first time.

With this aim, whole heparinized blood samples were taken from three healthy non-smoking donors. TiO2 was added to the cultures in concentrations of 5 and 10 µM. After the application of TiO2 and lichen extracts, seperate and together, the sister-chromatid exchange (SCE) test was used to assess DNA damage in lymphocytes.

None of the lichen extracts showed a significant genotoxicity alone. The extract of X. pulla did not show anti-genotoxic activity against the genotoxic effects induced by TiO2. However, D. intestiniformis, P. furfuracea, R. melanophthalma extracts caused significant decreases in titanium-induced SCE frequencies as dose dependent manner. The potency of anti-genotoxic activity was also in the following or-der: P. furfuracea > R. melanophthalma > D. intestiniformis.

Our findings indicated that lichens can be a new resource of therapeu-tic potential as recognized here against to adverse effects of drugs used.

P 003 Ref: 0005

IN VITRO EFFECTS OF ALUMINUM SULPHATE ON SISTER- CHROMATID EXCHANGES AND OXIDATIVE STRESS IN HUMAN BLOOD: PROTECTIVE ROLE OF BISMUTH SUBNITRATE Fatime GEYİKOĞLU, Hasan TÜRKEZAtatürk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, Turkey

Aluminum, which is found in several different forms and oxida-tion states, causes acute and chronic adverse health effects. Medici-nally, the treatment methods with bismuth compounds especially bismuth subnitrate (BSN) (as astringents, antacides, antiulcers and antidiarrheals) have been increased. The goal of the present study was to evaluate the genetic and oxidative effects of aluminum sul-phate (Al2(SO4)3) and BSN in human blood in vitro.

The various concentrations of Al2(SO4)3 (10 and 20 µg/mL) and BSN (0.75, 1.5, 3 and 5 µg/mL) were used. Evaluation of DNA dam-ages was carried out by using Sister-Chromatid Exchange (SCE) method in blood lymphocytes. Oxidative status of erythrocytes was assessed by measuring following oxidative stress markers: reduced glutathione (GSH), superoxide dismutase (SOD), glucose-6- phos-phate dehydrogenase (G6PDH) and catalase (CAT).

The SOD activity increased by Al2(SO4)3 (10 µg/mL) exposure but the ratio of SCEs didn’t change compared to the controls. On the other hand, the high dose of Al2(SO4)3 (20 µg/mL) caused oxidative stress and increased SCE frequency. When the concomitant treat-ment with Al2(SO4)3 of BSN were investigated, BSN exerted an anti-oxidant action at low doses (0.75 and 1.5 µg/mL). It also reduced the formation of SCEs.

This study suggests for the first time that BSN may be admin-istered as a potential protective against the effects of Al2(SO4)3 in which oxidative and genetic damages are clearly involved.

P 004 Ref: 0014

GST-CDNB ACTIVITIES IN GILTHEAD SEABREAM (SPARUS AURATA) LIVER CYTOSOL OF DIFFERENT AGES 1Hatice ARDAĞ-AKDOĞAN, 2Alaattin ŞEN1Pamukkale University, Science and Arts Faculty, Department of Chemistry, 20017 Kınıklı, Denizli, Turkey2Pamukkale University, Science and Arts Faculty, Department of Biology, 20017 Kınıklı, Denizli, Turkey

Lipophilic compounds such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, nitroaromatics, dioxins, drugs, various pesticides and natural residual are readily taken up into the tissues of aquatic organisms where biotransformation via Phase I and Phase II metabolism can in part, determine the fate and toxicity of the xenobiotics. The glutathione S-transferases (GST) represent a ma-jor group of detoxification enzymes. GSTs are a family of phase II detoxification enzymes. It is known that the important changes in drug metabolism occur with ageing because the various factors that have significant influences on xenobiotic metabolizing enzymes are altered by ageing and season.

In this study, gilthead seabream (Sparus aurata) fish liver cytosol of different ages were used as sample. The fish used in this study, Gilthead Seabream (Sparus aurata), were bought from Pinar Fish in İzmir, Aegean coast of Turkey. Glutathione S-transferase (GST) were determined from this Gilthead Seabream (Sparus aurata) fish livers cytosoles.

Though various factors that may have a significant impact on drug metabolism are affected by ageing, our results suggest that some im-

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portant age-related differences in xenobiotic metabolism do occur in Gilthead Seabream liver and are substrate specific, which might af-fect obtaining desired actions and/or responses to drugs, hormones and dietary supplements used during breeding.

GST-CDNB activity increased in gilthead seabream (Sparus au-rata) liver cytosol of different ages (ranging from 1.5 to 20 months). 1.5 months of the fish activity is 17,7 ± 0,5 pmol/dakika/mg protein, 20 months of the fish is 690,9 ± 32,0 pmol/dakika/mg protein.

P 005 Ref: 0015

THE PROTECTIVE ROLE OF PSEUDOVERNIA FURFURACEA AGAINST COLLOIDAL BISMUTH SUBCITRATE – INDUCED GENOTOXICITY IN HUMAN LYMPHOCYTE CULTURES Hasan TÜRKEZ, Fatime GEYİKOĞLUAtatürk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, Turkey

Bismuth compounds, especially, colloidal bismuth subcitrate (CBS) have been actively promoted for the treatment of diarrhoea, peptic and duedonal ulcer diseases. And the therapeutic bismuth compounds are now being marred by episodes of serious adverse reactions. On the other hand, the potential of lichens in cellular ac-tivities remains largely unexplored. The aim of this study was to as-sess the efficacy of the lichen Pseudovernia furfuracea (2.5, 5, 10 and 20 µg/mL) on the genotoxicity induced by CBS in the human blood cultures.

With this aim, whole heparinized blood samples were taken from three healthy non-smoking donors. Sister-chromatid exchanges (SCE) and Micronuclei (MN) tests were used for evaluating the ge-notoxicity.

SCEs and MN formations significantly increased by effect of 5 µg/mL CBS when compared with the the control group. However, the decreased rates of such formations indicated that P. furfuracea was anti-genotoxic agent. Our results also showed that the protective role of P. furfuracea was dose-related.

On the basis of data, it is thought that this lichen species can pro-vide anti-genotoxic effects as due to their antioxidant defenses al-though there is no evidence for the content of the lichen species in the present investigation.

P 006 Ref: 0016

QSAR MODELING ON SIGMA RECEPTOR LIGANDS Mine YARIM, Ece GÜRDAL, Dilek EROLYeditepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34755 İstanbul, Türkiye

Sigma (σ) receptors are functional, membrane-bound proteins distributed throughout the brain and peripheral organs. σ1 and σ2 receptor types are clearly established, and further pharmacologi-cal differentiation may be possible. σ1 receptors are implicated in central nervous system (CNS) disorders such as depression, schizo-phrenia and dementia. Further, σ1 receptor agonists have value as neuroactive agents, while antagonists may help alleviate cocaine ad-diction. The significance of the σ receptor system to human health is augmented through the overexpression of σ sites by a number of cancers. Thus, σ receptor ligands might be used for the detection and treatment of malignant neoplastic diseases. σ2 receptors may be of particular prognostic relevance, as the extent of their expression seems indicative of the proliferative status of tumors [1].

Structurally, σ ligands are represented by wide variety of chemical scaffolds, for example (+)-benzomorphans, phenylpiperidines, (+)-pentazocine and haloperidol [2].

Corbera et al. synthesized a series of novel tetrahydroindazole de-rivatives and tested for σ1 and σ2 receptor binding [3].

Since sigma receptors are membrane-bound proteins, isolating and resolving their three-dimensional structure has proven to be dif-ficult. QSAR (quantitative structure-activity relationship) methods assume that biological activity is correlated with chemical structure or properties and that as a consequence activity can be modeled as a function of computable physicochemical attributes. QSAR tech-niques are able to raise a predictive description of global structural requirements for interactions between substrates and receptor by us-ing binding data.

We have described a detailed QSAR study on tetrahydroindazole series, in order to give better picture of action and to rationalize se-lection of substituents.

The QSAR functions in the Molecular Operating Environment (MOE) and SYBYL were used to compute theoretical molecular de-scriptors related to physicochemical properties.

1. Lever JR, Gustafson JL, Xu R, Allmon RL, Lever SZ. Synapse, 59, 350-358, 2006.

2. Vangveravong S, Xu J, Zeng C, Mach RH. Bioorg. Med. Chem., 14, 6988-6997, 2006.

3. Corbera, J. et al. Chem. Med.Chem., 1, 140-154, 2006.

P 007 Ref: 0017

SYNTHESIS AND DNA-CLEAVING ACTIVITY OF A SERIES OF SUBSTITUTED ARENEDIAZONIUM IONS Murat KIZIL, Bircan ÇEKENDicle University, Faculty of Science and Art, Department of Chemistry, 21280 Diyarbakır, Turkey

Aryl radicals and biradicals are generated in biological systems as intermediates of some drugs targeted DNA. Cleavage of DNA via radical attack plays an important role in various biological processes, including chemoteraphy and carcinogenesis. Some antitumor drugs generate aromatic biradicals that are belived to cleave double-strand-ed DNA via hydrogen atom abstraction from the sugar moiety. How-ever, the chemical behavior of substituted phenyl radicals toward DNA has not been extensively investigated. We have investigated the ability of causing the supercoiled DNA cleavage and the free radical formation of the substituted aryl radicals and aryl cations derived from arenediazonium ions.

We prepared the substituted arenediazonium tetrafluoroborates in 22-74 % yields by reaction of the corresponding amine with isoamyl nitrite and aqueous fluoroboric acid in ethanol. The plasmid pBlue-script M13+ DNA was prepared and isolated according to stand-ard protocols using Qiagene plasmid mini preparation kit. Gel was scanned on Gel documentation system (Gel-Doc-XR, BioRad, Her-cules, CA, USA). Bands on the gels were quantified using discovery series Quantity One programme (version 4.5.2, BioRad Co.).

Systematic studies indicate that both aryl radicals as well as aryl cation participates in the DNA cleavage pathway.

It has been found that the substituted arenediazonium salts have capacity to cleave supercoiled DNA to form the open circular Form II DNA and linear Form III DNA.

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P 008 Ref: 0020

ANTIDEPRESSANT-LIKE EFFECT OF SOME NEW ARYL PROPANE DERIVATIVE COMPOUNDS 1Fulya MORAL, 1Meriç KÖKSAL, 1Mine YARIM, 2S. Sırrı BİLGE, 2S. Elif AKSÖZ 1Yeditepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34755 İstanbul, Turkey2Ondokuz Mayıs University, Ondokuz Mayıs University, Faculty of Medicine, Department of Pharmacology, 55139, Kurupelit, Samsun, Turkey

In the early 1970s, evidence of the role of serotonin (5-hydroxytry-tamine or 5-HT) in depression began to emerge and the hypothesis that enhancing 5-HT neurotransmission would be available mecha-nism to mediate antidepressant response was put forward. On the basis of this hypothesis, efforts to develop agents that inhibit the up-take of 5-HT from the synaptic cleft were initiated [1].

In recent years, selective serotonin reuptake inhibitors (SSRIs) have become a standard treatment because of their safety profile and few-er side effects than the other classes of antidepressants. Fluoxetine, (±)-N-methy-3-phenyl-3(α,α,α-trifluoro-p-tolyoxy)propylamine hydrochloride, is a potent selective serotonin reuptake inhibitor used for the treatment of major depression which marketed under the well-known trade name Prozac® [2]. Furthermore, some of the undesired effects such as sexual dysfunction, gastrointestinal intol-erance and activating effects such as nervousness, anxiety and im-sonia are showed with all available SSRIs [3, 4]. Therefore, one of the still therapeutic needs is the availability of antidepressants with a more rapid onset of action and less side effects.

In view of these literature results, we aimed modifications on Fluoxetine structure by changing the amine group with benzylpipe-ridine group to reach new antidepressant compounds with a faster onset of action, a better efficacy and less side-effects. The antide-pressant-like effect of this synthesized compounds was studied in comparison with other antidepressants (Fluoxetine, Sertraline, Imi-pramine) in forced swimming test (FST), a validated experimental model of depression in mice. According to antidepressant profile evaluation, three of six showed antidepressant-like effect some of which better than the standarts (fluoxetine, sertraline, imipramine) without changing any locomotor activity.

1. Wong DT, Perry KW, Bymaster FP. Nature Reviews Drug Discovery, 4, 764-774, 2005.

2. Takeuchi, K et al. Bioorg. Med. Chem. Lett., 13, 1903-1905, 2003. 3. Labbate A, Grimes JB, Arana GW. Biol. Psychiatry, 43, 904-907, 1998. 4. Takeuchi K, Kohn TJ, Honigschmidt NA, Rocco VP, Spinazze PG, At-

kinson ST, Hertel W, Nelson DL, Wainscott DB, Ahmad LJ, Shaw J, Threlkeld PG, Wong DT. Bioorg. Med. Chem. Lett., 13, 3939-3942, 2003.

P 009 Ref: 0023

COMPARATIVE ANALYSIS OF THE COFACTOR BINDING SITE OF PLASMODIUM VIVAX LACTATE DEHYDROGENASE WITH SOME OTHER KNOWN APICOMPLEXAN COUNTERPARTS 1Venhar ÇELİK, 2Dilek TURGUT-BALIK1University of Fırat, Faculty of Arts And Sciences, Department of Biology, 23169 Elazığ, Turkey2Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Davutpaşa Campus, 34210 Esenler-İstanbul, Turkey

Malaria is caused by the protozoan parasite Plasmodium from the phylum Apicomplexa that also includes the important pathogens Toxoplasma, Eimeria, Theileria, Cryptosporidium and Babesia. Ma-laria is one of the greatest causes of human misery and death. Despite continued efforts to control the disease, it remains a major health

problem in many regions of the world. The need for new antimalar-ials comes from the widespread resistance to those in current use. In this study, the use of parasite’s lactate dehydrogenase as an antima-larial drug target is evaluated. The cofactor binding site of Plasmo-dium vivax lactate dehydrogenase was compared to some other api-complexan counterparts by making a single residue change. Residue 163 is a leucine in Plasmodium, a serine in human and a methionine in other apicomplexans. Leucine 163 was replaced by methionine by site directed mutagenesis. It was observed that enzyme was tolerant when leucine 163 residue in its cofactor binding site was replaced with methionine like in its apicomplexan counterpart. This is a fea-ture that distinguishes Plasmodium and other apicomplexan lactate dehydrogenase enzymes from their human lactate dehydrogenase, supporting their suitability as targets in common drug design stud-ies.

P 010 Ref: 0024

MUTAGENIC ANALYSIS OF ACTIVE SITE LOOP OF LACTATE DEHYDROGENASE BY MIMICKING EIMERIA TENELLA IN PLASMODIUM VIVAX 1Venhar ÇELİK, 2Dilek TURGUT-BALIK1University of Fırat, Faculty of Arts and Sciences, Department of Biology, 23169 Elazığ, Turkey2Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Davutpaşa Campus, 34210 Esenler-İstanbul, Turkey

Malaria, the most prevalent and most pernicious parasitic diseases of humans, is estimated to kill approximately three million people each year. The increasing occurence of drug resistance in many en-demic areas emphasizes the need for antimalarial drugs. The gene encoding Plasmodium vivax lactate dehydrogenase is a functinal validation. We have isolated and cloned the lactate dehydrogenase, and overexpressed the protein from Plasmodium vivax. The en-zyme’s structure was also solved from P. vivax recently. Active site loop of the enzyme has been determined by crystal structure studies and shown to be an ideal site for the drug design studies. The active site loop of this enzyme was found to have a pentapeptide insertion and this insertion differentiates this enzyme from its human coun-terpart. Similar insertion sequence was also found in Eimeria tenella lactate dehydrogenase. It was observed in this study that replacing the pentapeptide insertion sequence in Plasmodium vivax with the insertion sequence from Eimeria tenella has unaffected the enzyme activity.

P 011 Ref: 0025

SYNTHESIS OF AMINO ALCOHOL BASED CHIRAL LIGANDS AND THEIR APPLICATIONS IN ENANTIOSELECTIVE DIETHYLZINC ADDITION TO BENZALDEHYDES Dilek Işık TAŞGIN, Canan ÜNALEROĞLUHacettepe University, Faculty of Science, Department of Chemistry, 06800 Beytepe-Ankara, Turkey

Among asymmetric catalysis of C-C bond-formation, the enanti-oselective addition of diorganozinc reagents to aldehydes in the pres-ence of a catalytic amount of a chiral ligand is a convenient method for the preparation of optically active secondary alcohols [1]. These structural features are important building blocks for the synthesis of many natural and biologically active compounds, and materials such as liquid crystals [2]. For this purpose, a wide variety of chiral cata-lysts, i.e. amino alcohols, amino thiols, diamines, disulfonamides,

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and diols have been synthesized [3]. Among the chiral ligands re-ported, β-amino alcohols are the most often used chiral ligands [4]. In this study, norephedrine based chiral ligands were synthesized starting from pyrrole carbaldehydes and norephedrine. The synthe-sized (1S,2R)-2-((1H-pyrrol-2-yl)methylamino)-1-phenylpropan-1-ol (1) and (1R, 2S)-2-((1H-pyrrol-2-yl)methyl amino)-1-phenyl-propan-1-ol (2) were used as chiral ligands in the enantioselective addition of diethylzinc to benzaldehyde.

Synthesis of Amino Alcohols: Synthesized imines (1.22 mmol) from pyrrolecarbaldehydes and norephedrine, and NaBH4 (1.35 mmol) in methanol was refluxed for 8 hours. When the reaction was completed, the mixture was cooled to room temperature and quenched with 15 mL water. It was extracted with ether (3x10 mL) and dried over MgSO4. The mixture was filtered and the solvent was evaporated. Crude products 1 and 2 were dissolved in benzene and filtered. The product was obtained after evaporation.

General Procedure for the Enantioselective Diethylzinc Addi-tion to Benzaldehyde: System was dried under vacuum by applying the Schlenk technique for five times. 0.05 mmol chiral ligand was dissolved in dry solvent (5 mL) under the argon atmosphere. Af-ter cooling to 0 oC, 2.2 mmol diethylzinc (1M in hexane) was added with a syringe over a period of 5 min. The mixture was stirred for 5 hours 0 oC and 1 mmol aldehyde was added. The reaction mixture was stirred for 16 hours at room temperature and monitored by TLC. The reaction was quenched with 5 mL of 1 N HCl solution and ex-tracted with ether and dried over anhydrous MgSO4 and the solvent was evaporated under reduced pressure. The crude product was pu-rified by flash column chromatography ( EtOAc:hexane, 1:6).

Amino alcohols were obtained by the reduction of imines with NaBH4 in methanol. Chiral ligands 1 and 2 were obtained in 65 % and 70 % yields, respectively. Characterization of these compounds was carried out by 1H-NMR and 13C-NMR techniques.

The enantioselective addition of diethylzinc to benzaldehyde was carried out in different solvents in the presence of 5 mol % of ligands under argon atmosphere at 0oC to room temperature. Catalytic ac-tivity of 1 and 2 were examined in toluene, hexane, toluene-hexane mixture and dichloromethane (Table 1). The best result was obtained with ligand 2 (70 % yield and 78 % ee) in toluene-hexane solvent mixture.

Table 1. The addition of diethylzinc to benzaldehyde with ligands 1 and 2.

entry ligand solvent yield (%) ee (%) conf.

1 1 Toluene 36 71 S

2 1 Hexane 85 58 S

3 1 Tol.+Hex. 67 66 S

4 2 Toluene 35 74 R

5 2 Hexane 77 64 R

6 2 Tol.+Hex. 70 78 R

7 2 Dichloromethane 17 27 R

1. Roudeau R, Pardo DG, Cossy J. Tetrahedron, 62, 2388, 2006. 2. Melo RPA, Vale JA, Zeni G, Menezes PH. Tetrahedron Lett., 47, 1829,

2006. 3. Xu Q, Zhu G, Pan X, Chan ASC. Chirality, 14, 716, 2002. 4. Cicchi S, Crea S, Goti A, Brandi A. Tetrahedron:Asymmetry, 8, 293,

1997.

P 012 Ref: 0028

ELECTROCHEMICAL INVESTIGATION OF INTERACTIONS BETWEEN DNA AND SOME CHEMICALS 1Görkem YALÇIN, 2Murat ÇİZMECİOĞLU, 1Özlem SÖĞÜT, 1Mehmet ÖZSÖZ1Ege University, Faculty of Pharmacy, Department of Analytical Chemistry, İzmir, Turkey2Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, İzmir, Turkey

Determinations of interactions between DNA and drugs are im-portant aspects of biological studies in drug discovery and pharma-ceutical development processes.

The interactions of some molecules with DNA have been inves-tigated by a variety of techniques. There has been growing interest to determine interacts between DNA and some molecules by using electrochemical methods.

In this study, interactions of some chalcone derivatives with DNA were investigated by using electrochemical methods. In recent years chalcone (1,3-diphenyl-2-propen-1-one) derivatives have been synthesized in order to develop active compounds against cancer, malaria, leishmaniase, tuberculosis and cardiovascular diseases. Therefore, determination of interactions between some chalcone derivatives and DNA will give some help to chalcone based drug de-velopment studies.

P 013 Ref: 0031

GLYCOSYLATION IN ROOM TEMPERATURE IONIC LIQUID (RTIL) USING UNPROTECTED AND UNACTIVATED DONORS 1Sultan N. BAYTAŞ, 2Tae-joon PARK, 1Robert J. LINHARDT1Rensselaer Polytechnic Institute, Department of Chemistry and Chemical Biology, Troy, NY, USA2Rensselaer Polytechnic Institute, Department of Chemical and Biological Engineering, Troy, NY, USA

Glycosylation occurs between a donor and an acceptor in the pres-ence of a promoter, which activates the donor. There are various fac-tors that need to be considered when carrying out the synthesis of glycosides including the manipulation of protecting groups in both

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donor and acceptor, the architecture of donor and acceptor and the solvent system [1]. These strategies involve often a large number of synthetic steps. The chemical synthesis of unprotected carbohydrates poses a number of challenges, including poor solubility in most con-ventional organic solvents. Only very polar organic solvents, such as formamide, dimethylformamide, dimethylsulfoxide, and pyridine, easily dissolve significant amounts of sugars. It is important to in-vestigate new solvent systems that dissolve carbohydrates, support glycosylation reactions of unprotected sugars, and facilitate product recovery. Room temperature ionic liquids (RTILs) are becoming increasingly used as solvents for a wide variety of chemical reac-tions [2]. RTILs also display desirable solvent properties and have the potential of replacing conventional volatile organic solvents in carbohydrate chemistry. In this work, we report the glycosylation of various simple, unprotected monosaccharides in RTILs to give ben-zyl glycosides, disaccharides and oligosaccharides. RTILs facilitate the use of unactivated and unprotected donors in these reactions, re-sulting in a simple synthetic strategy involving a single glycosylation step. The synthesis of galactose oligomers was also possible using these novel solvents.

1. Sasaki K, Nagai H, Matsumura S, Toshima K. A novel greener glycosida-tion using an acid-ionic liquid containing a protic acid, Tetrahedron Lett., 44, 5605-5608, 2003.

2. Murugesan S, Linhardt RJ. Ionic liquids in carbohydrate chemistry - cur-rent trends and future directions, Curr. Org. Synth., 2, 437-451, 2005.

P 014 Ref: 0033

DEVELOPMENT OF VALIDATED METHOD FOR RISEDRONATE BY HPLC-MS MS FROM BIOLOGICAL MATERIAL 2Zeynep İrem DİLER, 2Gülay ŞAHİN-KOÇ, 2Hüseyin YALÇINKAYA, 1Durişehvar ÖZER-ÜNAL, 1Dilek EROL1Yeditepe University, Faculty of Pharmacy, Department of Analytical Chemistry, İstanbul, Turkey2Yeditepe University, Yeditepe Sağlık Hizmetleri A.Ş., GLP Laboratory, Acıbadem-İstanbul, Turkey

Risedronate (RS) is a third generation of bisphosphonate class of drugs. It is widely used for the treatment of bone disorders such as osteopotrosis. Determination of RS from biological fluids have dif-ficulties because of its low level in urine and blood. A sentitive and reliable HPLC-MS MS method was developed and validated from human urine.

The extraction method was developed to analyse RS from bio-logical material. In this method TMS-diazomethan derivatization was used followed by solid phase extraction.The mobile phase was MeOH:H20 (80:20; v/v) containing 0.1 % formic acid. The best reso-lution was obtained by using Agilent Zorbax Eclipse XDB reversed phase C18 (150x4.6mm, 5µm) and alendronate was used as an inter-nal standart.

The developed method was applied succesfully to biological ma-terial. The limit of quantitation of RS from biological material was 5 ng/ml. The calibration curve was linear in between 5-400 ng/ml. Precision, recovery, accuracy and stability results were satisfactory for the method developed. The method is suitable for routine analy-sis of bioequivalence studies.

1. Zhu LS., Lapko VN., Lee JW et.al. Rapid Commun. Mass Spectrom., 20(22), 3421-6, 2006.

2. Vallano PT., Shugards SB., Kline WF. et. al. J. Chromatogr. B, 794, 23-33, 2003.

P 015 Ref: 0034

AMINO TERMINAL ANALYSIS OF THE ACTIVE SITE LOOP OF LACTATE DEHYDROGENASE FROM THE MALARIA PARASITE PLASMODIUM VIVAX 1Bünyamin ATMIŞ, 1Dilek SADAK, 1Venhar ÇELİK, 2Dilek TURGUT-BALIK1University of Fırat, Faculty of Arts And Sciences, Department of Biology, 23169 Elazığ-Turkey2Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Davutpaşa Campus, 34210 Esenler-İstanbul,Turkey

Malaria is parasitic disease that threatens nearly half the global population. It is caused by protozoan apicomplexan parasites of the genus Plasmodium; four species cause malaria in man. In this study, amino acid exchanges were made in the amino terminal end of the active site loop of Plasmodium vivax lactate dehydrogenase (LDH) by mimicking Toxoplasma gondii I ve II, Eimeria acervulina, Eimeria tenella ve Theileria parva LDH’s using the site directed mutagenesis method. Although enzymatic activity was decreased compared to that of the wild type protein, some enzymatic activity was present meaning that enzyme was still in contact with its substrate. Decrease in the enzymatic activity indicates that this region is sensitive to changes and this supports the idea that this site could be evaluated as an ideal target for the drug design studies for both Plasmodium and some other apicomplexans.

P 016 Ref: 0035

AN ANALYSIS TO BE USED IN SCTRUCTURE-BASED DRUG DESIGN STUDIES: COMPARISON OF ACTIVE SITE LOOPS OF ENZYMES, PLASMODIUM VIVAX AND TOXOPLASMA GONDII LACTATE DEHYDROGENASES 1Venhar ÇELİK, 2Dilek TURGUT-BALIK1University of Fırat, Faculty of Arts and Sciences, Department of Biology, 23169 Elazığ, Turkey2Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering , Davutpaşa Campus, 34210 Esenler-İstanbul, Turkey

Increasing resistance of Plasmodium to the antimalarial agents necessitates the development of new drugs which have different modes of action from the currently existing ones. Present study is targeted to lactate dehydrogenase (LDH) which is a key anaerobic metabolic pathway enzyme. LDH of Plasmodium has strikingly dif-ferent properties compared to its human counterparts. Inhibition of the activity of this enzyme has been shown to kill the parasites in the erythrocytes. Thus, we have isolated and cloned the LDH gene, and overexpressed the protein. The structure of the enzyme from P. vivax (PvLDH) recently reported. A five amino acids insertion in the ac-tive site formed a distinctive cleft on the surface of the PvLDH as did in P. falciparum LDH. This site has been identified as a potential tar-get for the structure based drug design studies. The site is not unique to Plasmodium. The five amino acid insertion was also observed in some other apicomplexan parasites. This site was analysed by mak-ing residue exchanges in the P. vivax LDH active site loop to mimick the same region of Toxoplasma gondii LDH1. It was observed that making residue exchanges in the active site loop of PvLDH was pos-sible without losing enzymatic activity. This observation emphasizes that the active site loop is a crucial region accross the apicomplexan LDHs.

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P 017 Ref: 0036

INTERACTION OF SOME 3,4-DIHYDROQUINOLIN-(1H)-2-ONE DERIVATIVES WITH RAT LUNG SEMICARBAZIDE-SENSITIVE AMINE OXIDASE (SSAO) 1Samiye YABANOĞLU, 2Sevil Görkem SUNAL, 2Akgül YEŞİLADA, 1Gülberk UÇAR1Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 16100 Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, 06100 Ankara, Turkey

Since semicarbazide-sensitive amine oxidase (SSAO) is shown to be involved in diabetes, Alzheimer’s and Parkinson diseases, heart and vascular diseases, the synthesis of new compounds as specific SSAO inhibitors suggested to be useful developing novel therapeutic agents. In view of the previous studies indicating that diazoheterocy-clic compounds have been introduced as promising class of revers-ible amine oxidase inhibitors, three compounds with 3,4-dihydro-quinoline-(1H)-2-one structure and their open ring derivatives were synthesized and the interaction of these compounds with SSAO pu-rified from rat lung were evaluated.

The compounds of Q (N-amino-3,4-dihydroquinoline-(1H)-2-one) and QB (1-(Benzyliden-amino)-3,4-dihiydroquinoline-(1H)-2-one) were synthesized by the reduction and the ring closure reaction of o-nitro-cinnamic acid whereas MBK (Tert-butyl-N-[cyclohexyl-carbamoyl-(3-hydroxyphenyl)methyl]-N-phenyl-carbamate), MG (Tert-butyl-N-[cyclohexylcarbamoyl-(3-hidroxyphenyl)methyl]-N-(2-benzoylphenyl)-carbamate) and PCN (2-(3-cyano-2-oxo-4-phe-nyl-2H-quinolin-1-yl-N-cyclohexyl-2-(4’-chlorophenyl)acetamide) were synthesized by one pot Ugi-Knovenagel reaction. The struc-tures of the novel compounds were evaluated using 1H NMR, 13C NMR and MS techniques.

Compound Q, which carries a free amine group appeared as a good substrate for rat liver SSAO suggesting that this relatively small compound may interact with the active site channel of the enzyme through its free amine group. QB, PCN and MG inhibited the en-zyme non-competitively and irreversibly suggesting that these com-pounds may interact with an another hydrophobic binding region outside of the active site of the enzyme. It is concluded that these compounds may have promising features as novel anti-parkinson/anti-Alzheimer agents in case their SSAO inhibitory effects can be supported by in vivo studies.

Keywords: 3,4-Dihydroquinoline-(1H)-2-ones, tissue-bound semicar-bazide-sensitive amine oxidase (SSAO), Ugi-Knovenagel condensation, substrate, inhibition

P 018 Ref: 0037

EFFECTS OF LIPOIC ACID AND VITAMIN C ADMINISTRATION ON STREPTOZOTOCIN INDUCED DIABETIC RAT LIVER CATALASE ACTIVITIES Gökhan SADİ, Tülin GÜRAYMiddle East Technical University, Department of Biochemistry, Ankara, Turkey

Diabetes mellitus which is a glucose metabolism disorder is as-sociated with consequences of oxidative stress due to non-enzymatic protein glycation, glucose autoxidation and polyol pathway, which augments the free radical production. In the tissues, cells evolve en-zymatic and non-enzymatic antioxidants to defend themselves for the oxidation potential of radicals. Superoxide dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx) are the major antioxidant enzymes, and reduced glutathione (GSH), ascorbic acid

(Vit C) and tocopherol (Vit E) are the major antioxidant molecules combating the bed effects of free radicals. Catalase, one of the major antioxidant enzyme, neutralize the hydrogen peroxide (H2O2) pro-duced form superoxide radical by dismutases.

In this study, effects of one water soluable antioxidant, ascorbic acid (Vit C), and one lipid soluable antioxidant, α-lipoic acid, on diabetic rat liver catalase activities were aimed to be studied. Furthermore, effects of both antioxidants given together were also analyzed. To do this, male Sprauge-Dawley rats were given streptozotocin (STZ) to induce diabetes, and groups were seperated as control (n=9), diabet-ic (n=9), diabetic+lipoic acid given (n=8), diabetic+vitamin C given (n=12) and diabetic+vitC and lipoic acid given (n=7). Four weeks after the development of diabetes, rats were decapitated and catalase activites were measured. It has been observed that catalase activities were significantly lowered in diabetic group (p<0.005) as compared to controls. Application of lipoic acid were increased the diabetic CAT activities but not up to the control level. Similarly, vitamin C rised the diabetic catalase activities and we observed that vitamin C was better for the restoration of diabetic CAT activities. Moreo-ver, combined antioxidant given groups’ catalase activities were also higher than those of the diabetics and this increament was as effec-tive as the one of the vitamin C group.

In conclusion, due to the increased oxidative stress, catalases are damaged or inhibited by the actions free radicals and administration of antioxidants help to reduce these side effects in streptozotocin in-duced diabetes.

P 019 Ref: 0038

SYNTHESIS AND ANALGESIC ACTIVITY OF SOME BENZIMIDAZOLE DERIVATIVES 1İlhan IŞIKDAĞ, 2Ümide DEMİR, 2Özgür Devrim CAN, 1Yusuf ÖZKAY, 2Yusuf ÖZTÜRK1Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskişehir, Turkey2Anadolu University, Faculty of Pharmacy, Department of Pharmacology, 26470 Eskişehir, Turkey

Heterocyclic compounds having two nitrogen atoms oriented in 1,3-positions in ring are endowed with broad spectrum of phar-maceutical properties. Imidazole and benzimidazole, shown as the instances for these heterocyclics, drugs have broaden scope in rem-edying various dispositions in clinical medicine [1]. Pharmaceutical properties concern antifungal and antimycotic, antiprotozoal, anti-neoplastic, antiulcer, antihistaminic and antiallergic, antihyperten-sive, anthelmintic, antioxidant, antiviral, antibacterial and antipara-sitic activities, all of which are unique characteristics known from imidazole and benzimidazole derivatives [2]. It is known well that synthetic chemical compounds especially lipophylic ones have vari-ous different effects on central nervous system. Benzimidazoles are examples of these derivatives with their reported pharmacological effects such as anesthetic and hypnotic[3], neuroleptic and antipsy-chotic [4], analgesic [5] and sedative [6] etc. These large research areas of benzimidazoles promted us to study with them.

We synthesized five 2-aryl-4,5-dichloro-(1H)-benzimidazoles via the condensation of 4,5-dichloro-o-phenylenediamine and cor-responding aldehyde derivatives in ethanol with the presence of sodium bisulfite. Their structures were elucidated with 1H-NMR, IR and MASS(Apci) spectral analysis. Tail-clip and tail-immersion tests, were applied in order to investigate probable analgesic effects. Synthesized compounds were applied to mices at a dose of 100 mg/kg (i.p). Morphine (1 mg/kg) was used as positive standart.

All of the synthesized compounds showed analgesic activities in tail-clip and tail-immersion tests. Compound 2 was found as the

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most active derivative in the series. 1H-NMR and IR spectral datas were obtained as expected. Mass (Apci) spectras of the compounds were agreed well with their molecular weight. The compounds (compounds 1, 2 and 4) including metoxy groups at metha posi-tions and/or hydroxyl group at para position of phenyl ring which is substituted at second position of benzimidazole ring, have higher analgesic activity. On the other hand, addition of nitro group at or-tho position of phenyl ring (compound 3) causes decrease in the an-algesic activity. These findings indicate the importance of chemical sturucture and pharmacological activity relationships of the synthe-sized compounds. In conclusion, it may be suggested that to obtain more active derivatives, containing the same structure with the title compounds, number of synthesis including substituted-p-hydroxy-benzaldehyde and substituted-m-methoxybenzaldehyde derivatives should be increased.

1. Kleeman A, Engel J, Kutscher B, Reichert D, Pharmaceutical Substances, 3rd ed.; Stuttgart: New York, 1999.

2. Nezhad AK, Rad MNS, Mohabatkar H, Asraria Z, Hemmateenejada B, Bioorg. Med. Chem., 13, 1931, 2005.

3. Janssen PAJ, Niemegeers CJE, Schellekens KHL, Lenaerts FM, Arzneim. Forsch., 21, 1234, 1971.

4. Sato M, Arimoto M, Ueno K, Kojima H, Yamasaki T, Sakurai T, Kasahara A, J. Med. Chem. 21, 1116, 1978.

5. Sondhi SM, Singh N, Kumar A, Lozach O, Meijer L, Bioorg. Med. Chem., 14, 3758, 2006.

6. Seredenim SB, Eur. Neuropsychopharm., 6, 111, 1996.

P 020 Ref: 0039

STUDIES ON THE SYNTHESIS AND ANTIPROLIFERATIVE ACTIVITY INVESTIGATION OF 2,2’-(3-METHOXYPHENYL) AND 2,2’-(3-HYDROXYPHENYL)-1H,1H’-[5.5’]-BIS-BENZIMIDAZOLES 1İlhan IŞIKDAĞ, 1Yusuf ÖZKAY, 2Zerrin İNCESU1Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskişehir, Turkey2Anadolu University, Faculty of Pharmacy, Department of Biochemistry, 26470 Eskişehir, Turkey

The incorporation of the imidazole and benzimidazole nuclei is an important synthetic strategy in drug discovery [1]. Previous observations suggest that substituted benzimidazoles and related heterocycles, which are the structural isosters of nucleotides ow-ing fused heterocyclic nuclei in their structures that allow them to interact easily with the biopolymers, possess potential activity with lower toxicities in the chemotherapeutic approach in man [2, 3]. The high therapeutic properties of the related drugs have en-couraged the medicinal chemists to synthesize the large number of novel chemotherapeutic agents. Antitumoral activities of benzimi-dazole and bisbenzimidazole compounds were reported in several studies. Furthermore, there are clinical anticancer drugs, known as Hoechst-33258 and Hoechst-33342 dyes including bisbenzimida-zole structure [4, 5]. Prompted above observations we synthesized the title compounds to investigate their possible antiproliferative effects.

We synthesized two bis-benzimidazoles via the condensation of 3,3’-diaminobenzidine and corresponding aldehyde derivatives in ethanol with the presence of sodium bisulfite. Their structures were elucidated with 1H-NMR, IR and MASS(Apci) spectral analysis. Their antiproliferative effects of the compounds will be determined on HDQ-P1 and HT-29 cancer cell lines by using MTT and BrdU assays.

Both of the bisbenzimidazole derivatives were synthesized and their spectral datas were recorded. 1H-NMR and IR spectral datas

were obtained as expected. Mass (Apci) spectras of the compounds were agreed well with their molecular weight. Our synthesis with bisbenzimidazole compounds have recently begun. Further studies are in progress to increase the number of compounds with smilar structures. After reaching adequate number of the compounds, anti-proliferative activity scaning will be started.

1. Townsend LB, Chem. Rev., 67, 533, 1976. 2. Haugwitz RD, Angel RG, Jacobs GA, J. Med. Chem., 25, 969, 1982. 3. Hisano T, Ichıkawa M, Tsumoto K, Tasaki M, Chem. Pharm. Bull., 30,

2996, 1982. 4. Kamal A, Ramul P, Srinivas O, Ramesh G, Kumar PP, Bioorg. Med. Chem.

Lett., 14, 4791, 2004. 5. Alper S, Arpacı, ÖT, Akı, EŞ, Yalçın İ, Farmaco, 58, 497, 2003.

P 021 Ref: 0041

SYNTHESIS OF 3-PHENETHYLAMINO-1-PHENYL/SUBSTITUTED PHENYL-1-PROPANONE HYDROCHLORIDES 1Ebru METE, 2H. İnci GÜL, 1Cavit KAZAZ1Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey

It has been reported that acetophenone derived several mono Mannich bases have cytotoxic, antifungal and anticonvulsant activi-ties [1-5]. In this study, synthesis of 1-aryl-3-phenethylamino-1-pro-panone hydrochlorides has been realised to investigate their biologi-cal activities in future.

Chemical structure of the compounds were confirmed by 1H NMR, 13C NMR, UV, IR and MS spectroscopies. Purity level of them was determined by elemental analyses.

1. Gül Hİ, Vepsalainen J, Gül M, Erciyas E, Hanninen O. Cytotoxic activi-ties of mono and bis Mannich bases derived from acetophenone against Renca and Jurkat cells, Pharm. Acta Helv., 74, 393-8, 2000.

2. Gül Hİ, Gül M, Erciyas E. Syntheses and stability studies of some Man-nich bases of acetophenones and evaluation of their cytotoxicity against Jurkat cells, Arzneimittel Forschung, 52, 628-35, 2002.

3. Gül Hİ, Çalış Ü, Vepsalainen J. Synthesis of some mono-Mannich bases and corresponding azine derivatives and evaluation of their anticonvul-sant activity, Arzneimittel Forschung, 54, 359-64, 2004.

4. Gül Hİ, Şahin F, Gül M, Öztürk S, Yerdelen KO. Evaluation of antimi-crobial activities of several Mannich bases and their derivatives, Arch. Pharm., 338, 335-8, 2005.

5. Gül Hİ, Das U, Pandit B, Li PK. Evaluation of the cytotoxicity of some mono-mannich bases and their corresponding azine derivatives against androgen-independent prostate cancer cells, Arzneimittel Forschung, 56, 850-4, 2006.

P 022 Ref: 0042

SYNTHESIS OF 1-PHENETHYL-3-AROYL-4-ARYL-4-PIPERIDINOLS 1Ebru METE, 2H. İnci GÜL, 1Cavit KAZAZ1Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey

It has been reported that piperidinol type of compounds have cy-totoxic, antifungal and anticonvulsant activities [1-4]. In this study, synthesis of 1-phenethyl-3-aroyl-4-aryl-4-piperidinols has been re-alised to investigate their biological activities in future.

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Chemical structure of the compounds were confirmed by 1H NMR, 13C NMR, UV, IR and MS spectroscopies. Purity level of them was determined by elemental analyses.

1. Gül Hİ, Çalış Ü, Vepsalainen J. Synthesis and evaluation of anticonvul-sant activities of some bis Mannich bases and corresponding piperidi-nols, Arzneimittel Forschung, 52, 863-9, 2002.

2. Gül M, Gül Hİ, Das U, Hanninen O. Biological evaluation and structure-activity relationships of bis-(3-aryl-3-oxo-propyl)-methylamine hydro-chlorides and 4-aryl-3-arylcarbonyl-1-methyl-4-piperidinol hydrochlo-rides as potential cytotoxic agents and their alkylating ability towards cellular glutathione in human leukemic T cells, Arzneimittel Forschung, 55, 332-7, 2005.

3. Gül Hİ, Şahin F, Gül M, Öztürk S, Yerdelen KO. Evaluation of antimi-crobial activities of several mannich bases and their derivatives, Arch. Pharm., 338, 335-8, 2005.

4. Gül M, Atalay M, Gül Hİ, Nakao C, Lappalainen J, Hanninen O. The ef-fects of some Mannich bases on heat shock proteins HSC70 and GRP75, and thioredoxin and glutaredoxin levels in Jurkat cells, Toxicol In Vitro, 19, 573-80, 2005.

P 023 Ref: 0043

SYNTHESIS AND ANTITUMOR ACTIVITY OF POLY(2,3-DIHYDROPYRAN-CO-MALEIC ANHYDRIDE-CO-VINYL ACETATE) 1Hatice KAPLAN-CAN, 2A. Lale DOĞAN, 3Zakir M. O. RZAEV, 2Ayşegül HASEGELİ-ÜNER, 1Ali GÜNER1Hacettepe University, Faculty of Science, Department of Chemistry, Ankara, Turkey2Hacettepe University, Institute of Oncology, Department of Basic Oncology, Ankara, Turkey3Hacettepe University, Faculty of Engineering, Department of Chemical Engineering, Ankara, Turkey

It is known that the water soluble anhydride-containing copoly-mers as polyanions and their functional derivatives have high biologi-cal and physiological activity, specially antimicrobial and antitumor properties. Polyanions are also known for their potential to stimulate the immune system and invoke activities against tumors, viruses and bacteria. Copolymers of dihydropyran and its derivatives with acrylic acid, which contain tetrahydropyran rings and free carboxylic groups on the polymer backbone, as well as an alternating cyclocopolymer of divinyl ether (acyclic analogy of dihydropyran) with maleic anhydride have exhibited antitumor activities in vitro [1, 2].

Radical-initiated terpolymerization of 3,4-dihydro-2H-pyran (DHP), maleic anhydride (MA) and vinyl acetate (VA) as a donor-acceptor-donor systems was carried out MEK in the presence of AIBN as initiator at 65oC in nitrogen atmosphere. Determination of some kinetic parameters, constants of thermal−copolymerization of complexed comonomers, terpolymer composition behavior re-lationships of synthesized polymers with alternating structure are described and discussed. Synthesized polymers were characterized by analytical methods (acid number), viscometer, FTIR and thermal (DSC and TGA) methods [3].

In vitro cytotoxicities of synthesized poly(DHP-alt-MA) and poly(DHP-co-MA-co-VA) polymers were evaluated using Raji cells (human Burkitt lymphoma cell line). Antitumor activity of prepared anion-active poly(DHP-alt-MA) and poly(DHP-co-MA-co-VA) polymers were studied by methyl-thiazol-tetrazolium (MTT) test-ing method using calorimetric measurements of chemotherapic ef-fect and quantitative evaluation of LD50 dose for the total number of tumor cells. Hydrolyzed copolymer has sufficiently high antitumor activity, which depends on the amount of hydrogen bonded car-boxylic groups and on their regular distribution in side chain of the functional macromolecules [3].

1. Stolfi RL, Martin DS. Therapeutic activity of maleic anhydride-vinyl ether copolymers against spontaneous, autochthonous murine mam-mary tumors, Cancer Treat Rep., 62(11),1791-6,1978.

2. Ottenbrite, RM. The antitumor and antiviral effects of polycarboxylic acid polymers in biological activities of polymers, ACS Symposium Se-ries 186, Washington DC, 1982.

3. Kaplan Can, H., Doğan, AL., Rzaev, ZMO, Üner, AH, Güner, A. Syn-thesis and Antitumor Activity of Poly(3,4-dihydro-2Hpyran-co-maleic anhydride-co-vinyl acetate), Journal Applied Polymer Science, 96, 2352-2359, 2005.

P 024 Ref: 0044

ANTIFUNGAL ACTIVITIES OF 3-PHENETHYLAMINO-1-ARYL-1-PROPANONE HYDROCHLORIDES AND 3-AROYL-4-ARYL-1-PHENETHYL-4-PIPERIDINOLS AGAINST SOME PLANT AND HUMAN PATHOGENIC FUNGI 1H. İnci GÜL, 1Canan ÖZELGÜL, 2Ebru METE, 3Fikrettin ŞAHİN, 3Dilşat YURDAKUL1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey3Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering, İstanbul, Turkey

1-Aryl-3-phenethylamino-1-propanone hydrochlorides (1, 3, 5, 7, 9, 11, 13, 15, 17, 19) and 3-aroyl-4-aryl-1-phenethyl-4-piperidinols (2, 4, 6, 8, 10, 12, 14, 16, 20) were synthesized. Aryl part was C6H5 for 1, 2; p-CH3C6H4 for 3, 4; p-CH3OC6H4 for 5, 6; p-ClC6H4 for 7, 8; p-FC6H4 for 9, 10; p-BrC6H4 for 11, 12; o, p-(Cl)2C6H3 for 13, 14; p-NO2C6H4 for 15,16; p-HOC6H4 for 17; C4H3S(2-yl) i.e. 2-Thienyl for 19, 20.

The compounds synthesized were tested against 8 plant patho-genic fungi (Rhizoctonia soloni-EB-ML, Fusarium oxysporium CE1, Sclerotinia sclerotiorum FD3, Aspergillus niger FS2, Aspergillus fla-vus Hak23, Alternaria alternata FS2002, Macrophamina phaseoli CE4, Botrytis cinerea MFD3) and 3 human pathogenic fungi (Mi-crosporum canis-AO5, Candida albicans EA-07, Candida parapsilosis EA-08) using agar dilution assay in the concentration range of 6,25 to 200 µg/ml [1]. Minimal Inhibition Concetrations (MIC) of the compounds were determined. Amphotericin-B was used as a refer-ence compounds. Amphotericin-B was only effective against Mac-rophamina phaseoli CE4, Aspergillus niger FS2 and Aspergillus flavus Hak23 at 100 mg/ml.

None of the compounds showed antifungal activity at the concen-tration range studied against Fusarium oxysporium-CE1, Aspergillus niger-FS7, Botrytis cinerea-MFD, Candida albicans-EA-07, which are plant pathogenic fungi. MIC values of the compounds (µg/ml) were as follows:

Compound (µg/ml): 13(25), 14(50) against Rhizoctonia soloni-2001; 10, 13, 15 (12.5) , 2, 3, 8, 9, 11, 19, 20 (25), 1 (50), 7 (200) against Microsporum canis-AÖ5, 11 (100) against Sclerotinia sclero-tiorum-FD3; 13(100), 15, 16 (200) against Aspergillus flavus Hak23; 8 (200), 14 (100) against Alternaria alternata-FS2002; 5, 7 (200) against Macrophamina phaseoli-CE4; 9, 13, 15, 17, 19 (100) against Candida parapisilosis EA-08.

1. Gül Hİ, Şahin F, Gül M, Öztürk S, Yerdelen KO. Evaluation of antimi-crobial activities of several mannich bases and their derivatives, Arch. Pharm., 338, 335-8, 2005.

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P 025 Ref: 0045

EVALUATION OF CYTOTOXICITY OF 1-ARYL-3-PHENETHYLAMINO-1-PROPANONE HYDROCHLORIDES AND 1-PHENETHYL-3-BENZOYL-4-ARYL-4-PIPERIDINOLS BY BRINE SHRIMP BIOASSAY 1Murat ÇİZMECİOĞLU, 2H. İnci GÜL, 3Ebru METE1Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 35100 İzmir, Turkey2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey3Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey

Synthesis of the novel 1-aryl-3-phenethylamino-1-propanone hydrochlorides (series 1) and 1-phenethyl-3-benzoyl-4-aryl-4-pip-eridinols (series 2) were carried out. Evaluation of the cytotoxicity of the compounds has been realised by brine shrimp bioassay as previously described [1]. Chemical structure of the compounds were confirmed by 1H-NMR, 13C-NMR, UV, IR and MS spectroscopic methods. Puririties of the synthesized compounds were determined by elemental analyses.

Of the compounds tested, compounds 13, 15, 17 (from the series 1), 4, 6, 8 (from the series 2) were found effective in brine shrimp bi-oassay (Table 1). Aryl part was (2,4-(Cl)2C6H3) for 13, (4-NO2C6H4) for 15, (4-HOC6H4) for 17, (4-CH3C6H4) for 4, (4-CH3OC6H4) for 6, (4-ClC6H4) for 8. The most effective compound was detected as compound 6 while the least effective one was found as compound 15. Cytotoxicities of these compounds were determined in the range of 13.85 - 3467.52 µg/ml while reference compound 5-fluorouracil exhibited 2.99 µg/ml of cytotoxicity.

Table 1. Cytotoxicity of the compounds in brine shrimp bioassay.

Code Aryl Cytotoxicity (µg/ml)

Series 1

13 2,4-(Cl)2C6H3 65.79 ± 0.083

15 4-NO2C6H4 3467.52 ± 0.469

17 4-HOC6H4 316.82 ± 0.122

Series 2

4 4-CH3C6H4 165.70 ± 0.186

6 4-CH3OC6H4 13.85 ± 0.191

8 4-ClC6H4 573.33 ± 0.156

1. Gül Hİ, Gül M, Hanninen O. Cytotoxic activities of some mono and bis Mannich bases derived from acetophenone in brine shrimp bioassay, Arzneimittel Forschung, 52, 840-3, 2002.

P 026 Ref: 0046

CRYSTAL STRUCTURE OF 3-(p-CHLOROBENZOYL)-4-(p-CHLOROPHENYL)-1-PHENETHYL-4-PIPERIDINOL 1Ertan ŞAHİN, 1Cavit KAZAZ, 1Ebru METE, 2H. İnci GÜL1Atatürk University, Faculty of Science & Arts, Department of Chemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey

NMR Study: The characterisation of compound 1, 3-(p-chlo-robenzoyl)-4-(p-chlorophenyl)-1-phenethyl-4-piperidinol, have

been elucidated on the basis of 1H and 13C NMR spectral data (DEPT, COSY, HMQC, HMBC and NOE). 1H NMR spectra of the com-pound was well resolved and the unambiguous proton chemical-shift assignments were based on the multiplicity pattern of proton reso-nances and also on the use of homonuclear 1H – 1H COSY spectra. The assignments of all carbon resonances of compound were based on the analysis of the HMQC and HMBC spectra.

X-Ray Study: For the crystal structure determination, the single–crystal of the molecule 3-(p-chlorobenzoyl)-4-(p-chlorophenyl)-1-phenethyl-4-piperidinol was used for data collection on a four–circle Rigaku R–AXIS RAPID–S diffractometer equipped with a two–di-mensional area IP detector. The graphite–monochromatized Mo Kα radiation (λ=0.71073 Å) and oscillation scans technique with ∆ω=5° for one image were used for data collection. The lattice parameters were determined by the least–squares methods on the basis of all reflections with F2>2σ(F2). Integration of the intensities, correction for Lorentz and polarization effects and cell refinement was per-formed using CrystalClear software [1]. The structures were solved by direct methods (SHELXS–97) [2] and non–H atoms were refined by full–matrix least–squares method with anisotropic temperature factors (SHELXL-97) [2].

Compound 1, 3-(p-chlorobenzoyl)-4-(p-chlorophenyl)-1-phene-thyl-4-piperidinol, crystallizes in the monoclinic crystal system with space group P21/a and the following unit cell parameters: a= 10.792.(4) Å, b= 12.863 (5)Å, c= 16.950 (6) Å; α =90°, β= 97.77(4)°, γ = 90°; V= 2331.3(16) Å3 , and Z=4. The compound 1 has intermo-lecular O1-H···N1 [O1-H; 0.820, H···N1; 2.11, O1···N1; 2.878(5) Å, O1···H-N1 156˚], C13···H-O2 [C13-H; 0.970, H···O2; 2.54, C13···O2; 3.373(6) Å, C13···H-O2 144˚] hydrogen bonds.

1. Rigaku (2005), CrystalClear, Version 1.3.6. Rigaku American Corpora-tion, 9009 New Trails Drive, The woodlands, TX 77381–5209, USA.

2. Sheldrick GM. SHELXS97 and SHELXL97, University of Göttingen, Germany, 1997.

P 027 Ref: 0047

STRUCTURE ELUCIDATION OF MONO MANNICH BASE 1-(p-METHOXYPHENYL)-3-PHENETHYLAMINO-1-PROPANONE HYDROCHLORIDE AND SEMICYCLIC MONO MANNICH BASE 3-(p-METHOXYBENZOYL)-4-(p-METHOXYPHENYL)-1-PHENETHYL-4-PIPERIDINOL USING 1D AND 2D NMR SPECTROSCOPY 1Cavit KAZAZ, 1Ebru METE, 2H. İnci GÜL1Atatürk University, Faculty of Science & Arts, Department of Chemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey

Mono Mannich base 1-(p-methoxyphenyl)-3-phenethylamino-1-propanone hydrochloride and semicyclic mono Mannich base 3-(p-methoxybenzoyl)-4-(p-methoxyphenyl)-1-phenethyl-4-piperidinol have been synthesized according to the literature process [1].

The structural elucidations of the compound was accomplished using extensive 1D-NMR (1H, 13C, NOE-diff, DEPT) and 2D-NMR (COSY, NOESY, HMQC and HMBC) spectroscopic techniques. Pro-ton and carbon-13 spectra were recorded at 27 °C in DMSO on a Varian mercury-plus (Palo Alto, USA) instrument at a frequency of 400 and 100 MHz, respectively, using a 5 mm ASW PFG probe.

1. Gül M, Gül Hİ, Das U, Hanninen O, Arzneimittel Forschung, 55(6), 332-7, 2005.

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P 028 Ref: 0048

INVESTIGATION OF CASEIN KINASE 2 (CK2) ENZYME AND ITS BINDING PROPERTIES WITH P53 PROTEIN, POLYLYSINE AND HEPARIN BY MALDI-MS 1Ömür ÇELİKBIÇAK, 1Cansel TAŞAĞIR, 2Wayne E. CRISS, 1Bekir SALİH1Hacettepe University, Faculty of Science, Department of Chemistry, 06532 Beytepe-Ankara, Turkey2Hacettepe Unıversity, Faculty of Medicine, Oncology Institute, Department of Biochemıstry, 06100 Sıhhiye-Ankara, Turkey

Matrix-assisted Laser Desorption/Ionization, along with electro-spray ionization, is now among the most important ionization meth-ods for nonvolatile, high molecular weight compounds, in particular peptides, proteins, oligonucleotides, oligosaccharides and synthetic polymers. MALDI has also been successfully used for the investiga-tion of fullerenes, fullerene derivatives, and synthetically prepared dendrimers. Under certain conditions, it has even been shown that MALDI can be used for the study of weakly bound noncovalent complexes [1]. Protein kinase CK2 (Casein Kinase 2) is a ubiquitous serine/threonine protein kinase which is composed of two regulatory β- and two catalytic α- or α’- subunits. Although its precise function in the cell is still unclear there is ample evidence that CK2 plays an important role in the regulation of cell proliferation [2]. The activity of CK2 is elevated in tissues with a high proliferation rate, such as tumors and embryonic tissue [2]. The p53 tumor suppressor protein is a potent transcription factor which is activated in response to a va-riety of DNA-damaging agents. Activation of p53 leads to cell growth arrest or the induction of apoptosis, thereby blocking the survival of genetically damaged cells [2]. In the absence of functional p53 genes, the cycle is not arrested and the apoptosis signal is not delivered, so a cell with abnormal DNA is allowed to replicate, thus increasing the chance of cancer developing [3]. P53 is phosphorylated at multiple sites in vivo and by several different protein kinases in vitro includ-ing the protein kinase CK2. In addition, it was shown previously by some researchers that mutation of p53 at the CK2 phosphorylation site abolishes the growth suppressor activity of p53 [2]. Because of the importance of the binding properties of p53 to the CK2 enzyme, MALDI-MS analysis of CK2 and p53 proteins were performed and interactions of p53 protein with α and β subunits of CK2 enzyme were investigated. Since CK2 was first discovered, many studies have showed us that the enzymatic activity of CK2 was stimulated by polyamines and inhibited by heparin in vitro [2]. According to these data, existence of interactions can be inferred between these mol-ecules and CK2 and/or between these molecules and CK2 substrates, which probably improve the susceptibility of substrates to be phos-phorylated or not. In order to get information about the mechanism of noncovalent interactions between these biomolecules, MALDI-MS was used by selecting suitable matrix, sample preparation and clean up procedures at appropriate pH and solvent conditions.

1. Zenobi R, Knochenmuss R. Ion Formation in MALDI Mass Spectrom-etry, Mass Spectrometry Reviews, 17, 337–366, 1998.

2. Schuster N, Götz C, Faust M, Schneider E, Prowald A, Jungbluth A, Mon-tenarh M. Wild-Type p53 Inhibits Protein Kinase CK2 Activity, Journal of Cellular Biochemistry, 81, 72-183, 2001.

3. Elliott, WH, Elliott, DC. Biochemistry and molecular biology, 3rd ed., Oxford University Press Inc., New York, 2001, p. 533-534.

P 029 Ref: 0049

CYTOTOXICITIES OF N,N’-BIS(3-DIMETILAMINO-1-ARYL-PROPYLIDENE)HYDRAZINE DIHYDROCHLORIDES 1Kaan KÜÇÜKOĞLU, 2Mustafa GÜL, 1H. İnci GÜL, 3Osmo HANNINEN, 3Mustafa ATALAY1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Erzurum, Turkey2Atatürk University, Faculty of Medicine, Department of Physiology, Erzurum, Turkey3University of Kuopio, Department of Physiology, Finland

Synthesis of N,N’-bis (3-dimethylamino-1-aryl-propylidene) hy-drazine dihydrochlorides were carried out according to the literature procedure [1] by using acetophenone or substituted acetophenones as the ketone component of the reactions while dimethylamine hydro-chloride was the amine component of the reactions. Substituents were methyl for 2, methoxy for 3, hydroxy for 4 and chloro for 5 at the para position of the phenyl ring while compound 1 was nonsubstituted.

Chemical structures of the compounds were confirmed by 1H NMR. Cytotoxicity of the compounds was determined against Jur-kat cells which is human T lymphocytes cells. Reference compounds were 5-fluorouracil and melphalane.

All compounds have shown more powerful activity than both references, cytotoxicity values of the compounds were in the range of 9.75-13.27µM. The most effective compound was 4 in five com-pounds studied.

1. Gül Hİ, Das U, Pandit B, Li PK, Evaluation of the cytotoxicity of some mono-mannich bases and their corresponding azine derivatives against androgen-independent prostate cancer cells, Arzneimittel Forschung, 56(12), 850-4, 2006.

P 030 Ref: 0050

SYNTHESIS OF 1-[3-(PIPERIDINOMETHYL)-4-HYDROXYPHENYL]-3-ARYL-2-PROPEN-1-ONES AND EVALUATION OF THEIR ANTICONVULSANT ACTIVITIES 1H. İnci GÜL, 1K. Özden YERDELEN, 2Ünsal ÇALIŞ1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Sıhhiye-Ankara, Turkey

In this study, Mannich bases with piperidine, 1-[3-(piperidinome-thyl)-4-hydroxyphenyl]-3-aryl-2-propen-1-one, B1-B5, were synthe-sized starting from the chalcones, 1,3-diaryl-2-propen-1-one, A1-A5.

Chemical structures of the compounds have been confirmed by 1H-NMR, 13C-NMR, IR, and UV spectra and elemental analysis. Anticonvulsant activities of the compounds were evaluated by MES, scMet tests. Neurotoxicities of the compounds were also evaluated by rotorod test [1, 2].

None of the compounds showed neurotoxicity at the screening of anticonvulsant activity. Compounds B1-B5 at MES test, compounds B1-B3 at scMet test have shown anticonvulsant activity at different dose levels (30-300 mg/kg) and time periods (1/2 h, 4 h).

To conclude, of the compounds B4, B5 against grand-mal epilep-sia, B1, B2 and B3 against both types of epilepsia, can be choosen as candidate compounds to develop new anticonvulsant compounds for further studies.

1. Gül Hİ, Çalış Ü, Vepsalainen J. Synthesis of some mono-Mannich bases and corresponding azine derivatives and evaluation of their anticonvul-sant activity, Arzneimittel Forschung, 54, 359-64 2004.

2. Gül Hİ, Çalış Ü, Vepsalainen J. Synthesis and evaluation of anticonvul-sant activities of some bis Mannich bases and corresponding piperidi-nols, Arzneimittel Forschung, 52, 863-9 2002.

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P 031 Ref: 0051

DEVELOPMENT AND OPTIMIZATION OF A SURFACE AIDED ENZYMATIC DIGESTION SYSTEM TO USE IN PROTEOMICS STUDIES 1Basri GÜLBAKAN, 1Aslı ÖZTÜRK-ÇAL, 2Talat YALÇIN, 1Bekir SALİH1Hacettepe Unıversity, Faculty of Science, Department of Chemistry, 06532 Beytepe-Ankara, Turkey2İzmir Institute of Technology, Faculty of Science, Department of Chemistry, 35430 İzmir, Turkey

Proteomics is the study of all methods covering isolation, sepa-ration, identification, characterization of all proteins in living or-ganisms and finding their functional roles and expression of the sequences of proteins in all tissues. The anomalies in the synthe-sis and function of the proteins are interrelated with several lethal diseases. Understanding the cause and the therapy of a disease is related to determining and understanding the proteins that are in-volved. After completion of the Human Genome Project, proteom-ics gained more importance. However, since proteins are more complex than that of genes, secondary cleavage reactions namely, enzymatic trypsin reactions are employed to obtain smaller peptide fragments and peptides are then analyzed to obtain structural in-formation about proteins. Thus the success of proteomics is closely related with the success of enzymatic trypsin cleavage. Isolation of proteins from their natural environment, which is a very complex matrix, leads to protein loss and in many cases necessitates the ad-dition of contaminating agents like SDS, CHAPS and sucrose. This also limits the information that is obtained from proteomics studies. The presented study was carried out in order to eliminate the intrin-sic difficulties of conventional in-solution and in-gel trypsin diges-tion protocols and to develop a new and effective digestion method. Proteins samples having different molecular weights and different structural complexity was preconcentrated and purified by using conventional reversed phase column packing materials that are C18 bonded silica, polystyrene-divinylbenzene and modified derivatives thereof and digested while bound to surface. The resulting peptides were then analyzed by MALDI mass spectrometry. The effect of dif-ferent MALDI matrices, sample preparation methods, effect of func-tional group loading onto poly (styrene-divinylbenzene) microbeads was studied. The results were compared by conventional methods using trypsin digestion to test the efficiency of the method.

1. Gevaert K, Vandekerckhove J. Protein identification methods in pro-teomics, Electrophoresis, 21, 1145-1154, 2001.

P 032 Ref: 0052

SYNTHESIS OF 1-[3-(DIBENZYLAMINOMETHYL)-4-HYDROXYPHENYL]-3-ARYL-2-PROPEN-1-ONES AND EVALUATION OF THEIR ANTIFUNGAL ACTIVITIES 1K. Özden YERDELEN, 1H. İnci GÜL, 2Fikrettin ŞAHİN1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey2Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering, 34755-Kayışdağı, İstanbul, Turkey

In this study, Mannich bases with dibenzylamine 1-[3-(diben-zylaminomethyl)-4-hydroxyphenyl]-3-aryl-2-propen-1-one, (C1-C5) were synthesized and the chemical structures of the compounds have been confirmed by 1H-NMR, 13C-NMR, IR, and UV spectra and elemental analysis.

Antifungal activities of the compounds have been tested and com-pared with their precursor chalcones (A1-A5) against 3 fungi species pathogenic in humans [Trichophyton rubrum (Hak-8), Trichophyton

mentagrophytes (Hak-9), Microsporum canis (Hak-4)] and 13 fungi species pathogenic in plants [Sclerotinia sclerotiorum, Sclerotinia minor, Alternaria alternate (AA-1121), Aspergillus flavus (Hak-23), Aspergil-lus variecolor (IO-Balik), Fusarium acuminatum, Fusarium oxysporum (ED-10), Fusarium solani (ED-1S), Fusarium tabacinum (ED-1T), Moniliania fructicola (FS-M), Penicillium spp. (P-TY), Rhizopus spp. (R-27), Rhizoctonia solani (EB-ML)] at the concetration range of 2-64 µg/ml using amphotericin–B as the reference compound (1).

Of the compounds, A1 against plant pathogens Sclerotinia scle-rotioruma and Rhizoctonia solani, while C5 against plant pathogen Fusarium oxysporum, have shown 2-4 times more powerful antifun-gal activity compared with amphotericin-B. Of the compounds syn-thesized, A1 and C5, against plant pathogenic fungi can be choosen as candidate compounds for further studies to develop new antifun-gal compounds.

1. Gül Hİ, Şahin F, Gül M, Öztürk S, Yerdelen KO. Evaluation of antimi-crobial activities of several mannich bases and their derivatives, 1, Arch. Pharm., 338(7), 335-8, 2005.

P 033 Ref: 0054

SYNTHESIS OF 1-[3-(PIPERIDINOMETHYL)-4-HYDROXYPHENYL]-3-ARYL-2-PROPEN-1-ONES AND EVALUATION OF THEIR CYTOTOXIC ACTIVITIES AGAINST HUMAN T LYMPHOCYTES (JURKAT) CELLS AND RAT SKELETAL MUSCLE DERİVED MYOBLAST CELLS (L6) 1H. İnci GÜL, 2Mustafa GÜL, 1K. Özden YERDELEN, 3Osmo HANNINEN, 3Mustafa ATALAY1Atatürk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Medicine, Department of Physiology, 25240 Erzurum, Turkey3University of Kuopio, Faculty of Medicine, Department of Physiology, 1627 Kuopio, Finland

In this study, Mannich bases with piperidine, 1-[3-(piperidinome-thyl)-4-hydroxyphenyl]-3-aryl-2-propen-1-one, B1-B5 were synthe-sized starting from the chalcones, 1,3-diaryl-2-propen-1-one, A1-A5. Chemical structures of the compounds have been confirmed by 1H-NMR, 13C-NMR, IR, and UV spectra and elemental analyses.

Cytotoxic activities of the compounds have been tested against rat skeletal muscle derived myoblast cells (L6) and transformed hu-man T lymphocytes (Jurkat). Melphalan and 5-fluorouracil were also tested as reference drugs [1].

All compounds have shown 1.28-5.40 times more powerful cyto-toxicity than 5-FU, and the compounds A3, B1, B2, B3 have shown 1.03-2.76 times more powerful cytotoxicity than melphalan against L6 cells, respectively. Preparation of Mannich bases with piperidine from the chalcones increased the cytotoxicity 1.55, 1.54, 1.33, 1.38 times at the compounds B1, B2, B4, B5 respectively, compared with their corre-sponding chalcones. While all compounds synthesized had 3.20-9.43 times more powerful cytotoxicity than 5-FU against Jurkat cells, ex-cept B1, all other compounds showed 1.11-2.38 times more powerful cytotoxicity than melphalan. Preparation of Mannich bases from the chalcones increased the cytotoxicity 1.42, 1.70, 1.43 times respectively at the compounds B2, B4 and B5 compared with the corresponding chalcones against Jurkat cells.The compounds synthesized have been found more selective against Jurkat cells compared with L6 cells.

Of the compounds synthesized, B2, B4 and B5 can be choosen as candidate compounds for further cytotoxicity studies to develop new cytotoxic compounds.

1. Gül M, Gül Hİ, Das U, Hanninen O, Arzneimittel Forschung, 55(6), 332-7, 2005.

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P 034 Ref: 0056

DETERMINATION OF PHYSICOCHEMICAL PROPERTIES OF SEVERAL SULFONAMIDES BY LIQUID CHROMATOGRAPHY IN ACETONITRILE-WATER BINARY MIXTURES 1Nurullah ŞANLI, 1Güleren ALSANCAK, 2Adil DENİZLİ1Süleyman Demirel University, Faculty of Science and Literature, Department of Chemistry, Isparta, Turkey2Hacettepe University, Faculty of Science, Department of Chemistry, Ankara, Turkey

Sulfonamides are antibacterial compounds commonly used to prevent and to treat diseases in medical and veterinary practice. A sulfonamide contains one basic amino group and one acidic amide group which correspond to pKa1 and pKa2 respectively. The degree of ionization of sulfonamides was strongly related with the in vitro bacteriostatic activity [1].

The use of HPLC retention parameters to determine pKa values has been widely applied [1-2]. Literature studies of the chromato-graphic determination of pKa values of sulfonamides and the effect of the organic modifier content on the pKa values of these com-pounds are scarce, but the investigation of Mengelers et al. should be mentioned [1].

In this study, the dissociation constants of related series of sulfon-amides (sulfodiazine, sulfothiazole, sulfomerazine, sulfomethazine, sulfomonomethoxine, sulfodoxine, sulfomethoxazole) were deter-mined by LC methodology and the effects of the ACN percentage on the pKa values were investigated. The prodigy C-18 was used as stationary phase. On changing the mobile phase of the system, the column was thoroughly equilibrated with studied new mobile phase. The electrode system was calibrated with potassium acid phthalate in organic mixture of the same composition as the mobile phase ac-cording to IUPAC rules. The pH of the mobile phase was measured after the addition of the ACN to properly investigate the effect of pH on the retention of ionizable compounds. The effect of pH on sulfonamides retention was investigated in the range of pH 1.7-9.7. The retention time was plotted against pH value of the mobile phase to calculate pKa2 values. The sigmoidal behaviors of studied sulfona-mides are shown in Figure 1.

Figure 1. Plots of retention times of sulphonamides against mobile phase pH (4.5-9.0) at 30% (v/v) ACN. Symbols indicate: ■ sulfodiazine, ♦ sulfothiazole, ▲ +sulfomerazine, х sulfomethazine, ж sulfomonomethoxine, • sulfodoxine, sulfomethoxazole).

Their pKa values in ACN – water mixture (50% v/v) were also potentiometricaly determined [3] and compared with the results ob-tained by LC methodology.

The retention data of sulfonamides obtained by Reversed Phase Liquid Chromatography (RPLC) were correlated with the molecu-

lar partition coefficient, Po, obtained in octanol-water systems by Countercurrent Chromatography (CCC) [4], and the correlated data fits perfectly.

1. Mengelers MJB, Hougee PE, Janssen LHM, Van Miert AS. Structure-ac-tivity relationships between antibacterial activities and physicochemi-cal properties of sulfonamides, J. Vet. Pharmacol. Therap., 20, 276-283, 1997.

2. Botsoglou NA, Fletouris DJ, Simeonidou EJ, Psomas IE. Retention be-havior of multiple sulfonamides in various liquid chromatographic sys-tems, Chromatographia, 46, 9/10, 477-782, 1997.

3. Qiang Z, Adams C. Potentiometric determination of acid dissociation constants (pKa) for human and veterinary antibiotics, Water Research, 38, 2874-2890, 2004.

4. Carda-Broch S, Berthord A. Countercurrent chromatography for the measurement of the hydrophobicity of sulphonamide amphoteric com-pounds, Chromatographia, 59, 79-87, 2004.

P 035 Ref: 0057

CONTROLLED RELEASE OF NAPROXEN FROM POLY (VINYL ALCOHOL) / SODIUM ALGINATE MICROSPHERES 1Oya ŞANLI, 2Ebru KONDOLOT-SOLAK1Gazi University, Arts and Sciences Faculty, Department of Chemistry, 06500 Ankara, Turkey2Gazi University, Atatürk Vocational College, Department of Chemistry, Ankara, Turkey

In this study microspheres of poly(vinyl alcohol)/sodium algi-nate (PVA/Na-Alg) and sodium alginate (Na-Alg) were prepared to encapsulate naproxen sodium drug. Microspheres were prepared by liquid curing method by crosslinking with glutaraldehyde, then characterized by fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC) and scanning electron mi-croscopy (SEM).

Microspheres were also characterized by measuring the particle diameter, equilibrium swelling values and determining release pro-files. Equilibrium swelling experiments indicated that the swelling of the spheres decreased with an increase in crosslinking time and concentration however diameter of the spheres was not affected con-siderably.

The release studies were carried out at three pH values 1.2, 6.8 and 7.4 respectively. The release of diclofenac from the micropheres increased as the drug/polymer ratio decreased. Optimum condition for the preparation and release of the spheres were determined as PVA/Na-Alg: 1/2, drug/polymer: 1/4 and pH: 7.8. The release of these conditions was found as 80.3 % at the end of 6th hour.

P 036 Ref: 0058

SPECTROPHOTOMETRIC ANALYSIS OF CABERGOLINE IN PHARMACEUTICAL PREPARATIONS 1Nursabah E. BAŞÇI, 2Demet SALMAN1Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, Turkey2Turkish Republic Instutition of Social Security, Ankara, Turkey

Cabergoline is a synthetic dopamine agonist having high affinity to D2 receptors and used for early and advanced Parkinson’s patients and hyperprolactinemy disorders [1]. Chromatographic and radio-immunoassay methods have been reported for quantification of ca-bergoline in body fluids [2-5], but there was not any spectrophoto-metric analysis of cabergoline in pharmaceutical preparations in the literature. In this study, simple, fast, reliable and validated UV-VIS and 2nd derivative spectroscopy methods were developed for deter-mination of cabergoline in pharmaceutical preparations.

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Determination of cabergoline was performed by UV-VIS spectro-photometry at 280 nm wavelength and by 2nd derivative spectropho-tometry at a range of 227-232 nm. HPLC (Shimadzu SCL-10AVP) connected to an electrochemical detector (Decade) was used in com-parison analysis. Cabergoline substance was donated by Pharmacia. Dostinex® (0.5 mg Cabergoline, Pfizer) and Cabaser® (1, 2 and 4 mg Cabergoline, Pfizer) tablets were used for pharmaceutical applica-tions. Developed methods were validated according to the regula-tions and guidelines of ICH, EMEA and FDA. Validation results were statistically evaluated using related methods at a significant level of 95%.

Developed UV-VIS and 2nd derivative spectrophotometry meth-ods were linear over the range of 1-125 µg mL-1. The limit of detec-tion for the methods was 0.5 µg mL-1 (RSD = 0.75%, Bias = 0.30) and the limit of quantification was 1.0 µg mL-1 (RSD = 0.67%, Bias = 0.33). The highest relative error and relative standard deviation in inter-day and intra-day study for UV-VIS spectrophotometry were 1.10% and 0.63%, respectively. The highest relative error and relative standard deviation in inter-day and intra-day study for 2nd derivative spectrophotometry were 1.10% and 0.70%, respectively. The methods were applied to the analysis of cabergoline in pharma-ceutical preparations and there was no statistically significant differ-ence when the results were compared with the results of comparison method (HPLC/ECD). In conclusion, the developed UV-VIS and 2nd derivative spectrophotometry methods were accurate, sensitive, pre-cise and repeatable and can be applied to the analysis of cabergoline in pharmaceutical preparations as direct, fast and simple methods.

1. Gottwald MD, Bainbridge JL, Dowling GA, Aminoff MJ, Alldredge BK. New pharmacotherapy for parkinson’s disease, Annals of Pharmacothera-phy, 31(10), 1205-17, 1997.

2. Persiani S, Pianezzola E., Broutin F, Fonte G, Benedetti MS. Radioim-munoassay for the synthetic ergoline derivative cabergoline in biological fluids, Journal of Immunoassay, 13(3), 457-76, 1992.

3. Pianezzola E, Bellotti V, Croix RL, Benedetti MS. Determination of ca-bergoline in plasma and urine by high-performance liquid chromatog-raphy with electrochemical detection, Journal of Chromatography, 574, 170-174, 1992.

4. Allievi C, Dostert P. Quantitative determination of cabergoline in human plasma using liquid chromatography combined with tandem mass spec-trometry, Rapid Communications in Mass Spectrometry, 12, 33-39, 1998.

5. Igarashi K, Hotta K, Kasuya F, Abe K, Sakoda S. Determination of caber-goline and L-dopa in human plasma using liquid chromatography-tan-dem mass spectrometry, Journal of Chromatography B, 792, 55-61, 2003.

P 037 Ref: 0059

INVESTIGATION OF ANTIMICROBIAL ACTIVITES OF NEW SULFONYLHYDRAZONE DERİVATİVES ON SOME MICROORGANISMS Ümmühan ÖZDEMİR-ÖZMEN, Fatma HAMURCUGazi University, Arts and Sciences Faculty, Department of Chemistry, 06500 Ankara, Turkey

The chemistry of hydrazones has been intensively investigated in recent years, owing to their coordinating capability, pharmacological activity, antibacterial and antifungal properties, Sulfonamide drugs are widely used chemotherapeutic agents with large spectrum of ac-tivity [1]. Methane sulfon amide residue has appeared as a suitable pharmacophoric equivalent to replace functional groups in drug de-sign . Having hydrophilic character, like the sulfonyl group is con-sidered as a suitable pharmacophoric equivalent for replacing func-tional groups in drug design [2]. In previous paper, we reported the antibacterial and cytotoxic effect of methanesulfonic acid hydrazide (msh) [3].

Ethanesulfonic acid hydrazide, C2H5SO2NHNH2, (esh) ; 5-methyl-2-hydroxyacetophenoneethanesulfonylhydrazone (5mafesh) and its Ni(II) ,Co(II) complexes containing sulfonamide and hydrazine fragments were synthesized and their structures were determined by using elemental analysis, NMR, FT-IR, LC-MS, magnetic and conductivity studies. Their antimicrobial activities were investigated against to Escherichia coli ATCC 11230, Bacillus subtilis RSKK 244, Bacillus cereus RSKK 863, Bacillus magaterium RSKK 5117, Salmo-nella enteritidis ATCC 13076, Staphylococcus aureus ATCC 25923 by using MIC’s method. MIC’s was defined as the lowest concentrations of compounds which inhibit the growth of microorganisms.

The antimicrobial results evidently showed that the sulfonamide derivatives possessed a broad spectrum of activity against the tested bacteria (MIC’s values g/mL). All compounds exhibited the most ac-tivity against to Staphylococcus aureus between 60-672µg. The pres-ence of the NH group in the sulfonamides contributes positively to the increase of the activity of compounds against bacteria. In addi-tion the negative charges on the other donor atoms show a tendency to increase the activity [4].

1. Albert A. Selective Toxicity, Chapman and Hall, London, New York, 1985.

2. Topiol S, Sabio M, Erhardt PW, J. Chem. Soc.Perkin Trans., II, 437, 1988. 3. Dodoff NI, Özdemir Ü, Karacan N et al., Z. Naturforsch., 54 b , 1553,

1999. 4. Zanatta N, Alues SH, Coelho HS et al., Bioorganic & Medicinal Chemis-

try, 15, 1947, 2007.

P 038 Ref: 0060

DETERMINATION OF DISSOCIATION CONSTANTS OF SEVERAL SULFONAMIDES BY POTENTIOMETRY IN METHANOL, 2-PROPANOL, TETRAHYDROFURAN-WATER BINARY MIXTURES 1Senem ŞANLI, 1Ebru ÇUBUK-DEMİRALAY, 2Hale SEÇİLMİŞ, 3Jose Luis BELTRAN1Süleyman Demirel University, Science & Literature Faculty, Department of Chemistry, 32260 Isparta, Turkey2Süleyman Demirel University, Research Centre, 32260 Isparta, Turkey3Barcelona University, Department of Analytical Chemistry, 08028 Barcelona, Spain.

The pKa value is a main item in the biophysical characterization of a drug and may be helpful in predicting the behavior of a drug under in vivo conditions. Sulfonamides (SAs) are typical amphoteric compounds and dissociation pathways of sulfonamides are given in Figure 1. Ka1 and Ka2 are the dissociation constants of the aromatic amine and sulfonic groups respectively.

Fig 1. Scheme of dissociation equilibrium of sulfonamides.

Literature studies of the potentiometric determination of pKa val-ues of sulfonamides are scarce, but the investigation of Qiang and Adams should be mentioned [1]. In addition only a few pKa values

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of sulfonamides in organic solvent-water binary mixtures can be ob-tained from the literature [2].

Among the pKa determination techniques, the potentiometric titration is most economical of time and if due care is taken; this technique is accurate and has good reproducibility. This paper inves-tigated the potential of potentiometric method to determine the pKa values of sulfonamides that show poor solubility. In this study the effect of the organic modifier type and content on the pKa values of these compounds were also investigated. These solvent mixtures can dissolve drugs more effectively than water and in many cases they are more suitable solvent for the determination of the dissociation constants. Methanol and 2-propanol are closest to water in structure and properties. THF is one of the most widely used dipolar aprotic solvent and is much better differentiating solvent than water.

The pKa1 and pKa2 values of sulfonamide were determined by up-ward titration using 0,025 M KOH. The typical titration curve for sulfametaxazole is shown in Fig 2. In this study PKPOT program was used to correct the effect of ionic strength on pKa determina-tion [3].

Fig 2. Potentiometric titration curve of sulfomethaxazole in MeOH-H2O, 30% (v/v).

1. Qiang Z, Adams C. Potentiometric determination of acid dissociation constants (pKa) for human and veterinary antibiotics, Water Research, 38, 2874-2890, 2004.

2. Mengelers MJB, Hougee PE. Janssen LHM, Van Miert AS. Structure-ac-tivity relationships between antibacterial activities and physicochemi-cal properties of sulfonamides, J. Vet. Pharmacol. Therap., 20, 276-283, 1997.

3. Barbosa J, Barron D, Beltran JL, Sanz-Nebot V. PKPOT, a program for the potentiometric study of ionic equilibria in aqueous and non-aqueous media, Anal. Chim. Acta, 317, 75-81, 1995.

P 039 Ref: 0061

IMPROVED ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF INDAPAMIDE IN HUMAN PLASMA Zeliha ATEŞ, Sami EREN, Selma ÖZİLHAN, Tuncel ÖZDENNovagenix Bioanalytical R&D Centre, Ankara, Turkey

Indapamide, 3-(aminosulfonyl)-4-chloro-N-(2,3-dihydro-2-me-thyl-1H-indol-1-yl)-benzamide, is an oral antihypertensive and diuretic agent [1]. Indapamide inhibits carbonic anhydrase en-zyme, which reduces the vascular response to noradrenalin and an-giotensin II by inhibiting the transportation of Ca²+ into vascular smooth muscle. It shows diuretic effect by acting at the first parts of distal tubules [2].

A simple, rapid, sensitive and selective method for the analysis of indapamide in human plasma, utilizing ultra performance liquid chromatography (UPLC), has been developed and validated to fulfill FDA guidelines for bioanalytical methods [3]. The analyte and the internal standard, sulfamethazine, were isolated from plasma sam-ples by liquid-liquid extraction with diethyl ether. The assay exhib-ited a linear dynamic range of 1 to 100 ngmL-1 for indapamide in human plasma.

The limit of quantification (LOQ) was 1 ngmL-1 with a relative standard deviation of less than 12.2 %. Inter and intra-day preci-sion (CV %) and accuracy (%) for quality control samples (3, 50, 80 ngmL-1) ranged from 0.55 % to 8.48 % and from 94.62 % to 107.56 % respectively. Furthermore, this method was successfully applied to the pharmacokinetic and bioequivalence study of indapamide tab-lets in healthy male volunteers within 96 h period.

1. Caruso FS, Szabadi RR, Vukovich RA. Am. Heart, 106, 212-220, 1983. 2. Johnston MM, Rosenberg MJ, Yeung AK, Grebow PE. J. Pharm. Sci., 69,

1158-1160, 1980. 3. FDA Guidance for Industry, Bioanalytical Method Validation, May

2001.

P 040 Ref: 0062

THE EFFECT OF TAUROLIDINE ON THICKNESS OF SCAR TISSUE IN RABBIT MODEL 1Ali HAYAT, 2Füsun TEMAMOĞULLARI, 3Füsun BABA1University of Harran, Faculty of Veterinary Medicine, Department of Surgery, Şanlıurfa, Turkey2University of Harran, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Şanlıurfa, Turkey3University of Harran, Faculty of Medicine, Department of Pathology, Şanlıurfa, Turkey

Clinicans have used numerous strategies to combat wound infec-tions, including topical and systemic administration of antibiotics and various antiseptic agents [1]. The antimicrobial properties of taurolidine have been ascribed to the biological active methylol tau-rinamide which reacts with cell wall constituents of microbial patho-gens via methylene iminium ions preventing bacterial adhesion to biological surfaces. Taurolidine has a short half-life and is metabo-lised to taurine, carbon dioxide and water. It has been shown to be non-toxic to human and animals [4]. Povidone iodine is commonly used in clinical practice but dermal hypersensitivity is associated with the use of povidone iodine in humans and small animals [2]. In this study, we have investigated efficacy of 2% taurolidine solu-tion on healing wound standing on the clinical and histopathologic parameters comparing with 10% povidine iodine solution and 0.9% sodium chloride.

Six male and six female rabbits (mean weight: 2500±200) were taken for the study. All rabbits were anesthetized with intramuscu-lar administration of xylazine hydrochloride 10 mg/kg (Rompun, Bayer) and ketamin hydrochloride 50 mg/kg (Ketanes, Alke) Right and left costal regions of rabbits were clipped and the skin was pre-pared for aseptic surgery. Then full-thickness skin wounds (3 cm in diameter) as two cranial and one caudally located were created on each animal using a template prepared from x-ray film. Daily, 2% taurolidine, 10% povidine iodine solution and 0.9% sodium chloride were applied on wounds. Macroscopically wounds were examined from point of the exudation during the postoperative days. Biopsy specimens which were collected on the 4th, 8th, 12th and 16th PODs. Specimens were evaluated according to several histopathologic pa-rameters, such as the thickness of scar tissue.

Macroscopically, the wounds treated with 10% povidine iodine so-lution and 0.9% sodium chloride appeared to have marked fibrinous

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exudate and crust formation which caused adherence to the gauze. On the other hand. 2% taurolidine gauze applied wounds showed lesser degree of exudate and lesser adherence. Daily applied 2% tau-rolidine reduced the thickness of scar tissue when compared to other two solution. The degree of attachment between dressing material and the wound surface is important [3]. The subjective examination showed that the 2% taurolidine conformed well to the wound surface and it was readily separeted from underlying wound. As a result, 2% taurolidine application to full thickness skin wounds in rabbits posi-tively effected wound healing but the mechanism underlies this fact still needs furter investigations.

1. Burks, RI. Povidone-iodine solution in wound treatment, Phys. Ther., 78, 212-218, 1998.

2. Farstvedt, E, Stashak, TS., Othic, A. Update on Topical Wound Medica-tions, Clin. Tech. Equine Pract., 3, 164-172, 2004.

3. Kılıç, S, Timurkan, N, Ünsaldı, S, Günay, C, İstek, Ö, Yılmaz, B. Compari-son of the Effects of Some Wound Healing Materials on Full Thickness Skin Wounds in Rabbits, Turk. J. Vet. Anim. Sci., 26, 263-272, 2002.

4. Koldehoff, M, Zakrzewski, JL. Taurolidine is effective in the treatment of central venous catheter-related bloodstream infections in cancer pa-tients, International Journal of Antimicrobial Agents, 24, 491-495, 2004.

P 041 Ref: 0063

THE QUANTITATIVE DETERMINATION OF FEXOFENADINE IN HUMAN PLASMA BY LC/MSD SYSTEM Müberra ŞEN, Evren İŞLEYEN, Selma ÖZİLHAN, Suna TOPTAN, Tuncel ÖZDENNovagenix Bioanalytical R&D Centre, Ankara, Turkey

Fexofenadine hydrochloride, (±)-4-[1-hydroxy-4-[4-(hydroxy-diphenylmethyl)-1-piperidinyl]-butyl]-α, α-dimethyl benzeneacetic acid hydrochloride has an empirical formula, C32H39NO4.HCl with a molecular weight of 538.1 which is the active metabolite of terfena-dine and is a second-generation histamine H1-receptor antagonist in piperidine-class drugs. Fexofenadine is a H1-receptor antagonist that blocks peripheral histamine H1-receptors selectively [1, 2].

A simple, rapid, sensitive and selective LC-MS method was de-veloped and validated for quantification of fexofenadine in human plasma. The LC-MS system was operated under the positive elec-trospray ionisation mode (ESI). After liquid-liquid extraction, fex-ofenadine analysis was performed through a C18 column with a mo-bile phase of acetonitrile: 10 mM ammonium acetate: formic acid, 70:30:0.1 (v/v/v) at a flow rate of 1 mLmin-1 by using loratadine as an internal standard. The lower limit of quantitation was 3 ngmL-1 for fexofenadine.

Results of analysis showed after five days validation process, co-efficient correlation was 0.9993 – 0.9999. In quality control sam-ples, with-in-batch and batch-to batch accuracy ranges were 86.51 – 113.50% and 97.92 – 106.06% respectively; precision ranges were 3.89 – 13.62% and 8.40 – 11.81% respectively. In calibration stand-ard samples, batch-to batch accuracy ranges were 96.40 – 104.17%; batch-to batch precision ranges were 2.44 – 5.80%. Our whole study was conducted according to FDA regulations about bioanalytical method validation process [3]. The presented analytical method that is developed originally and validated in our laboratory was used to evaluate the bioequivalency of two different brand name fexofena-dine products.

1. Simpson K, Jarvis B. Drugs, 59, 301-321, 2000. 2. Dollery C. Therapeutic Drugs 2nd edition Churchill Livingstone, United

Kingdom, A151-A154. 1999. 3. FDA, Bioanalytical Method Validation, Guidance for Industry, May

2001.

P 042 Ref: 0064

COMPARISON OF THE ROYAL JELLY AND POVIDONE IODINE ON WOUND HEALING IN RABBITS 1Füsun TEMAMOĞULLARI, 2Ali HAYAT, 3Füsun BABA1University of Harran, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Şanlıurfa, Turkey2University of Harran, Faculty of Veterinary Medicine, Department of Surgery, Şanlıurfa, Turkey3University of Harran, Faculty of Medicine, Department of Pathology, Şanlıurfa, Turkey

Royal jelly (RJ) has been used worldwide for many years as medi-cal products, health foods and cosmetics [1]. A number of biological and immuno-regulatory actions attributed to RJ have been report-ed. In this study, we have investigated the efficacy of RJ on healing wound standing on the clinically and histopathologically comparing with 10 % povidone iodine and 0,9% sodium chloride.

Six male and six female rabbits weighing about 2500 ± 200 g were anesthetized with i.m. administration of 10mg/kg xylazine hydro-chloride (Rompun, Bayer) and 50mg/kg ketamine hydrochloride (Ketanes, Albe). On dorsal aspect of each animal, two cranially and one caudally located full-thickness skin wounds in 3,14 cm diam-eter were created using a template prepared from an X-ray film. Fol-lowing incision different wounds of each animal were treated with RJ (83 mg/ml Royal Jelly-Arıjel Co.,Ltd. ), 10% povidon iodine and 0,9% sodium chloride as the control respectively. Then the wounds were closed with sterile gauze and fixed with circular adhesive bands. Wounds were examinated macroscopically and by exudation. The beginning of the wound contraction as the indicator of the begin-ning of healing, granulation of the tissue and the first day of epithe-lization were noted regularly. SPSS 11.0 for Windows was used for statistical analyses.

Whole control wound surface were covered by a thin gelatinous exudate (POD 4). After this exudate was removed, an ongoing healthy granulation and epithelization tissues were determined. RJ gauze-applied wounds showed strong adherence and a lesser degree exudate. Therefore, they required greater tearing force for removal of the dressing. According to other groups, the acceleration of epi-thelization in the RJ treated group appeared to occur between 7 and 9 days clinically as well as histologically. However, adhered strongly and the frequent dressing may delay the healing. The epithelization was completed on POD 16 on RJ and 10 % povidone iodine gauze-applied wounds, whereas it wasn’t completed on 0.9 % sodium chlo-ride gauze-applied wounds and ulceration in central wounds was seen. The granulation tissues on all wounds were noted on PODs 3-5, while epithelization was seen on PODs 6-8. The expansion proc-ess (to POD 4) was followed by the contraction process (to PODs 6-8). The contraction in RJ gauze-applied wounds (to PODs 16) be-came more than other wounds. The thickness of scar tissue was sig-nificantly different on PODs 4 and 12 between RJ groups (P< 0.05). In our study, we did not observe any adverse effects of antiseptics on the thickness of scar tissue, the density of vascular proliferation and the degree of inflammatory cell infiltration in full thickness skin wounds. According to current study, RJ gauze-applied wounds was showed strong adherence, the dressing every day and required an extra force to separate it from them. This force could cause epithelial damage and thus may increase the thickness of scar tissue. In conclu-sion, RJ application in full-thickness skin defects in rabbits acceler-ated wound healing.

1. Hidaka S, Okamoto Y, Uchiyama S, Nakatsuma A, Hashimoto K, Ohni-shi ST, Yamaguchi M. Royal jelly prevents osteoporosis in rats: Beneficial effects in ovariectomy model and in bone tissue culture model, Evid. Based Complement. Alternat. Med., Sep 3(3), 339-48, 2006.

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P 043 Ref: 0065

INVESTIGATION OF CYTOTOXIC EFFECTS OF ANHYDRIDE CONTAINING WATER-SOLUBLE COPOLYMERS ON L929 MOUSE FIBROBLASTS 1Esin AKBAY, 1Handan SEVİM, 1Özer Aylin GÜRPINAR, 2Hatice KAPLAN-CAN, 1Mehmet Ali ONUR, 3Zakİr M. O. RZAEV, 2Ali GÜNER1Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe-Ankara, Turkey2Hacettepe University, Faculty of Science, Department of Chemistry, 06800 Beytepe-Ankara, Turkey3Hacettepe University, Department of Chemical Engineering, Faculty of Engineering, 06800 Beytepe-Ankara, Turkey

Synthetic polymers, water-soluble or in the form of hydrogels, na-noparticles, dendrimers or microspheres, are materials with which we are in daily contact or which are under development as materi-als for medical applications. At the end of the last century, synthetic polymers successfully replaced a number of natural materials, either because the latter were in short supply or because the physicochemi-cal characteristics of synthetic polymers exceeded those of materials available from natural sources [1].

In this study, complex-radical copolymerization of maleic anhy-dride (MA), and acrylic acid (AA) and ternary polymerization of maleic anhydride (MA), vinyl acceptor−donor−acetate (VA) and acrylic acid (AA) and, considered as acceptor systems, were carried out in 1,4-dioxane with benzoyl peroxide (BPO) as an initiator at 70 oC under a nitrogen atmosphere. The co- and terpolymer synthe-sized by the use of 1: 1 and 1: 2: 1 molar ratio of initial monomers, re-spectively. Polymer samples were purified by several reprecipitating from anhydrous acetone, n-hexane, diethyl ether and were dried in vacuo at 60 oC to a constant weight with quantitative yields [2]. The cytotoxic effects of poly(MA-co-AA) and poly(MA-co-VA-co-AA) polymers samples were investigated in cell culture. The cytotoxicity was observed on L929 mouse fibroblasts. In the first step, fibroblasts were cultured in DMEM at initial density of 50.000 cells/ml. Follow-ing a 24 hour of incubation, the cell culture medium was removed and fresh medium containing poly(MA-co-AA) and poly(MA-co-VA-co-AA) was added. Poly(MA-co-AA) and poly(MA-co-VA-co-AA) were prepared in five different dilutions (Dilution 1: 0.00114 g/mL; Dilution 2: 0.00057 g/mL; Dilution 3: 0.00028 g/mL; Dilution 4: 0.00014 g/mL; Dilution 5: 0.00007 g/mL). Untreated cells served as controls. The cells were incubated during 5 days. Cell number and cell morphology were investigated at the 1st and 5th days. Propidium iodide/acridine orange (PI/AO) staining was used to assess apopto-sis of treated cells and of the control group.

The results showed that there were differences between poly(MA-co-AA) and poly(MA-co-VA-co-AA) in view of cell proliferation. In poly(MA-co-VA-co-AA) group cell number was higher than poly(MA-co-AA). A relatively few number of apoptotic cells were observed in the Poly(MA-co-VA-co-AA) on day 5. Therefore it can be said that toxicity of Poly(MA-co-AA) was related with the pro-liferation characteristics of cells. Cytotoxicity of results can be ex-plained polyanionic character of the co- and ternary polymers and also poly(MA-co-VA-co-AA) depicts the low cytotoxicity behavior. Vinyl acetate fragments in the terpolymer gives the immobility to the polymer chains and lower polyanionic character [2].

1. Ottenbrite RM, Kaplan AM. Some biologically active copolymers of maleic anhydride, macromolecules as drugs and as carriers for biologi-cally active materials, Annals of the New York Academy of Sciences, 446 (1), 160–168, 1985.

2. Kaplan CH, Doğan AL, Rzaev ZMO, Uner AH, Güner A. Synthesis, characterization and antitumor activity of poly(maleic anhydride-co-vi-nyl acetate-co-acrylic acid), Journal Applied Polymer Science, 100, 3425-3432, 2006.

P 044 Ref: 0066

TOPIC ADMINISTRATION OF YARROW EXTRACT ON WOUND IN RABBITS 1Füsun TEMAMOĞULLARI, 2Ali HAYAT, 3Füsun BABA1University of Harran, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Şanlıurfa, Turkey2University of Harran, Faculty of Veterinary Medicine, Department of Surgery, Şanlıurfa, Turkey3University of Harran, Faculty of Medicine, Department of Pathology, Şanlıurfa, Turkey

Many infectious diseases are known to be treated with herbal remedies throughout the history of mankind. Yarrow belongs to the Asteraceae family and contains aquileic acid, essential oils, tannins, flavonoids and acids. The application of infusions showed positive ef-fects on wound healing and hemorrhages [1]. The aim of the present study was to investigate efficacy of yarrow extract on healing wound standing on the clinical comparison with 10 % povidone iodine and 0.9 % sodium chloride.

Six male and six female rabbits (2250 ± 100g) were taken for the study. All rabbits were anesthetized with i.m. administration of 10 mg/kg xylazine hydrochloride (Rompun, Bayer) and 50 mg/kg ketamine hydrochloride (Ketanes, Albe). Then full-thickness skin wounds were created (n: 3) on costal sides. Yarrow methanolic ex-tract was prepared by infusion of the aerial parts of the plant (10 days) in methanol at 1:5, w/v. The infusion filtered [2] with gauze was applied to the defect on the right cranial side (yarrow ektract group), and 10 % povidone iodine was applied to the defect on the left cranial side, as for control 0.9 % sodium chloride was applied to the defect on the left caudal side of the same animal. Wound surfaces were examined macroscopically and microscopically from the points of exudation, bleeding, thickness of scar, contraction and epitheliza-tion during the postoperative days (PODs).

Macroscopically, there was much less bleeding, and thicker scar and more contraction was observed in yarrow extract group compared to others. The epithelization in this group was completed on PODs 12. But the wounds treated with 0.9 % sodium chloride and 10 % povidon iodine was not completed on PODs 12. The density of vascular prolif-eration progression was significantly different on PODs 4 and 16 with-in yarrow extract group. Such a relation was not found on PODs 8-12 ((P> 0.05). The degree of inflammatory cell infiltration was signifi-cantly different on PODs 8-12 within the yarrow extract group. Such a relation was not found on PODs 4,12 and 16 (P> 0.05). 10% povidone-iodine is a microbicidal, antiseptic agent. However, it is inactivated by organic material and blood [3]. Candan et.al [4], observed that yarrow possess strong antioxidative activity but low antimicrobial activitiy in vitro. In this study, the degree of inflammatory cell infiltration was more decreased in yarrow extract than 10 % povidone iodine applied wounds on PODs 8-12. This might be a result of reduced bleeding due to yarrow extract Conclusively, we suggested that yarrow extract led limited bleeding, better contraction and decrease of inflammatory cell infiltration in wound treatment process.

1. Teixeira RO, Camparoto ML, Mantoovani MS, Vicentini, VEP. Asses-ment of two medicinal plants Psidium guajava L. and Achillea millefo-lium L. in vitro and in vivo assays, Genetics and Moleculer Biology, 26(4), 551-555, 2003.

2. Baytop, T. Türkiye’de Bitkiler ile Tedavi (Geçmişte ve Bugün), İstanbul Üniversitesi Eczacılık Fakültesi, İstanbul, s.166-167, 1999.

3. Frastvedt E., Stashak TD, Othic A. Update on Topical wound medica-tions, Clinical Techinique Practice, 3, 164-172, 2004.

4. Candan F, Ünlü M, Tepe B, Daferera D, Polissiu M, Sökmen A, Akpulat HA. Antioxidant and antimicrobial activity of the essential oil and meth-anol extracts of Achillea millefolium Subs. Millefolium Afan, Journal of Ethnopharmacology, 87, 215-220, 2003.

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P 045 Ref: 0067

INVESTIGATION OF CELL PROLIFERATION OF DEXAMETHASONE ON HUMAN PULP AND GINGIVAL FIBROBLASTS 1Handan SEVİM, 1Esin AKBAY, 1Özer Aylin GÜRPINAR, 2Zafer C. ÇEHRELİ, 1Mehmet Ali ONUR, 1Aşkın TÜMER1Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe-Ankara, Turkey2Hacettepe University, Faculty of Dentistry, Department of Pediatric Dentistry, 06100 Sıhhiye-Ankara, Turkey

Although previous studies have suggested that topical use of dex-amethasone in replanted animal teeth enhances healing and results in fewer resorption complications, the effect of dexamethasone on the types of human cells involved in the periodontal healing process remains unknown [1, 2].

This study investigated the effects of dexamethasone on cultured human pulp fibroblasts (HPF) and gingival fibroblasts (HGF). HPF and HGF were cultured in DMEM at initial density of 20.000 cells/ml and 30.000 cells/ml, respectively. Following 24h incubation, the cell culture medium was removed and fresh medium containing three different dilutions of dexamethasone (Dilution 1: 0.00001 mM ; Dilution 2: 0.0001mM; Dilution 3: 0.05 mM) were added separate-ly. Untreated cells served as controls. The cells were incubated for 5 days. Cell number and cell morphology were investigated at the 1st, 2nd, 3rd, 4th and 5th days. Propidium iodide/acridine orange (PI/AO) staining was used to assess apoptosis of treated cells and of the control group at 1st and 5th days. In Dilution 1, cell number was significantly higher than those of Dilutions 2 and 3. Compared to other dilutions, the number of apoptotic cells observed in the 3rd dilution was relatively higher than those of other dilutions.

The proliferation of HPF was significantly lower than HGF. The results of this study indicate that the effect of topical administered dexamethasone in an avulsion-type dental trauma varies for both cell-type and concentration.

1. Cabral MC, Costa MA, Fernandes MH. In vitro models of periodontal cells: a comparative study of long-term gingival, periodontal ligament and alveolar bone cell cultures in the presence of beta-glycerophosphate and dexamethasone, Journal of Materials Science, 2007 Feb 1, in press.

2. Soury B, Hentzen D, Vignal M, Christeff N, Doly J. Induction of inter-feron-beta gene expression by dexamethasone in murine L929 cells, Mo-lecular Endocrinology, 9, 199-207, 1995.

P 046 Ref: 0069

INVESTIGATION OF INTERACTIONS IN POLY(MA-ALT-AA)/PVP BLENDS 1Hatice KAPLAN-CAN, 1Serap KAVLAK, 1Ali GÜNER, 2Zakir M. O. RZAEV1Hacettepe University, Faculty of Science, Department of Chemistry, Ankara, Turkey2Hacettepe University, Faculty of Engineering, Department of Chemical Engineering, Ankara, Turkey

In the last few decades the development of various hydrophilic materials based on blends and interpolymer complexes of poly(carboxylic acid)s and non-ionic water-soluble polymers is of great importance because of their unique properties and possible applications in medicine [1]. Hydrophilic synthetic polymers have been widely investigated as carrier substances. PVP is an amorphous and biocompatible polymer with a high affinity for water and its in-teraction with water has became the topic of a large body of research [2]. It has been known that some maleic anhydride based polymers with high carboxylic acid content exhibit high biological activities such as inhibitory effect on viruses, bacteria and tumors [3].

In this present study, poly(maleic anhydride-alt-acrylic acid) co-polymer, poly(MA-alt-AA), and poly(N-vinyl-2-pyrrolidone), PVP, are used in the preparation of blends. Poly(MA-alt-AA)/PVP blends, covering a full range of compositions, were prepared by dissolution of both of the polymers in common solvent followed by the solvent removal by drying at ambient temperature. Characterization of blends was carried out by FTIR and Raman spectroscopy.

The FTIR and Raman measurements prooved the establishment of the interactions between copolymer and PVP. The significant chemical shifts in the characteristic frequencies in FTIR and Raman spectra support the strong hydrogen bond formation. Thus compat-ible blends were obtained due to this strong hydrogen bond forma-tion between copolymer and PVP.

1. Khutoryanskiy VV, Cascone MG, Lazzeri L, Nurkeeva ZS, Grigory MA, Mangazbaeva RA. Phase behaviour of methylcellulose-poly(acrylic acid) blends and preparation of related films, Polym. Int., 52, 62-67, 2003.

2. Feldstein MM, Kuptsov SA, Shandryuk GA, Plate NA, Chalykh AE. Coherence of thermal transitions in poly(N-vinyl pyrrolidone)-poly(ethylene glycol) compatible blends 3. Impact of sorbed water upon phase behavior, Polymer, 41, 5349-5359, 2000.

3. Abd El-Rehim HA, El-Hag Ali A, Mostafa TB, Farrag HA. Anti-micro-bial activity of anhydride copolymers and their derivatives prepared by ionizing radiation, Eur. Polym. J., 40, 2203-2210, 2004.

P 047 Ref: 0070

KINETIC EVALUATION OF THE SUBSTITUTIONS ON BISBENZIMIDAZOL DERIVATIVES ON THEIR INHIBITORY ACTIVITIES OF MAMMALIAN DNA TOPOISOMERASE I 1Sevil ZENCİR, 2A. Selcen ALPAN, 2Pınar ALCIL, 2Güneş ÇOBAN, 2H. Semih GÜNEŞ, 1Zeki TOPÇU1Ege University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, 35100 İzmir, Turkey2Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 35100 İzmir, Turkey

1H-Benzimidazole derivatives are known to have antibacterial, an-tifungal, antimicrobial, antiprotozoal and antihelmintic activities [1]. We have previously identified a considertable inhibition exerted by a number of 1H-benzimidazole derivatives on the mammalian type I DNA topoisomerases [2]. Topoisomerases are ubiquitous enzymes that regulate the conformational changes in DNA topology by cata-lyzing the concerted breakage and rejoining of DNA strands during many genetic processes including DNA replication, transcription, re-combination and transposition [3]. Because of the increased aware-ness on the targetting of these enzymes as an effective approach for the development of chemotherapeutics, we extended our analysis on the 1H-benzimidazole-generated topoisomerase inhibition. In this study, we synthesized 1,2-bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)ethane and showed a significant interference of this compound on the mammalian type I topoisomerase using in vitro plasmid super-coil relaxation assays [4-6]. A known topoisomerase I poision, Camp-tothecin, was used as the reference compound in the evaluation of the inhibition. Our report also includes the effects of the substitutions of the hydrogen atom at the 5- and/or 6- position on 1H-benzimidazol ring with chloro, nitro and methyl hydrogen acceptor or donor atoms/groups on the inhibitory activity of the compound.

1. Güneş HS, Coşar G. Arzneimittel-Forschung/ Drug Res., 42, 1045-1048, 1992. 2. Alpan AS, Güneş HS, Topçu Z. 1H-Benzimidazole Derivatives As Mam-

malian DNA Topoisomerase I Inhibitors, Amnusc., submitted 2007. 3. Wang JC. Ann Rev Biochem, 65, 635-692, 1996. 4. Shriner RL. Upson RW. J Am Chem Soc, 63, 2277-2278, 1941. 5. Topçu Z, Castora FJ. Biochim. Biophys. Acta, 1264, 377-387, 1995. 6. Topçu Z. J. Clin. Pharm. Ther., 26, 405-416, 2001.

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P 048 Ref: 0071

LC-DAD METHOD FOR THE DETERMINATION OF pKA VALUES OF SULFONAMIDES AND OPTIMIZING CHROMATOGRAPHIC SEPARATION BY VARYING SOLVENT COMPOSITION 1Nurullah ŞANLI, 1Güleren ALSANCAK, 2Zerrin ERDEMGİL, 3Jose Luis BELTRAN, 3Jose BARBOSA1Süleyman Demirel University, Science & Literature Faculty, Departament of Chemistry, 32260 Isparta, Turkey2Anadolu Unıversity, BİBAM, 32260 Eskişehir, Turkey3Barcelona University, Department of Analytical Chemistry, 08028 Barcelona, Spain

Systematic optimization of liquid chromatographic (LC) meth-ods requires an accurate knowledgement of the parameters that influence the separation of the compounds. The linear correlation between the logarithm of retention factor and Reichardt’s polarity parameter have been used to predict the chromatographic behavior of the compounds. Nowadays, acetonitrile-water mixtures are widely used in high performance liquid chromatography (HPLC) and LC retention parameters of the compounds strongly depend on the dis-sociation behavior of the compounds and the pH of the acetonitrile-water mobile phases [1].

The aim of this work is to analyze sulfodiazine, sulfothiazole, sul-fomerazine, sulfomethazine, sulfodoxine, sulfomonometoxine, sulfo-methoxazole like sulfonamides efficiently using HPLC method. The separation conditions have been optimized using the relationships between Reichardt’s polarity parameter and the capacity factors of the sulfonamides. The experimental region was selected in a such way that the capacity factors of the sulfonamides would stay within the limits 1< k < 10 [2]. These limits have been provided when the organic modi-fier content of the mobile phase was in the range of 25 -16% (v/v).

The sulfonamides investigated are ampholytes with weakly basic and acidic characteristic. Because of this molecular structure the re-tention of sulfonamides is expected to pass through a maximum at an intermediate mobile phase pH. The pH dependent retention pro-files have been investigated and the results obtained demonstrated that the changes in retention are consistent with this expectation. Before the optimization of chromatographic separation dissociation constants of these compounds have been determined by LC-DAD methodology [3]. The results obtained show that pKa1 and pKa2 val-ues are between approximately 2 and 7. The pH of the mobile phase was kept constant at pH 4.50 where the neutral form is predominant. Thus they will present a maximum hydrophobicity around this pH [4]. The greatest retention was obtained for sulfonamides bearing additional methyl or methoxy groups on the R side chain. This sug-gests that the R side chain plays an important role in the hydropho-bic interaction of the compounds with the reversed phase column.

1. Botsoglou NA, Fletouris DJ, Psomas IE. Retention behavior of multiple sulfonamides in various liquid chromatographic systems, Chromato-graphia, 46, 477-481, 1997.

2. Augiar de PF, Bourguignon B, Massart DL. Comparison of models and designs for optimization of the pH and solvent strength in HPLC, Anal. Chim. Acta, 356, 7-18, 1997.

3. Jiménez-Lozano E, Marqués I, Barrón D, Beltrán JL, Barbosa J. Determi-nation of pKa values of quinolones from mobility and spectroscopic data obtained by capillary electrophoresis and diode array dedector, Anal. Chim. Acta, 464, 37-45, 2002.

4. Carda-Boch S, Berthod A. Countercurrent chromatography for the measurement of hydrophobicity of sulfonamide amphoteric compounds, Chromatographia, 59, 79-87, 2004.

P 049 Ref: 0072

SYNTHESIS AND CHARACTERZATION OF NOVEL DENDRITIC POLYMERS FOR ANTI-CANCER DRUG ENCAPSULATION AND RELEASE 1Esra GÜÇ, 2Güngör GÜNDÜZ, 1Ufuk GÜNDÜZ1Middle East Technical University, Department of Biological Sciences, Ankara, Turkey2Middle East Technical University, Department of Chemical Engineering, Ankara, Turkey

Controlled drug delivery by biocompatible artificial systems has be-come one of the most interested research areas because of the ability to reduce the problems of conventional chemotherapy. By dendritic poly-mer architectures, controlled drug delivery systems gain a new strategy. Particularly dendritic polyesters are highly investigated due to their unique properties including high degree of branching, nontoxicity and water solubility [1]. Fatty acids are good candidates for polymeric sys-tems with their encapsulation stability for hydrophobic drugs and with their biosafety properties [2]. The aim of this study is to design hyper-branched polyesters with fatty acids, to encapsulate anti-cancer drugs and to study their release for chemotherapeutic purposes.

Dendritic polyesters were based on dipentaerythritol (used as core molecule) and dimethylolpropionic acid (used as repeated end groups) and they were used in stoichiometrical ratios [3]. Ricinoleic acid, a C18 fatty acid was hydrolyzed from castor oil and was con-jugated to the end groups of polyesters for effective encapsulation of hydrophobic drug. Dendritic polyesters were characterized by fourier transform infrared spectroscopy (FTIR) and size exclusion chromatography (SEC) analysis. For encapsulation of hydrophobic anti-cancer drug, idarubicin was used. Encapsulation of the drug was followed with spectrophotometric measurements in the wave-lenght regions at 540 nm and 578 nm. Interactions of idarubicin and dendritic polyester were shown by FTIR analysis. For drug release profiles samples are placed into dialysis bags and drug delivery were applied against phosphate buffer saline (PBS). Releasing medium were analyzed spectrophotometrically to measure the released drug. For the next study, toxicity effect of unloaded and drug loaded den-dritic polyesters on cell culture (MCF-7 breast cancer cell lines) will be investigated by XTT tests and IC 50 values will be determined.

The characterization by FTIR of the obtained hyperbranched pol-ymer have indicated the expected hydroxyl end groups. SEC studies have shown the molecular weight distribution of the dendritic poly-mer. According to the molecular characterization results, various polymer to drug ratios were tested for encapsulation studies.The en-capsulation efficieny variations with respect to hyperbranched poly-mer (HBR) to drug ratio is shown in the Figure 1. The results showed that encapsulation efficiency of the drug was increased by increasing the polymer amount. Conversely efficiency of encapsulation was de-creased with increasing drug ratio. Sustained release profiles of den-dritic nanoparticles were obtained and analyzed successfully. The next step in this study will be the determination of cytotoxicity of empty and drug loaded dendritic nanoparticles on MCF-7 cell line. These studies will bring new insights to successful cancer therapy by controlled delivery of anti-cancer drugs.

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Figure 1. Encapsulation efficiency of idarubicin at 25mg, 50mg, 75mg of hyperbranched polymer (HBR). Encapsulation efficiency = Encapsulated drug weight(mg)/Initial drug weight(mg)*100a. aMean ± SEM (n=2)

1. Dhanikula RS, Hildgen P. Synthesis and evaluation of novel dendrimers with a hydrophilic interior as nanocarriers for drug delivery, Bioconju-gate Chem., 17 (1), 29, 2006.

2. Slivniak R, Ezra A, Domb AJ. Hydrolytic degredation and drug release of ricinoleic acid-lactic acid copolyesters, Pharmaceutical Research, 23, 6, 2006.

3. Bat E, Gündüz G, Kısakürek D, Akhmedov İ. M. Synthesis and charac-terization of hyperbranched and air drying fatty acid based resins, Prog. Org. Coat., 55, 330-336, 2006.

P 050 Ref: 0073

DETERMINATION OF CATECHOL USING MODIFIED ELECTRODE WITH A COMPOSITE OF POLY(VINYLFERROCENE) AND POLYANILINE Muammer KAVANOZ, Nuran ÖZÇİÇEK-PEKMEZ, Kadir PEKMEZ, Attila YILDIZHacettepe University, Faculty of Science, Department of Chemistry, Beytepe-Ankara, Turkey

The determination of phenolic compounds has a great interest in many fields, such as neurochemistry, pharmaceutical and clini-cal chemistry. Some phenols as catechol, resorcinol and chlorogenic acid are found in plants, fruits and herbs. Among them, the catechol has acquired an increasing interest, not only to be a model molecule for bi-phenolic compounds such as dopamine, adrenaline, isopre-nalin, dobutamin and phenylethylamine but also have important pharmacological activities. Therefore, it is very important to develop a sensitive analytical method for the determination of catechol in biological studies. The interest in the determination of this phenolic compound has proportionated the development of several methods for their quantification, such as the chromatographic methods with different detection systems. These methods are very important, but time and reagent consuming are, generally, high. Thus, the develop-ment of new methods, that makes possible the minimal use of rea-gent and a lower time of analysis are very important. In this sense, electrochemical methods involving the development of chemical sensors have been utilized [1, 2].

Polyaniline (PANI) is one of the most promising conducting poly-mers due to its high conductivity, good redox reversibility and good stability in aqueous solutions and air. These properties provide favo-rable conditions for its potential applications in super capacitor and electrocatalysis. Poly(vinylferrocene) (PVF) is also an electroactive polymer. In this work, PVF in deposited composite film was used as an electron transfer mediator in the electrochemical oxidation of catechol due to its reversible redox.

In this study, electropolymerization of aniline in the presence of PVF was carried out by cyclic voltammetry in non-aqueous methyl-ene chloride medium. Thin and more adhesive films were obtained

when PANI and PVF+ClO4- were codeposited. The peaks belonging to both PVF and PANI were observed clearly from their cyclic vol-tammograms. It was also proven that film contains both PANI and PVF using FT-IR spectra. This PANI/PVF+ codeposited film on Pt electrode were used for determination of catechol without using en-zyme. The experimental results indicate that the anodic peak potential of catechol at the PANI/PVF+ modified electrode is lower than that at the Pt electrode in a solution consisting of catechol. The OH group on the PANI chain in the composite film plays an important role in the electron transfer between PANI and catechol in the solution. Opti-mum conditions for the determination of the relationship between the response current and the concentration of catechol are that the poten-tial was set at 0.55 V and the pH of the solution controlled at 4.0. Fig. 1 shows the change in the response current with the concentration of catechol from 3,91 to 500 µM and from 3.91 to 64 000 µM, respective-ly. This modified electrode has a lower working potential and a good operational stability due to reducing the electrode fouling, compared with the direct oxidation of catechol at the bare Pt electrode.

Figure 1. a, b) Current time for different catechol concentrations. The relationship between the response current and the concentration of catechol, c) from 3.91 µM to 500 µM, d) from 3.91 µM to 64000 µM.

1. Rita de Cassia Silva Luz, Flavio Santos Damos, Adriano Bof de Oliveira, Jo-hannes Beck, Lauro Tatsuo Kubota. Development of a voltammetric sensor for catechol in nanomolar levels using a modified electrode with Cu(phen)2 (TCNQ)2 and PLL, Sensors and Actuators, B 117, 274-281, 2006.

2. Shaolin M. Catechol sensor poly (aniline-co-o- aminophenol) as an electron transfer mediator, Biosensors and Bioelectronics, 21, 1237-1243, 2006.

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P 051 Ref: 0074

THE UV-SPECTROSCOPIC METHOD FOR DETERMINATION OF DISSOCIATION CONSTANTS OF SEVERAL SULFONAMIDES IN WATER AND ACETONITRILE-WATER BINARY MIXTURES 1Senem ŞANLI, 1Güleren ALSANCAK, 2Jose Luis BELTRAN, 2Jose BARBOSA1Süleyman Demirel University, Science and Literature Faculty, Department of Chemistry, 32260 Isparta, Turkey2Barcelona University, Department of Analytical Chemistry, 08028 Barcelona, Spaın.

Sulfonamides are anti-bacterial and anti-infective drugs com-monly used to treat in medicine and veterinary practice. The physi-cochemical profiling of drugs includes the determination of their dissociation constants. Data on proton dissociation of pharmacolog-ically active substances are of utmost interest for the understanding of stability and solubility of drugs. A major factor of drug permea-tion is their aqueous pKa.

Very often, the main difficulty in the determination of dissociation constants of drugs is their aqueous insolubility that forces the use of spectroscopic method. UV spectroscopy is an excellent method for pKa determination. This method requires very low analyte con-centration and allows suitable absorbance measurement in aqueous solution even for products with low aqueous solubility. Acetonitrile (ACN)-water mixtures are usually employed for pKa determina-tion of water insoluble drugs. Literature shows several extrapolation equations to estimate aqueous pKa from the pKa values determined in acetonitrile-water mixtures [1].

In this study, approximately 1.0 x 10-5 M solution of sulfonamides (sulfadiazine, sulfamethazine, sulfatiazole, sulfamethoxazole, sul-famonomethoxine, sulfamerazine, sulfadimethoxine) in water and ACN – water mixtures were titrated with NaOH in the range of pH 2-11 for determination of dissociation constants [2].

The data evaluation was performed by using STAR program [3]. The spectrum recorded for sulfadiazine at various pH values in ACN-water, 30 % (v/v) is shown in Fig 1. The aqueous pKa obtained from UV data and extrapolations of the data in ACN-water medium is consistent between them and agree with those from literature [4].

Fig 1. UV-spectrum of sulfadiazine obtained at various pH in ACN-water mixture (30 % v/v).

1. Ruiz R, Roses M, Rafols C, Bosch. Critical validation of a new simpler approach to estimate aqueous pKa of drugs sparingly soluble in water, Anal. Chim. Acta, 550, 210-221, 2005.

2. Polster J, Lachmann H. Spectrometric Titrations, VCH, 1989. 3. Jiménez-Lozano E, Marqués I, Barrón D, Beltrán JL, Barbosa J. Determi-

nation of pKa values of quinolones from mobility and spectroscopic data obtained by capillary electrophoresis and a diode array detector, Anal. Chim. Acta, 464, 37-45, 2002.

4. Lin CE, Lin WC, Chen YC, Wang SW. Migration behavior of sulfona-mides in capillary electrophoresis, J. Chromatogr. A, 792, 37-47, 1997.

P 052 Ref: 0075

DETERMINATION OF PHYSICOCHEMICAL PARAMETERS AND SAR STUDY ON THE SOME BENZIMIDAZO[1,2-a]PYRIMIDINE DERIVATIVES Asiye MERİÇAnadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskişehir, Turkey

Condensed compounds bearing bridgehead nitrogen atom have various activity, ranging from antihelmintic and anticonvulsant to antitumor and antiviral. Last study in our research group was re-vealed the cytotoxicities of some benzimidazo[1,2-a]pyrimidine compounds on non-cancer and cancer cell lines [1]. It is necessary to evaluate their SAR in order to understand the activity mechanism of this type fused azole compounds, properly.

Initially, physicochemical parameters of synthetic compounds were calculated theoretically. Steric {molecular weight (MW), mo-lecular refraction (MR) [2], molecular volume (MV) [3], molecular connectivity index (MCI) [4], paracor (Par) [5-7]}, hydrophobic {partition coefficient [8], hydrophobic substituent constant (π) [9]} and electronic{electronic substituent constant [10]} parameters of 2,4-di- and 2,3,4-trisubstituted benzimidazo[1,2-a]pyrimidine de-rivatives were determined firstly. Then, correlations were constituted between cytotoxicities (IC50) and structural properties of compounds. Some computerized programs were also used for further evaluation.

Table . Training Set of Compounds

Compound R1 R2 R3 Compound R1 R2 R3

1 OH H Me 6 Ph H Me

2 OH H Ph 7 Ph H Ph

3 OH H n-Pr 8 Me H Me

4 OH Ph Me 9 Me Me Me

5 OH Me Me 10 Me Et Me

The SAR results of compounds can be summarized as follows:At positions R1, R2 and R3; hydrophobic, electronic and steric

properties were found important. The existence and abundance of methyl substituent on the structure increase the cytotoxic activity. It can be concluded that the substances abundantly bearing methyl subtituent may evaluate as promising compounds for potential anti-neoplastic activity. 1. Meriç A, İncesu Z, Karayel A, Özbey S. Synthesis of some 2,4-di- and

2,3,4-trisubstituted benzimidazo[1,2-a]pyrimidines and evaluation of their cytotoxicities toward F2408 and 5RP7 cells, Revista de Chimie, 57(11), 1090-1097, 2006.

2. Dunn III WJ. Molar refractivity as an independent variable in quantita-tive structure-activity studies, Eur. J. Med. Chem.-Chim. Ther., 12, 109-112, 1977.

3. Berkem AR, Baykut S., Fizikokimya Kitabı, İstanbul Üniversitesi Yayınları, Sayı: 2735, Kimya Fakültesi, No. 42, 1980.

4. Kier LB, Hall LH. Derivation and significance of valence molecular con-nectivity, J. Pharm. Sci. 70, 583-9, 1981.

5. Quayle QR. The parachors of organic compounds, Chem. Rev., 53, 439-89, 1953.

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6. Sugden S. A relation between surface tension, density, and chemical composition, J. Chem. Soc., 125, 1177, 1924.

7. Vogel AI. Physical properties and chemical constitution. Part IX. Aliphat-ic hydrocarbons, J. Chem. Soc., 133, 1946.

8. Rekker RF, De Kort HM. The hydrophobic fragmental constant, an ex-tension to a 1000 data point set, Eur. J. Med.-Chim. Ther., 14(6), 479-488, 1979.

9. Hansch C, Leo A, Unger SH, Kim KH, Nikaitanı D, Lien EJ. Aromatic substituent constant for structure activity correlations, J. Med. Chem., 16, 1207-26, 1973.

10. Akı-Şener E, Yalçın İ. Kantitatif Yapı-Etki İlişkileri Analizleri (QSAR), Ankara Üniversitesi Eczacılık Fakültesi Yayınları No: 86, 2003. (ISBN 975-482-585-8)

P 053 Ref: 0076

ENHANCED ENZYME ACTIVITY OF IMMOBILIZED LIPASE AS BIOCATALYST FOR SYNTHESIS OF ESTERS IN ORGANIC MEDIA Taylan K. ÖZTÜRK, Funda KARTAL, Ali KILINÇEge University, Faculty of Science, Department of Biochemistry, İzmir, Turkey

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and the synthesis of esters of glycerol and long-chain fatty acids. The many applications of lipases include special organic syntheses, hydrolysis of fats and oils, modification of fats, flavor en-hancement in food processing, resolution of racemic mixtures and chemical analyses [1].

Research on lipase catalyzed production of various kinds of ester has increased tremendously in the recent past. Esters are present in fats and oils and in natural and synthetic polymers. They are useful intermediates or end products in the chemical industry. Enzymatic production of esters can be achieved either by reaction between free acid and hydroxyl groups of alcohol or by ester exchange or transes-terification (include alcoholysis, acidolysis and interesterification).

Lipase catalized esterification reactions have been actively pur-sued to produce various kinds of commercially important esters. Esters of short chain fatty acids are extremely important aromatic compounds. Esters of short chain alcohols and long chain fatty acids are valuable oleochemicals that may be used as lubricants, diesel fuel and antistatic reagents. Esters of long chain fatty acids and polyhy-dric alcohols like glycerol, sorbitol and other carbohydrates (called-emulsifiers/surfactants) find immense application in food and phar-maceutical industries [2].

In this work the immobilized form of lipase was prepared by covalent attachment of enzyme on crosslinked polyvinyl alcohol [3]. Optimization of reaction conditions for ester synthesis was made by experimenting with different chain length acids and alcohols and ef-fects of organic solvent type on esterification activity were studied. The effects of reaction time and reaction temperature were also stud-ied. Esterification activity of immobilized lipase in organic solvents was measured by GC-FID.

1. Hari Krishna S, Karanth N.G. Production, purification, characterization and applications of lipases. Catalysis Reviews, 44, 499-591, 2002.

2. Rohit S, Yusuf C, Uttam B. Lipases and lipase-catalyzed esterification reactions in nonaqueous media, Biotechnology Advances, 19,627-662, 2001.

3. Kılınç A, Önal S, Telefoncu A. Chemical attachment of porcine pancre-atic lipase to crosslinked poly(vinyl alcohol) by means of adipoyldichlo-ride, Process Biochemistry, 38, 641-647, 2002.

P 054 Ref: 0077

EFFECT OF USNIC ACID ON TISSUE NITRIC OXIDE SYNTHASE ACTIVITY AND GLUTATHIONE LEVEL IN TITANIUM-IMPLANTED SUBJECTS 1Fehmi ODABAŞOĞLU, 2Hayati AYGÜN, 2Ömer Selim YILDIRIM, 3Zekai HALICI, 1Mesut HALICI, 4Zafer OKUMUŞ, 5Ali ASLAN, 6Ahmet ÇAKIR, 7Cavit KAZAZ1Atatürk University, Faculty of Pharmacy, Department of Biochemistry, 25240 Erzurum, Turkey2Atatürk University, Faculty of Medicine, Department of Orthopedics and Traumatology, 25240 Erzurum, Turkey3Atatürk University, Faculty of Medicine, Department of Pharmacology, 25240 Erzurum, Turkey4Atatürk University, Faculty of Veterinary Medicine, Department of Surgery, 25240 Erzurum, Turkey5Atatürk University, Kazım Karabekir Education Faculty, Department of Biology, 25240 Erzurum, Turkey6Atatürk University, Kazım Karabekir Education Faculty, Department of Chemistry, 25240 Erzurum, Turkey7Atatürk University, Faculty of Science, Department of Chemistry, 25240 Erzurum, Turkey

Debris due to the frictions as well as biochemical and magnetic re-actions following orthopedic implantations may play a role in aseptic loosening through initiating a series of complex cellular reactions between bone and implant. Loosening may be related to cytotoxic-ity [1, 2]. Usnic acid (UA) (Fig. 1) is a dibenzofuran derivative bio-synthesised by lichens. Previously, it has been shown that UA has various biological activities [3]. The present study was conducted to evaluate the effect of UA on nitric oxide synthase (NOS) activity and glutathione level (GSH) in Ti-implanted tissues of rabbits.

Fig. 1. Molecular structure of usnic acid (UA)

UA was isolated from a lichen species, Usnea longissima [3]. Eight-een New Zealand rabbits divided into 6 groups, of which femurs of rabbits in 5 groups were subperiostally implanted with Ti. Then, they received UA (30 mg/kg) and olive oil (OO) orally or locally every 3 d for 21 d or received none. Rabbits from the other group served as control. Following euthanasia, tissues around the implant were scrapped and then ground within liquid nitrogen for NOS activity and GSH level.

In the present study, 2.17, 1.6, and 1.7-fold increases were deter-mined in the activities of iNOS (inducible), cNOS (constitutive), and tNOS (total) respectively in Ti-implanted rabbits compared to con-trol rabbits (Table 1). However, both local and oral administration of OO and oral administration of UA decreased NOS activities. Olive oil was more effective than UA when administered locally, whereas UA was more effective than OO when administered orally. Surgi-cal intervention was associated with a 31% reduction in GSH level. Local and oral administration of UA and only local administration of OO alleviated this reduction. In conclusion, UA and OO admin-istration may affect cytotoxicity via suppressing iNOS activity and increasing GSH level.

Keywords: Titanium implant, usnic acid, nitric oxide synthase, glutath-ione

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Table 1. Effects of local and oral-administrated usnic acid (UA) and olive oil (OO) on the activities of nitric oxide synthase enzymes (tNOS, cNOS and iNOS) and amount of glutathione (GSH) in titanium-implanted subperiostal tissues of rabbits. Titanium group (TIT) was compared with healthy group. The 30 mg/kg dose of UA and OO treated groups were compared with TIT group.

Trea

tmen

ts

N

NITRIC OXIDE SYNTHASE (NOS) ACTIVITY

(µmol/min/mg tissue)a

Amount of GSH

(nmol/mg tissue)a

tNOS cNOS iNOS

TIT+UA (local) 3 6.84±0.27** 3.07±0.17** 3.77±0.19** 3.44±0.02*

TIT+UA (oral) 3 2.58±0.19** 2.47±0.18** 0.12±0.07** 3.96±0.04**

TIT+OO (local) 3 3.65±0.27** 3.52±0.25* 0.13±0.02** 3.52±0.12*

TIT+OO (oral) 3 3.40±0.37** 2.85±0.40** 0.55±0.05** 2.51±0.13*

TIT (control) 3 5.43±0.21** 4.39±0.22** 1.04±0.03** 2.91±0.08**

Healthy tissue 3 3.19±0.08 2.72±0.06 0.48±0.02 4.21±0.01

a Means±SEM of tissues of six legs in each group. N: The number of rabbits. *Significant at p<0.05; **Significant at p<0.01.

1. Stea S, Visentin M, Granchi D, Cenni E, Ciapetti G, Sudanese A, Toni A. Apoptosis in peri-implant, Biomaterials, 21, 1393-1398, 2000.

2. Raha S, Robinson BH. Mitochondria, Oxygen free radicals, and apopto-sis, Am. J. Med. Gen., 106, 62-70, 2001.

3. Odabaşoğlu F, Çakır A, Süleyman H, Aslan A, Bayır Y, Halıcı M, Kazaz, C. Gastroprotective and antioxidant effects of usnic acid on indometha-cine-induced gastric ulcer in rats, J. Ethnopharmacology, 103 (1), 59-65, 2006.

P 055 Ref: 0078

SYNTHESIS AND CHARACTERIZATION OF SOME NOVEL ISATIN DERIVATIVES IN WHICH ANTICONVULSANT ACTIVITY IS PREDICTED Ebubekir SEPTİOĞLU, Mutlu DİLSİZ-AYTEMİR, Ünsal ÇALIŞHacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100-Sıhhiye, Ankara, Turkey

The restrictive treatment of epileptic seizures of the patients lets the researches to find the new agents with more efficient activity and less toxicity [1]. Recently, the anticonvulsant activities of isatin derivatives were reported by various studies [2-4]. In this study, we have synthesized some new isatin derivatives as shown below and will evaluated the anticonvulsant activities of all compounds in fu-ture work.

The synthesized compounds were prepared by Tacconi and col-leagues’ method [5]. Treatment of isatin with sodium hydride in N,N-dimethyl formamide (DMF) at room temperature gave isatin sodium salt. This isatin salt was alkylated with 1,2-dibromoethane in DMF. Reaction of the alkylated isatin as named 1-(2-bromoe-thyl)-1H-indole-2,3-dione with benzoxazol-2-one derivatives gave compounds 1 and 2. Final compounds 3 and 4 which have phenyl-hydrazone group were prepared by heating compounds 1 or 2 with phenylhydrazine in ethanol.

The basic structures of these compounds were confirmed by IR, 1H-NMR, mass spectral and elemental analyses data. The reaction products were assigned structure that were in accordance with their spectroscopic and chemical behaviors. Thus, compounds 1 and 2 showed two strong band at 1771-1766 and 1745-1746 cm–1 in their IR spectra assignable to C=O groups. Also, compounds 3 and 4 have a one stretching band at 1783-1779 cm–1. The ethylene group protons in the 1H-NMR spectra of all compounds appeared as a triplet at 3.31-4.07 ppm for -N-CH2-, a triplet signal at 4.23-4.14 ppm for -N-CH2-CH2-. Characteristic singlet peak of –NH for compounds 3 and 4 were observed at 12.4 ppm.

1. Malawska B. New Anticonvulsant Agents, Curr Top Med Chem 5(1), 69-85, 2005.

2. Popp FD. Potential anticonvulsants. IX. Some isatin hydrazones and re-lated compounds, J. Heteroc. Chem. 21, 1641-1645, 1984.

3. Pandeya SN, Sriram D, Yogeeswari P, Stables JP. Anticonvulsant and neurotoxicity evaluation of 5-(un)-substituted isatin-imino derivatives, Pharmazie, 56, 875-876, 2001.

4. Pandeya SN, Smitha S, Stables JP. Anticonvulsant and sedative-hypnotic activities of N-substituted isatin semicarbazones, Arch Pharm (Weinhe-im), 335(4), 129-134, 2002.

5. Tacconi G, Righetti PP, Desimoni G. Einfache Darstellung von N-substi-tuierten Isatinen, J. Prakt. Chem., 315(2), 339-344, 1973.

P 056 Ref: 0079

PHENOLIC COMPOUNDS OF SIDERITIS OZTURKII AND THEIR IN VIVO ANTI-INFLAMMATORY AND ANTINOCICEPTIVE ACTIVITIES 1Pınar ŞAHİN, 2Esra KÜPELİ, 1İhsan ÇALIŞ, 1Nurten EZER, 3Erdem YEŞİLADA1Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Ankara, Turkey2Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, Ankara, Turkey3Yeditepe University, Faculty of Pharmacy, Department of Pharmacognosy, İstanbul, Turkey

In Turkey, the genus Sideritis L. (Lamiaceae) is represented by 46 species [1] and some of which are used in traditional medicine for their beneficial and curative effects [2]. Sideritis species growing in Turkey are known to be rich in essential oils, diterpenes, flavonoids and phenylethanoid glycosides [3-5]. In a continuation of our phyto-chemical studies on Turkish Sideritis species, we now report the iso-lation of phenolic compounds from S. ozturkii Aytaç & Aksoy, which is endemic to Turkey, through in vivo bioassay-guided fractionation procedures.

Acetone extract from aerial parts of S. ozturkii and its fractions were investigated for its in vivo anti-inflammatory and antino-ciceptive activities. For the anti-inflammatory activity assessment, carrageenan-induced hind paw edema and for the antinociceptive activity, p-benzoquinone-induced abdominal constriction tests were used [6].

Acetone extract of the plant and its phenolic fraction were found to possess significant inhibitory activity on these models in mice. Ozturkosides A-C were isolated from the active phenolic fraction.

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The structures of isolated compounds were elucidated by spectro-scopic techniques (UV, IR, 1D- and 2D-NMR, MS). Ozturkoside C showed notable antinociceptive and anti-inflammatory activities without inducing any apparent acute toxicity or gastric damage. Al-though the activity of ozturkosides A and B were found insignifi-cant in statistical analysis, some inhibitory effects were observed. Accordingly, it is suggested that these components in phenolic fraction might possibly share the antinociceptive and anti-inflam-matory activities together.

1. Aytaç Z, Aksoy A. A new Sideritis species (Labiatae) from Turkey, Flora Mediterranea, 10, 181-184, 2000.

2. Baytop T. Therapy with Medicinal Plants in Turkey (Past and Present), Nobel Tıp Publications, İstanbul, p.375, 1999.

3. Ezer N, Vila R, Cañigueral S, Adzet T. Essential oil composition of four Turkish species of Sideritis, Phytochem., 41, 203-205, 1996.

4. Akcoş Y, Ezer N, Özçelik B, Abbasoğlu U. Iridoid glycosides from Sideri-tis lycia Boiss & Heldr. and its antimicrobial activities, FABAD J. Pharm. Sci., 23, 99-103, 1998.

5. Şahin FP, Taşdemir D, Rüedi P, Ezer N, Çalış İ. Three new acylated flavon glycosides from Sideritis ozturkii Aytaç & Aksoy, Phytochem., 65, 2095-2099, 2004; 66, 125, 2005.

6. Küpeli E, Harput UŞ, Varel M, Yeşilada E, Saracoğlu İ. Bioassay-guided isolation of iridoid glucosides with antinociceptive and anti-inflamma-tory activities from Veronica anagallis-aquatica L., J. Ethnopharmacol., 102, 170–176, 2005.

P 057 Ref: 0080

ANTI-INFLAMMATORY EFFECTS OF THE ALPHA-LIPOIC ACID ON CARRAGEENAN-INDUCED ACUTE AND COTTON PELLET-INDUCED CHRONIC INFLAMMATION MODELS IN RATS AND ITS RELATION WITH NITRIC OXIDE SYNTHASE ACTIVITY 1Fehmi ODABAŞOĞLU, 2Zekai HALICI, 3Hayati AYGÜN, 1Mesut HALICI, 1Yasin BAYIR, 4Ahmet ÇAKIR, 5Elif ÇADIRCI, 1Fadime ATALAY1Atatürk University, Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey2Atatürk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey3Atatürk University, Faculty of Medicine, Department of Orthopedics and Traumatology, Erzurum, Turkey4Atatürk University, Kazım Karabekir Education Faculty, Department of Biology, Erzurum, Turkey5Atatürk University, Faculty of Pharmacy, Department of Pharmacology, Erzurum, Turkey

Alpha-lipoic acid (ALA) is a dithiol that is found naturally in mitochondria as the coenzyme for pyruvate dehydrogenase and al-pha-ketoglutarate dehydrogenase. ALA has been shown to combat oxidative stress by quenching a variety of intracellular reactive oxy-gen species (ROS) [1]. ALA has been demonstrated to be effective in preventing pathology in various experimental models in which ROS have been implicated [2]. Mediators such as free radicals, ni-tric oxide, prostaglandins, and cytokines play important roles in the development of acut or chronic inflammation [3]. The present study was conducted to evaluate the effect of ALA on acute and chronic inflammation models in Dawley rats. Additionally, we have investi-gated the alterations in the activity of nitric oxide synthase following oral administration of ALA, indomethacine (IND) and diclofenac (DIC) in CAR-induced paw edema tissues of rats.

The present study was carried out in a total of 72 male Sprague-Dawley rats, weighing 180-190 g. The animals were grouped before the experiments and kept under standard conditions [4]. Animals were assigned randomly for acute inflammation to the control groups receiving either CAR (0.1 ml of 1% per animal) or water and expamintal groups receiving CAR plus ALA (50, 100 and 200 mg/

kg), indomethacine (IND, 25 mg/kg) or diclofenac (DIC, 25 mg/kg). In other series of experiments, the effect of ALA on the proliferative phase of inflammation was investigated using cotton pellet test [5].

In the present study, we found that 1) All doses of ALA, IND and DIC have significantly decreasing effect on the mean weight of the cotton pellets. The anti-proliferative effect of ALA was found as 67.7, 68.9 and 69.9 %, of IND was 83.8 % and of DIC was 76.1 %; 2) ALA reduced the development of CAR-induced paw edema, at a smaller magnitude for ALA than for IND and DIC; and 3) There were 2.57-fold increase in activity of inducible nitric oxide synthase (iNOS) in CAR-induced rats compared to control rats. However, oral adminis-tration of ALA, IND and DIC significantly decreased iNOS activity. All doses of ALA were more effective than IND and DIC for de-creasing iNOS activity. These results suggest that the anti-inflamma-tory effect of ALA on CAR-induced acute and cotton pellet-induced chronic inflammations can be attributed to its decreasing effect on activities of inducible nitric oxide synthase.

Table 1. Effects of ALA, indomethacine (IND) and diclofenac (DIC) on carrageenan (CAR)-induced paw edema and cotton pellet granuloma test in rats.

Treatment Num

ber o

f ani

mal

s

Dos

e m

g/kg

bod

y w

t

Acu

te

antii

nfla

mm

ator

y ef

fect

(Inh

ibiti

on %

)

Chr

onic

an

tiinf

lam

mat

ory

effe

ct (I

nhib

ition

%)

ALA 2x6 50 18.5 67.7

- 2x6 100 29.6 68.9

- 2x6 200 40.7 69.9

DIC 2x6 25 44.4 76.1

IND 2x6 25 55.5 83.8

CONTROLS 2x6 - - -

Table 2. Effects of ALA, IND and DIC on changes in activities of cNOS, iNOS and tNOS in CAR-induced paws (5th hour) of rats.

Treatment Num

ber o

f an

imal

s

Dos

e m

g/kg

bo

dy w

t.

cNO

S

iNO

S

tNO

S

ALA 6 50 2.70±0.53* 0.17±0.02** 2.86±0.55

- 6 100 3.10±0.25* 0.13±0.05** 3.23±0.29*

- 6 200 3.23±0.37* 0.13±0.03** 3.37±0.37*

CAR+DIC 6 25 2.28±0.07 0.18±0.03** 2.46±0.07

CAR+IND 6 25 2.04±0.05 0.22±0.01** 2.25±0.05

CAR 6 - 2.22±0.25 0.36±0.06** 2.58±0.24

HEALTHY 6 - 2.51±0.37 0.14±0.03 2.65±0.37

1. Bilska A, Wlodex L. Lipoic acid- the drug of future?, Pharmacol Report, 57, 570-577, 2005.

2. Odabaşoğlu F. Alpha lipoic acid, Pharma Şark, 1 (4), 12-15, 2006.

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3. Gualillo O, Eiras S, Lago F, Dieguez C, Casanueva FF. Evaluated serum leptin concentrations induced by experimental acute inflammation, J. Ethnopharmacol., 75, 213-218, 2001.

4. CCAC. 1993. Guide to the care and use of experimental animals, Vol I, 2nd Ed. Canadian Council on Animal Care. Bradda Printing Services Inc., Ottawa, ON, Canada.

5. Süleyman H, Odabaşoğlu F, Aslan A, Çakır A, Karagöz Y, Göçer F, Halıcı M, Bayır Y. Antiinflammatory and antiulcer effects of aqueous extract of Lobaria pulmonaria, Phytomedicine, 10 (6-7), 552-557, 2003.

P 058 Ref: 0081

GASTROPROTECTIVE EFFECT OF MONTELUKAST (SINGULAIRE) ON INDOMETHACINE-INDUCED GASTRIC ULCER IN RATS AND ITS RELATION WITH SOME GLUTATHIONE METABOLISM PARAMETERS 1Günnur ÖZBAKIŞ-DENGİZ, 2Zekai HALICI, 3Fehmi ODABAŞOĞLU, 4Elif ÇADIRCI, 2Halis SÜLEYMAN1Karaelmas University, Faculty of Medicine, Department of Pharmacology, Zonguldak, Turkey2Atatürk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey3Atatürk University, Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey4Atatürk University, Faculty of Pharmacy, Department of Pharmacology, Erzurum, Turkey

Non-steroidal anti-inflammatory drugs (NSAID) are widely used in the treatment of pain, fever and inflammation. However, these drugs have some side effects, especially on the gastrointestinal tract. Recently, reactive oxygen species (ROS) have also been shown to play a critical role in gastric ulceration process. The role of ROS in the development of pathogenesis in acute experimental gastric le-sions induced by stress, ethanol and NSAID’s is well known [1]. The glutathione (GSH), glutathione S-transferase (GST) and glutathione reductase (GR) play an important role in the prevention of the gas-tric damages [2]. On the other hand, montelukast (MLK), a selec-tive reversible cysteinyl leukotriene-1 receptor (LTD4 receptor) an-tagonist is used in the treatment of asthma [3] and presently nothing known about its effects on the gastro-intestinal system. We have in-vestigated alterations in the GSH level and the activities of antioxida-tive enzymes (GST and GR), following oral administration of MLK, lansoprazole (LAN), famotidine (FAM) and ranitidine (RAN) in rats with indomethacin (IND)-induced ulcer.

48 male Sprague-Dawley rats, weighing 180–190 g were used in the present study. The animals were grouped before the experiments and kept under standard conditions [4]. MLK (5, 10 and 20 mg/kg), LAN (30 mg/kg), FAM (25 mg/kg) and RAN (25 mg/kg) were administrated orally. 5 min after MLK, LAN, FAM and RAN administrations, IND (25 mg/kg) was administrated to all animals orally. Control group re-ceived only water. After 6 h of all treatments, animals were sacrificed using sodium thiopental (50 mg/kg). The stomachs were removed and opened along the greater curvature and washed with saline [2]. The wideness of ulcer areas were determined using a magnifier and a mil-limeter paper. Tissues were grounded under liquid nitrogen for the determinations of GSH level and GST and GR activities.

In the present study, we found that 1) MLK, LAN, FAM and RAN reduced the development of IND-induced gastric damages, at a greater magnitude for MLK, FAM and LAN than for RAN; 2) MLK and RAN caused an increase in the activity of GST possibly resulted from gastric injury; and 3) MLK and RAN ameliorated depressions in the GSH levels and the GR activity possibly caused by IND ad-ministration. These results suggest that the gastroprotective effect of MLK on IND-induced ulceration can be attributed to its ameliorat-ing effect on the oxidative damage.

Table 1. Effects of the MLK, FAM, RAN and LAN on IND-induced gastric damage in rats. IND was compared with healthy and treated groups with IND group.

Treatment N Dose mg/kg body wt.

Ulcer index (UI)

% Inhibition

IND+MLK 6 5 2.98±0.88** 59.1

IND+MLK 6 10 2.21±0.91** 69.7

IND+MLK 6 20 2.03±0.49** 72.2

IND+FAM 6 25 0.16±0.08*** 97.8

IND+RAN 6 25 2.54±0.76** 65.2

IND+LAN 6 30 0.0±0*** 100

IND 6 25 7.29±0.43*** -

Healthy 6 - - -

Table 2. Effects of the MLK, RAN and LAN on GSH, GST and GR in rat’s IND-induced tissues. IND was compared with healthy and treated groups with IND.

Treatment N Dose (mg/kg)

GST ACTIVITY

(EU)

GR ACTIVITY

(EU)

GSH LEVEL

IND+MLK 6 5 26,29±0,5* 34.5±0.6* 3.31±0.06*

IND+MLK 6 10 28,22±0,7* 29.6±0.8* 3.76±0.04*

IND+MLK 6 20 33,08±0,8** 27.6±0.7** 4.24±0.05**

IND+LAN 6 30 33,65±0,4** 43.1±0.6* 4.85±0.03**

IND+RAN 6 25 17,33±0,8* 25.2±0.2** 4.17±0.10**

IND 6 25 23,53±0,3* 37.7±0.9** 3.05±0.04**

HEALTHY 6 - 28,60±0,4 25.6±0.5 4.12±0.04

1. Das, D, Bandyopadhyay, D, Bhattacharjee, M, Banerjee, RK. Hydroxyl radical is the major cousative factor in stress-induced gastric ulceration, Free Radical Biol. Med., 23, 8-18, 1997.

2. Odabaşoğlu F, Çakır A, Süleyman H, Aslan A, Bayır Y, Halıcı M, Kazaz, C. Gastroprotective and antioxidant effects of usnic acid on indometha-cine-induced gastric ulcer in rats, J. Ethnopharmacology, 103 (1), 59-65, 2006.

3. Wenzel, SE. Leukotriene receptor antagonists and related compounds, Can. Respir. J., 6, 189-193, 1999.

4. CCAC. Guide to the care and use of experimental animals, Vol I, 2nd Ed. Canadian Council on Animal Care. Bradda Printing Services Inc., Ottawa, ON, Canada, 1993.

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P 059 Ref: 0082

GASTROPROTECTIVE EFFECT OF AMIODARONE ON INDOMETHACIN-INDUCED GASTRIC ULCER IN RATS AND ITS RELATION WITH MYELOPEROXIDASE AND SOME ANTIOXIDANT ENZYMES 1Günnur ÖZBAKIŞ-DENGİZ, 2Fehmi ODABAŞOĞLU, 3Zekai HALICI, 4Elif ÇADIRCI, 2Mesut HALICI, 3Halis SÜLEYMAN1Karaelmas University., Faculty of Medicine, Department of Pharmacology, Zonguldak, Turkey2Atatürk University, Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey3Atatürk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey4Atatürk University, Faculty of Pharmacy, Department of Pharmacology, Erzurum, Turkey

Amiodarone (AMD){2-butyl-3-(3’;5’diiodo-4’α-diethyl-amino-ethoxy-benzoyl) -benzofuran} is a multiple ion (Ca++, Na+, K+) chan-nel blocker drug and is also a non competitive α- and β-blocker in cardiac cell. It is an effective anti-arrhythmic and is used to treat a wide variety of ventricular and supraventicular tachyarrhytmias [1]. Clinical use of AMD is limited because of its potential for develop-ing numerous adverse side effects. Of greatest concern is AMD-in-duced pulmonary toxicity (AIPT), due to the potential for mortality. However, hepatotoxicity and other adverse effects are also of clinical importance. This drug can also modulate thyroid function and phos-pholipid metabolism [1-3]. Non-steroidal anti-inflammatory drugs (NSAID) are widely used in the treatment of pain, fever and inflam-mation. However, these drugs have some side effects, especially on the gastrointestinal tract. Recently, reactive oxygen species (ROS) have also been shown to play a critical role in gastric ulceration proc-ess. The role of ROS in the development of pathogenesis in acute experimental gastric lesions induced by stress, ethanol and NSAID’s is well known [4]. ROS damage membrane proteins via causing lipid peroxidation in membranes by attacking to unsaturated fatty acids [5]. Oxygen-handling cells have antioxidant enzymes that are able to protect them against. The enzymatic antioxidant defenses include superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPx), as marker of ulceration process [6, 7]. These antioxidants also play an important role in the prevention of the gastric damages. We have investigated alterations in the activities of SOD, CAT and MPx, following oral administration of AMD, lansoprazole (LAN) and ranitidine (RAN) in rats with indomethacin (IND)-induced ul-cer.

42 male Sprague-Dawley rats, weighing 180–190 g were used in this study. The animals were grouped before the experiments and kept under standard conditions [8]. AMD (25, 50 and 100 mg/kg body weight doses prepared as suspension in water) and positive controls [LAN (30 mg/kg body weight) and RAN (25 mg/kg body weight)] were administrated orally to the assigned groups of rats. 5 min after AMD, LAN and RAN administrations, IND (25 mg/kg body weight) was administrated to all animals orally. One group was assigned as control group, which received only water. After 6 h of all treatments, animals were sacrificed using sodium thiopental (50 mg/kg). The rats’ stomachs were removed and opened along the greater curvature and then washed with serum physiological solution. The wideness of ulcer areas was determined using a magnifier and a mil-limeter paper. Then tissues grounded within liquid nitrogen for as-says of SOD, CAT and MPx activities.

In the present study, we found that 1) AMD, LAN and RAN re-duced the development of IND-induced gastric damages, at a greater magnitude for AMD and LAN than for RAN; 2) AMD and RAN al-leviated increase in the activity of CAT enzyme resulting from ulcer; 3) AMD and RAN ameliorated depression in the activities of SOD enzyme caused by IND administration; and 4) All doses of AMD caused an amplification in MPx activity resulting from induced gas-

tric ulcers. These results suggest that the gastroprotective effect of AMD on IND-induced ulceration can be attributed to its ameliorat-ing effect on the oxidative damage, but can not be attributed to its effect on MPx activity, because of high iodine content, amiodarone can activate MPx activity in rat stomach [9].

Keywords: Amiodarone; Indomethacin; Gastroprotective effect; Myeloperoxidase; Antioxidant enzymes

Table 1. Effects of different doses of the amiodarone and single dose of ranitidine and lansoprazole on indomethacin-induced gastric damage in rats. Three doses of amiodarone, lansoprazole and ranitidine treated groups were compared with indomethacin group.

Treatment N Dose mg/kg body wt.

Ulcer index (UI)a

[Ulcerated area / Total stomach area] x 100

% Inhibitionb

6 25 2.75 ±0.70 * 54.5

Amiodarone 6 50 2.28 ±0.83 ** 62.3

6 100 0.89 ±0.46 *** 85.3

Ranitidine 6 25 1.64 ±0.85 ** 72.8

Lansoprazole 6 30 0 ±0 *** 100

Indomethacin 6 25 6.04 ±1.27 *** -

Healthy group 6 - 0 ±0 -

aMean damage index ±SEM of six animals in each group. N: The number of rats.

b% Inhibition in ulcer index in relation to indomethacin group. *Significant at p<0.05; **Significant at p<0.01; *** Significant at p<0.005. as compared with indomethacin group.

Table 2. Effects of different doses of the amiodarone and single dose of ranitidine on the activity of superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPx) enzymes in rat’s indomethacin (IND)-induced gastric tissue.

Treatment N Dosemg/kg body

wt

CAT(mmol/min/

mg tissue)

SOD(mmol/min/mg

tissue)

MPx Activity(µmol/min/mg

tissue)

6 25 115,99±1,20 94,20±0.98* 10,22±0.08

IND+AMD 6 50 90,17±1,20* 107,45±0.81** 11,47±0.26*

6 100 74,05±1,10* 125,92±0.28** 13,51±0.14*

IND+RAN 6 25 50,68±0,84* 116,08±0.06** 5,73±0.11*

IND 6 25 117,89±0,36* 74,57±0.52** 9,75±0.07*

Healthy rats 6 - 74,93±0,95 122,35±1.13 7,65±0.61

N: The number of rats. Results are means ± SE of three measurements. IND group was compared with healthy group. Treated groups were compared with IND group. *Significant at p<0.05; **Significant at p<0.01 as compared with IND group.

1. Mason JW. Amiodarone, New. Eng.. J. Med. 316, 455-466. 1987. 2. Vrobel TR, Miller PE, Mostow ND, Rakita, L. A general overview of

amiodarone toxicity: its prevention, detection, and management, Prog. Cardiovasc. Dis. 31, 393-426, 1989.

3. Figge HL, Figge J. The effects of amiodarone on thyroid hormone func-tion: A review of the physiology and clinical manifestations, J. Clin. Phar-macol., 30, 588-595, 1990.

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4. Das D, Bandyopadhyay D, Bhattacharjee M, Banerjee RK. Hydroxyl radi-cal is the major cousative factor in stress-induced gastric ulceration, Free Radical Biol. Med. 23, 8-18, 1997.

5. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging, Proc. Natl. Acad. Sci., 90, 7915-7922, 1993.

6. Mates JM, Perez-Gomez C, Nudez de Castro I. Antioxidant enzymes and human diseases, Clinical Biochemistry, 32, 595-603, 1999.

7. Elliot SN, Wallace JL.. Neutrophil-mediated gastrointestinal injury, Ca-nadian Journal of Gastroenterology, 12, 559-568, 1998.

8. CCAC. 1993. Guide to the care and use of experimental animals, Vol I, 2nd Ed. Canadian Council on Animal Care. Bradda Printing Services Inc., Ottawa, ON, Canada.

9. Magnusson RP, Taurog A, Dorris ML. Mechanisms of thyroid peroxi-dase- and lactoperoxidase catalyzed reactions involving iodide, J. Biol. Chem., 259, 13783-13790, 1984.

P 060 Ref: 0083

SYNTHESIS OF SOME 5-CHLORO-6-(THIAZOL–4-YL)-2-OXO-3H-BENZOTHIAZOLE DERIVATIVES AS POTENTIAL COX-2 INHIBITORS Selcen ADAK, Deniz S. DOĞRUER, Serdar ÜNLÜGazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey

It is known that inhibition of the enzyme cyclooxygenase (COX) is the principal mechanism for the efficacy of NSAIDs. Two distinct and independently COX isoforms have been identified COX-1 and COX-2 [1]. Interruption of COX-1 activity can lead to life-threating gatro-intestinal (GI) toxicity of perforation, ulceration and bleeding. In the meantime, COX-2 is induced upon inflammatory stimuli and is responsible for progression of inflammation [2]. Therefore, many studies have been focused on COX-2 inhibitors to reduce the risk of GI complications. The greatest research activity in the field of COX-2 inhibitors has been made in the synthesis and pharmacological testing of the class of diaryl heterocyclics [3].

Some compounds bearing 5-chloro-2-oxo-3H-benzothiazole ring have been reported to have anti-inflammatory activity [4]. In addi-tion, potent selective COX-2 inhibitors which bear thiazole struc-ture as a central ring have been reported in the literature [5, 6]. In the design of new compounds, development of the hybrid molecules through the combination of different pharmacophores in one struc-ture may lead to compounds with increased analgesic and anti-in-flammatory activities.

Therefore, these observations prompted us to synthesize six new compounds which bear a central thiazole ring whose 2,4-positions are substituted with phenyl and 5-chlorobenzothiazolone moieties, respectively. Structures of the synthesized compounds have been confirmed by IR, 1H-NMR and elemental analysis. Their COX-2 in-hibitory potency will be evaluated by using in vitro human whole blood assay.

1. Menozzi G, Merello L, Fossa P, Mosti L, Piana A, Mattioli F. 4-Substitut-ed 1,5-diarylpyrazole, analogues of celecoxib: synthesis and preliminary evaluation of biological properties, IL Farmaco, 58, 795-808, 2003.

2. Shin SS, Noh MS, Byun YJ, Choi JK, Kim JY, Lim KM, Ha JY, Kim JK, Lee CH, Chung S. 2,2-Dimethyl-4,5-diaryl-3(2H)furanone derivatives as selective cyclooxygenase-2 inhibitors, Bioorg. Med. Chem., 11, 165-168, 2001.

3. Dannhardt G, Laufer S. Structural approaches to explain the selectivity of COX-2 inhibitors: Is there a common pharmacophore, Curr. Med. Chem., 7, 1101-1112, 2000.

4. Önkol T, Doğruer DS, Ito S, Şahin MF. Synthesis and antinociceptive activity of (5-chloro-2-benzothiazolinon-3-yl)acetamide derivatives, Ar-chiv der Pharmazie, 333(10), 337-340, 2000.

5. Kontogiorgis CA, Hadjipavlou-Litina DJ. Non steroidal anti-inflamma-tory and anti-allergy agents, Curr. Med. Chem., 9, 89-98, 2002.

6. Kalgutkar AS, Zhao Z. Discovery and design of selective clooxygenase-2 inhibitors as non-ulcerogenic, anti-inflammatory drugs with potential utility as anti-cancer agents. Curr. Drug Targets, 2, 79-106, 2001.

P 061 Ref: 0085

SYNTHESIS AND ANTIMICROBIAL EVALUATION OF 6-SUBSTITUTED-3(2H)-PYRIDAZINONE-2-YL ACETOHYDRAZİDE DERIVATIVES 1Mehtap GÖKÇE, 1Gülay YELKEN, 2Serpil POLAT, 3Seda TEZCAN, 2Mehmet SERİN1Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey2Mersin University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Mersin, Turkey3Mersin University, Faculty of Medicine, Department of Medicinal Microbiology, Mersin, Turkey

Several antibiotics have been prescribed and found to be effective on various infectious disorders. However, the appearance of multi-drug resistant Gram-positive bacteria, in particular, methicillin-re-sistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) is causing a serious menace. Moreover, the emer-gence of vancomycin resistant MRSA can be anticipated in foresee-able future. For the treatment of these intractable infections, a new antibacterial agent is needed. Furthermore Synthetic antibiotics are widely prescribed drugs because of their safety, good tolerance, broad antibacterial spectrum and less resistance.

Due to favorable presence a pyridazinone moiety in known active structures, pyridazinone derivatives provoked a special interest in the search for new antibacterial agents [1-4]. Meanwhile hyrazide-hydrazones have been claimed to exhibit appreciable antimicrobial activity [5-9]. On the basis of these observations a new series of 6-substituted-3(2H)-pyridazinone-2-yl acetohydrazide (I) was syn-thesized using an appropriate synthetic route. The structure eluci-dation of new compounds was based on the relevant 1H-NMR and IR spectral characteristics and purity of the products was confirmed both by TLC and elemental analyses. All the target compounds were evaluated for their in vitro antimicrobial activity against Sta-phylococcus aureus, Bacillus subtilis as examples for Gram positive bacteria, Escherichia coli, Pseudomonas aeruginosa as examples for gram negative bacteria and Candida albicans, Candida parapsilosis as representative of fungi. The minimum inhibitory concentration (MIC) was determined for test compounds as well as for reference standards.

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1. Sönmez M., Berber I., Akbaş E. Eur. J. Med. Chem., 41(1), 101-5, 2006. 2. Fuks B, Talaga P, Huart C, Henichart JP, Bertrand K, Grimee R, Lorent G.

Eur. J. Pharmacol., 519(1-2), 24-30, 2005. 3. Akbaş E, Berber I, Şener A, Hasanov B. Farmaco, 60(1), 23-6, 2005. 4. Takaya M. Yakugaku Zasshi., 113(9), 676-81, 1993. 5. Koçyiğit-Kaymakçıoğlu B, Orçun E, Ünsalan S, Kandermirli F, Shvets N,

Rollas S, Antony D., Eur. J. Med. Chem., 41(11), 1253-61, 2006. 6. Bijev A, Arzneimittel Forschung, 56(2), 96-103, 2006. 7. Küçükgüzel G, Kocatepe A, De Clercq E, Şahin F, Gulluce M. Eur. J. Med.

Chem., 41(3), 353-9, 2006. 8. Sriram D, Yogeeswari P, Madhu K. Bioorg. Med. Chem. Lett., 16(4), 876-

8, 2006. 9. Grover G, Kini SG. Eur. J. Med. Chem., 41(2), 256-62, 2006.

P 062 Ref: 0086

SYNTHESIS AND ANTIMICROBIAL EVALUATION OF 5-CHLORO-2(3H)-BENZOXAZOLINONE-3-ACETYL-2-(p--SUBSTITUTED BENZAL)HYDRAZONE DERIVATIVES 1Tijen ÖNKOL, 1Mehtap GÖKÇE, 1Ali Ulvi TOSUN, 2Serpil POLAT, 2Mehmet SERİN, 3Seda TEZCAN1Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey2Mersin University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Mersin, Turkey.3Mersin University, Faculty of Medicine, Department of Clinical Microbiology, Mersin, Turkey

Since it is a common agreement of multidrug-resistant bacteria are major cause of failure in the treatment of infectious diseases, the need for the synthesis novel antibiotics is a reality.

The structural and therapeutic diversity coupled with commercial viability of small molecules has fascinated organic and medicinal chemists. It has been reported in the literature that hydrazide-hydra-zones have been demonstrated to possess antibacterial, and antifun-gal activities [1-4].

Furthermore, it is reported in the literature that 2(3H)-benzoxa-zolinone derivatives can exhibit diverse activities. Particularly antibac-terial [3] and antifungal [4] activities have been scrutinized intensively. In addition, it has been published that chlorinated 2(3H)-benzoxa-zolinone compounds have valuable fungicidal properties [5].

These observations led us to synthesize a series of Schiff bases combining 5-chloro-2(3H)-benzoxazolinone, hydrazide and benzal-dehyde moieties in the same molecule. Synthesized 5-chloro-2(3H)-benzoxazolinone-3-acetyl-2-(p-substituted benzal)hydrazone I de-rivatives were evaluated for screening antibacterial and antifungal activities on microorganisms respectively on four bacteria and two Candida species.

R1= H, F, Cl, Br, CH3, OCH3, OH

1. Metwally KA, Abdel-Aziz LM, Lashine el-SM, Husseiny MI, Badawy RH., Bioorg. Med. Chem., 14(24), 8675-8682, 2006.

2. Koçyiğit-Kaymakçıoğlu B, Orçun E, Ünsalan S, Kandemirli F, Shvets N, Rollas S, Antony D., Eur. J. Med. Chem., 41(11), 1253-1261, 2006.

3. Mincheva ZP, Kalcheva VB, Golovinsky EG., Pharmazie, 48(11), 859-860, 1993.

4. Wang HX, Liu F, Ng TB., Comp. Biochem. Physiol., 30(3), 379-388, 2001. 5. Basel EM, Riehen JB., US Patent 2,922,794 (Cl. 260-304), January 26,

1960.

P 063 Ref: 0087

SYNTHESIS OF 6-(SUBSTITUE THIAZOL–4-YL)-2-OXO-3H-BENZOXAZOLE DERIVATIVES AS POTENTIAL COX-2 INHIBITORS Şeyma CANKARA, Serdar ÜNLÜGazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey

Selective inhibition of cyclooxygenase-2 (COX-2) which is being induced during inflammation is off interest since the resulting drugs lack the gastric ulceration side effects associated to classical NSAIDs. Since 2-oxo-3H-benzoxazole [1] and thiazole [2]ring system bear good anti-inflammatory property and the 2,4-diaryl/heteroaryl structures might also be important for selective COX-2 inhibitory activity, we hereby describe the biological consequences of incorpo-ration of a 2-oxo-3H-benzoxazole ring as one of the aryl substituents and the effect of a 2,4-diarylsubstitution pattern around the thiazole ring on the in vitro activity of the resulting derivatives towards COX-2 inhibition.

Thus, we have designated a series of 6-(substitute thiazole–4-yl)-2-oxo-3H-benzoxazole derivatives and these compounds were pre-pared by the reaction of 6-bromoacetyl-2-oxo-3H-benzoxazole and substituted thiobenzamides. The result of inhibitory potency against COX was evaluated using human whole blood assay [3].

1. Ünlü S, Önkol T, Dündar Y, Ökcelik B, Küpeli E, Yeşilada E, Noyanalpan N, Şahin MF. Synthesis and Analgesic and Anti-inflammatory Activity of Some New (6-Acyl-2-benzoxazolinone and 6-Acyl-2-benzothiazolinone Derivatives with Acetic Acid and Propanoic Acid Residues, Arch. Pharm. Pharm. Med. Chem., 336, 353–361, 2003.

2. Narender M, Reddy MS, Sridhar R, Nageswar YVD, Rama Rao K. Aque-ous phase synthesis of thiazoles and aminothiazoles in the presence of β-cyclodetrin, Tetrahedron Letters, 46, 5953-5955, 2005.

3. Patrignani P, Panara Mr, Greco A, Fusco O, Natoli C, Iacobelli S, Cipol-lone F, Ganci A, Creminon C, Maclouf J, Patrono C. Biochemical and Pharmacological Characterization of the Cyclooxygenase Activity of Hu-man Blood Prostaglandin Endoperoxide Synthases, The Journal of Phar-macology and Experimental Therapeutics, 271, 1705-1712, 1994.

P 064 Ref: 0088

INHIBITION EFFECT OF NEW SULFONAMIDE DERIVATIVES ON CARBONIC ANHYDRASE Ümmühan ÖZDEMİR-ÖZMEN, Fatma ARSLAN, Fatma HAMURCUGazi University, Arts and Sciences Faculty, Department of Chemistry, 06500 Ankara, Turkey

Sulfonamides have been known as powerful inhibitors of carbonic anhydrase (CA) [1]. Since sulfonamides interact with the active site of CA, the active site charge requirements and the orientations of sulfonamide groups are essential for the inhibitory powers of these drugs. Understanding the mechanism of the inhibition of CA isoen-

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zymes with sulfonamide derivatives at the molecular level have been reported to be helpful for the rational design of novel derivatives with minimum side effects [2].

In the present study, inhibitory effects of ethanesulfonic acid hy-drazide (esh), 5-methyl-2-hydroxyacetophenone ethanesulfonylhy-drazone (5mafesh) and its Ni(II) complex on CAII have been inves-tigated. Enzyme activity was determined using p-nitrophenylacetate as substrate. According to the corresponding IC50 and Ki values, 5mafesh was found as the most potent inhibitor of CAII.

1. Arslan O, Küfrevioğlu Öİ, Nalbantoğlu B., Bioorg. & Med. Chem., 5(3), 515, 1997.

2. Chakravarty S , Kannan KK., J. of Mol. Biol., 243, 298, 1994.

P 065 Ref: 0089

DESIGN AND SYNTHESIS OF NOVEL TIAZOLE DERIVATIVES AS POTENTIAL CYCLOOXYGENASE- 2 (COX-2) INHIBITORS Leyla UZUN, Tijen ÖNKOL, Serdar ÜNLÜGazi University, Faculty of Pharmacy Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey

The major side effects associated with the currently available NSAIDs are gastrointestinal (GI) hemorrhagia and ulceration which result from the nonselective inhibition of COX enzymes, namely COX-1 and COX-2. After the discovery of the second isoform (COX-2) that is expressed in inflammatory cells, but not in gastric mucosa, novel NSAIDs with selective COX-2 inhibitory activity resulted to be more useful for the treatment of inflamatory diseases [1] without gastric side effects.

For this reason, the series of 2,4-diarylthiazole derivatives were designed and synthesized for their evaluation as selective COX-2 in-hibitors in an enzymatic assay using human whole blood. We selected thiazole ring to synthesize the title compounds since thiazole ring have been reported to have analgesic and anti-inflammatory activities [2]. The synthesis of 6-(substituted thiazol-4-yl)-2- oxo-3H-benzo-thiazoles were accomplished by the reaction of 6- bromoacetyl-2-oxo-3H-benzothiazole with o/p-substituted thiobenzamides using microwave-assisted synthesis. Physical and chemical properties of synthesized compounds have been confirmed by using their melting points, IR, 1H-NMR and elemental analysis.

1. Ulbrich H, Fiebich B, Dannhardt G. European Journal of Medicinal Chemistry, 37, 953-959, 2002.

2. Norender M, Reddy MS, Sridhar R, Nageswar YVD, Rao KR. Aqueous phase Ssynthesis of thiazoles and aminothiazoles in the presence of β-cyclodextrin, Tetrahedron Letters, 46, 5953-55, 2005.

P 066 Ref: 0090

SYNTHESIS OF 5-CHLORO-2-OXO-6-(2-SUBSTITUTED PHENYL-1,3-THIAZOL-4-YL)-3H-BENZOXAZOLE DERIVATIVES AND EVALUATION OF THEIR COX-1/COX-2 INHIBITORY ACTIVITIES Şölen URLU, Serdar ÜNLÜGazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey

The therapeutic efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) results from the inhibition of cyclooxygenases (COX) which were shown to exist as two distinct isoforms, namely, COX-1 and COX-2. COX-1 is constitutively expressed as a house keeping enzyme in nearly all tissues and mediates physiological responses. On the other hand, COX-2 is expressed by cells involved in inflam-mation and has emerged as the isoform that is primarily responsi-ble for the synthesis of prostanoids involved in acute and chronic inflammatory states. It was hypotesised that selective inhibition of COX-2 might have therapeutic actions similar to those of NSAIDs, but without causing gastrointestinal side effects [1].

Our ongoing studies towards the derivatives of chlorzoxazone

[2]and thiazole with anti-inflamatory activities [3] prompted us to design new compounds which could be selective COX-2 inhibitors. For this purpose some 5-chloro-2-oxo-6-(2-substituted phenyl-1,3-thiazol-4-yl)-3H-benzoxazole derivatives were synthesized by con-densation of 6-bromoacetyl-5-chloro-2-oxo-3H-benzoxazole and appropriate thiobenzamides derivatives in diethylenglicoldimethyl-ether. The activities of the compounds for possible COX inhibitory activity will be performed by enzyme immunoassay method.

1. Brune K, Hinz B. Selective cylooxygenase-2 inhibitors: similarities and differences, Scand. J. Rheumatol., 33, 1-6, 2004.

2. Park, JY, Kim KA, Park PW, Ha JM. Effect of high-dose aspirin on CYP2E1 activity in healty subjects measured using chlorzoxazone as a probe, Journal of Clinical Pharmacology, 46, 109-114, 2006.

3. Kazzouli Sl, Berteina-Raboin S, Mouaddib A, Guillaumet G. Solid support synthesis of 2,4-disubstituted thiazoles and aminothiazoles, Tet-rahedron Letters, 43, 3193-3196, 2002.

P 067 Ref: 0091

PHYSICOCHEMICAL CHARACTERIZATION OF BIODEGRADABLE CARDIOVASCULAR STENTS Can SARISÖZEN, Betül ARICA, Sema CALIŞ, A. Atilla HINCALHacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100 Ankara, Turkey

The aim of our study was to realize physicochemical characteriza-tion and in vitro evaluation of prepared biodegradable cardiovascular stents for preventing restenosis of blood vessels. Stents were prepared from solution-cast biodegradable formed polymeric films and a model drug prednisolone acetate (PA) was incorporated into the stents.

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For this purpose, two types of synthetic biopolymer (PLGA 75:25 and PLGA 50:50) were dissolved in dichloromethane separately to have a final concentration of 25% (w/v) in which PEG 4000 was add-ed as a plasticizer. The polymer solutions cast onto the aluminum pans (in 3x2 cm area). After evaporation of dichloromethane, the polymer films were cut into strips and coiled on the cylindrical rods to form a helical shaped stents.

Two types of stents were used in our study: stents formed by a polymer film including PA and stents formed by a polymer film in-cluding PA containing spray-dried chitosan microspheres which has been developed by our group previously. The characteristics of these prepared stents such as wall thickness, diameter, length, morphology were measured and evaluated.

Differential Scanning Calorimetry (DSC) and Attenuated Total Reflectance (ATR)-FTIR studies were also performed. Samples were heated under nitrogen atmosphere in aluminum hermetic pans and thermograms recorded a range of (20°C to 300°C at a heating rate of 10°C/min). ATR-FTIR studies were performed to determine if there were drug present on the surface of the stents or not. Stents which were prepared by PLGA 75:25 polymer were analyzed for specific surface area analyzes. In vitro drug release testing was performed to evaluate the drug release form the stents. The test was also performed in PBS solution containing 0.5%(w/v) sodium lauryl sulphate and 0.05%(w/v) sodium azide at pH 7.4.

The prepared stents had outer diameters approximately 3mm and their length were 1.5cm. The polymer wall thicknesses of empty stents were measured using a micrometer and determined as 136µm±0.05. Based on ease of handling for perivascular placement, this film thick-ness was assessed to be optimum. Thin films (100µm) tended to fold and 200µm films were not elastic and easy to crack. Incorporation of drug or drug loaded microspheres did not interfere during the forma-tion of homogenous films and helical stent structure. Prepared stents seemed to have smooth surface without any visible defects and cracks. Incorporated drug or microspheres were homogenously distributed in the polymer matrix. The ATR-FTIR spectrums showed that biode-gradable stent surfaces were free of drug and microspheres.

Thermograms of the formulations are given in Figure 1. As it can be seen from the graphics, melting peak of PA disappeared after it was incorporated into the stents. The results for the specific surface area analyzes are given in Table 1. Although the release of PA from the stents seemed to be very slow following the initial release pe-riod which was about 1-5 days in PBS buffer, a significant amount of the drug was determined to be released at the second release period which started on the 5th day and on the 63th day. This might be ex-plained by the slow degradation of the PLGA polymer in the release medium. Nevertheless, approximately 5-62% of the PA from the PA-loaded stents and 10-13% of the PA from the chitosan microspheres containing stents were released in 63 days.

Table 1. Characterization of biodegradable stent formulations.

Polymer PA or Microspheres

Length (cm)

Diameter (mm)

Thickness (µm)

Specific Surface

Area

PLGA 75:25

None (Empty Stent)

1.5 3 136.5±5 µm 1,32 m2/g

PLGA 75:25

PA Incorporated

1.5 3 140±3.6 µm 2,14 m2/g

PLGA 75:25

Microsphere Incorporated

1.5 3 145±4 µm 0,44 m2/g

PLGA 50:50

None (Empty Stent)

1.5 3 109±8 µm -

PLGA 50:50

PA Incorporated

1.5 3 119.3±4.2 µm -

PLGA 50:50

Microsphere Incorporated

1.5 3 118.7±3.2 µm -

P 068 Ref: 0092

CO-ADMINISTRATION OF A NITRIC OXIDE SYNTHASE INHIBITOR AND MELATONIN EXERTS AN ADDITIVE ANTIDEPRESSANT-LIKE EFFECT IN THE MOUSE FORCED SWIM TEST 1Yusuf ERGÜN, 2Ufuk Güney ERGÜN, 3Özlem ORHAN, 4Ekrem KÜÇÜK1Kahramanmaraş Sütçü İmam University, School of Medicine, Department of Pharmacology, 46100 Kahramanmaraş, Turkey2Kahramanmaraş Sütçü İmam University, School of Medicine, Department of Family Medicine, 46100 Kahramanmaraş, Turkey3Kahramanmaraş Sütçü İmam University, School of Medicine, Department of Psychiatry, 46100 Kahramanmaraş, Turkey4Osmaniye High School of Science, Osmaniye, Turkey

Previous studies have shown that nitric oxide synthase inhibi-tors and melatonin are as efficacious as conventional antidepres-sant drugs in pre-clinical antidepressant screening procedures such as Porsolt swim test. The aim of the present study was to assess the combination therapy of these two distinct arrays of drugs.

Porsolt swim test was conducted to resemble the symptomatology of major depressive disorder, and an open filed locomotor activity was also used.

NG-nitro-L-arginine (L-NNA) at doses 3 to 30 mg/kg and me-latonin at 3 and 10 mg/kg were examined in the forced swim test in mice. 3 mg/kg L-NNA slightly but not significantly reduced the duration of immobility, and increasing the dose to 10 mg/kg was suf-ficient to attain a significant reduction (Fig. 1). On the other hand, the maximal dose of L-NNA (30 mg/kg) had no effect although a non-significant small increase was observed (Fig. 1). The results obtained with L-NNA were in accordance with a U-shape effect. 3 mg/kg melatonin was found to be ineffective while a statistically sig-nificant decrease in the duration of immobility was found at the dose of 10 mg/kg (Fig. 1). The combination of ineffective doses (3 mg/kg, each) of L-NNA and melatonin revealed no further inhibition in the duration of immobility and the most effective doses (10 mg/kg, each) caused a more pronounced reduction when compared to those of each drug alone (Fig. 1). None of the drugs effected locomotor activity over the dose range applied (Fig. 2).

In conclusion, the combined therapy with L-NNA and melatonin seems to have an additive effect and may be considered as a feasible candidate in attenuating the symptoms of major depressive disorder.

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Figure 1. The effects of saline, 1% alcohol, NG-nitro-L-arginine (L-NNA: 3, 10 and 30 mg/kg) and melatonin (3 and 10 mg/kg) on the duration of immobility (sec) in forced swim test. Values presented as mean ± SEM, n=10. * P<0.05 (versus control, saline or 1% alcohol), ** P<o.o5 (versus L-NNA or melatonine alone) analysis of variance (ANOVA) with Bonferroni correction.

Figure 2. The effects of saline, 1% alcohol, NG-nitro-L-arginine (L-NNA: 3, 10 and 30 mg/kg) and melatonin (3 and 10 mg/kg) on the squares entered in locomotor activity test. Values presented as mean ± SEM, n=10.

1. Trullas R, Skolnick P. Functional antagonists at the NMDA receptor complex exhibit antidepressant actions, Eur. J. Pharmacol., 185, 1-10, 1990.

2. Harkin AJ, Bruce KH, Craft B, Paul IA. Nitric oxide synthase inhibitors have antidepressant-like properties in mice. 1. Acute treatments are ac-tive in the forced swim test, Eur. J. Pharmacol., 372, 207-213, 1999.

3. Karolewicz B, Paul IA, Antkiewicz-Michaluk L. Effect of NOS inhibitor on forced swim test and neurotransmitters turnover in the mouse brain, Pol. J. Pharmacol., 53, 587-596, 2001.

4. Overstreet DH, Pucilowski O, Retton MC, Delangrange P, Guardiola-Le-maitre B. Effects of melatonin receptor ligands on swim test immobility, Neuroreport, 9, 249-253, 1998.

5. Mantovani M, Pértile R, Calixto JB, Santos ARS., Rodrigues ALS. Me-latonin exerts an antidepressant-like effect in the tail suspension test in mice: evidence for involvement of N-methyl-D-aspartatereceptors and the L-arginine-nitric oxide pathway, Neurosci. Lett., 343, 1-4,2003.

6. Heiberg IL, Wegener G, Rosenberg R. Reduction of cGMP and nitric oxide has antidepressant-like effects in the forced wimming test in rats, Behav. Brain Res., 134, 479-484, 2002.

P 069 Ref: 0093

A UTILITY OF FURAN CORED COMPOUNDS; SYNTHESIS OF NITROGEN TETRACYCLES, ISOBENZOFURANOL AND ISOBENZOTHIOPHENOL Muhsin KARAARSLAN, Ersen GÖKTÜRK, Aydın DEMİRCANUniversity of Niğde, Faculty of Sciences & Letters, Department of Chemistry, Kampus, 51100 Niğde, Turkey

Intramolecular Diels–Alder (IMDA) reactions involving furane as a diene form oxanorbornenes that have been used in the synthesis of numerous complex targets and as an intermediate in the synthesis

of natural products such as carbohydrates and prostaglandins [1]. A series of key precursors to the IMDA reaction of furane diene (2a–c) have been prepared via facile alkylation and protection. While the cycloaddition process for (3a–c) was afforded in hot toluene, a com-mercial microwave (2450 MHz) was used for the synthesis of (5a–b) (Figure 1).

Figure 1. i. Br2, Et3N, DCM, 0°C, 98%; NaBH4, CeCl3.7H2O, MeOH, 0°C, 97%; PBr3, Pyridine, PhH, 0°C reflux, 68%; KOtBu, CHBr3, Pentane, 0°C; Heat 155°C, 1h, 78%; 2,3-dibromocyclopentene or 2,3-dibromocyclohexene, K2CO3, THF, reflux, 3d; (Boc)2O, DMAP, DCM, 0°C, 2h.

Treatment of fused oxy- and thio-heterotricycles (5a–b) with bo-rontriluoride-etherate in dichloromethane at –78°C cleaved Epoxy-Bridge and concomitant aromatisation gave the isobenzo-furanol and thiophenol (6a–b) in 72–76% yields respectively (Figure 2) [2].

Figure 2.

1. Lipshutz BH. Chem. Rev., 86, 795, 1986. Wang Q, Padwa A, Rh(I)-Cata-lyzed ring opening of an IMDAF-derived oxabicyclo cycloadduct as the key step in the synthesis of ±-epi-Zephyranthine, Org. Lett., 6, 2189, 2004.

2. Demircan A, Karaarslan M, Turac E.. A facile synthesis of heterotricycles from furfurylbromoalkenes using thermal IMDA cycloaddition. Hetero-cyclic Commun., 3&4, 233, 2006. Karaarslan M, Göktürk E, Demircan A. Thermal intramolecular Diels–Alder reaction of furan; Synthesis of ni-trogen tetracycles, isobenzofuran and isobenzothiophene, J. Chem. Res., 2, 117, 2007.

P 070 Ref: 0094

THE DETERMINATION OF ANTHOCYANINS DELPHINIDIN AND PEONIDIN IN FRESH FRUITS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Nafiz Öncü CAN, Göksel ALTIOKKAAnadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskişehir, Turkey

Anthocyanins constitute one of the major groups of natural pig-ments and are responsible for many of the colours of fruits and vegetables [1]. It has been demonstrated that anthocyanins possess some positive therapeutic effects [2, 3] and are in use as nutritional supplements in many countries [4]. The aim of this study was to de-velop a simple and precise procedure for the determination of two anthocyanins, delphinidin and peonidin, and indicate their levels in some fresh fruits consumed in Turkey.

11 fruit samples, including pomegranate, cherry and grape, were examined using high performance liquid chromatography coupled

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to photodiode array detection. Two anthocyanins and cyanidin (in-ternal standard) were separated using a gradient elution technique by a reversed phase column. Pumping the mobile phase at a flow rate of 0.8 mL.min-1, analytes were detected at 520 nm wavelength within an average time of 18 min. Samples were prepared from fresh fruits by taking their juices with direct pressing which was followed by filtration through 0.2 µm PVDF filters, and injected into the sys-tem with no further extraction or concentrating steps.

Figure 1. A typical chromatogram of pomegranate fruit sample (DP: Delphinidin, PN: Peonidin, IS: Internal standard)

The method was found to be linear between the ranges about 80 – 400 ngmL-1, giving symmetrically sharp and repeatable analyte peaks. The overall limits of quantitation (LOQ) were 58.4 and 50.1 ngmL-1 for delphinidin (DP) and peonidin (PN), respectively. It was observed that the amount of anthocyanins detected in fruits reach to the maximum due to freshness and exist especially in dark coloured fruits.

1. Kong JM, Chia LS, Goh NK, Chia TF, Brouillard R. Analysis and biologi-cal activities of anthocyanins, Phytochemistry, 64, 923-933, 2003.

2. Dell’Agli M, Busciala A, Bosisio E. Vascular effects of wine polyphenols, Cardiovasc. Res., 63, 593-602, 2004.

3. Kowalczyk E, Krzesinski P, Kura M, Szmigiel B, Blaszczyk J. Anthocy-anins in medicine, Pol. J. Pharmacol., 55, 699-702, 2003.

4. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutri-tional significance, Nutr. Rev., 56, 317-333, 1998.

P 071 Ref: 0096

SCREENING OF ANTHOCYANINS PELARGONIDIN AND MALVIDIN IN COMMERCIAL FRUIT JUICES USING LIQUID CHROMATOGRAPHY COUPLED TO DIODE ARRAY DETECTION Göksel ALTIOKKA, Nafiz Öncü CANAnadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskişehir, Turkey

Commercial fruit juices are one of the most common consumed food products that contain many naturally occurring compounds, such as anthocyanins, which have therapeutic effects against cancer and cardiovascular diseases [1, 2]. Development of a cheap and re-producible method for the determination of two anthocyanins, pel-argonidin and malvidin, in fruit juices was introduced in this study.

8 samples of different fruit juices were examined using liquid chromatography coupled to photodiode array detection. Utilizing the sugar-free aglycone cyanidin as an internal standard, two an-

thocyanins were resolved by reversed phase technique within about 16 minutes. Analytes were detected at 520 nm wavelength and mo-bile phase was pumped through the column at a flow rate of 0.8 mL.min-1. Samples were injected to the system directly, following a simple filtration through 0.2 µm PVDF filters, without any extrac-tion or concentration steps.

Figure 1. A typical chromatogram of grape juice sample (PG: Pelargonidin, MV: Malvidin, IS: Internal standard).

Anthocyanins were detected in ppb levels with adequate chroma-tographic resolution. Linearities were obtained between the ranges of 78.1 – 390.7 ppb for pelargonidin and 88.15 – 440.7 ppb for mal-vidin. On the whole, malvidin and pelargonidin levels were found to be relatively high in grape products, such as wine and grape juice, in accordance with the previous studies on these compounds.

Keywords: Pelargonidin, malvidin, food analysis, liquid chromatography

1. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutri-tional significance. Nutr. Rev., 56, 317-333, 1998.


P 072 Ref: 0097

RAPID DETERMINATION OF ACRYLAMIDE IN POTATO CHIPS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE ARRAY DETECTION Nafiz Öncü CAN, Göksel ALTIOKKAAnadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskişehir, Turkey

Acrylamide is one of the heat-generated food toxicants, which was frequently found in various foodstuffs such as potato chips, biscuits and coffee [1, 2]. It has been demonstrated that acrylamide possesses some neurotoxic and carcinogenic effects [3, 4] and is classified in Group 2A as a possible carcinogen to humans by International Agency for Research on Cancer [5]. The aim of this study was to develop an inexpensive, simple and sensitive method for determi-nation of acrylamide and indicate acrylamide levels of some potato products produced in Turkey.

Acrylamide concentration of the samples was examined using high performance liquid chromatography combined to diode ar-ray detection. Acrylamide and methacrylamide (internal standard) were separated using isocratic elution technique by a reversed phase column (GL Sciences, Inertsil® ODS-3). Mobile phase (acetonitrile:water, 2:98, v/v) was pumped at 0.5 mL.min-1 flow rate and analytes

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were detected at 200 nm wavelength within an average retention time of 15 min. Samples were prepared by solid phase extraction and injected into the system with no further extraction or pre-concentra-tion steps.

Figure 1. A typical chromatogram of homemade potato chip sample (AA: Acrylamide, MAA: Methacrylamide)

Acrylamide was detected in ppb levels with adequate chromato-graphic resolution. The developed method was validated according to the recommendations of International Conference of Harmoni-zation – Q2(R1) and system suitability parameters were tested ac-cording to United States Pharmacopeia. The method was found to be linear in the ranges of 2.9-14.2 ppb, giving LOD and LOQ con-centrations of 0.93 and 2.82 ppb, respectively. As a result, it was ob-served that the amount of acrylamide in potato chips varies due to cooking conditions and was found to be relatively high in many of the samples.

Keywords: acrylamide, food analysis, liquid chromatography, Turkish foods

1. Tareke E, Rydberg P, Karlsson P, Eriksson S, Tornqvist M. Acrylamide: a cooking carcinogen? Chem. Res. Toxicol., 13, 517-522, 2000.

2. Tareke E, Rydberg P, Karlsson P, Eriksson S, Tornqvist M. Analysis of acr-ylamide, a carcinogen formed in heated foodstuffs, J. Agric. Food Chem., 50, 4998-5006, 2002.

3. JECFA - Joint FAO/WHO Expert Committee on Food Additives, Sum-mary and conclusions of the sixty-fourth meeting, JECFA/64/SC, p. 7-17, World Health Organization WHO, Rome, Italy, 2005.

4. Shipp A, Lawrence G, Gentry R, McDonald T, Bartow H, Bounds J, Mac-donald N, Clewell H, Allen B, van Landingham C. Acrylamide: Review of toxicity data and dose-response analyses for cancer and noncancer ef-fects, Crit. Rev. Toxicol., 36, 481-608, 2006.

5. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 60: Some Industrial Chemicals, p. 389-433, International Agen-cy for Research on Cancer (IARC),Geneva, Switzerland, 1997.

P 073 Ref: 0098

IMPROVED HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF CYANIDIN AND PETUNIDIN IN FRESH VEGETABLES USING AN ODS-3 REVERSED-PHASE COLUMN Göksel ALTIOKKA, Nafiz Öncü CAN, Zeki ATKOŞARAnadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskişehir, Turkey

As a subgroup of the polyphenols family, anthocyanins are known as one of the best natural antioxidant compounds that can be found

in many of the vegetables such as red cabbage and turnip [1]. An-thocyanins, which act as colouring pigments in plants, have been shown to be strong antioxidants with potential health benefits [2]. These properties make these compounds preferable as nutritional supplements and possible therapeutic agents against cancer and car-diovascular diseases [3, 4]. The aim of this study was to investigate the levels of two major anthocyanins, cyanidin and petunidin, in vegetables using an improved liquid chromatographic method.

Anthocyanin content of some commonly consumed vegetables was determined using an ODS-3 reversed phase column (GL Sci-ences, Inertsil ODS-3, 3 µm, 150 x 4.6 mm) with excellent accuracy (recovery more than 99%) and precision (RSD% < 0.9). Both com-pounds were separated with adequate resolution factor which was higher than 3. Cyanidin and petunidin signals were found to be line-ar between the ranges of 80.8 – 404.0 ng/mL and 85.8 – 429.0 ng/mL, giving limit of detection levels of 7.9 and 9.1 ng/mL for cyanidin and petunidin, respectively. No intensive extraction steps were applied in order to keep the methodology as simple as possible; direct pressing was applied to the homogenized samples, and extracted juice was injected to the system after filtration.

Figure 1. A typical chromatogram of turnip sample (CY: Cyanidin, PT: Petunidin, IS: Internal standard)

Developed method was validated according to the recommenda-tions of International Conference of Harmonization. As a result, it can be concluded that both anthocyanins exist at high levels espe-cially in red cabbage and turnip, while there is no evidence was ob-served in tomato and red pepper. Further investigations are advised to construct a general profile of anthocyanins in vegetables.

1. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutri-tional significance, Nutr. Rev., 56, 317-333, 1998.

2. Kong JM, Chia LS, Goh NK, Chia TF, Brouillard R. Analysis and biologi-cal activities of anthocyanins, Phytochemistry, 64, 923-933, 2003.

3. Kowalczyk E, Krzesinski P, Kura M, Szmigiel B, Blaszczyk J. Anthocy-anins in medicine, Pol. J. Pharmacol., 55, 699-702, 2003.


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P 074 Ref: 0100

MOLECULAR MODELLING STUDIES OF BENZO[α]CARBAZOLE DERIVATIVES AS ESTROGEN RECEPTOR INHIBITORS Tuğba TAŞKIN, Fatma SEVİNHacettepe University, Faculty of Science, Department of Cemistry, Ankara, Turkey

There have been some researches about a number of 5,6-dihydro-11-alkylbenzo[α]carbazoles derivatives with one or two hydroxy groups in the aromatic rings for their binding affinities for the estro-gen receptor [1, 2]. According to analysis, the best conditions for the receptor binding were provided by one hydroxy group at C-3 and a second one at position 8 or 9. These results prompted us to elucidate and visualize this experimental researches by using computational and molecular docking methods.

Our conformatinal analysis consist of first generating conformers by using Gaussian 03 software and then optimizing with AM1, PM3, DFT-B3LYP/6-31G* methods. QSAR [3] is used to determine which molecular descriptors preferences that can be rapidly computed and describe the structure, size, topology and other properties of a mol-ecule. The most statistically significant descriptors like substituent hydrophobicity constant (π), electrophilicity index (ω) and heat of formation energy (HF) are selected using stepwise multiregression analysis (SMLR) to validate biochemical activity. According to the results of analysis, the most fitting equation is determined to describe the relationship between Log1/C and three independent variables for each method. In addition, predicted biological activities have been compared with observed activities.

Furthermore, to visualize the interactions with 5,6-dihydro-11-alkylbenzo[α]carbazole derivatives-binding sites on selected a-chain of estrogen receptor, ICM Pro.3.4. was performed. Results are used to compare physical and chemical properties in determining the conformational preference. Then the most stable conformers, 2 and 3 compounds were brought onto the ER and than the system are fully optimized allowing both systems interactions by using the potential virtual screening of this method. The results are discussed in terms of the nature of the these compounds, ligands recognition on the estrogen receptor.

1. Angerer E, Prekajac J. Journal of Medicinal Chemistry, 29, 3, 1986. 2. Katzenellenbogen JA, Katzenellenbogen BS. Chem. Biol., 3, 529, 1996. 3. Pahsa FA, Srıvastava HK, Singh PP. International Journal of Quantum

Chemistry, 104, 87–100, 2005.

P 075 Ref: 0101

RAPID AND SENSITIVE VOLTAMMETRIC DETERMINATION OF ASCORBIC ACID IN TABLET DOSAGE FORMS AND SOME FRUIT JUICES 1Selehattin YILMAZ, 1Gökçe AŞKIN, 1Sultan YAĞMUR, 1Gülen TÜRKER, 1Gülşen SAĞLIKOĞLU, 1Hüseyin ERDUĞAN1Çanakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of Chemistry, 17020 Çanakkale, Turkey2Çanakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of Biology, 17020 Çanakkale, Turkey

Ascorbic acid, a water-soluble vitamin, is important in forming collagen, a protein that gives structure to bones, muscles, and blood vessels. Also it helps to take of ferrum into body. Ascorbic acid is one of the most ubiquitous vitamins ever discovered. Besides play-ing a paramount role as an antioxidant and free radical scavenger, it has been suggested to be an effective antiviral agent. Ascorbic acid is usually administered orally. Because of the notable loss of the in-fused ascorbic acid in the urine, daily doses of 200 mg are needed to

maintain normal concentrations of about 60 M in plasma It is the most common electroactive biological compound [1-4]. So, the elec-trochemical oxidation of ascorbic acid has been carried out at glassy carbon electrode in various aqueous solution in the pH range of 0.64-10.15 (Britton-Robinson, acetat, phosphate buffers and 0.5 M sulfu-ric acide solution) by cyclic (CV) and differential pulse voltammetry (DPV). The best results were obtained for the quantitative determi-nation of ascorbic acid by DPV technique in 0.2 M acetate buffer ( pH 3.49) at 0.342 V. The diffusion controlled nature of the peak was established. This electroanalytical procedure enabled to determine ascorbic acid in the concentration range 8x10-6-8x10-4 M. The regres-sion equation was obtained as Ip (µA) = 5.29 x10-3 C (M) + 0.035 (r = 0.998). Limit of detection (LOD) and limit of quantitation (LOQ) were obtained as 5.16x10-7 M and 1.72x10-6 M, respectively. Based on this study, simple, rapid, selective, and sensitive DPV technique was developed for the quantitative determination of ascorbic acid in pharmaceutical dosage forms and some fruit juices. The proposed voltammetric technique was validated and good recoveries were ob-tained in tablet dosage forms and some fruit juices.

1. Erdurak-Kılıç CS, Uslu B, Doğan B, Özgen U, Özkan SA, Coşkun M. Anodic voltammetric behaviour of ascorbic acid and its selective deter-mination in pharmaceutical dosage forms and some rosa species of Tur-key, J. Analytical Chemistry, 61, 1113-1120, 2006.

2. Sweetman SC. in Martindale: The Complete Drug Reference, 33rd ed., London: Pharmaceutical, 2002, p. 1389.

3. Hardman JG, Limbird LE. Goodmann and Gilmann’s the pharmacologi-cal basis of therapeutics, 9th ed. [CD-ROM], New York: McGraw-Hill, 1996.

4. Pamuk F. Biochemistry (in Turkish), 1st ed. Ankara, Gazi press, 2000, p.138-139.

P 076 Ref: 0102

ONE-POT SYNTHESIS AND STRUCTURAL ELUCIDATION OF SOME NEW BIGINELLI COMPOUNDS İnci Selin DOĞAN, Selma SARAÇHacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Ankara, Turkey

3,4-Dihydropyrimidin-2(1H)-ones(thiones) have been reported to possess diverse pharmacological properties, such as antibacte-rial, antiviral, antitumor, antiinflammatory, antihypertensive, cal-cium channel blocker, -1a-antagonist and neuropeptide Y (NPY) antagonist effects. The biological activity of some alkaloids isolated recently has also been attributed to the dihydropyrimidine moiety [1, 2]. Therefore, synthesis of these compounds is still of great in-terest. Earlier, we have described the synthesis and calcium antago-nistic activity of some 5-acetyl-3,4-dihydro-6-methyl-4-(substituted phenyl)-2(1H)-pyrimidinone derivatives [3]. In continuation of our study, we aimed to report the synthesis of some new 5-acetyl–6-me-thyl-4-(substituted phenyl)-3,4-dihydropyrimidin-2(1H)-thiones which are expected to have calcium channel blocker and antibacte-rial activities.

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The title compounds were prepared by using the Biginelli three-component cyclocondensation reaction of acetylacetone, substituted benzaldehyde and thiourea under strongly acidic conditions [4]. The reaction was carried out simply by stirring the mixture of the three components dissolved in absolute ethanol with a catalytic amount of hydrochloric acid at room temperature. The solid products were isolated after cooling the reaction mixture.

The reactions were completed within 15-20 hours in 30%-40% yields. The structures of the compounds were confirmed by IR, 1H-NMR, 13C-NMR, mass spectra and elemental analysis. The calcium channel blocker and antibacterial activities of the com-pounds are in progress.

1. Kappe CO. Biologically active dihydropyrimidones of the Biginelli-type – A literature survey, Eur. J. Med. Chem., 35, 1043-1052, 2000.

2. Zorkun İS, Saraç S, Çelebi S, Erol K. Synthesis of 4-aryl-3,4-dihydropyri-midine-2(1H)-thione derivatives as potential calcium channel blockers, Bioorg. Med.Chem., 14, 8582-8589, 2006.

3. Yarım M, Saraç S, Ertan M, Batu ÖS, Erol K. Synthesis, structural elu-cidation and pharmacological properties of some 5-acetyl-3,4-dihydro-6-methyl-4-(substituted phenyl)-2(1H)-pyrimidinones, Il Farmaco, 54, 359-363, 1999.

4. Biginelli P.Aldehyde-urea derivatives of aceto- and oxaloacetic acids. Gazz. Chim. Ital., 23, 360-416, 1893.

P 077 Ref: 0103

AN EXPERIMENTAL DESIGN APPROACH TO SELECTING THE OPTIMUM LIQUID CHROMATOGRAPHIC CONDITIONS FOR THE DETERMINATION OF LOCAL ANESTHETICS 1Aysun DİNÇEL, 2Nursabah E. BAŞÇI1Hacettepe University, Faculty of Medicine, Department of Pharmacology, Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, Turkey

A sensitive high performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of four local anesthetics: lidocaine, proparacaine, bupivacine and oxybuprocaine. A full factorial design was used. The chromatographic separation was achieved using a Bondesil C8 (4.6 x 2.5 mm i.d., particle size 5 µm) analytical column. An optimised mobile phase consisted of acetonitrile and sodium dihydrogen phos-phate (pH=3.0, 20 mM) (30:70, v/v) at a flow rate of 1.2 ml min-1. Local anesthetics detection was performed by UV/Vis detector at 220 nm. The retention times for lidocaine, proparacaine, bupivacine and oxybuprocaine 5.74, 9.28, 16.84 and 26.26 min, respectively. This method was linear in the range of 50-5000 ng ml-1 for lidocaine and proparacaine and 100-5000 ng ml-1 for bupivacine and oxybu-procaine. The limit of detection (LOD) was 25 ng ml-1 for lidocaine, proparacaine and 30 ng ml-1 for bupivacine and oxybuprocaine. The limit of quantification (LOQ) was found to be 50 ng ml-1 for lido-caine, proparacaine and 100 ng ml-1 for bupivacine, oxybuprocaine (RSD ≤ 15 %, n=6).

Keywords: Local anesthetics, validation, experimental design and meth-od optimization

P 078 Ref: 0104

STUDIES ON NEW OXIME ESTER DERIVATIVES OF NAFIMIDONE ALCOHOL AS POTENTIAL ANTICONVULSANT COMPOUNDS 1Burcu SELİMOĞLU, 2Arzu KARAKURT, 1Sevim DALKARA1Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Ankara, Turkey2İnönü University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 44280 Malatya, Turkey

Nafimidone [1-(2-naphthyl)-2-(imidazol-1-yl)ethanone] is one of the two representatives of (arylalkyl)imidazole anticonvulsants [1] and nafimidone alcohol is a major and active metabolite of nafimidone. Nafimidone oxime ethers also have anticonvulsant activities [2]. In this project we prepared ten new nafimidone oxime ester derivatives. These compounds (I-X) have been synthesized by esterification of nafimidone alcohol with benzoyl and substituted benzoyl chlorides.

Total synthesis scheme starting from 2-acetylnaphthalene is given below:

Their structural elucidation were realized by IR, 1H-NMR, Mass spectral data and elemental analysis.

1. Walker KAM, Wallach BM, Hirschfeld RD. 1-(Naphthylalkyl)-1H-imi-dazole Derivatives, a New Class of Anticonvulsant Agents. J. Med. Chem., 24, 67-74, 1981.

2. Karakurt A, Dalkara S, Özalp M, Özbey S, Kendi E, Stables JP. Synthesis of Some 1-(2-naphthyl)-2-(imidazole-1-yl)ethanone Oxime and Oxime Ether Derivatives and Their Anticonvulsant and Antimicrobial Activi-ties, Eur. J. Med. Chem., 36, 421-433, 2001.

Acknowledgement This project was supported by Hacettepe University Scientific Research

Fund, Project no: 06 D03 301 001.

P 079 Ref: 0105

SEPARATION OF SEVEN QUINOLONES USING HPLC: DETERMINATION OF LEVOFLOXACIN IN PLASMA AND AMNIOTIC FLUID 1Emirhan NEMUTLU, 1Sedef KIR, 2Sinan BEKSAÇ1Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 Ankara, Turkey2Hacettepe Unıversity, Faculty of Medicine, Department of Obstetrics and Gynecology, 06100 Ankara, Turkey

A reversed-phase high-performance liquid chromatographic method is described for the separation and determination of seven quinolones (ciprofloxacin, levofloxacin, oxolinic acid, lomefloxacin moxifloxacin, enoxacin, and pefloxacin) in plasma and amniotic fluid. Chromatographic separation of the quinolones was performed on a Zorbax Eclipse XDB-C18 column (150 mm x 4.6 mm i.d.) in connec-tion to a Phenomenex C18 column (4 mm x 3.0 mm i.d.). The op-timum assay conditions were found with experimental designs. Op-timum conditions were 15 mM citrate buffer, pH 3.2, 9 % MeCN, 5

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% MeOH, 5 mM TMAB, 1.5 mL min-1 flow rate and 40 °C column temperature. Photodiode array detector was set to 280 nm. Marbo-floxacin (MAR) was used as internal standard. The retention times of the quinolones in optimum conditions were 6.0 min for MAR, 6.7 min for enoxacin, 7.8 min for levofloxacin, 8.5 min for pefloxacin, 9.3 min for ciprofloxacin, 11.2 min for lomefloxacin, 16.3 min for moxi-floxacin and 17.3 min for oxolonic acid. A simple and efficient solid phase extraction was applied for sample preparation of plasma and amniotic fluid. The validation studies of the method for the analysis of levofloxacin was performed according to FDA guidelines and the linearity range was found as from LOQ to 30 µg mL-1. The LOD and LOQ were 0.010 and 0.035 µg mL-1, respectively. The absolute recover-ies from plasma and amniotic fluid were 98.0 and 95.9, respectively. In intra-day and inter-day studies, the developed method was found to be accurate and precise with a bias value less than 2.6 % and the RSD value less than 5 %. The robustness studies were performed with an experimental design. All validation data were showed that the method was accurate, precise, linear, robust, and specific. Finally, the method has been applied to quantification of levofloxacin in amniotic fluid and plasma for investigation fetal transformation of levofloxacin.

P 080 Ref: 0106

INVESTIGATION FOR NATURAL QUORUM SENSING INHIBITORS: ALTERNATIVE TO ANTIBIOTICS? 1Gülgün BOŞGELMEZ-TINAZ, 1Seyhan ULUSOY, 1Fatma Filiz COŞKUN-ARI, 2Aysel UĞUR, 2Özgür CEYLAN1Süleyman Demirel University, Faculty of Arts and Sciences, Department of Biology, Isparta, Turkey2Muğla University, Faculty of Arts and Sciences, Department of Biology, 480000 Muğla, Turkey

As the bacterial resistance to multiple antibiotics increases, it is becoming more difficult to treat infections and, in many cases, the available therapeutic options are severely limited. Hence, there is a growing urgency to search for novel targets and the development of new antimicrobials. Millions of dollars are devoted to antibiotics that almost immediately start to become less therapeutically useful when they finally reach the marketplace. For this reason, pharma-ceutical companies are now focusing on finding novel bacterial tar-gets and new ways to control and eradicate bacterial infections [1]. To infect a host and cause a disease, bacteria produce an array of viru-lence determinants that contribute to pathogenesis. It is now known that many different Gram-negative pathogens communicate via production of small, diffusible N-acyl homoserine lactone (AHL) derivatives as signaling molecules to coordinate virulence determinant production. This event is called “quorum sensing (QS)”. A variety of physiologi-cal process in a range of bacterial species is regulated by QS, including antibiotic biosynthesis, biofilm differentiation and the production of virulence determinants in animal and plant pathogens [2]. Since many pathogens use QS to regulate virulence, strategies intended to interfere with signaling systems will likely have many potential applications. Biotechnological research is now focused on the development of AHL antagonists. In medicine, usage of such molecules represents a novel therapeutic approach offering the opportunity to attenuate virulence, and thus infection, by blocking cell-to-cell communication [3]. This approach is highly attractive because it does not impose harsh selective pressure for the development of resistance as with antibiotics [1].

Various plant samples were screened for anti-QS activities using bioassays with Chromobacterium violaceum (ATCC 12472) and Erwinia carotovora. Plant materials were dried and extracted using chloroform, methanol and water.

In this study, we tested 46 terrestrial plants materials for their ability to inhibit QS-regulated behaviors in different bacterial spe-

cies. The chloroform-soluble compounds extracted from Scor-zonera sandrasica were found to inhibit violacein production, a QS-regulated behavior in C. violaceum which is used as a model system for QS inhibition studies. This extract was also able to in-hibit QS-regulated carbapenem antibiotic production in an im-portant plant pathogen E. carotovora. In addition, some of these plant materials such as Origanum onites L and Nigella sativa have strong anti-bacterial effect against some bacterial species. As the regulation of many bacterial processes is controlled by QS systems, the finding of natural compounds acting as QS inhibitors suggests an attractive tool to control and handle detrimental infec-tions caused by human, animal and plant pathogens.

1. Hentzer M, Giskov M. Pharmacological inhibition of quorum sensing for the treatment of chronical bacterial infections, J. Clin. Invest., 112, 1300-1307, 2003.

2. Fuqua C, Parsek MR, Greenberg EP. Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing, Annu. Rev. Genet., 35, 439-468, 2001.

3. Finch RG, Pritchard DI, Bycroft BW, Stewart GSAB. Quorum sensing-a novel target for anti-infective therapy, J. Antimicrob. Chemother., 42:569-571, 1998.

P 081 Ref: 0107

DEVELOPMENT OF A CAPILLARY ZONE ELECTROPHORESIS METHOD FOR THE SIMULTANEOUS ANALYSIS OF OLMESARTAN MEDOXOMIL AND HYDROCHLOROTHIAZIDE Mustafa ÇELEBİER, Sacide ALTINÖZHacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, Turkey

Olmesartan medoxomil is a selective AT1 subtype angiotensin II receptor antagonist [1]. Hydrochlorothiazide is a thiazide diuretic that is commonly used in combination with other antihypertensive agents. It has been reported that the combination of olmesartan medoxomil and hydrochlorothiazide is a safe, well tolerated, and ef-fective option for antihypertensive therapy, demonstrating greater blood pressure reduction than monotherapy [2]. In the literature, there hasn’t been any method described for the simultaneous analy-sis of olmesartan medoxomil and hydrochlorothiazide. A capillary zone electrophoresis method with ultraviolet detection for the simul-taneous analysis of olmesartan medoxomil and hydrochlorothiazide in synthetic tablets was developed. A fused silica capillary (50 µm internal diameter, 48.5 cm total length, 40 cm effective length) was used and the separation was obtained by 40 mM pH 9.5 borate buffer followed by detection with an ultraviolet detector at 210 nm. The analysis were performed at 30 °C with the application of 3 seconds hydrodynamic injection at 50 mbar pressure and a applied potential of 30 kV. Diflunisal was used as internal standard. The developed method was validated according to the literature [3].

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1. Mire DE, Silfani TN, Pugsley MK. A review of the structural and func-tional features of olmesartan medoxomil, an angiotensin receptor block-er, Journal of Cardiovascular Pharmacology, 46, 585-593, 2005.

2. Steven GC, Michael AW, Antonia CW, Donald JH. Evaluation of antihy-pertensive therapy with the combination of olmesartan medoxomil and hydrochlorothiazide, AJH, 17, 252–259, 2004.

3. ICH Steering Committee. Text and Methodology Q2 (R1). Harmanized Tripartite Guideline, 2005.

P 082 Ref: 0110

CYP1A2, CYP2A6, NAT2 AND XANTHINE OXIDASE ACTIVITIES IN TURKISH VOLUNTEERS 1Aysun DİNÇEL, 2Nursabah E. BAŞÇI, 1Atila BOZKURT1Hacettepe University, Faculty of Medicine, Department of Pharmacology, 06100 Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 Ankara, Turkey

A RP-HPLC method has been developed for the determination of caffeine metabolites in urine for the evaluation of the CYP1A2, CYP2A6, xanthine oxidase (XO) and N-acetyl-transferase-2 (NAT-2) activities in 101 Turkish volunteers (47 males and 54 females). Spot urine samples were analyzed 5 h after 100 mg caffeine con-sumption. The urinary caffeine metabolites, 1,7-dimethylxanthine (17X), 1,7-dimethyluric acid (17U), 1,3-dimethyluric acid (13U), 3-methylxanthine (3X), 1-methylxanthine (1X), 1-methyluric acid (1U), theobromine (37X), and 5-acethylamino-6-formylamino-3-methyluracil (AFMU) were analysed. CYP1A2, CYP2A6, XO and NAT-2 activities were estimated from the metabolic ratios (AFMU + 1U + 1X)/17U, 17U/17X, 1U/(1X + 1U) and AFMU/(AFMU + 1U + 1X), respectively. Urine samples were extracted with chloro-form/isopropanol (85:15, v/v) and separated on a reversed phase C18 (4.6 x 2.5 mm i.d., particle size 5 µm) analytical column with acetic acid/tetrahydrofuran/acetonitrile/water (1:2.5:44:925, v/v/v/v). Peaks were monitored with UV detection at 280 nm. The en-zyme activities of CYP1A2, CYP2A6, NAT2 and XO were found as 5.28±6.12, 0.22±0.11, 0.33±0.17, and 0.65±0.16 (mean±SD), respec-tively. Smoking and gender were not affected CYP1A2, and CYP2A6 activities. The developed RP-HPLC method was validated and suc-cessfully applied for the evaluation of CYP1A2, CYP2A6, XO and NAT-2 activities. These results are comparable to those reported for Caucasian populations previously.

Keywords: CYP1A2, CYP2A6, XO, NAT-2, HPLC, caffeine

P 083 Ref: 0112

DETERMINATION OF PAEONIFLORIN AND OXYPAEONIFLORIN IN PAEONIA SPECIES USING VALIDATED HPLC METHOD 1Semra KOYUNOĞLU, 2Sedef KIR, 3Ali A. DÖNMEZ, 4İhsan ÇALIŞ1Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, 06100 Sıhhiye-Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 Sıhhiye-Ankara, Turkey3Hacettepe University, Faculty of Science, Department of Biology, 06100 Sıhhiye-Ankara, Turkey4Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, 06100 Sıhhiye-Ankara, Turkey

Paeonia L., the largest genus in the family Paeoniacea, is repre-sented by 7 species in the flora of Turkey. Paeonia Radix is one of the most important herbal drugs in traditional Chinese medicine, and as well as in different countries. Significant chemical and pharmaco-

logical studies have been conducted on the roots of Paeonia species. Monoterpen glycosides are the active constituents for this plant spe-cies. Among the terpen glycosides, paeoniflorin (1) and oxypaeoni-florin (2) are the main compounds.

In the present study, reversed-phase high performance liquid chromatographic (HPLC) method with diode array detection for the determination of the 1 and 2 has been developed. Compounds 1 and 2 were isolated from Paeonia species using chromatographic meth-ods. The structure elucidations of 1 and 2 were achieved by combina-tion of 1D and 2D-NMR experiments and mass spectrometry. The HPLC separation was achieved on a Nucleosil 100-5 C18 (5 µm, 250 x 4.6 mm) column using a mobile phase composed of acetonitrile:10 mM pH 3.5 phosphate buffer (20:80 v/v) at a flow rate of 1 mL min-1 . A wavelength of 230 nm for diode array detection was selected. The internal standard was metronidazol.

The retention times were 4.1 min for 1 and 7.8 min for 2. The developed method was optimized by changing the chromatographic parameters such as organic solvent ratio and pH of mobile phase. Optimal conditions were selected by calculating capacity and tail-ing factors of each peaks and by identifying the resolution for the separation. Low retention time, symmetric peak, high peak area and high resolution were performed by optimized method. The validated method was found to be linear, accurate, precise, sensitive, rugged and robust. The linear ranges were 0.25-80 µg/ml for 1 and 0.25-60 µg/ml for 2. The developed method was found to be useful for de-termination of compounds 1 and 2 in Paeonia species. By the help this method, 3.47 %- 5.46 % paeoniflorin (1), and 0.049 % -0.422 % oxypaeoniflorin (2) were found from 2 g of the crude samples of P. mascula, P. daurica, P. peregrina, P. tenuifolia, P. mascula subsp. bod-urii collected from the flora of Turkey.

P 084 Ref: 0114

STABILITY OF LIQUID AND SOLID FORMS OF RECOMBINANT HUMAN INTERFERON Bilgen ÖZBATIR, Filiz ÖNERHacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, 06100 Ankara, Turkey

Due to the patient compliance problems of injectable formulations, alternative routes for peptide-protein drugs are under investigation in recent years. Mucosal route seems advantageous for administra-tion of peptide drugs due to the direct systemic effect and avoidance of first hepatic elimination [1]. The aim of this study was to develop a stable lyophilized solid lozenge form for mocosal application of rHuIFN-α2b which has a molecular mass of 19.269 kDa and has a significant therapeutic potential in treatment of some cancers and infectious diseases.

Studies on the therapeutic effects of low dose oral mucosal for-mulations of interferon present promising results in some references [2]. In this study we prepared a solid lozenge form and liquid form of rHuIFN-α2b (recombinant human interferon alfa 2b) and exa-mined the stability of the active substance in vitro by comparing with the commercial product. Composition of the formulated forms of interferon alpha is shown in Table I.

Experiments were performed in an artificial saliva medium (pH 6.7) in order to simulate the medium in the oral cavity. Liquid and solid forms were kept at 4oC and 25oC for 1 and 4 months and the amount of active substance was determined quantitatively by HPLC and qualitatively by SDS-PAGE. No degradation product was found and rHuIFN-α2b was kept stable for 1 month at both 4oC and 25oC temperatures.

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Table I. Composition of the liquid and solid interferon formulations used in the study

Components

Liquid formulation Solid formulation

rHuIFN-α2b rHuIFN-α2b

Sodium phosphate dibasic anhydrous Gelatin

Sodium phosphate monobasic monohydrate Glycerol

Disodium EDTA Sucrose

Polysorbate 80 (Tween 80) Water for injection

Phenol

Sodium chloride

Water for injection.

1. Shojaei AH. Buccal mucosa as a route for systemic drug delivery: A re-view, J. Pharm. Pharmaceut. Sci.,1(1), 15-30, 1998.

2. Ship JA., Fox PC, Michalek JE, Cummins MJ, Richards AB. Treatment of pimary Sjögren’s syndrome with low-dose natural human interferon- α administered by the oral mucosal route: a phase II clinical trial, Journal of Interferon and Cytokine Research, 19, 943-951, 1999.

P 085 Ref: 0116

HPLC-ECD DETERMINATION OF EPINEPHRINE PLASMA CONCENTRATIONS IN VARIOUS DENTAL PATIENTS 1Şeyda DEMİRCAN, 2Ayşe Gül AKALIN, 1Filiz SAYIN, 2Gökçe MERAL, 2Ferda TAŞAR, 1Sedef KIR, 1Nursabah E. BAŞÇI1Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100, Sıhhiye-Ankara, Turkey2Hacettepe University, Faculty of Dentistry, Department of Oral Surgery, 06100-Sıhhiye-Ankara, Turkey

A sensitive and reliable reversed-phase high performance liquid chromatographic (RP-HPLC) method with electrochemical detector (ECD) has been developed for the determination of local anesthetic agent added epinephrine in plasma obtained during dental surgery. For this aim, the changes of plasma levels of epinephrine have been measured in the blood samples taken at five different times (0, 3, 15, 30 and 60 minutes) after administration of the local anesthetic. The method was based on the use of a Nucleosil 100-5 C18 (5 µm, 250 x 4,6 mm i.d.) as analytical column with a mobile phase containing methanol: 0.1 mol L-1 citrate buffer (10:90, v/v, pH 2.5) with a flow rate of 1.2 mL min-1. Electrochemical detector was set to 800 mV. Retention times of epinephrine and isoprotrenole (internal standard, IS) were 8.0 and 19.0 min, respectively. The plasma assay was vali-dated for the parameters such as specificity, accuracy and extraction recovery and was applied to three different patient groups. The re-covery of epinephrine after extraction from spiked plasma was 97 ± 0.03 % (mean±SD). The method was linear over the range of 15-200 ng mL-1 (r2 = 0.9992). The detection limit (LOD) was found to be 5 ng mL-1 (signal-to-noise ratio = 3) and the quantitation limit (LOQ) was 15 ng mL-1 for epinephrine. The described HPLC-ECD method is simple, selective and can successfully be applied with high degrees of precision and accuracy for the quantitative determination of epinephrine in plasma samples. The pharmacokinetic profiles for epinephrine in the healthy young, healthy older and ASA II CVS di-seased older dental patients after the administration of local anesthe-sia with epinephrine were obtained.

Keywords: Epinephrine; HPLC; Electrochemical Detector (ECD); Vali-dation; Plasma; Dental Patients

P 086 Ref: 0118

ANTIMICROBIAL ACTIVITY OF 3-HYDROXY-6-METHYL-2-SUBSTITUTED 4H-PYRAN-4-ONE DERIVATIVES 1Ekrem KILIÇ, 2Mutlu DİLSİZ-AYTEMİR1Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, 06100 Sıhhiye-Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Sıhhiye-Ankara, Turkey

Over the past several years the emergence of organisms resistant to nearly all the class of antimicrobial agents has become a serious public health concern. For these reasons the research of new antimi-crobial agents with novel modes of action represents a main target in chemotherapy. It has been shown that hydroxypyran-4H-one deriva-tives have antimicrobial activity [1, 2]. In our previous studies, we synthesized some Mannich bases of 3-hydroxy-6-methyl-4H-pyran-4-one derivatives and investigated their antimicrobial activity [3].

Antifungal activities of the compounds evaluated in vitro against fungi such as Candida albicans (ATCC 90028), C. krusei (ATCC 6258) and C. parapsilosis (ATCC 90018). They were also tested against Gram-positive bacteria such as Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212) and Gram-negative bac-teria such as Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) by using microdilution broth method recommended by National Committee for Clinical Laboratory Standards (NCCLS)

[4, 5]. The minimum inhibitory concentrations (MIC) were defined as the lowest concentrations of the compounds that prevented visible growth. Fluconazole and Ampicillin were used as the standards for antifungal and antibacterial tests, respectively. The screening data indicates that compound 5 (MIC: 16 µg/ml), which was carrying (4-chlorophenyl)phenylmethyl substituent showed antibacterial activity against S. aureus. The other compounds had no valuable inhibitory activity. Compound 5 (MIC: 16 µg/ml) was also the most effective compound towards C. albicans than the others (MIC: 32-64 µg/ml).

1. Aytemir MD, Erol DD, Hider RC, Özalp M. Synthesis and evaluation of antimicrobial activity of new 3-hydroxy-6-methyl-4-oxo-4H-pyran-2-carboxamide derivatives, Turk J. Chem., 27(6), 757-764, 2003.

2. Kayahara H, Shibata N, Tadasa K, Kotani T. Amino acid and peptide de-rivatives of kojic acid and their antifungal properties Agric. Biol. Chem., 54(9), 2441-2442, 1990.

3. Aytemir MD, Çalış Ü, Özalp M. Synthesis and evaluation of anticonvul-sant and antimicrobial activities of 3-Hydroxy-6-methyl-2-substituted 4H-pyran-4-one derivatives. Arch. Pharm. Pharm. Med. Chem. 337, 281-288, 2004.

4. National Committee for Clinical Laboratory Standards (NCCLS), Meth-ods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, Approved Standard M7-A, 37(2), Villanova, PA. (1997).

5. National Committee for Clinical Laboratory Standards (NCCLS), Refer-ence method for broth dilution susceptibility testing of yeast, Approved Standard M27-A, 17(9) Villanova, PA. (1997).

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P 087 Ref: 0119

INHIBITION OF RAT LIVER MONOAMINE OXIDASE (MAO)-A AND –B BY GENTIANELLA CAUCASEA (Loddiges ex Sms) HOLUB EXTRACTS 1Tayfun ERSÖZ, 1Zeliha Ş. AKDEMİR, 2Samiye YABANOĞLU, 1, 3Funda Nuray YALÇIN, 1Duygu KAYA, 4İ. İrem ÇANKAYA, 2Gülberk UÇAR1Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, 06100 Ankara, Turkey2Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 06100 Ankara, Turkey3Hacettepe University, Faculty of Pharmacy, Department of Pharmacy Management, 06100 Ankara, Turkey4Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Botany, 06100 Ankara, Turkey

Monoamine oxidase (MAO), which is found in two forms desig-nated as MAO-A and -B, plays an essential role in the metabolism of the biogenic amines [1]. MAO-B inhibitors are shown to be useful in the treatment of Parkinson’s and Alzheimer’s diseases [2] while MAO-A inhibitors are known as antidepressant and antianxiety agents [3]. Since severe side-effects have been observed with some classical MAO inhibitors [4], new inhibitors devoid of these adverse effects are needed. The presence of plant-derived MAO inhibitors suggests that such plant extracts might be useful as potential neu-roprotectans in the treatment or prevention of depression, psychosis, schizophrenia, Alzheimer’s and Parkinson’s diseases [5].

Gentianella caucasea is a biennial stem to 30 cm grown in North-ern and Northeastern Anatolia [6]. Preliminary works on Gen-tianella caucasea showed the presence of secoiridoid, flavonoid and xanthone compounds in the chemical composition. Xanthones are known to possess MAO inhibitor activity [7].The present study was designed to investigate the MAO inhibitory activities of the metha-nol, petrolum ether, chloroform and the remaining water extracts prepared from the aerial parts of Gentianella caucasea.

MAO was purified from the rat liver and MAO-A and –B activities were determined according to a previous method [8]. Kinetic data for interaction of the enzyme with the abstracts were determined us-ing Microsoft Excel package program. IC50 values were determined from plots of inhibitory activity percentage, calculated in relation to a sample of the enzyme treated under the same conditions wit-hout extracts, versus extract concentration. All Gentianella extracts tested inhibited rat liver MAO isoforms in a dose-and time-depen-dent manner. Incubation of the enzyme with the extracts at 37 0C for 60 min. increased the inhibitory action of the extracts. Metha-nol extract inhibited MAO-B isoform selectively with IC50 value of 38.2±2.26 µg dry weight/mL whereas chloroform, petrolum ether, and water extracts inhibited MAO-A selectively with IC50 values of 58.7±4.31, 64.33±5.06 and 102.13±7.55 µg dry weight/mL, respec-tively. Xanthones of Gentianella caucasea may be a possible source of pharmaceuticals for the treatment and prevention of depression, Parkinson’s and Alzheimer’s diseases. Phytochemical study on the title plant is carrying on.

1. Loscher W, Lehman H, Teschendorf H, Traut M, Gross, G. Inhibition of monoamine oxidase type A, but not type B, is an effective means of in-ducing anticonvulsant activity in the kindling model of epilepsy. J Phar-macol Exp Ther 288: 984-992, 1999.

2. Uçar G, Gökhan N, Yeşilada A, Bilgin A. 1-N-substituted thiocarbamoyl-3-phenyl-5-thienyl-2-pyrazolines: A novel cholinesterase and selective monoamine oxidase-B inhibitors for the treatment of Parkinson’s and Alzheimer’s diseases. Neurosci Lett 382: 327-331, 2005.

3. Ertuğrul A, Uçar G, Başar K, Demir B, Yabanoglu S, Uluğ B. Influence of clozapine on platelet serotonin, monoamine oxidase and plasma seroto-nin levels. Pscyhiatr Res 149: 49-57, 2007.

4. Power MB, Hackett LP, Dusci LJ, Ilett KF. Antidepressant toxicity and the need for identification and concentration monitoring in overdose. Clin Pharmacokin 29: 154-171, 1995.

5. Haraguchi H, Tanaka Y, Kabbashi A, Fujioka T, Ishizu T, Yagi A. Mono-amine oxidase inhibitors from Gentiana lutea. Phytochemistry 65: 2255-2260, 2004.

6. Pritchard NM, Gentianella “in Flora of Turkey and the East Aegean Is-lands “(Ed. Davis PH) University Press, Vol 6 Edinburg, 1978.

7. Suzuki O, Katsumata Y, Oya M, Chari VM, Vermes B, Wagner H, Hos-tettmann K. Inhibition of type A and B monoamine oxidases by naturally occuring xanthones Planta Med 42: 17-21, 1981.

8. Holt A, Sharman DF, Baker GB, Pelcic MM. Continuous spectrophoto-metric assay for monoamine oxidase and related enzymes in tissue ho-mogenates. Anal Biochem 244: 384-392, 1997.

P 088 Ref: 0120

NEW MOLECULARLY IMPRINTED MICROSPHERES FOR COLONIC DELIVERY OF 5-AMINOSALICYLIC ACID Yeliz TUNÇ, Ersin BAYKARA, Kezban ULUBAYRAMHacettepe University, Faculty Pharmacy, Department of Basic Pharmaceutical Sciences, Ankara, Turkey

Molecular imprinting has become an increasingly active field of study for the construction of new highly stable molecularly imprin-ted polymers (MIPs) that possess selective molecular recognition properties [1]. MIPs have a large number of potential applications such as seperation process, immunoassays and antibody mimics, biosensor recognition elements, and catalysis and artifical enzymes [2]. Recently, researchers have applied the molecular recognition properties of imprinted polymers for enhanced control in the release of pharmaceutical compounds [3].

In this study, feasibility of molecularly imprinted polymeric mi-crospheres (MIPs) has been investigated for colonic delivery of 5-aminosalicylic acid (5-ASA), an active agent used in treatment of ulcerative colitis and Crohn’s disease. For the first time, 5-ASA im-printed microspheres were prepared by a single step precipitation polymerization of 2-(diethylamino) ethyl methacrylate (DEAEMA; functional monomer) and trimethylolpropane trimethacrylate (TRIM; crosslinker). The microspheres were prepared in the diame-ter range between 1.2 and 2.7 by varying polymerization conditions. The release characteristics of 5-ASA imprinted and non-imprinted microspheres were determined and molecular imprinting effect on release behaviors was evaluated. We present a precipitation po-lymerization method for preparing uniform molecularly imprinted microspheres in micron range, quickly and cleanly. Monodisperse polymer particles with good spherical shapes and smooth surfaces were obtained. Furthermore, the results show that the imprinted mi-crospheres have a slower 5-ASA release in the initial stages than the non-imprinted microspheres, because of the interaction of the drug molecules with the recognition sites in the imprinted microspheres. This result indicates that molecular imprinting may have a potential for controlled delivery of drugs.

1. Tunç Y, Hasırcı N, Yeşilada A, Ulubayram K. Comonomer effects on binding performances and morphology of acrylate-based imprinted polymers, Polymer, 47, 6931-6940, 2006.

2. Zhang H, Ye L, Mosbach K. Non-covalent molecular imprinting with emphasis on its application in separation and drug development, J. Mol. Recognit., 19, 248-259, 2006.

3. Pepppas, N.A, Intelligent therapeutics: Biomimetic systems and nanote-chnology in drug delivery, Adv. Drug Del. Rev., 56, 1529-1531, 2004.

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P 089 Ref: 0121

BIOLOGICALLY ACTIVE COMPOSITE SCAFFOLD FOR TISSUE ENGINEERING APPLICATIONS 1Sinan GÜVEN, 2Nesrin HASIRCI, 3Sevda MÜFTÜOĞLU, 1Kezban ULUBAYRAM1Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, Ankara, Turkey2Middle East Technical University, Faculty of Arts and Sciences, Department of Chemistry, Ankara, Turkey3Hacettepe University, Faculty of Medicine, Department of Histology and Embrology, Ankara, Turkey

Tissue engineering plays a vital role in regenerative medicine in order to create new tissues and organs from cultured cells for trans-plantation [1]. In scaffold oriented tissue engineering, the scaffolds should mimic the structure and biological function of native extra-cellular matrix which provide mechanical support and regulate cell activities [2].

In the present study biologically active composite scaffolds were developed from natural polymers by tissue engineering approach and tested in vitro. Freeze-dried scaffolds composed of chitosan, gelatin and dermatan sulfate were treated with different stirring rates, freez-ing temperatures and molding. Among the prepared scaffolds at dif-ferent conditions, scaffolds (SC) prepared at 500 rpm and frozen at -80°C were chosen for further studies. These scaffolds achieved 0.512 MPa tensile strength, 9.165 MPa tension modulus and 3.428 MPa compression modulus. Besides in lysozyme containing degradation medium they conserved their integrity and lost about 30% of their initial weight in 30 days period. Mechanical and enzymatic degrada-tion tests showed that scaffolds have physical integrity for the tissue engineering applications. To mimic the natural tissue and enhance cell growth, biologically active arginine-glycine-aspartic acid-serine (RGDS) peptides and platelet derived growth factor-BB (PDGF-BB) were immobilized on selected scaffolds. Fibroblast cells were seeded on the scaffolds containing RGDS, (SC-RGD), and PDGF-BB, (SC-RGD-GF), and incubated in media either free of serum or contain-ing serum. Scaffolds immobilized with RGDS and PDGF-BB had the highest attached cell number by the day 15. According to scan-ning electron microscopy (SEM) results, cells on modified scaffolds demonstrated better cell morphology and attachment. Based on the obtained results, it can be concluded that RGDS-PDGF immobilized chitosan-gelatin-dermatan sulfate systems have a great potential to be used as a scaffold for soft tissue engineering applications.

1. Langer R, Vacanti JP, Tissue Engineering, Science, 260, 920-926, 1993. 2. Marler JJ, Upton J, Langer R, Vacanti JP. Transplantation of cells in matri-

ces for tissue regeneration, Advanced Drug Delivery Reviews, 33, 65–182, 1998.

P 090 Ref: 0122

EFFECT OF CORIANDER (Coriander sativum L) AND PARSLEY (Petroseleinum sativum Hoffm.) EXTRACTS ON PLATELET AGGREGATION AND BLOOD COAGULATION Muhammad Jamal SHAMMOUTUniversity of Jordan, Analytical Toxicology, Amman, Jordan

Certain herbs may cause pharmacological effects on some car-diovascular aspects such as platelet aggregation and blood coagula-tion during homeostasis processes. In this study, the volatile oils and crude extracts of Coriander (Coriander sativum L) and Parsley (Pet-roseleinum sativum Hoffm.) were isolated using steam distillation and heating under reflux techniques. The effects of herb’s prepara-

tions on platelets aggregation were investigated in vitro using adeno-sine-5’-diphosphate (ADP) and arachidonic acid (AA) as agonists.

The volatile oil of coriander seeds and the ethanolic extracts of leaves of coriander and parsley exhibited inhibitory effects on plate-let aggregation induced by ADP and AA in dose response manner.

The effects of aspirin and dimethylsulfoxide (DMSO) on platelet aggregation induced by ADP and AA were also studied. It was found that Aspirin exhibited a strong inhibitory effect on platelet aggrega-tion induced by ADP or AA, while DMSO exhibited an inhibitory effect on platelets.

A combination of aspirin with coriander and parsley extracts was studied in terms of its effect on platelet aggregation. İt was found that the combination of aspirin and coriander seed’s volatile oil extract were approximately additive effect on platelet aggregation.

The effects of coriander and parsley extracts on blood coagulation were also investigated. The effects of plant preparations were inves-tigated by determination of their effects on activated partial thromo-plastine time (APTT). None of tested extracts affected prothrombine time (PT) values.

As a result, the coriander’s seeds volatile oil and parsley leaves extracts are of the interest for open new approaches towards expla-nation of the beneficial effects on several manifestations of cardio-vascular diseases like hypertension, arteriosclerosis and ischemic diseases and it could be assessed as useful natural source of pharma-ceutical agents.

Author Index

93

AU

THO

R IN

DEX



A

AKALIN, Ayşe Gül 87

AKDEMİR, Zeliha Ş. 88

YEŞİLADA, Akgül 56

ŞEN, Alaattin 51

ASLAN, Ali 71

GÜNER, Ali 58, 66, 67

HAYAT, Ali 64, 65, 66

KILINÇ, Ali 71

ONUR, Mehmet Ali 66

KARAKURT, Arzu 84

MERİÇ, Asiye 70

TÜMER, Aşkın 67

ÖZTÜRK-ÇAL, Aslı 61

BOZKURT, Atila 86

HINCAL, A. Atilla 78

YILDIZ, Attila 69

DEMİRCAN, Aydın 80

DEMİR, Ayhan 48

GÜRPINAR, Özer Aylin 66, 67

HASEGELİ-ÜNER, Ayşegül 58

UĞUR, Aysel 85

DİNÇEL, Aysun 84, 86

B

BARBOSA, J. 8

BAŞÇI, Nursabah E. 86, 87

GÜLBAKAN, Basri 43, 61

BAYKARA, Ersin 88

BAYRAM, Hakan 25

SALİH, Bekir 43, 60, 61

BENAVENTE, F. 8

ARICA, Betül 78

ÇEKEN, Bircan 52

BRASH, Douglas 47

ATMIŞ, Bünyamin 55

SELİMOĞLU, Burcu 84

C

C. ÇEHRELİ, Zafer 67

ÇALIŞ, Ünsal 72

ÖZELGÜL, Canan 58

ÜNALEROĞLU, Canan 53

ÇANKAYA, İ. İrem 88

TAŞAĞIR, Cansel 60

SARISÖZEN Can 78

KAZAZ Cavit 57, 59, 71

CROOKS, Peter A. 35

ÇAKIR, Ahmet 71, 73

D

SALMAN, Demet 62

DEMİR, Ayhan S. 36

DEMİRCAN, Şeyda 87

DENİZLİ, Adil 62

CAN, Özgür Devrim 56

DAYANGAÇ-ERDEN Didem 48

EROL, Dilek 52, 55

SADAK, Dilek 55

TURGUT-BALIK, Dilek 53, 55

YURDAKUL, Dilşat 58

DİLSİZ-AYTEMİR, Mutlu 72, 87

DÖNMEZ, Ali A. 86

ÖZER-ÜNAL Durişehvar 55

E

BAŞÇI, Nursabah E. 62, 84

CRISS, Wayne E. 60

ÇUBUK-DEMİRALAY, Ebru 63

KONDOLOT-SOLAK, Ebru 62

METE Ebru, 57, 58, 59

GÜRDAL, Ece 52

KÜÇÜK, Ekrem 79

AKSÖZ, S. Elif 53

ÇADIRCI, Elif 73, 74, 75

NEMUTLU, Emirhan 84

ERCAN, Ayşe 47

YEŞİLADA, Erdem 72

ERİLHAN, İsmail 46

GÖKTÜRK, Ersen 80

ERSÖZ, Tayfun 88

ŞAHİN, Ertan 59

AKBAY, Esin 66, 67

GÜÇ, Esra 68

KÜPELİ, Esra 72

İŞLEYEN, Evren 65

F

ATALAY, Fadime 73

GEYİKOĞLU, Fatime 51, 52

ARSLAN, Fatma 77

HAMURCU Fatma 63, 77

SEVİN Fatma 83

ODABAŞOĞLU Fehmi 71, 73, 74, 75

ŞAHİN, Fikrettin 58, 61

COŞKUN-ARI, Fatma Filiz 85

FILLET, M. 14

ÇAĞLAR, Fulya 43, 44

MORAL, Fulya 53

KARTAL, Funda 71

BABA, Füsun 64, 65, 66

TEMAMOĞULLARI, Füsun 64, 65, 66

G

BORA, Gamze 48

GEURTS, P. 14

GIMÉNEZ, E. 8

AŞKIN, Gökçe 83

SADİ, Gökhan 56

ALTIOKKA, Göksel 80, 81, 82

SUNAL, Sevil Görkem 56

YALÇIN, Görkem 54

GREF, Ruxandra 18

ŞAHİN-KOÇ, Gülay 55

YELKEN, Gülay 76

UÇAR, Gülberk 47, 56

TÜRKER, Gülen 83

ALSANCAK, Güleren 62, 68, 70

BOŞGELMEZ-TINAZ, Gülgün 85

SAĞLIKOĞLU, Gülşen 83

ÇOBAN, Güneş 67

ERGÜN, Ufuk Güney 79

GÜNDÜZ, Güngör 68

ÖZBAKIŞ-DENGİZ, Günnur 74, 75

GÜVEN, Sinan 89

H

SEÇİLMİŞ, Hale 63

SÜLEYMAN, Halis 74, 75

SEVİM, Handan 66, 67

TÜRKEZ, Hasan 51, 52

HASIRCI, Nesrin 89

ARDAĞ-AKDOĞAN, Hatice 51

KAPLAN-CAN, Hatice 58, 66, 67

AYGÜN, Hayati 71, 73

ERDEM-YURTER, Hayat 48

ERDUĞAN, Hüseyin 83

YALÇINKAYA, Hüseyin 55

I

ÇALIŞ, İhsan 72, 86

IŞIKDAĞ, İlhan 56, 57

GÜL, H. İnci 47, 57, 58, 59, 60, 61

DİLER, Zeynep İrem 55

İŞÇİ, Hüseyin 46

İŞGÖR, S. Belgin 46

J

J. LINHARDT, Robert 54

BARBOSA , Jose 68, 70

K

ÖZTÜRK, Taylan K. 71

KÜÇÜKOĞLU, Kaan 60

KABANOV, A. V. 39

PEKMEZ, Kadir 69

KANSU, Emin 3

KAYA, Duygu 88

YELEKÇİ, Kemal 48

KILIÇ, Ekrem 87

KIR, Sedef 87

L

DOĞAN, A. Lale 58

UZUN, Leyla 78

LOUIS, Ed. 14

BELTRAN, Jose Luis 63, 68, 70

M

MALAISE, M. 14

MARCO, José Luis 4

ÖZSÖZ, Mehmet 54

SERİN, Mehmet 76, 77

GÖKÇE, Mehtap 76, 77

MERAL, Gökçe 87

94

AU

THO

R IN

DEX

May 17-20, 2007


KÖKSAL, Meriç 53

MERVILLE, M-P. 14

HALICI, Mesut 71, 73, 75

MEUWIS, M-A. 14

YARIM Mine 52, 53

KAVANOZ, Muammer 69

ŞEN, Müberra 65

MÜFTÜOĞLU, Sevda 89

KARAARSLAN, Muhsin 80

ÇİZMECİOĞLU, Murat 54, 59

KIZIL, Murat 52

ATALAY, Mustafa 60, 61

ÇELEBİER, Mustafa 85

GÜL, Mustafa 60, 61

N

BAYTAŞ, Sultan N. 54

GÖKHAN-KELEKÇİ, Nesrin 47

ÖZÇİÇEK-PEKMEZ, Nuran 69

EZER, Nurten 72

ŞANLI, Nurullah 62, 68

O

RZAEV, Zakir M. O. 58, 67, 66

ÖĞÜŞ, İ. Hamdi 47

ÇELİKBIÇAK, Ömür 60

CAN, Nafiz Öncü 80, 81, 82

ÖNER, Filiz 86

ONUR, Mehmet Ali 67

HANNINEN, Osmo 60, 61

ŞANLI, Oya 62

ÖZALP-YAMAN, Şeniz 46

ÖZBATIR, Bilgen 86

YERDELEN, K. Özden 60, 61

KÜL, Özge 43, 44

CEYLAN, Özgür 85

ÖZKAN, Serkan 27

DEMİREL, Özlem 43

ORHAN, Özlem 79

SÖĞÜT, Özlem 54

ÖZTÜRK, Recep 22

P

PARINI, Angelo 7

AYHAN, Peruze 48

ALCIL,” Pınar 67

ŞAHİN, Pınar 72

PİŞKİN, Erhan 38

S

DOĞRUER, Deniz S. 76

ALTINÖZ, Sacide 85

ŞAHİN, Fethi 28

YABANOĞLU, Samiye 47, 56

EREN, Sami 64

SANYAL, Rana 45

SANZ, Elisenda 4

SANZ-NEBOT, V. 8

SAYIN, Filiz 87

TEZCAN, Seda 76, 77

KIR, Sedef 84, 86

ADAK, Selcen 76

ALPAN, A. Selcen 67

YILMAZ, Selehattin 83

YILDIRIM, Ömer Selim 71

DOĞAN, İnci Selin 83

ÖZİLHAN, Selma 64, 65

SARAÇ, Selma 83

CALIŞ, Sema 78

GÜNEŞ, H. Semih 67

KOYUNOĞLU, Semra 86

ŞANLI, Senem 63, 70

SENY, D. de 14

SEPTİOĞLU, Ebubekir 72

KAVLAK, Serap 67

ÜNLÜ, Serdar 76, 77, 78

POLAT, Serpil 76, 77

ZENCİR, Sevil 67

DALKARA, Sevim 48, 84

ULUSOY, Seyhan 85

CANKARA, Şeyma 77

SHAMMOUT, Muhammad Jamal 89

BEKSAÇ, Sinan 84

SIPPL, Wolfgang 17

BİLGE, S. Sırrı 53

URLU, Şölen 78

YAĞMUR, Sultan 83

TOPTAN, Suna 65

T

PARK, Tae-joon 54

YALÇIN, Talat 61

TAŞAR, Ferda 87

TAŞGIN, Dilek Işık 53

ÖNKOL, Tijen 77, 78

YALÇINKAYA, Timuçin 43

TAŞKIN, Tuğba 83

GÜRAY, Tülin 56

TUNÇ, Yeliz 88

ÖZDEN, Tuncel 64, 65

U

UÇAR, Gülberk 88

GÜNDÜZ, Ufuk 68

BÜYÜKDEMİRCİ, Uğur 44

ULUBAYRAM, Kezban 88, 89

TOSUN, Ali Ulvi 77

DEMİR, Ümide 56

ÖZDEMİR-ÖZMEN, Ümmühan 63, 77

AKKAYA, Engin Umut 45

SALGIN-GÖKŞEN, Umut 47

ÜNAL, Serhat 20

ÇALIŞ, Ünsal 60

UNZETA, Mercedes 4

UZUN, Ömrüm 24

V

ÇELİK, Venhar 53, 55

ADAR, Vildan 43, 44

W

WEHENKEL, L. 14

Y

YABANOĞLU, Samiye 88

YALÇIN, Funda Nuray 88

BAYIR, Yasin 73

YEŞİLADA, Erdem 29

ÇAPAN, Yilmaz 45

ERGÜN, Yusuf 79

ÖZKAY, Yusuf 56, 57

ÖZTÜRK, Yusuf 56

Z

OKUMUŞ, Zafer 71

HALICI, Zekai 71, 73, 74, 75

ATKOŞAR, Zeki 82

TOPÇU, Zeki 67

ATEŞ, Zeliha 64

ERDEMGİL, Zerrin 68

İNCESU, Zerrin 57

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