DDR 2007

INTERNATIONAL SYMPOSIUM on DRUG RESEARCH and DEVELOPMENT
From Chemistry to Medicine

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TRKYE May 17-20, 2007
Hacettepe University Medicinal Chemistry Research, Development and Application Center (MAGUM)
From Chemistry to Medicine DRD 2007
A big THANK YOU

Supporting Organizations
ASSOCIATION OF RESEARCH - BASED PHARMACEUTICAL COMPANIES
THE SOCIETY OF PHARMACEUTICAL SCIENCES OF ANKARA
TURKISH PHARMACEUTICAL INDUSTRIES ASSOCIATION
TURKISH PHARMACEUTICAL TECHNOLOGY SCIENTISTS ASSOCIATION
TURKISH PHARMACEUTICAL MANUFACTURERS ASSOCIATION
TURKISH ASSOCIATION OF PHARMACEUTICAL AND MEDICINAL CHEMISTRY
Sponsoring Organizations
THE SCIENTIFIC & TECHNOLOGICAL RESEARCH COUNCIL OF TRKYE
Pharmaceutical Companies
ADEKA LA SAN. A.. ABBOTT LABORATUVARLARI THALAT HRACAT ve TC.LTD.T.
ABD BRAHM LA SANAY ve TC. A..
BEM TIBB CHAZ ve LA SANAY LTD. T.
Kervansaray Convention Center & Hotel, LARA, ANTALYA / TRKYE
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International Symposium on Drug Research and Development
BLM LA SANAY ve TC. A..
PFIZER LALARI LTD. T.
FAKO LALARI A..
ROCHE MSTAHZARLARI SAN. A..
GLAXOSMITHKLINE LALARI A..
SANOF-AVENTS LALARI LTD.T.
JOHNSON & JOHNSON SIHH MALZEME SAN. ve TC. LTD. T.
SANOVEL LA SAN. VE TC. A..
SANTA FARMA LA SANAY A.. LLLY LA TCARET LMTED T.
SCHERING PLOUGH TIBB RNLER TC. A.. MUSTAFA NEVZAT LA SANAY A..
SERVIER LA ve ARATIRMA A.. NOVAGENIX BO ANALTK LA AR-GE MERKEZ SAN. TC. A..
NOVARTIS SALIK GIDA ve TARIM RNLER SAN. VE TC. A..
ULKAR KMYA SAN. VE TC. A..
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May 17-20, 2007
Institute
TURKISH PATENT INSTITUTE
Manufacturers/Distributors of Analysis Products and Equipments
ALBO KMYEV MADDELER THALAT ve TC. A..
HACETTEPE TEKNOKENT A..
ANAMED ANALTK ve MEDKAL SSTEMLER A..
IIN TIP ARATIRMA RNLER TC. LTD. T.
ANT TEKNK CHAZLAR PAZ. ve DI TC. LTD.T.
NCEKARA TEKNK CHAZLAR ENDSTRS ve TC. A..
ATOMKA MAKNA TC. LTD. T.
NFO KMYA LABORATUVAR CH. TC. LTD. T.
BETA LABORATUVAR CHAZLARI LTD. T.
NTERLAB LABORATUVAR RNLER SANAY ve TCARET A..
FARGEM, FARMASTK ARATIRMA GELTRME MERKEZ SAN. ve TC.A..
KUTAY LABORATUVAR CHAZLARI TCARET A..
REFERANS KMYA LTD. T. LABOR LDAM LAB. MALZ. TC. LTD. T
SARTONET SEPERASYON TEKNOLOJLER LTD. T.
MEDSANTEK LABORATUVAR MALZEMELER SANAY ve TCARET LTD.T. SESA ELEKTRONK SAN. TC. A..
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Others
BOSTAR
SOYDANLAR KIRTASYE
LEDA PASTAHANES ZEK KIRTASYE
SAYDAN AS TEKSTL
SAYDAN AS TEKSTL
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May 17-20, 2007
Prof. Tunalp ZGEN, MD President of Hacettepe University
Honorary Chairman
Organizing Committees
Chairman Scientific Secretary Treasurer Members : : : : Prof. nsal ALI, Ph. D. Prof. Selma SARA, Ph. D. Assoc. Prof. Kezban ULUBAYRAM, Ph. D Prof. Sedef KIR, Ph. D. Prof. Glberk UAR, Ph. D. S. Kutay DEMRKAN, Pharm. D.
Scientific Committee
Analytical Chemistry Sacide ALTINZ Jos BARBOSA Nursabah BACI Nuran ZALTIN Biochemistry Hamdi nci ZER Nazmi ZER Angelino PARINI Kevser PKN Mercedes UNZETA Biology Nilfer AKSZ Vasf HASIRCI Akn TMER Hacettepe University, Trkiye Middle East Technical University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Hacettepe University,Trkiye Rangueil Institute of Molecular Medicine, France Hacettepe University, Trkiye Barcelona Autonoma University, Spain Hacettepe University, Trkiye University of Barcelona , Spain Hacettepe University, Trkiye Hacettepe University, Trkiye Marianne FILLET Alexander V. KABANOV Ningur NOYANALPAN Sekin ZDEN Wolfgang SIPPL Ljubica SUTURKOVA Fethi AHN Pharmaceutical Technology Sema ALI Nevin ELEB Ruxandra GREF Seda HEKMOLU A. Atilla HINCAL Filiz NER Levent NER A. Yekta ZER Nilfer TARIMCI Pharmaceutical Microbiology Ufuk ABBASOLU Sulhiye YILDIZ Gazi University, Trkiye Ankara University, Trkiye Hacettepe University, Trkiye Gazi University, Trkiye University of Paris-Sud, France Hacettepe University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Ankara University, Trkiye University of Liege, Belgium University of Nebraska, USA Gazi University, Trkiye Ankara University, Trkiye Martin Luther University, Germany Ss. Cyril and Methodius University Skopje, R. Macedonia Gazi University, Trkiye
Chemistry and Chemical Engineering Ayhan S. DEMR Adil DENZL Emir Baki DENKBA Metin BALCI Meneme GMDERELOLU Olgun GVEN Nesrin HASIRCI Yunus KARA Erhan PKN Bekir SALH Muzaffer TALU Middle East Technical University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Middle East Technical University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Middle East Technical University, Trkiye Atatrk University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Gazi University, Trkiye
Pharmacognosy and Pharmaceutical Botany Ahmet BAARAN mr DEMREZER Nurten EZER Erdem YELADA Pharmacology smail Hakk AYHAN E. Rt ONUR nci AHN Serdar UMA Toxicology Sema BURGAZ Filiz HINCAL Gnl AHN Gazi University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Ankara University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Hacettepe University, Trkiye Yeditepe University, Trkiye
Pharmaceutical and Medicinal Chemistry Peter CROOKS Sevim DALKARA eref DEMRAYAK Erin ERCYES Dilek EROL Mevlt ERTAN University of Kentucky, USA Hacettepe University, Trkiye Anadolu University, Trkiye Ege University, Trkiye Yeditepe University, Trkiye Hacettepe University, Trkiye
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May 17-20, 2007
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
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Exhibitor A - Z
A3 ALBO KMYEV MADDELER THALAT VE TC. A.. ............................................ C 11 ANAMED ANALTK VE MEDKAL SSTEMLER A.. ........................................... C 10 ANT TEKNK CHAZLAR PAZ. VE DI TC. LTD.T. ............................................... A 4 ATOMKA TEKNK CIH. TC. LTD. T. .............................................................. A 10 BEM TIBB CHAZ VE LA SANAY LTD. T. ..................................................... B 9 BLM LA SANAY VE TC. A.. ....................................................................... B 6 FABAD .......................................................................................................... A 8 FARGEM, FARMASTK ARATIRMA GELTRME MERKEZ SAN. VE TC.A.. ....... B 5 HACETTEPE TEKNOKENT A. . ......................................................................... A 7 IIN TIP ARATIRMA RNLER TC. LTD. T. .................................................. B 7 NCEKARA TEKNK CHAZLAR ENDSTRS VE TC. A.. .................................... C 12 NFO KMYA LABORATUVAR CH. TC. LTD. T. ............................................... A 12 NTERLAB LABORATUVAR RNLER SANAY VE TCARET A.. ......................... A 18 LABOR LDAM LAB. MALZ. TC. LTD. T. .......................................................... B 8
ABD BRAHM LA SANAY VE TC. A.. ..........................................................
MEDSANTEK LABORATUVAR MALZEMELER SANAY VE TCARET LTD.T. ......... A 19 NOVAGENIX BO ANALTK LA AR-GE MERKEZ SAN. TC. A.. ........................ A 17 NVE SANAY MALZEMELER MALAT VE TC. A.. ............................................. C 9 ONDRT MAYIS DERGS ............................................................................. A 14 PATENT ENSTTS ............................................................................... A 15,16 PFIZER LALARI LTD. T. .............................................................................. A 5 REDOKS KMYASAL BYOLOJK MAD. VE LAB. CH. PAZ. TH. HR. SAN. VE TC.LTD.T ........................................... A 1 REFERANS KMYA LTD. T. ............................................................................. C 7 SESA ELEKTRONK SAN. TC. A.. ................................................................... A 20 TERRALAB LABORATUVAR MALZEMELER SANAY VE TCARET A:. ................... C 8 TUBTAK ....................................................................................................... A 6 TFTAD ........................................................................................................ A 9 TRK KMYA DERNE ................................................................................. A 13 ULKAR KMYA SAN. VE TC. A.. ...................................................................... B 4
May 17-20, 2007
www.drd2007.org
PROGRAMME
MAY 17, 2007 THURSDAY 16.16-16.0 Hall A 16.00-17.30 Registration OPENING CEREMONY Plenary Lecture Smart molecules in targeted therapies Emin Kansu (Hacettepe University, Trkiye) WELCOME COCKTAIL MAY 18, 2007 - FRIDAY Hall A 09.00-10.20 Chairpersons: 09.00-09.40 09.40-10.20 SESSION I Simultaneous Turkish translation will be provided. (New Approaches to Neurodegenerative Diseases) Peter Crooks (University of Kentucky, USA) Emin Kansu (Hacettepe University, Trkiye) Propargylamines as neuroprotective agents in neurodegenerative diseases Mercedes Unzeta (Universitat Autonoma de Barcelona, Spain) Oxidative stress and monoamine oxidases: From basic studies to novel therapeutical interventions Angelo Parini (Rangueil Institute of Molecular Medicine, France) Coffee Break SESSION II Simultaneous Turkish translation will be provided. (Analytical Methods in Medicine) Wolfgang Sippl (Martin Luther University, Germany) Nursabah Ba (Hacettepe University, Trkiye) Characterization of glycoproteins by capillary electrophoresis electrospray mass spectrometry (CE-ES-MS). Applications to diagnosis in biomedicine Jos Barbosa (University of Barcelona, Spain) Biomarker discovery for chronic inflammatory diseases using proteomic serum profiling Marianne Fillet (University of Liege, Belgium) Investigation of noncovalent protein-protein interactions by matrix- assisted llaser desorption/ ionization mass spectrometry Bekir Salih (Hacettepe University, Trkiye) LUNCH
19.19-19.0
10.20-10.50 Hall A 10.50-12.10 Chairpersons: 10.50-11.30 11.3012.10 12.1012.35
12.35-13.30
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Poster Hall 13.30-14.30 Industry Hall Chairperson: 13.30-14.00 14.00-14.30 Satellite Hall Chairperson: 13.30-14.00 14.00-14.30 Hall A 14.30-16.00 Chairpersons: 14.30-15.10 15.10-15.35 15.35-16.00 Hall B 14.30-16.00 Chairpersons: 14.30-15.10 15.10-15.35 15.35-16.00 16.00-16.30 Anamed Hall 16.00-16.30 Chairperson:
Poster Session All presentations will be in Turkish. (Mission and Vision of the Pharmaceutical Companies) Nilfer Tarmc (Ankara University, Trkiye) Bilim la Sanayii ve Ticaret A.. (Trkiye) Farmastik Aratrma Gelitirme Merkezi Sanayii ve Ticaret A..- FARGEM (Trkiye) All presentations will be in Turkish. Sekin zden (Ankara University, Trkiye) New Technology: Near Infrared (NIR) Analysis Systems ncekara Holding/FOSS Analytical A.. (Trkiye) XRD Applications in Pharmaceutical Industry: Determination of Crystal Structure Ant Teknik Ltd. ti./Shimadzu Corp. (Trkiye) SESSION III Simultaneous Turkish translation will be provided (Molecular Modeling) Jos Barbosa (University of Barcelona, Spain) Sevim Dalkara (Hacettepe University, Trkiye) In silico medicinal chemistry Understanding biological effects through molecular docking and molecular dynamics simulations Wolfgang Sippl (Martin Luther University, Germany) Molecular dynamics simulation of thrombin: Target for anticoagulant drugs zge Kl (Hacettepe University, Trkiye) Molecular dynamics simulaton of hirudins: Specific thrombin inhibitor isolated from medicinal leech Fulya alar, (Hacettepe University, Trkiye) SESSION IV (Current Approaches for Development of Novel Drug Delivery Systems) Alexander V. Kabanov (University of Nebraska, USA) Nevin elebi (Gazi University, Trkiye) Surface-modified nanoparticles for drug entrapment Ruxandra Gref (University of Paris-Sud, France) Dendrimers as drug delivery agents to bone Rana Sanyal (Boazii University, Trkiye) Recent advances in brain drug delivery Ylmaz apan (Hacettepe University, Trkiye) Coffee Break Presentations will be in Turkish. Coffee Break Session Ayhan S. Demir (Middle East Technical University, Trkiye) See the Future:Microwave Synthesis from Ls to Ls Nejla Kl (ANAMED & ANALTK Grup, Trkiye) SESSION V Simultaneous Turkish translation will be provided (Design and Synthesis of New Anticancer Drugs) Fethi ahin (Gazi University, Trkiye) Vildan Adar (Hacettepe University, Trkiye) Rational design of novel photosensitizers as potential photodynamic therapy reagents Engin Umut Akkaya (Middle East Technical University, Trkiye) A novel potential antitumor active drug: Platinum blue complex containing sulfur-donor ligand eniz zalp-Yaman (Atlm University, Trkiye) Synthesis of acetophenone and substituted acetophenone derived Mannich bases and their biological activities H. nci Gl (Atatrk University, Trkiye) May 17-20, 2007
Hall A 16.30-18.15 Chairpersons: 16.30-16.55 16.55-17.20 17.20-17.45
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MAY 19, 2007 - SATURDAY Hall A 09.00-10.30 Chairpersons: 09.00-09.30 09.30-10.00 10.00-10.30 SESSION VI Simultaneous translation will be provided (Experiences of Clinicians: New Horizons and Expectations From Research & Development in Antimicrobial Agents) Angelo Parini (Rangueil Institute of Molecular Medicine, France) Ahmet Baaran (Hacettepe University, Trkiye) Challenges in treatment of gram positive bacteria Serhat nal (Hacettepe University, Trkiye) Challenges in treatment of gram negative bacteria Recep ztrk (stanbul University, Trkiye) Challenges in antifungal agents mrm Uzun (Hacettepe University, Trkiye) Coffee Break
10.30-11.00
Info Kimya Hall Presentations will be in Turkish. 10.30-11.00 Coffee Break Session Chairperson: Filiz ner (Hacettepe University, Trkiye) Technological Applications in Pharmaceutical QA/QC and R&D Ouzhan Ay (Info Kimya Laboratuar Cihazlar Tic. Ltd. ti, Trkiye) Hall A 11.00-12.10 Chairperson: 11.00-11.40 11.40-12.10
SESSION VII Simultaneous translation will be provided (Patenting in Health and Pharmaceutics) Levent ner (Hacettepe University, Trkiye) National and international patent protection, free patent search tools (espacenet) and strategies Hakan Bayram (Turkish Patent Institute, Trkiye) Patentability of health and pharmaceutical inventions Serkan zkan (Turkish Patent Institute, Trkiye) LUNCH
12.10-13.30 Poster Hall 13.30-14.30 Industry Hall Chairperson: 13.30-14.00 14.00-14.30 Satellite Hall Chairperson: 13.30-14.00 14.00-14.30
Poster Session All presentations will be in Turkish. (Mission and Vision of the Pharmaceutical Companies) H. nci Gl (Atatrk University, Trkiye) Novartis (Trkiye) Abdi brahim la Sanayii ve Ticaret A.. (Trkiye) All presentations will be in Turkish. Ylmaz apan (Hacettepe University, Trkiye) New Horizons in Drug Industry: Technological Platforms Ahu Ycesoy (The Scientific & Technological Research Council of Trkiye-TBTAK) Bioavailability and Bioequivalency Studies in Trkiye Tuncel zden (Novagenics, Trkiye)
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Hall A 14.30-16.25 Chairpersons: 14.30-15.10 15.10-15.35 15.35-16.00 16.00-16.25 Hall B 14.30-16.00 Chairpersons: 14.30-15.10 15.10-15.50 15.50-16.30 16.30-17.00 Atomika Hall 16.30-17.00 Chairperson:
SESSION VIII Simultaneous Turkish translation will be provided (Pharmacology and Molecular Pharmacology) Mercedes Unzeta (Universitat Autonoma de Barcelona, Spain) nci Erdemli (Hacettepe University, Trkiye) Importance of polymorphism in drug research and development M. Fethi ahin (Gazi University, Trkiye) Studies on histone deacetylase inhibitory activity of some carboxylic acid derivatives and their structure-activity relationships Gamze Bora (Hacettepe University, Trkiye) Free-radical scavenger activities of newly synthesized 2-benzoxazolinone derivatives containing thiosemicarbazide, triazole, thiadiazole and hydrazone Samiye Yabanolu (Hacettepe University, Trkiye) UVB-Induced apoptotic effect of 11 P53 actinic keratosis mutations Aye Ercan (Hacettepe University, Trkiye) SESSION IX (Natural Compounds as Drug Leads) Engin Umut Akkaya (Middle East Technical University, Trkiye) Bekir Salih (Hacettepe University, Trkiye) A vast source for the discovery of novel drug leads: Traditional medicines Erdem Yeilada (Yeditepe University, Trkiye) Parthenolide analogs as antileukemic agents with clinical potential Peter Crooks (University of Kentucky, USA) Synthesis and biological evaluation of novel camptothecin class topo-isomerase inhibitors Ayhan S. Demir (Middle East Technical University, Trkiye) Coffee Break Presentations will be in Turkish. Coffee Break Session Fsun Acartrk (Gazi University, Trkiye) 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 Saraolu (Atomika Teknik Ltd., Trkiye) PANEL Simultaneous English translation will be provided. (Expectations of Pharmaceutical Industry from Drug Research & Development Studies) Panelists: Altan Demirdere (Novartis, Trkiye) (Moderator) Erdal Akaln (Pfizer, Trkiye) Vedat Eilmez (Abdi brahim la Sanayi ve Ticaret A.., Trkiye) GALA DINNER
Hall A 17.00-18.30
20.00-24.00
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May 17-20, 2007
MAY 20, 2007 - SUNDAY Hall A 10.10-10.10 Chairpersons: 09.30-10.20 10.20-11.00 11.00-11.20 SESSION (Nanomedicine and Nanomaterials) Ruxandra Gref (University of Paris-Sud, France) Sema al (Hacettepe University, Trkiye) Nanomaterials in medicine Erhan Pikin (Hacettepe University, Trkiye) Nanomedicine for central nervous system (CNS) drug delivery Alexander V. Kabanov (University of Nebraska, USA) CLOSING REMARKS MAY 18, 2007 - FRIDAY TFTAD SATELLITE SYMPOSIUM organized by Turkish Pharmaceutical Technology Scientists Association (TFTAD) Projects and Support Sources in the Development of Active Pharmaceutical Ingredients and Drug Formulations in University and Industry Cooperation TFTAD Hall All presentations will be in Turkish. Simultaneous English translation will not be provided. la Etkin Maddesi ve Formulasyon Gelitirmede niversite Sanayi birlii Projeleri ve Destek Kaynaklar TBTAK Sanayi AR-GE Projeleri Destekleri Dr. Blent gen (Trkiye Bilimsel ve Teknolojik Aratrma Kurumu -TEYDEB) Eczacba zgn Kimyada Faydalanlan AR-GE Tevikleri Dr. Mustafa Adyaman (Eczacba zgn Kimya) Sanayide AR-GEnin Rol Do.Dr. Tuncer Aslan (Ulkar la Sanayi ve Tic. A..) TBTAK Sanayi AR-GE Destekleri ve Etkilerinin Deerlendirilmesi Uzm. Ecz. Ece Kut (Abdi brahim la Sanayi) TBTAK-TEYDEB Projelerinde Hakemlik ve zleyicilik Deneyimleri Prof. Dr. A.Atilla Hncal (H.. Eczaclk Fakltesi) Prof. Dr. Nilfer Tarmc (A.. Eczaclk Fakltesi)
30.30-30.30
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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
L 01
SMART MOLECULES IN TARGETED THERAPIES

Emin KANSU
Hacettepe University, Institute of Oncology, Ankara, Turkey
n recent years there has been a significant progress in the field of cancer cell biology and molecular oncology. Many researchers outlined very important processes of cancer genesis, growth, invasion and metastasis. As a result of this progress a large number of molecular abnormalities that are specific to cancer cells and that are a critical feature of cancer phenotype 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 targeted therapies, because these structures target specific cellular molecular and/or abnormalities. Tumour cells acquire the ability to proliferate uncontrollably, resist apoptosis, sustain angiogenesis and evade immune surveillance. Different from conventional chemotherapy agents, which mainly kill proliferating cells by interacting with general cellular processes, targeted agents are expected to affect only cells in which the specific molecular alteration is present, induce predominantly antiproliferative effects, and be specific for cancer cells versus normal tissues. In this context, there have been very significant progress in the anti-cancer targeted molecules including tyrosine kinase inhibitors, angiogenesis inhibitors, modulators of cell matrix interactions, agents that interact with the cell cycle and cell death (apoptosis) and protein trafficking regulators. Several of these new therapeutic agents are showing promise in the clinic and many more are being developed. The RAS proteins control signalling pathways that are key regulators of several aspects of normal cell growth and malignant transformation. They are aberrant in most human tumours due to activating mutations in the RAS genes themselves or to alterations 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 p53MDM2 interaction is a promising approach for activating p53, because this association is well characterized at the structural and biological levels. MDM2 inhibits p53 transcriptional activity, favours its nuclear export and stimulates its degradation, so inhibiting the p53MDM2 interaction with synthetic molecules should lead to p53-mediated cell-cycle arrest or apoptosis in p53-positive stressed cells. Angiogenesis inhibitors are a new class of drugs, for which the general rules involving conventional chemotherapy might not apply. The successful translation of angiogenesis inhibitors to clinical application depends partly on the transfer of expertise from scientists who are familiar with the biology of angiogenesis to clinicians. STAT proteins especially STAT3 and STAT5 regulate all of these processes and are persistently activated in a surprisingly large number of human cancers. Consequently, STAT proteins are emerging unexpectedly 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, including cancer. Six antibodies including Trastuzumab (Herceptin), Rituximab (Mabthera), Alemtuzumab (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 radioimmunoconjugates, Bexxar (tositumomab; 131iodine) and Zevalin (ibritumomab tituxetan; 90yttrium), and Mylotarg (anti-CD33-calicheamicin conjugate) are approved for cancer therapy and currently in clinical practice.
LECTURES
LECTURES
L 02
PROPARGYLAMINES AS NEUROPROTECTIVE AGENTS IN NEURODEGENERATIVE DISEASES

Elisenda SANZ, Jos Luis MARCO, Mercedes UNZETA
Departament Bioquimica y Biologia Molecular, Facultad de Medicina-Instituto de Neurociencias, Universitat Autonoma de Barcelona, 8193 Barcelona, Spain
arkinsons disease is a neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons in the nigrostriatal system. Different factors have been suggested to induce Parkinsonism: environmental toxins, alteration of the intracellular calcium homeostasis, mitochondrial dysfunction, genetic factors, oxidative stress etc., [1]. All them are able by themselves to induce apoptotic cell death [2]. Oxidative stress, arising from an imbalance between the production of reactive oxygen-containing freeradical 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, furthermore, it is metabolised by monoamine oxidase in a reaction that forms H2O2. Furthermore, elevated levels of MAO-B activity have been reported in the SN of patients with Parkinsons disease [3]. Thus, the increase of the oxidation of dopamine by MAOB, 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 dopamine, 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 Parkinsons patients [6, 7]. Apoptosis (Programmed cell death), is a highly regulated physiological process that occurs in all vertebrates and controls the cellular turnover from fetal development to aging [8]. Apoptosis is morphologically characterized by chromatin condensation, 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 cleavage and activation of different cellular substrates.The apoptotic death is under genetic control and is characterized by the expression of some genes that enhance the apoptosis (bax, bad ) and others that in4
hibits it (bcl-2, bcl-xL, bcL-w, mcl-1). Defects in the physiological apoptotic pathway, leading to inappropriate cell death underlies in some neurodegenerative diseases. The programmed cell death may contribute to human neurodegeneration. It has been reported that apoptosis is involved in the death of the dopaminergic neurons in Parkinsons disease [6, 7, 9]. In humans, MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) produces a behavioural syndrome similar to the neuropathological features of idiopathic Parkinsons disease [10] and involves a selective destruction of nigrostriatal dopaminergic neurons. Although MPTP itself displays low chemical reactivity, it is biotransformed by glial MAO-B, to form MPP+ (1-methyl-4-phenylpyridinium) as the effective toxin [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 human neurodegeneration probably due to the free radicals formation as a consequence of the mitochondrial dysfunction [13]. The mitochondrial process implicated in the toxic and neurodegenerative conditions is related with the opening of the mitochondrial pore in the inner membrane, that leads to the membrane depolarization, release of small proteins such as Cytochrome c, apoptosome complex formation, activation of the executer caspase 3, poly (ADP-ribose) polymerase (PARP) activation, DNA damage and cell death. Recent studies have demonstrated that ER stress induced by protein missfolding as a consequence of the inhibition of protein glycosylation, trafficking or alteration of the Ca+ homeostasis in the endoplasmic reticulum (ER), may contribute to the pathogenesis and neurodegeneration in Parkinson and Alzheimer diseases. It has been reported that ER stress can induce apoptotic cell death in mice trough another molecular pathway different to mitochondrial one that involves the activation of an ER-specific Caspase 12 [14]. Furthermore, pharmacological agents able to inMay 17-20, 2007
hibit the perturbation of the mitochondria and ER function may represent a potential therapeutically approach for the prevention of neurotoxin-induced Parkinsons disease. In this context, the design and synthesis of new antiapoptotic molecules, able to interfere with different apoptotic molecular pathways, inhibiting the opening of the mitochondrial pore, preserving the membrane 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 potential 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 selegyline (l-deprenyl), widely used as coadjuvant of lDOPA in PD therapy [20]. Moreover, PF 9601N did not show the amphetaminergic side-effects of l-deprenyl [21] and was able to inhibit the dopamine uptake in human and rat striatum. PF 9601 N showed a neuroprotective effect in vivo, using several dopaminergic toxins in different experimental models such as MPTP, [22] and 6-hydroxydopamine-striatal lesion [23]. Furthermore, PF 9601N also enhanced the duration of l-DOPA-induced contralateral turning in 6hydroxydopamine lesioned rats [24]. A structurally different non-amphetamine compound, Rasagiline, has also been reported to be neuroprotective [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 transport inhibition is an important factor in the neuroprotective 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 antiapoptotic effect of PF 9601N using human neuroblastoma SHSY5Y cells lesioned with MPP+ as a toxin that induces the apoptotic mitochondrial pathway, and lesioned as well with Brefeldin A, Tunicamycin and Thapsigargin, as toxins inducers of the ER stress apoptotic pathway. The neuroprotective properties of PF9601N in these different apoptotic cell death models, were assessed using several viability assays (Alamar Blue reduction and Calcein-AM staining). PF9601N pretreat-
ment significantly reduced MPP+ induced cell death and diminished the activation of one of the main executioner caspases, Caspase-3 and also inhibited the PARP activation. PF9601N showed the same neuroprotective 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 transcription factor and transactivation of p53 response elements. Additionally, p53 inhibitor pifithrin-alpha partially prevented MPP+-induced apoptosis, suggesting that activation of p53 contributes to cell death. PF9601N pre-treatment was able to partially avoid MPP+ induced cell death through preventing an increasing p53 expression and thus reducing transcriptional 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 pharmacological 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 dependent 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 Parkinsons disease, Ann Rev Neurosci., 22, 123-144, 1999. 2. Simonian NA, Coyle JT. Oxidative stress in neurodegenerative 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), 22938, 1990. 5. Kragten E, Lalande I, Zimmermann K, Roggo S, Schindler P, Mller D, Oostrum J van, Waldemeir P, Frst P. Glyceraldehide-3-phosphate deshydrogenase, the putative target of the antiapoptotic compounds CGP3466 and l-deprenyl, J Biol Chem., 273, 5821-5828, 1998. 6. Hartmann A, Hirsch EC. Parkinsons disease. The apoptosis hiptesis revisited, Adv Neurol., 86, 143-153, 2001. 7. Mochizuki H, Goto K, Mori H, Mizuno Y. Histochemical detection of apoptosis in Parkinsons 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 method to detect and confirm apoptotic nuclei in Parkinsons 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 Parkinsons Research (Hefti F, eds), Plenum Press New York pp 145-152. 13. Beal MF. Mitochondria, free radicals and neurodegeneration, 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-reticulum-specific apoptosis and cytotoxicity by amyloid-beta, Nature, 403, 98-103, 2000. 15. Djaldetti R, Melamed E. New drugs in the future treatment of Parkinsons disease, J Neurol., 249 (Suppl), II30II35, 2002. 16. Wellington CL, Hayden MR. Caspases and neurodegeneration: on the cutting edge of new therapeutic approaches, Clin Genet., 57, 1-10, 2000. 17. Tajrena A, Cesari V, Borlongan RLM, Faull CEW, Ros GC, Gluckman PD, Hughes PE. Neuroprotective strategies for basal ganglia degeneration: Parkinsons and Huntingtons diseases, Prog Neurobiol., 60:5, 409-470, 2000. 18. Cruces MA, Elorriaga C, Fernandez-Alvarez E. Acetylenic 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. Indolalkylamines derivatives as antioxidant and neuroprotective agents,in a experimental model of Parkinsons disease, Med Sci Monit., 10, BR477-484, 2004. 20. Perez V, Marco JL, Fernandez-Alvarez E, Unzeta M. Relevance of benzyloxy group in 2-indolyl methylamines in the selective MAO-B inhibition, Br J Pharmacol., 127, 869-76, 1999. 21. Lees AJ. Parkinsons disease research group of the United Kingdom. Comparison of therapeutic effects and mortality data of levpdopa and levodopa combined with ldeprenyl in patienst with early, mild Parkinsons disease, Br Med J., 311, 1602-1607, 1995. 22. Perez V, Unzeta M. PF 9601N [N-(2-propynyl)-2-(5-benzyloxy-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-(2propynyl)-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-hydroxydopamine 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-aminoindan (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 transporter (DAT) expression and amphetamine-induced dopamine release in vivo, Br J Pharmacol., 126, 997-1002, 1999.
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L 03
OXIDATIVE STRESS AND MONOAMINE OXIDASES: FROM BASIC STUDIES TO NOVEL THERAPEUTICAL INTERVENTIONS
Angelo PARINI
INSERM U858, Institut de Mdecine Molculaire de Rangueil I2MR, Toulouse, France
iogenic amines, including catecholamine and serotonin, regulate a variety of cell functions through the interaction of G-coupled membrane receptors. During the last years, we described a novel mechanism of action of dopamine and serotonin that occurs independently of membrane receptor stimulation and requires hydrogen peroxide generation by monoamine oxidases (MAO). Using different models of renal and cardiac cells we showed that, in addition to the classical receptor-dependent effects, dopamine and serotonin induces cell proliferation and hypertrophy by a mechanism independent of receptor activation. At higher concentrations (up to 10 M), 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 fragmentation. Both proliferative and apoptotic effects of dopamine and serotonin were not inhibited by specific 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 receptor-independent mechanism requiring amine internalisation into the cells, their degradation by MAOs and hydrogen peroxide production. Based on these findings, we next investigated the potential role of hydrogen peroxide generated by MAOs on cell death in vivo. Our results showed that MAO inhibition largely reduced renal and myocardial damage induced by ischemia/reperfusion in rat. The protective 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 biogenic amines and propose these enzymes as a pharmacological target for prevention of organ damages.
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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. GIMNEZ
Departament de Qumica Analtica, Facultat de Qumica, C/Mart i Franqus 1, 08028 Barcelona, Spain
enomics and, even more important, the wide field of proteomic and related clinical applications like biomarkers, dramatically increase the demand of sensitive and selective analytical tools for the analysis of biological samples. The sugar content 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 incubation culture conditions [1]. The polymorphism associated with the amount, the size, and the structure of the carbohydrate chains is known as microheterogeneity, 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 glycosylation patterns observed in sera from healthy individuals and patiens. Glycosylation is the most common posttranslational modification in proteins carbohydrates participate in many biological processes and encode information 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 processes [3]. For example, patients with Congenital Disorders of Glycosylation (CDG) or chronic alcoholism 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 erythropoiesis 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 account of their extensive misuse as performance enhancing agents in endurance sports [6]. Until recently, isoelectric focuosing electrophoresis was the reference method for glycoforms analysis due to its high selectivity allowing an easy detection of genetic glycoprotein variants. However, this
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complex and time consuming procedure favoured the introduction of new alternative methods. Capillary electrophoresis (CE), has become one of the most important techniques for glycoform separation, combining high resolution power under nondenaturing conditions [7]. In previous studies Tf sialoforms were resolved by CE-UV (4). Also the two commercial ready-to-use pharmaceuticals of recombinant human EPO (rHuEPO), epoetin- and epoetin-, and the hyperglicosilated EPO derivated NESP were analysed [8, 9]. Separation and characterization of the different glycoforms from each glycoprotein are presented. However endogenous EPO are generally found at subnanomolar levels such as other endogenous hormones and the poor concentration detection sensitivity of CE preclude the direct analysis of these hormones at the levels found in biological fluids. The high values of the concentration limits of detection in CE are closely related with the small volume capacity of the capillary columns. Several instrumental, electrophoretic and chromatographic modifications have been described in order to overcome this limitation. In solid phase extraction coupled on-line to capillary electrophoresis (SPE-CE), a microcartridge or analyte concentrator is inserted near the inlet of the separation capillary [10]. The analyte concentrator contains a solid phase extraction sorbent which retains the target analyte, enabling large volumes of sample to be intruduced. The captured analyte is eluted in a small volume of an appropiate solution, resulting in sample clean-up and concentration enhancement, with minimum sample handling. Several researchers have perceived the suitability of SPE-CE to perform selective and sensitive analysis of proteins and peptides in complex diluted samples. In addition, the use of solid-phase extraction coupled on-line to capillary electrophoresis electrospray mass spectrometry (SPE-CE-ESI-MS) has demonstrated improved capabilities for characterization 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. HowevMay 17-20, 2007
er, direct analysis of intact glycoproteins by MALDITOF and conventional electrospray ionization mass spectrometry (ESI-MS) has presented some problems in order to resolve microheterogenous structures. Thus, a previous glycoform separation is mandatory to obtain valuable information about the carbohydrate heterogeneity of glycoproteins. CE-ESI-MS has been successfully used for this purpose [11-13]. However, volatile background electrolytes (BGE) are necessary to provide suitable electrospray ionization and therefore, obtain good sensitivity. The non-volatile buffer used in the current CE-UV methods for glycoform 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 developed. The CE-ESI-MS separation method for intact rHuEPO has been improved as a consequence of the use of a novel acrylamide-based coating that provides a stable suppression of EOF and allows a succesful glycoform separation [7, 17]. Also a method for the separations of Tf sialoforms has been developed, that permits the diagnostic of Congenital Disorders Glycosylation and chronic 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 studied for the analysis of peptide hormones in dilute solution [10]. This resulted in sample clean-up and concentration enhancement, with minimum sample handling. The CE-ESI-MS developed methods are applied for the characterization of rHuEPO glycoforms. The achieved separation and the highly mass-resolving time of fligh (TOF) mass detection allows to establish the most probable rHuEPO glycoforms.
EXPERIMENTAL PART Chemicals All chemicals used in the preparation of buffers and solutions are analytical reagent grade. Standard human transferrin (partially saturated, min 98%), insulin, sodium dextran sulphate (M=500000), and hexadimethrin 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). Standard rHuEPO was obtained as BRP from Pharmacopoeia (EDQM, European Pharmacopoeia, Council of Europe, Strasbourg, France). Epoetin- (Eprex) 6000
IU from Janssen-Cilag (Neuss, Germany) and epoetin- (NeoRecormon) 4000 IU from Roche (Mannheim, 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 ultrasonication before use.
Instrumental CE-ESI
CE was performed on a Hewlett Packard CE (Agilent Technologies, Waldbronn, Germany). For CEMS coupling, a coaxial sheath-liquid sprayer was used (Agilent Tecnologies). For intact EPO glycoprotein analysis, separation was performed in capillaries coated with polybren (PB) or ultra Tof Dynamic 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 Biosystems, Framingham, MA, USA) coupled to CE systems whereas the hormones spectrometric investigations were performed with the CE system compled to a MSD Ion Trap mass spectrometer (Agilent Technologies) and characterization of glycoprotein were performed using a CE-microTOF (Bruker Daltonik), an orthogonal accelerated TOF mass spectrometer (oaTOF-MS).
RESULTS AND DISCUSSION Transferring glycoform analysis by CE-UV and CE-ESI-MS Application to CDG chronic alcoholism diagnosis A CE-UV separation method has been developed using 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 Ionic 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 interactions between Tf and capillary walls improving separation 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 applied in clinical diagnosis.
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Using CE-ESI-MS methodologies the best sensitivity 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 order to achieve good sensitivity and separation .Separation 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 serum (Figure 2).
Analysis of hormones by on line SPE-CE-ESI-MS
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.
Endogenous hormones are generally found at subnanomolar levels, e.g. between 100 and 1 ng L depending on the hormone, in biological samples. In our studies, solid-phase extraction coupled on-line to capillary electrophoresis electrospray mass spectrometry (SPE-CE-ESI-MS) is explored for the preconcentration and separation of dilute solutions of peptide hormones. First, a CE-ESI-MS methodology was developed 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 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 capillary. (Figure 3). After optimizing the on-line preconcentration methodology, LOD between 10 and 0.1 ng mL-1 were achieved. The preconcentration methodologies have been applied to rHuEPO analysis using an in-line inmunoaffinity solid phase extraction(IACE-ESI-MS).The preliminary results obtained using a custom-made inmunoaffinity sorbent prepared from an anti-human EPO polyclonal antibody and glutaraldehyde-glass beads show the potential of this novel approach.
Characterization of rHuEPO glycoforms by CE-ESI-MS
Several recombinant human erythropoietins (rHuEPO) from different origen have been analysed. Coated capillaries are mandatory in order to decrease or suppress the adsorption of glycoproteins to the silica capillary wall. In our works two different capillary 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 sensitivity has been obtained in sheath liquids containing 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, numerous and complex data are obtained and therefore, extracting useful and valuable information
Figure 3. Scheme of the manufacturing procedure for the C18 analyte concentrator (C18AC).
from spectra is not as obvious as in more straightforward 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 necessary. In our work a CE-ESI-MS separation method 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 g/L 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).
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Figure 5. Typical tetra-antennary N-glycan of rHuEPO.
ure 5. Also the sialic acid content of O-glycosylation site has been characterized based on the glycopeptides 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 percentages of the N-glycans and the O-glycans depending 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 molecular mass of 29888.2 Da, consist of the protein backbone (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 molecular masses and the respective carbohydrate composition.
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, NeusB C, Sanz-Nebot V, Barbosa J. Electhrophoresis, (2006) in press. 4. Sanz-Nebot V, Gonzlez P, . Toro I, Ribes A, Barbosa J. J. Chromatogr.B, 798, 1-7, 2003.
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 ().
Table 1. Main intact rHuEPO glycoforms and the respective carbohydrate composition obtained in a LN coated capillary.
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5. Fisher JW. Exp. Biol. Med., 228, 1-14, 2003. 6. Sanz-Nebot V, Benavente F, Vallverd A, Guzmn NA, Barbosa J. Anal. Chem., 756, 5220-5229, 2003. 7. Balaguer E, Demelbauer U, Pelzing M, Sanz-Nebot V, Barbosa J, NeusB C. Electrophoresis, 27, 2638-2350, 2006. 8. Sanz-Nebot V, Benavente F, Gimnez E, Barbosa J. Electrophoresis 26, 1451-1456, 2005. 9. Gimnez E, Benavente F, Barbosa J, Sanz-Nebot V.(2006) in press. 10. Benavente F, Vescina MC, Hernndez 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. Neusss 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. Hernndez-Borges J, NeusB C, Cifuentes A, Pelzing M. Electrophoresis, 25, 2257-2281, 2004. 17. Benavente F, Gimenz E, Olivieri AC, Barbosa J, SansNebot V. Electrophoresis, 27, 4008-4015, 2006.
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L 05
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 Lige, and CHU, 4000 Lige, Belgium
he development of new technologies in the last decade in the field of genomic and proteomic analysis has brought novel optimism to the discovery of new biomarkers. One of the novel strategies employed for the discovery of new biomarkers is the analysis of the peptides and proteins in plasma or serum samples by mass spectrometry. Protein differential display techniques such as two-dimensional gel electrophoresis (2-DE), one- or two dimensional liquid chromatographic approaches (LC-MS) or surface-enhanced laser desorption and ionization time of flight (SELDI-TOF) mass spectrometry have become increasingly useful to establish fingerprint profiles 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 expression level of proteins. Proteins common to both groups are ignored when up- and downregulated proteins become of the main interest and potential biomarkers. SELDIs ProteinChip Arrays distinguish this technology from other mass spectrometry-based analytical systems. ProteinChip Arrays provide a variety of surface chemistries that allow researchers to optimize protein capture and analysis. The surface chemistries of the arrays include a series of classic chromatographic chemistries and specialized affinity capture surfaces. Classic chromatographic surfaces include normal phase for generic protein binding; hydrophobic surfaces for reversed-phase capture; cation- and anion-exchange surfaces; and immobilized metal affinity capture (IMAC) for metal-binding proteins. Specific proteins of interest can be covalently immobilized on pre-activated surface arrays, enabling customized 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 easily accessible and reflects secondary systemic chang14
es, as it perfuses all the tissues of the body, sothat it carries not only specific blood proteins but also proteins or fragments shed by diseased tissue. In order to analyse circulating proteins and peptides, the cellular components of blood are removed, either in the presence 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-seven percent of the proteins found in plasma belong to one of the seven major groups of high-abundant plasma proteins: albumin, immunoglobulin, fibrinogen, alpha 1-antitrypsin, alpha 2-macroglobulin, transferring and lipoproteins. The remaining 3% is a complex mixture of middle and low abundance proteins, including proteins from the family of complements, hormones, other proteins originated from normal tissue 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 orders of magnitude) is insufficient to cover the range present in blood sample. To overcome this disadvantage, two non-exclusive strategies have been developed. 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 sample. The use of antibodies for the albumin, immunoglobulins and other high-abundance proteins is at present the most efficient and specific method. The major drawback is the high cost. These resins are robust, highly specific and yield to reproducible results. However, it is known that albumin and other highabundance proteins bind some less abundant peptides in the blood and act as their carriers. Moreover, the depletion shows a certain degree of cross-reactivity with non-targeted proteins. In order to prevent the finding of artefactual biomarkers, it is important to eliminate as many of variables. The all procedure has to be standardized from the medical 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 influences 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 inflammatory bowel diseases (IBD) outcome. The aim is to accurately distinguish patients with Rheumatoid Arthritis or Crohn disease from patients with other inflammatory diseases and from healthy controls. Chronic inflammatory diseases like Rheumatoid Arthritis (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 endoscopic tissue histology for CD. Biomarkers for early diagnosis or prognosis do not exist and clinicians often manage the patients empirically and secondarily adapt the therapeutic strategy according to the clinical evolution. The project requires the establishment of a biobank including serum, tissue samples and epidemiological data, from large cohort of patients afflicted with RA, CD and related diseases (Psoriatic Arthritis PsA- and Ulcerative rectocolitis -UC-). The number of samples and types of samples is one of the most important parameters that determine the success of a project. Usually, we profile at least 30 samples in each classification group (e.g., disease versus healthy or treated versus untreated). This number of samples is usually enough to give us > 90% statistical confidence in single marker with p values < .01 and is also enough to use different forms of multivariate analysis. Control sera were selected on the basis of matching for age, sex and Caucasian race. All samples were aliquoted and frozen at -80C until thawed specifically 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 example, the sera freeshly collected have been aliquoted, stored at 80C and were only ones unfreezed. Our quality control serum allowed us to detect any unusual features during the process. All those precautions 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 SELDI experiment. Several chip arrays such as strong anion-exchange (SAX), weak cation-exchange (CM10) and hydrophobic (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 washing buffers were optimized using ion-exchange arrays 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, normalization or peak detection. Baseline subtraction was achieved by employing a varying-width segmented convex hull algorithm that eliminates any baseline signal caused mostly by matrix distortions. Normalization 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 current normalization function by following the software instruction. Peak detection was performed using the ProteinChip Biomarker software version 3.0 (Ciphergen Biosystems, Inc.). The part of the spectrum m/z values < 1000 was not used for analysis, as the energy absorbing matrix signal generally interfered with peak detection in this area. Peaks between 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 sources of fluctuation and coefficient of variations (CVs) were calculated to evaluate the reproducibility of experiments using SELDI-TOF-MS approach. Moreover, we chose to include patients with a variety of very close pathologies to RA (osteoarthritis, PsA) in our control groups to mimic real life diagnostic difficulties. We also wanted to include heterogeneous control groups, because we wanted to differentiate markers that are inflammatory in nature from RA specific molecules. Data were analyzed with an original multivariate statistical method based on multiple decision trees algorithms. One of the challenges in the analysis of
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SELDI mass spectrometry-generated data is avoiding the false discovery of proteins peaks, of which the discrimatory power is due to random variation. The statistical approach we used to analyse the SELDI profiles reduces this problem by ranking all of the detected protein peaks according to their relative contribution 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 exhibited a reproducibly high ranking in both sets of analysis were used. For most comparisons, bioinformatics analysis yielded a panel of 10 or more distinguished between patients groups. Once the optimum discriminatory decision process is created, blinded samples can then be subjected to analysis to confirm the utility of the algorithm. In a first approach, we compared RA spectra versus control spectra (inflammatory controls and non-inflammatory controls). According to the boosting statistical 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 clinical 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 performed. 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 Laser Desorption Ionization-Time Of Flight-Mass Spectrometry 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 understanding of chronic inflammatory diseases.
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May 17-20, 2007
L 06
IN SILICO MEDICINAL CHEMISTRY UNDERSTANDING BIOLOGICAL EFFECTS THROUGH MOLECULAR DOCKING AND MOLECULAR DYNAMICS SIMULATIONS
Wolfgang SIPPL
Institute of Pharmaceutical Chemistry, Martin-Luther-University of Halle-Wittenberg, 06120 Halle/Saale, Germany
rotein homology models have been used in conjunction with structure-based approaches to successfully identify novel inhibitors over the last few years. It is widely accepted that for example 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 accurate docking programs, high quality protein models are needed. The present talk will highlight the results obtained for two protein targets where we used a combination of comparative protein modelling and structure-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 inhibitors as well as CAR agonists the validated homology 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 biological effects.
References
1. Windshgel B, Jyrkkrinne 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. Jyrkkrinne J, Windshgel B Mkinen J, Ylisirni M, Perkyl M, Poso A, Sippl W, Honkakoski P. Amino acids important for ligand specificity of the human constitutive androstane receptor, J. Biol. Chem., 280, 5960-5971, 2005. 3. Spannhoff A, Heinke R, Bauer I, Trojer P, Metzger E, Gust R, Schle R, Brosch G, Sippl W, Jung M. Targetbased approach to inhibitors of histone arginine methyltransferases, 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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L 07
SURFACE-MODIFIED NANOPARTICLES FOR DRUG ENTRAPMENT AND TARGETING

Ruxandra GREF
UMR CNRS 8612, Paris-Sud University, Chtenay-Malabry, France
ver the last decades, there has been a growing interest in the development of a drug carrier that is small enough for intravenous administration 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 controlled drug release into the vascular compartment, or targeted delivery. There are numerous potential applications for such a system, for example, the protection of highly sensitive 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 successful administration of protein drugs is also often problematic. Hydrolyzed or denaturated in the gastrointestinal tract, most proteins cannot be administered orally, and usually, they are quickly metabolized if intravenously injected. Their typical half-lives,ranging from 2 to 30 min, could be improved by encapsulating 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 carriers 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 macromolecular materials in which the active principle is dissolved, entrapped, and encapsulated and/or to which the drug is adsorbed or attached. The strategy of choice in the design of submicronic long-circulating drug carriers was to sterically exclude the cellular immune system from recognizing and responding to the particles artificial surfaces. To achieve this, polymers were covalently bound or adsorbed on surfaces. Among these, poly (ethylene gly18
col) (PEG) were the most commonly used polymers for surface modification. Core-shell (PEG) nanoparticles revealed to be of outmost interest; the biodegradable core is able to entrap and release a variety of active molecules, whereas the hydrophilic PEG surface governs the in vivo interactions and determines the fate after intravenous administration. Examples of applications will be given, including the entrapment of proteins and lipophilic drugs. The accent will be put on the challenge of busulfan entrapment. This small fragile molecule with a strong tendency 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 haematological malignancies and non-malignant disorders such as immunodeficiency. For a long time, busulfan has been available only in oral form and a wide intra-patient and inter-patient bioavailability variability 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 concentration-time curve. In order to overcome these problems, intravenous formulations of busulfan were developed, using cosolvent mixtures [2]. However, these organic solvents have their own well-documented toxicity. Therefore, to avoid the massive use of organic solvents, injectable 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 nanoparticles able to combine the ability of the biodegradable polymeric cores to efficiently encapsulate busulfan, with the excellent steric repulsive properties of the diblock copolymer, poly (-caprolactone)-poly (ethylMay 17-20, 2007
Figure 1. Typical transmission electron microscopy studies of PCL-PEG (left) and composite PIBCA/ PCL-PEG (right) nanoparticles.
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 polymer able to encapsulate the highest amounts of busulfan. For this, busulfan was entrapment by nanoprecipitation 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 cyanoacrylate) (PECA). Molecular modelling along with energy minimization process was employed to identify the nature of the interactions occurring between busulfan and PIBCA. Further, optimization studies enabled to obtain PIBCA nanoparticles displaying 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 performed 1H-NMR spectroscopy experiments showing that chemical integrity of the drug was preserved after loading into nanoparticles. The in vitro release studies 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 nanoparticles 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 microscopy studies revealed a particular core-shell structure typical of the composite nanoparticles (Fig. 1). The chemical composition of the top layers was determined by X-ray photo-electron spectroscopy (XPS). 3H-labelled busulfan was used in order to determine the drug loading efficiency and the invitro drug release by liquid scintillation counting. Physico-chemical techniques such as Zeta potential determination and XPS analysis provided evidence about a pref-
erential surface distribution of the PCL-PEG polymer. Therefore, composite nanoparticles have a coreshell-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 loading increase, in comparison to the single PCL-PEG nanoparticles. In addition, the complement activation 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 nanoparticles could serve as long-circulating busulfan carriers.
References
1. Hassan M, Oberg G, Bekassy AN, Aschan J, Ehrsson H, Ljungman P, Lonnerholm G, Smedmyr B, Taube A, Wallin I, et al.. Pharmacokinetics of high-dose busulphan in relation to age and chronopharmacology, Cancer Chemother. Pharmacol., 28(2), 130-134, 1991 2. Bhagwatwar HP, Phadungpojna S, Chow DSL, Andersson BS. Formulation and stability of Busulfan for intravenous 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 crystalline 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 coreshell nanoparticles as busulfan carriers, J. Contr. Release, 111(3), 271-80, 2006.
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L 08
CHALLENGES IN THE TREATMENT OF GRAM-POSITIVE INFECTIONS

Serhat NAL
Hacettepe University, Faculty of Medicine, Department of Medicine, Section of Infectious Diseases, Ankara, Turkey
he introduction of benzylpenicillin in the 1940s was the start of an era in the treatment of bacterial infections but is soon became apparent that some strains of Staphylococcus aureus were resistant due to production of -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 Grampositive bacteria which might have resulted from the emphasis placed on controlling Gram-negative infections. Today we have to deal with infections caused by multidrug-resistant organisms, particularly methicillin resistant staphylococci, penicilin- and erythromycin-resistant pneumococci and vancomycin-resistant enterococci. The emergence and rapid spread of methicillin- resistant S.aureus (MRSA), which were resistant not only to all -lactams but also to the main antibiotic classes, resulted a an increase use of glycopeptide antibiotics, namely vancomycin and teichoplanin. 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 antimicrobial agents, the oxazolidinones. It was approved by the U.S. Food and Drug Administration (FDA) in 2000 for the treatment of uncomplicated and complicated skin and soft tissue infections, including diabetic foot infections without concomitant osteomyelitis, community acquired and nosocomial pneumonia and vancomycin-resistant Enterococcus faecium infections including cases with concurrent bacteremia. Although clinical trials have shown linezolid to be as effective as best established therapy, resistance has been reported. In addition, high rates of adverse effects, including thrombocytopenia and anemia, are seen with prolonged courses of therapy. Serotonin syndrome, potentially irrevesible peripheral neuropathy and lactic acidosis are also reported. Daptomycin (Cubicin) is the first agent of a new class of cyclic lipopeptides The drug exhibits a calcium
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dependent antimicrobial effect against Gram-positive microorganisms, including MRSA. Its oncedaily dosing and and the favourable safety profile (except some concerns about rhabdomyolysis and neuropathy) are making daptomycin an attractive option for the treatment of Gram-positive infections. Succesful results had been reported for the treatment of bacteremia and right-sided infective endocarditis but not for the treatment of community acquired pneumonia. Tigecycline (Tygacil) is a new, semisynthetic glycylcycline that was approved by US FDA for the treatment of skin, soft tissue and intra-abdominal infections It appears to be very active not only against MRSA but also against glycopeptide-resistant staphylococci and enterococci. Nausea and vomitting are the most common side-effects. Quinupristin-dalfopristin (Synercid) is fixed micture of semisynthetic streptogramin derivatives. It is bacteriostatic against Enterococcus faecium and bactericidal against methicillin-susceptible and methicillin-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 patients 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 staphylococci, including penicilin-resistant strains of pneumococci and MRSA, while maintaining the activity of extended-spectrum cephalosporins against Gramnegative organisms. In vivo, screening models predict good activity for ceftobiprole against Gram-positive and Gram-negative bacteria. Changing patterns of resistance have compounded and exacerbated the need for new antimicrobial agents. The appropriate indications and cost-effecMay 17-20, 2007
ctiveness of these molecules will determine our future treatment options. Until then the prudent use of existing antibiotics with strict reinforcement of infection 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 Microbiol Infec., 12 (Supp 2), 3-10, 2006.
2. Pace JL, Yang G. Glycopeptides: update on an old successful 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 vancomycin-resistant enterococci, Curr Opin nfec 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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L 09
CHALLENGES IN TREATMENT OF GRAM-NEGATIVE BACTERIA

Recep ZTRK
stanbul University, Cerrahpaa Medical Faculty, Infectious Diseases and Clinical Microbiology, stanbul, Turkey
he issue of resistance among nosocomial or even community acquired gram-negative micoorganisms including primarily extended-spectrum beta-lactamase producing Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae), multidrug-resistan (MDR) Pseudomonas, and MDR Acinetobacter produce a challenge for their tratement. Recently carbapenem-resistant, ESBL producing enteric bacilli and panresistant Pseudomonas and Acinetobacter 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. Discovery 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-infectives is apparent. However especially develeoping drugs for the agents especially for the multidrugor 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 microorganisms. Recently available drugs against gram-negatives with ongoing studies are as follows:
Tigecycline It is the first member of the glycylcycline class antibiotics, which are the synthetic analogues of classical tetracyclines. 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 tetracycline resistance. It exhibits activity against grampositive bacteria including MRSA and VRE as well as gram-negative bacteria such as ESBL producing enteric rods and multidrug-resistant Acinetobacter spp. -lactamase negative and positive strains of Haemophilus influenzae and Moraxella catarrhalis are within its spectrum. Moreover, it is active against the anaerobic bacteria and MOTT (Mycobacterium other than tuberculosis) rods. It has limited activity against Pseudomonas aeruginosa and Proteus spp. It is indicated in 22
complicated skin, soft tissue and intra-abdominal infections. Its activity in the treatment of communityand hospital-acquired pneumonias is under investigation. It is approved by the Ministry of Health in our country. Nausea, vomiting and diarrhea are among the side effects [2, 5].
Ceftobiprole It exhibits activity against resistant gram-positive bacteria such as MRSA and penicillin-resistant Streptococcus pneumoniae as well as gram-negative bacteria such as E. coli, K. Pneumoniae and Enterobacter cloacae. It has oral and parenteral formulations. Complicated skin and soft tissue infections, and community- and hospital-acquired pneumonias are among the probable indications for its usage [2, 6]. Doripenem It is a carbapenem class antibiotic with activity against gram-positive, gram-negative and anaerobic bacteria. It exhibits activity against ESBL-producing enteric rods similar to imipenem and meropenem. It has partial activity against carbapenem-resistant P. aeruginosa. 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]. Faropenem It is a carbapenem class antibiotic with oral formulation available. It has wide-spectrum of activity similar to other carbapenems [2]. Garenoxacin It is a new quinolone antibiotic undergoing phase III clinical trials. It exhibits activity against gram-positive, 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 inhibitors There is ongoing research on these new class antibiotics with wide spectrum (A-72310 and A-692345). This class exhibits activity against gram-positive, gram-negative 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 structure. As they have a different mechanism of action, they are not affected by cross-resistance to other ribosomal inhibitors such as macrolides, tetracyclines, chloramphenicol, aminoglycosides and oxazolidinones. Other developments There 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-negative bacteria: multidrug resistance to the maximum, Am J Infect Control., 34(5 Suppl 1), S29-37, 2006.
2. Bosso JA. The Antimicrobial Armamentarium: Evaluating Current and Future Treatment Options, Pharmacotherapy, 25(10 Pt 2), 55S62S, 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 doripenem (S-4661) against drug-resistant clinical pathogens, Antimicrob Agents Chemother., 48, 313640, 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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L 10
CHALLENGES IN ANTIFUNGAL THERAPY

mrm UZUN
Hacettepe University School of Medicine, Department of Medicine, Section of Infectious Diseases, Ankara, Turkey
he incidence of invasive fungal infections has increased, and the spectrum of etiologic agents has widened in recent years. Recent advances in newer classes of antifungals havepromised better outcome, especially in difficult-to-treat invasive mould infections; however this has not proven to be true. The empirical use of antifungal therapy in patients with neutropenia who remain febrile in spite of adequate antibacterial therapy has been a standard approach for almost two decades. However, thestatistical weakness of data, the relatively low incidence of IFI in patients not undergoing allogeneic hematopoietic 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 questionable. Recently, new developments in fungal diagno-
sis have rompted a reappraisal of the concept of empirical antifungal therapy. The time period between the biologicalstart of a fungal infection and the appearance 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 facilitate rapid and early diagnosis of invasive fungal infections. On the other hand, the concept of combination therapy has become appealing, but there is still a long way to go.
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May 17-20, 2007
L 11
NATIONAL AND INTERNATIONAL PATENT PROTECTION, FREE PATENT SEARCH TOOLS (ESPACENET) AND STRATEGIES
Hakan BAYRAM
Patent Examiner, Turkish Patent Institute, Hipodrom Street 115, Yenimahalle, Ankara, Turkey
About PCT The 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). Instruments of ratification or accession must be deposited with the Director General of WIPO. The Treaty makes it possible to seek patent protection for an invention simultaneously in each of a large number of countries by filing an international patent application. 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 foreign 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 processing of the application; on the basis of the international 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 international 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 offices can be considerably reduced or virtually eliminated thanks to the international search report, the written opinion and, where applicable, the international preliminary examination report that accompany the international application; (iii) since each international application is published together with an international search report, third parties are in a better position to formulate a well-founded opinion about the patentability of the claimed invention.
About EPC The Convention on the Grant of European Patents of 5 October 1973, commonly known as the European Patent Convention (EPC), is a multilateral treaty 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 granted by the EPO, after grant such a patent is not a unitary right, but a group of essentially independent nationally-enforceable, nationally-revocable patents, subject to revocation and/or narrowing as a group pursuant a time-limited, unified, post-grant opposition procedure.
The EPC provides a legal framework for the granting of European patents, via a single, harmonized procedure before the European Patent Office. A single patent application in one language, may be filed at the European Patent Office at Munich, at its branches at The Hague or Berlin or at a national patent office 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 application 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 accessible 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 accessible through electronic means. In several technical fields, Patents are the most efficient source of information for the following reasons.
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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. 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. 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.
Concentration: Full patent collections are often present in national Patent Office archives. From a relevant list of patent documents you will
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
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 accessible that should be considered when drawing conclusions from the result of a search. Commercial patent databases usually 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 document 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 classification schemes covering all possible technical domains. When conducting a search, it is essential to systematically 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.
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L 12
PATENTABILITY OF MEDICAL AND PHARMACEUTICAL INVENTIONS

Serkan ZKAN
Patent Examiner, Turkish Patent Institute, Hipodrom Street 115, Yenimahalle, Ankara, Turkey
ntellectual 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. Intellectual 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 design rights, utility model rights, and patent rights. Patents are given for any inventions, whether products or processes, in all fields of technology without discrimination, subject to the normal tests of novelty, 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. According to subparagraph (e) of this article 6, methods of diagnosis, therapy and surgery applying to human or animal body shall remain outside the scope the patent protection. The provision under subparagraph (e) of the Article 6, shall apply neither to the products and compositions (per se) used in connection with these methods nor to their process of manufacturing. 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 breeding/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 development 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 major user of the patent system. While only a small - and declining - number of new chemical entities are approved annually, thousands of patents are applied for to protect variants of existing products, processes of manufacture or, where admitted, second uses of known pharmaceutical products. The number of international patent applications continues to rise with impressive growth. The greatest number of international applications (PCT) published in 2006 were in telecommunications (10.5%), pharmaceuticals (10.4%), and information technology (10.4%). The fastest growing technology areas are semiconductors (28% increase), information technology (22%) and pharmaceuticals (21%). There is a remarkable growth rate in the number of patent applications received by the Turkish Patent Institute (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. Similar to the increase in the total number of patent applications, number of pharmaceutical applications increased from 320 to 467 between 2005 and 2006.
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L 13
IMPORTANCE OF POLYMORPHISM IN DRUG RESEARCH AND DEVELOPMENT

Fethi AHN
Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey
olymorphism is the property of a compound found in different crystal forms. Different crystal forms exhibit different dissolution profile and polymorphs of recently discovered drug substances show different lipophilicity. During the development of an original active principal all the polimorphs should be determined before clinical investigations. The more stable olymorph which is discovered 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 industry can be classified as follows:
Thermodynamic stability Chemical stability Mechanical features Solubility
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L 14
A VAST SOURCE FOR THE DISCOVERY OF NOVEL DRUG LEADS: TRADITIONAL MEDICINES
Erdem YELADA
Yeditepe University, Faculty of Pharmacy, Kayda 34755 stanbul, Turkey
iscovery of new drug leads may be defined as a SACRED ART. In order to create MASTERPIECES in this field, scientists should have to embroider each detail obtained through recent computational techniques, SAR studies, ethiological studies 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 phytoallexins have been synthesized in each plant specimen in the nature. Therefore, plants are well-known as an important source of many biologically active compounds. Since ancient times of civilizations, people have been relying on plants as either prophylactic 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 expensive task and various tools are developed in order to increase success. Traditional medicine is among the most extensively implemented approach for this purpose. 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 interdisciplinary 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 ethnobotanical knowledge. A careful evaluation of ethnobotanical inquiries based on field surveys are compulsory to attain an idea about the selection of the correct material, 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 successful results. The latter issue is of special importance for the elaboration of pharmacological studies. The diffuser 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 location was reported that the decoction of hawthorn (Crataegus tanacetifolia) roots was used against rheumatic pain. Actually such utilization was interesting since was not previously reported for hawthorn. We prepared aqueous and methanol extracts from the roots of the plant and tested for their effects on carrageenaninduced hind paw edema model in mice, a well-known technique for the assessment of anti-inflammatory activity. 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 activity 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 convenient technique for the investigation of nonsteroidal anti-inflammatory agents which act through cyclooxygenase 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, however was found totally ineffective. Water immersion-induced 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 effective remedy for wound healing, which suggests that the activity of the extract might possibly, at least partially, through a cytoprotective mechanism. Therefore we tested the effects of the extracts on indomethacin-in29
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duced ulcer model as a representative model for assessing cytoprotective activity and a potent antiulcer activity was found (Grbz et al., 2000). Im always enthusiastic that how the people had discovered 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 mentioned 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 models 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 triterpenoid saponin with potent antiulcer activity (Yeilada et al., 2000a). Actually, because of the lowest alkaloid content inhabitants have used the flowers of plant in therapy without any apparent toxicity. If leaves or other parts of the plant were used, instead of flowers, might possibly cause death, due to the higher alkaloid content.
Bioassay-guided fractionation and isolation procedures The most effective way for the discovery of drug leads from the natural sources in ethnopharmacology is the Bioassay-guided Fractionation and Isolation procedures (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 subjected to successive chemical fractionation and isolation procedures and each fraction and/or components is then submitted to bioassay model, which eventually yield the active ingredient(s). The chemical structure of the isolated constituent is elucidated through detailed spectroscopic analysis, i.e., mass spectrometry and 1H- and 13C-NMR techniques. Although, this procedure 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 chemical 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 fractionation, or may be spread among many fractions and compounds. This may be, in particular, due to the synergistic interaction where many constituents in the extract/fraction of sometimes even in different chemical classes contributing to a pharmacological effect. Another problem is the possible decomposition of the active ingredient(s) during the chemical separation processing; due to the loss of co-metabolites responsible for the stability of the active ingredient or wrong extraction or chromatographic conditions employed for the isolation of constituents. 30
In particular, in the last two decades, extensive researches on BGFI procedures have been carried out for the identification and isolation of pharmacologically 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 molecules Turkey has a rich flora as well as cultural diversity which yielded the accumulation of notable tradition originated remedic information. Through extensive field surveys between 1986 and 1994, we have documented this wealth of knowledge using scientific 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 information and through application of BGFI procedures a number of molecules have been defined with anti-inflammatory, antinociceptive, antidiabetic, antiHelicobacter, IL-2 inducing, antihepatotoxic, antiulcerogenic, 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 externally on affected joints. Through BGFI techniques three catalpol derivative of iridoids were isolated. The antiinflammatory (carrageeenan-induced hind paw edema) and antinociceptive (p-benzoquinone-induced writing) activities were found to be shared by three compounds but, in particular, catalposide and verproside showed higher activity and were safe as regard to gastric toxicity (Kpeli et al., 2005).
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An isoflavone derivative, scandenone, was isolated from osage orange (Maclura pomifera) (Moraceae) fruits by BGFI techniques showed comparable anti-inflammatory (carrageenan-induced hid paw edema, TPA-induced ear edema) and antinociceptive (PBQ-induced writhings) activity to indomethacin, without inducing any gastric lesion (Kpeli et al., 2006a).
which is occasionally removed to drain the accumulated inflammation out. On the other hand, for curing this open wound a fresh leaf of Plantago major ssp. major L. is applied. Moreover, C. vitalba branches are also used to stop toothache by smoking like a cigarette in Northwestern Anatolia. Recently we have isolated a potent anti-inflammatory, antinociceptive and antipyretic principle from the dried herbs of Clematis vitalba, as a new C-glycosylflavon, 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-, serotonin-induced paw edema, inhibitory activity on acetic acid-induced vascular permeability, castor oil-induced diarrhea; subacute inflammation test: Air-sac test; and chronic inflammation test:, Freunds complete adjuvant (FCA)-induced chronic arthritis; antipyretic tests: yeast- and FCA-induced pyrexia; antinociceptive test: p-benzoquinone-induced writings without inducing any apparent gastric toxicity (Yesilada and Kpeli, 2007). Another C-glycosylflavon with potent anti-inflammatory 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 gastric toxicity (Kpeli et al., 2004). This compound was also found to possess a potent antihepatotoxic (Kpeli 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 saponin, ilwensisaponin A, were isolated as the potent anti-inflammatory and antinociceptive components without inducing any apparent acute toxicity or gastric damage (Kpeli et al., 2007).
Due to widespread distribution of Clematis species 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 rheumatic 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 Turkish species of Clematis are used as remedy among public of Anatolia. The most salient application related 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 provide 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,
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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 activity guided fractionation and isolation techniques 17 active constituents were isolated and their structures were chemically elucidated. Diterpenes [genkwadaphnin, gnidilatin, gnidicin and 20-palmitates of these compounds and 1,2-dehydrodaphnetoxin], a coumarin derivative [daphnetin], Lignan derivatives [eudesmin, wikstromol, matairesinol]. Effects of these molecules were studied against a group of cytokines known to play key role in inflammatory disorders; interleukin-1 and 1 and tumor necrosis factor-. Genkwadaphnin, 1,2-dehydrodaphnetoxin 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 immunomodulatory and have been implemented against various diseases 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 species, A.oleifolius, A.cephalotes, A.trojanus, were studied using ELISA tests against cytokines induced by bacterial lipopolysaccharide stimulation; IL-1, IL-8, TNF- , and phorbolacetate stimulation; IL-2, IL-4, INF-. Among the compounds studied only Astragaloside VII was isolated from Astragalus oleifolius showed a potent IL-2 stimulatory activity (142%). Since a few local healers claimed that several Astragalus species (not revealed) from southeast Anatolia to possess healing effect on cancers (personal note), it is supposed that this result may partially support the above assertions (Yesilada et al., 2005).
Cistus laurifolius (Cistaceae), rockrose, is a widespread 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-1, an inflammatory cytokine (Yesilada et al., 1997).
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Internal use of flowers for treatment of peptic ulcer was also reported. In a recent study, to evaluate antirheumatic activity in TFM, we isolated 16 compounds mainly flavonoids and lignans through activity-guided procedures and major component 3O-methyl quercetin was found to possess potent prostaglandin inhibitory and antioxidant activity; inhibitory 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-Helicobacter pylori activity (stn et al., 2006). Other flavonoids, kaempferol-3,7-O--dirhamnoside and quercetin-3,7-O--dirhamnoside from the leaves of Tilia argentea showed antinociceptive (34% and 40% inhibition, respectively) and anti-inflammatory (36% for both) activities without inducing any apparent gastric lesions (Toker et al., 2004).
techniques yielded two sesquiterpenes as the active components; Chlorojanerin (90%***), 13-acetyl solstitialin A (89%*), while Solstitialin A showed a weak activity (31%) (Yesilada et al., 2004; Grbz and Yesilada, 2007).
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 possess 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 triterpenoid, spartitrioside. This compound showed potent 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 flowers (Yesilada et al., 2000b). Fresh flowers of yellow starthistle (Centaurea solstitialis ssp.solstitialis) (Asteraceae) are swallowed to treat ulcers in TFM. Methanol extract showed 100% inhibition against EtOH-induced lesions in rats. BGFI
Ethnopharmacological studies have been conducted on TFM so far revealed that flavonoids are the most promising phytochemicals as drug leads. A wide activity 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 flavonoid type compounds. Among the isolated flavonoids in the course of ethnopharmacological studies from TFM; 3-O-methyl quercetin (isorhamnetin) deserves further detailed attention. This compound showed a potent anti-Helicobacter pylori activity (MIC 3.9 g/ml; AMP 0.06 g/ml) (stn et al., 2006), anti-inflammatory and antinociceptive activities (Kpeli and Yesilada, 2007), prostaglandin inhibitory and antioxidant activities (Sadhu et al., 2006) and antihepatotoxic activity (Kpeli 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 Products as Medicinal Agents. J.L. Beal, E. Reinhard (Eds). Hippokrates Verlag: Stuttgart, pp.18-19. 2. Grbz, ., Akyz, ., Yeilada, E., ener, B., 2000. Antiulcerogenic Effect of Momordica charantia L. fruits on Various Ulcer Models in Rats. Journal of Ethnopharmacology 71, 77-82.
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3. Grbz ., Yeilada, E., 2007. Evaluation of anti-ulcerogenic effect of sesquiterpene lactones from Centaurea solstitialis L. ssp.solstitialis by using various in vivo and biochemical techniques. Journal of Ethnopharmacology (in press). 4. Kpeli E., Aslan M., Grbz ., Yeilada E., 2004. Evaluation of the in vivo biological activity profile of isoorientin. Zeitschrift fr Naturforshung C 59c, 787-89. 5. Kpeli E., Harput ., Varel M., Yeilada, E., Saraolu, .., 2005. Bioassay-guided isolation of iridoid glucosides with antinociceptive and anti-inflammatory activities from Veronica anagallis-aquatica. Journal of Ethnopharmacology 102, 170-176. 6. Kpeli E., Orhan ., Toker G., Yesilada E., 2006a. Antiinflammatory and antinociceptive potential of Maclura pomifera (Rafin.) Schneider fruit extracts and its major isoflavonoids, scandenone and auriculasin. Journal of Ethnopharmacology 107, 169-174. 7. Kpeli E., Orhan Deliorman D., Yeilada E., 2006b. Effect of Cistus laurifolius L. leaf extracts and isolated flavonoids on acetaminophen-induced hepatotoxicity in mice. Journal of Ethnopharmacology 103, 455-60. 8. Kpeli, E., Tatl, .., Akdemir, Z.S., Yeilada E., 2007. Bioassay-guided isolation of anti-inflammatory and antinociceptive glycoproteins from the flowers of Verbascum lasiniathum Boiss. ex Bentham.. Journal of Ethnopharmacology 110, 444-450. 9. Sadhu S.K., Okuyama E., Fujimoto H., Ishibashi M., Yesilada 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., Yeilada E., Ito S., 2005. Hypoglycaemic activity of Gentiana olivieri and isolation of the active constituent through Bioassay-guided fractionation techniques. Life Sciences 76, 1223-38. 11. Toker G., Kpeli E., Temizer Memiolu M., Yesilada E., 2004. Flavonoids with antinociceptive and anti-inflammatory activity from the leaves of Tilia argentea (Linden). Journal of Ethnopharmacology 95, 393-7.
12. stn O., zelik B., Akyn Y., Abbasolu U., Yeilada E., 2006. Flavonoids with anti-Helicobacter pylori activity from Cistus laurifolius leaves. Journal of Ethnopharmacology 108, 457-61. 13. Yeilada, E., stn, O., Sezik, E., Takaishi, Y., Ono, Honda, G., 1997. Inhibitory effects of Turkish folk remedies on inflammatory cytokines: interleukin-1a, interleukin-1 and tumor necrosis factor . Journal of Ethnopharmacology 58, 59-73. 14. Yeilada, E., Takaishi, Y., Fujita, T., Sezik, E., 2000a. Antiulcerogenic effects of Spartium junceum flowers on in vivo test models in rats. Journal of Ethnopharmacology 70, 219-226. 15. Yeilada, E., Tsuchiya, K., Takaishi, Y., Kawazoe, K., 2000b. Isolation and characterization of free radical scavenging flavonoid glycosides from the flowers of Spartium junceum by activity-guided fractionation. Journal of Ethnopharmacology 73, 471-78. 16. Yeilada 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 constituents. Cytokine 13, 359-64. 17. Yeilada E., Grbz ., Bedir E., Tatl ., Khan I.A., 2004. Isolation of antiulcerogenic sesquiterpene lactones from Centaurea solstitialis L. ssp.solstitialis through bioassayguided fractionation procedures in rats. Journal of Ethnopharmacology 95, 213-9. 18. Yeilada, E., Bedir, E., al, ., Takaishi, T., Ohmoto, 2005. Effects of triterpene saponins from Astragalus species on in vitro cytokine release. Journal of Ethnopharmacology 96, 71-7. 19. Yeilada E., Kpeli 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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L 15
PARTHENOLIDE ANALOGS AS ANTILEUKEMIC AGENTS WITH CLINICAL POTENTIAL

Peter A. CROOKS
University of Kentucky, College of Pharmacy, 501A Pharmacy Building, Rose Street, Lexington, KY 40536-0082
he 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 initiation of AML and relapse. Thus, novel compounds such as parthenolide, that selectively target LSCs, represent potential novel clinical agents for the treatment of AML. Unfortunately, parthenolide has poor physicochemical properties that preclude its development as an effective clinical agent. Utilizing classical 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-B inhibition, p53 activation, and induction of oxidative stress responses. Pharmacokinetic and metabolic studies in rodents indicate that DMAPT fumarate has ~70% bioavailability via the oral route, while studies in mouse xenograft models and in dogs suffering from spontaneous acute leukemias indicate that DMAPT has in vivo bioactivity. Thus, based upon the above preclinical data, DMAPT fumarate appears to be a potential clinical candidate for the treatment of human LSCs.
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L 16
SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL CAMPTOTHECIN CLASS TOPOISOMERASE I INHIBITORS

Ayhan S. DEMR
Middle East Technical University, Department of Chemistry, 06531 Ankara, Turkey
amptothecin (CPT) is a pentacyclic alkaloid 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 implicated in DNA replication and RNA transcription. A camptothecin drug stabilizes and forms a reversible enzyme-camptothecin-DNA ternary complex, designated the cleavage complex. The formation of the cleavable complex specifically prevents the reunion step of the breakage/union cycle of the topoisomerase reaction.1 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 after injection to form the parent camptothecin compound. 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 interest as a result their ability to halt the growth of a wide range of human tumors. For example, these drugs exhibit unprecedented high levels of antitumor activities against human colon cancer.3 Camptothecin has also been shown to be effective against other experimental cancer types such as lung, breast, and malignant melanoma. A need continues to exist for the development of new and better camptothecin compounds having still higher anti-tumor activity and still more improved watersolubility 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 converted to active form by physical methods and subMay 17-20, 2007
The natural fluorescent properties of CPT and its derivatives have been exploited to monitor its concentration 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 structurally-related camptothecin analogs are water insoluble. 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.
36
stitution of the 7-position of camptothecin or homocamptothecin to form water-soluble derivatives containing a 7- aminoalkyl moiety. Generally, the camptothecins serving as starting materials are suspended in organic solvents or water. To the mixture is added the following compounds which give equivalent 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 hydrogen peroxide. The mixture is then stirred to complete the reaction. Any precipitate which forms is removed by filtration and the product is isolated after removal of the solvent. We have evaluated the abilities of the novel camptothecins described here to inhibit the topoisomerase I enzyme. In these experiments the oligonucleotide was labeled at the 3 -end of the upper (scissile) strand with 32P--cordycepin and terminal transferase.4 Topoisomerase I-mediated cleavage generates DNA fragments that can be readily detected by electrophoresis and quantitated by phosphorimager 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 distribution of cleavage sites are comparable to those
generated by camptothecin or 7-ethyl-10-hydroxycamptothecin (SN-38). This slower reversal may correspond to more stable cleavable complexes. The guanadino 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.; Lakowicz, 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.
37
LECTURES
LECTURES
L 17
NANOMATERIALS IN MEDICINE
Erhan PKN
Hacettepe University, Chemical Engineering and Bioengineering; Biyomedtek: Society for Biomedical Technologies, Beytepe, Ankara, Turkey
anotechnology deals with materials and systems 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, polymeric 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, plasmid DNA, etc., for effective therapy of a wide range of diseases. Nanofibers can be electrospun and form porous nonwoven films as potential dressings and scaffolds for tissue engineering. This presentation will cover some diverse medical applications of nanostructured materials including authors contributions and ongoing studies.
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May 17-20, 2007
L 18
NANOMEDICINE FOR CENTRAL NERVOUS SYSTEM (CNS) DRUG DELIVERY

A. V. KABANOV
Department of Pharmaceutical Sciences, and Center for Drug Delivery and Nanomedicine, University of Nebraska Medical Center, 985830 Nebraska Medical Center, Omaha, Nebraska 68198-5830
eurodegenerative and infectious disorders including Alzheimers and Parkinsons diseases, amyotrophic lateral sclerosis, and stroke are rapidly increasing as populations age. Alzheimers disease alone currently affects 4.5 million Americans, and more than $100 billion is spent per year on medical and institutional care for affected people. Such numbers will double in the ensuing decades. Currently disease diagnosis for all disorders is made, in large measure, on clinical grounds as laboratory and neuroimaging tests confirm what is seen by more routine examination. Achieving early diagnosis would enable improved disease outcomes. Drugs, vaccines or regenerative proteins present real possibilities for positively affecting disease outcomes, but are limited in that their entry into the brain is commonly restricted across the blood-brain barrier. This presentation will review highlights how these obstacles can
be overcome by polymer science and nanotechnology. 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 hydrophobic anchors groups and amphiphilic polymers to enhance their transport across the blood-brain barrier; 3) development of nanogels capable of carrying antisense oligonucleotides and siRNA across the bloodbrain barrier; 4) cell-mediated delivery of nanozymes to the brain. Such approaches may improve diagnostic 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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LECTURES
Oral Presentations
O 01
Ref: 0053
INVESTIGATION OF NONCOVALENT PROTEINPROTEIN INTERACTIONS BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY

Bekir SALH, Basri GLBAKAN, zlem DEMREL
Hacettepe University, Faculty of Science, Department of Chemistry, 06532 BeytepeAnkara, Turkey
Matrix-Assisted Laser Desorption/Ionization-Mass spectrometry (MALDI-MS) is a very important and rapidly evolving analytical tool that is used in many different areas, including protein interaction 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 solution are maintained throughout the sample preparation and ionization process. Noncovalent interaction between A 1-42 peptide and A 1-42 antibody is studied. A 1-42 peptide and A 1-40 antibody was used as negative control to determine the specificity. A 1-42 peptide was digested with trypsin and digestion products were incubated with A 1-42 antibody and A 1-40 antibody respectively. Fibril form of the A 1-40 peptide was studied. The effect of TFA and A 1-40 antibody onto disaggregation of A 1-40 fibril peptide was followed by capillary electrophoresis (CE) and MALDI-MS. Interaction of bovine serum albumin (BSA) with A 142 was investigated with varying working concentrations by MALDIMS 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 interactions. 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, Journal of Mass Spectrometry, 33(8), 697-704, 1998. 2. Frenkel D, Solomon B, Benhar I. Modulation of Alzheimers amyloid neurotoxicity by site-directed single-chain antibody, Journal of Neuroimmunology, 106, 2331, 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 amyloid peptide, Proceedings of National Academy of Science USA, 93, 452455, 1996.
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 dynamic 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 databank.
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 substrates including pdb code and number of residues are given in Table 1. All water molecules and ligands from X-Ray structures are removed 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 No.of atoms (chain A) No.of atoms (chain B) No.of solvent molecles Total charge in the system Box geometry Box volume (nm3) Temperature Simulation time 1qj6 287 2093 8559 3.000e octahedron 126.84 300 K 20 ns. 1qj7 287 2093 8545 3.000e octahedron 124.07 300 K 20 ns. 1qhr 287 2093 8501 3.000e octahedron 126.27 300 K 20 ns. 1ppb 287 2093 8695 3.000e octahedron 148.15 300 K 20 ns.
O 02
Ref: 0010
MOLECULAR DYNAMICS SIMULATION OF THROMBIN: TARGET FOR ANTICOAGULANT DRUGS

1
1
zge KL, 1Fulya ALAR, 1Vildan ADAR, 2Timuin YALINKAYA
Hacettepe University, Faculty of Science, Department of Chemistry, Computer-aided Drug Design Group, Ankara, Turkey 2 Forte Technology, Simulation Group, Ankara, Turkey
Thrombin is a primary target for the development of novel anticoagulants, since it plays two important and opposite roles in hemostasis 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
43
ORAL PRESENTATIONS
ORAL PRESENTATIONS
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 message-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 Scientific, 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/ We would like to thank Turkish Scientific and Technical Research Association (TUBITAK) for the financial support (Grant no: TBAG-106T088) in this research. This work was carried out under the HPC-EUROPA project( RH3-CT2003-50607), with the support of the European Communitys Research Infrastructures action of the European Research Area Programme.
Acknowledgement
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 trajocteries the VMD [8]1.8 and PyMOL [9] respectively were used. MD simulations have been performed to determine time-dependent characteristic of hirudin variants (HV1, HV2, HV3). All systems were minimized prior to simulation using the steepest descent method 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) No. of residues No. of atoms No. of solvent molecules Total charge Box geometry Box volume (nm^3) Temperature (K) Simulation time (ns) Methods 65 483 10404 -9 1hrt (HV1) 49 347 2881 -3 5hir (HV1) 49 348 3183 -3 1hrt (HV2) 4htc (HV2) 65 478 10284 -7 65 479 10751 -6
O 03
Ref: 0011
MOLECULAR DYNAMICS SIMULATION OF HIRUDINS: SPECIFIC THROMBIN INHIBITOR ISOLATED FROM MEDICINAL LEECH
1
1
Fulya ALAR, 1zge KL, 1Vildan ADAR, 2Uur BYKDEMRC
Hacettepe University, Faculty of Science, Department of Chemistry, Computer-aided Drug Design Group, Ankara, Turkey 2 Forte Technology, Simulation Group, Ankara, Turkey
octahedron octahedron octahedron octahedron octahedron 326,893 300 20 X-Ray Diffraction 93,46 300 20 X-Ray Diffraction 104,041 300 20 NMR 321,3 300 20 X-Ray Diffraction 341,39 300 20 X-Ray Diffraction
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 inhibitor 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 isolated from Hirudo medicinalis [2-4]. In Figure 1 x-ray structure of HV1 obtained from 1hrt is given.
A detailed comparison of simulations were examined by computing 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 disulfide bridges) and a flexible C-terminal tail (residues 48-65), and it has a disordered region made up of residues 31-36.
We would like to thank Turkish Scientific and Technical Research Association (TUBITAK) for the financial support (Grant no: TBAG-106T088) in this research. This work was carried out under the HPC-EUROPA Project (RH3-CT2003-50607), with the support of the European Communitys Research Infrastructures action of the European Research Area Programme. Figure 1. Structure of HV1 taken from complex, 1hrt. 1. Markwardt F. Untersuchungen uber Hirudin, Naturwissenschaften, 42, 537538, 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, 180184, 1984.
Acknowledgement
X-Ray structures of hirudin variant 1 (HV1) taken from the Brook Protein Data Bank [5], with the pdb codes 5hir and 1hrt.5hir
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May 17-20, 2007
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 medicinalis. Proc Natl Acad Sci USA, 83, 10841088, 1986. 4. Tripier D. Hirudin: a family of iso-proteins. Isolation and sequence determination of new hirudins, Folia Haematol (Leipzig), 115, 3035, 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 message-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
Rana SANYAL
Ref: 0113
DENDRIMERS AS DRUG DELIVERY AGENTS TO BONE

Boazii University, Faculty of Arts and Sciences, Department of Chemistry, 34342 Bebek, stanbul, Turkey
Dendrimers have emerged as promising candidates for targeted drug carriers since they can be tailored to accommodate a high density 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 application of drug molecules to the diseased tissue or organ increases the effect of the therapy and decreases the side effects, targeted drug delivery 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 delivery [2, 3]. Synthesis of different generations of biodegradable dendritic 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 macromolecular therapeutics, Adv. Drug Delivery Rev., 57, 1049-1076, 2005. 3. Gittens SA, Bansal G, Zernicke, RF, Uluda, H. Designing proteins for bone targeting, Adv. Drug Delivery Rev., 57, 1011-1036, 2005.
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 tumor tissue to provide effective and sustained local drug concentrations without causing systemic side effects. Thus, in rats treated with MTZloaded microspheres, tumor volumes were significantly reduced. No tumor formation was observed when glioma cells and MTZ-loaded 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 distance from the injection site. The data demonstrate that MTZloaded PLGA microspheres can deliver therapeutic concentrations of the drug to the tumor and prevent glioma growth without causing 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 significantly reduces vulnerability to the neuronal cell death. The inhibition 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 efficacy 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 nanoparticulate drug delivery system was able to translocate into the brain tissue after iv administration. Consequently, these novel nanoparticles 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
O 05 RECENT ADVANCES IN BRAIN DRUG DELIVERY

Yilmaz APAN
Ref: 0111
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 developed to overcome the BBB and to achieve successful brain drug delivery. Here, we describe two different approaches for drug delivery to the brain. In the first approach, we describe the formulation of mitoxantrone (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 PEGBIO-SA/ OX26 for the delivery of a caspase-3 inhibitor.
Photodynamic therapy (PDT) is a noninvasive method of treating malignant tumors and age-related macular degeneration, and particularly 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 administration. The sensitizer is then excited with red or NIR light, generating 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 compounds 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 bodys therapeutic window (650-800 nm, low absorptivity region in typical mammalian tissues). Therefore, there is a significant impetus to develop novel and better efficiency sensitizers 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.
45
ORAL PRESENTATIONS
ORAL PRESENTATIONS
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 extended 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 absorptions in the 650-680 nm region. The singlet oxygen generation efficiency was studied using the singlet oxygen trap 1,3-diphenylisobenzofuran (DPBF). In order to facilitate comparison to previously 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].
the dose that can be administered to patients. The clinical inconvencies 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 produced 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 nephrotoxicity than cis-DDP [6]. Various platinum blues complexes containing nitrogen and oxygen 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 measurements made on this dark blue colored compound suggest that it is a new platinum blue type complex.
Thus, we demonstrated that novel di-styrylborodiazaindacene dyes with bromo system are very efficient singlet oxygen-substituents on the fluorochrome pi- generators. In addition, these water soluble 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 including functionalized carbon nanotubes.
1. Atlgan S, Ekmeki Z, Doan AL, G, D, Akkaya EU. Water soluble distyryl-boradiazaindacenes as efficient photosensitizers for photodynamic therapy. Chem. Commun., 4398-4400, 2006.
DNA binding studies of the complex were carried out by voltammetric and UV titrations supporting the electrostatic binding of ctDNA with suggesting preferential stabilization of PtIII over PtII on binding to DNA. The effect of platinum blue complex on GST, one of the most important enzymes involved in drug conjugation and biotransformation reactions ,were also investigated.
1. 2. 3. 4. 5. 6. Rosenberg B, VanCamp L., Nature, 205, 698, 1965. Tejel C, Ciriano MA, Oro LA., Chem. Eur. J., 5, 1131, 1999. Matsumoto K, Sakai, K. Adv. Inorg. Chem., 49, 375, 2000. Hoffmann KA, Bugge G., Berichte, 41, 312, 1908. Gillard RD, Wilkinson G., J. Chem. Soc., 2835, 1964. Davidson JP, Faber PJ, Fisher RG., Cancer Chemother. Rep., 59, 287, 1975.
O 07
Ref: 0115
A NOVEL POTENTIAL ANTITUMOR ACTIVE DRUG: PLATINUM BLUE COMPLEX CONTAINING SULFURDONOR LIGAND
1
1 2
eniz ZALP-YAMAN, 1smail ERLHAN, 1S. Belgin GR, 2Hseyin
Atlm University, Engineering Faculty Chemistry Group, 06836-ncek, Ankara, Turkey Middle East Technical University, Chemistry Department, Ankara, Turkey
Cisplatin (cis-diamminedichloroplatinum(II), Pt(NH3)2Cl2 (CDDP) was chemically described in 1845, but its antitumor properties were only found accidentally by Rosenberg in 1965 [1]. Decipite the success of cisplatin in chemotherapy, serious side effects limits
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May 17-20, 2007
O 08
Ref: 0055
O 09
Ref: 0117
SYNTHESIS OF ACETOPHENONE AND SUBSTITUTED ACETOPHENONE DERIVED MANNICH BASES AND THEIR CYTOTOXIC ACTIVITIES
H. nci GL
Atatrk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey
UVB-INDUCED APOPTOTIC EFFECT OF 11 P53 ACTINIC KERATOSIS MUTATIONS

1
1
Aye ERCAN, 2. Hamdi , 3Douglas BRASH
Mannich bases are generally formed by the reaction between a compound containing a reactive hydrogen atom, formaldehyde and a secondary 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 synthesis 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-aryl3-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), piperidinols, 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, Nbis(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, piperidine, 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 deaminate faster comparing to their corresponding nonquaternary derivatives. 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 isomers. 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 representative compounds on glutathione and related enzymes were tested in order to determine the mechanism of thiol alkytion to confirm the stability study of some representative compounds [7, 8]. A preliminary 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.Gl H, Vepsalainen J, Gl M, Erciyas E, Hanninen O. Pharm. Acta Helv., 4, 393-8, 2000. 3.Gl H, Gl M, Erciyas E. Arzneimittel Forschung, 52, 628-35, 2002. 4.Gl H, Gl M, Vepsalainen J, Erciyas E, Hanninen O. Biol. Pharm. Bull. 26, 631-7, 2003. 5.Gl H, Das U, Pandit B, Li PK. Arzneimittel Forschung, 56, 850-4, 2006. 6.Gl M, Gl H, Das U, Hanninen O. Arzneimittel Forschung, 55, 332-7, 2005. 7.Gl M, Atalay M, Gl H, Nakao C, Lappalainen J, Hanninen O. Toxicol. In Vitro, 19, 573-80, 2005. 8.Gl H, Gl M, Erciyas E. Arzneimittel Forschung, 52, 628-35, 2002. 9.Gl M, Gl H, Hanninen O. Toxicol. In Vitro, 16, 107-12, 2002.
Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 06100 Ankara, Turkey 2 Hacettepe University, Faculty of Medicine, department of Biochemistry, 06100 Ankara, Turkey 3 Yale 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 tumor 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 missense 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
1 2
1
Samiye YABANOLU, 2Umut SALGIN-GKEN, Nesrin GKHAN-KELEK, 1Glberk UAR
Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 06100 Ankara, Turkey 2 Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Ankara, Turkey
It has been reported that some benzoxazolinone, triazole, thiadiazole and hydrazone derivatives showed significant analgesic and antiinflammatory activities [1-3]. It has been speculated that non-steroidal 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 pharmacological profiles and the effects of thiosemicarbazide, triazole, thiadiazole and hydrazone bearing 2-benzoxazolinone were investigated on free radical scavenging activity.
47
ORAL PRESENTATIONS
ORAL PRESENTATIONS
O 11
Ref: 0108
STUDIES ON HISTONE DEACETYLASE INHIBITORY ACTIVITY OF SOME CARBOXYLIC ACID DERIVATIVES AND THEIR STRUCTURE-ACTIVITY RELATIONSHIPS
Gamze BORA, 1Didem DAYANGA-ERDEN, 2Peruze AYHAN, Kemal YELEK, 4Sevim DALKARA, 2Ayhan DEMR, 1 Hayat ERDEM-YURTER
1 3
Hacettepe University, Faculty of Medicine, Department of Medical Biology, Ankara, Turkey 2 Middle East Technical University, Department of Chemistry, Ankara, Turkey 3 Hadir Has University, Faculty of Arts and Sciences, stanbul, Turkey 4 Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey
1
Figure 1.
Thiosemicarbazide, triazole, thiadiazole and hydrazone derivatives (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 reducing 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-hydroxyl 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 antiinflammatory activity of the compounds.
1. Kksal M, Gkhan N, Kpeli E, Yeilada E, Erdoan H. Synthesis, analgesic and antiinflammatory properties of certain 5-/6-acyl-3-(4-substituted-1-piperazinylmethyl)-2-benzoxazolinones derivatives, Arch. Pharm., 338, 117-125, 2005. 2. Labanauskas L, Kalcas E, Udrnait E, Gaidelis P, Bruktus A, Daukas, 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 exhibiting anti-inflammatory activity, Pharmazie, 56, 617-619, 2001. 3. Sondhi SM, Dinodia M, Kumar A. Synthesis, anti-inflammatory and analgesic activity evaluation of some amidine and hydrazone derivatives, Bioorg. Med. Chem., 14, 4657-4663, 2006. 4. Salgn U et al. Unpublished data 5. Ferreira ICFR, Baptista P, Vilas-Boas M, Barros L. Free-radical scavenging capasity and reducing power of wild edible mushrooms from northeast Portugal: individual cap and stipe activity, Food Chem., 100, 15111516, 2007. 6. Kumaran A, Karunakaran RJ. In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India, LWT, 40, 344-352, 2007.
Histone deacetylation is responsible for transcriptional repression whereas histone hyperacetylation facilitates gene expression. Therefore histon deacetylating enzymes (HDACs) are popular targets 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 acids. 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 analogous to understand the structural requirements for HDAC inhibitory 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 results were evaluated to establish structure-activity relationship. Acknowledgement
This project was supported by The Scientific & Technological Research Council of Turkey, Project No: 105G014.
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May 17-20, 2007
Poster Presentations
P 001
Ref: 0003
P 003
Ref: 0005
PRICEITE DOES NOT INDUCE GENOTOXICITY IN HUMAN LYMPHOCYTES IN VITRO

Hasan TRKEZ, Fatime GEYKOLU
Atatrk University, Faculty of Arts and Sciences, Department of Biology, 25240 Erzurum, Turkey
IN VITRO EFFECTS OF ALUMINUM SULPHATE ON SISTERCHROMATID EXCHANGES AND OXIDATIVE STRESS IN HUMAN BLOOD: PROTECTIVE ROLE OF BISMUTH SUBNITRATE
Fatime GEYKOLU, Hasan TRKEZ
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 industrial, 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 concentrations 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 GEYKOLU, Hasan TRKEZ
Aluminum, which is found in several different forms and oxidation states, causes acute and chronic adverse health effects. Medicinally, 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 sulphate (Al2(SO4)3) and BSN in human blood in vitro. The various concentrations of Al2(SO4)3 (10 and 20 g/mL) and BSN (0.75, 1.5, 3 and 5 g/mL) were used. Evaluation of DNA damages 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- phosphate dehydrogenase (G6PDH) and catalase (CAT). The SOD activity increased by Al2(SO4)3 (10 g/mL) exposure but the ratio of SCEs didnt change compared to the controls. On the other hand, the high dose of Al2(SO4)3 (20 g/mL) caused oxidative stress and increased SCE frequency. When the concomitant treatment with Al2(SO4)3 of BSN were investigated, BSN exerted an antioxidant action at low doses (0.75 and 1.5 g/mL). It also reduced the formation of SCEs. This study suggests for the first time that BSN may be administered as a potential protective against the effects of Al2(SO4)3 in which oxidative and genetic damages are clearly involved.
P 004
Ref: 0014
Despite the increasing use of factory-made synthetic drugs, natural 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 evaluate 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 purpose 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 M. 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 order: P. furfuracea > R. melanophthalma > D. intestiniformis. Our findings indicated that lichens can be a new resource of therapeutic potential as recognized here against to adverse effects of drugs used.
GST-CDNB ACTIVITIES IN GILTHEAD SEABREAM (SPARUS AURATA) LIVER CYTOSOL OF DIFFERENT AGES
1
1
Hatice ARDA-AKDOAN, 2Alaattin EN
Pamukkale University, Science and Arts Faculty, Department of Chemistry, 20017 Knkl, Denizli, Turkey 2 Pamukkale University, Science and Arts Faculty, Department of Biology, 20017 Knkl, 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 major 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 zmir, 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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POSTER PRESENTATIONS
portant age-related differences in xenobiotic metabolism do occur in Gilthead Seabream liver and are substrate specific, which might affect obtaining desired actions and/or responses to drugs, hormones and dietary supplements used during breeding. GST-CDNB activity increased in gilthead seabream (Sparus aurata) 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 TRKEZ, Fatime GEYKOLU
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 activities remains largely unexplored. The aim of this study was to assess the efficacy of the lichen Pseudovernia furfuracea (2.5, 5, 10 and 20 g/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 genotoxicity. SCEs and MN formations significantly increased by effect of 5 g/ 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 provide anti-genotoxic effects as due to their antioxidant defenses although there is no evidence for the content of the lichen species in the present investigation.
Structurally, 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 derivatives and tested for 1 and 2 receptor binding [3]. Since sigma receptors are membrane-bound proteins, isolating and resolving their three-dimensional structure has proven to be difficult. 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 techniques are able to raise a predictive description of global structural requirements for interactions between substrates and receptor by using binding data. We have described a detailed QSAR study on tetrahydroindazole series, in order to give better picture of action and to rationalize selection of substituents. The QSAR functions in the Molecular Operating Environment (MOE) and SYBYL were used to compute theoretical molecular descriptors related to physicochemical properties.
1. Lever JR, Gustafson JL, Xu R, Allmon RL, Lever SZ. Synapse, 59, 350358, 2006. 2. Vangveravong S, Xu J, Zeng C, Mach RH. Bioorg. Med. Chem., 14, 69886997, 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 EKEN
Dicle University, Faculty of Science and Art, Department of Chemistry, 21280 Diyarbakr, Turkey
P 006
Mine YARIM, Ece GRDAL, Dilek EROL
Ref: 0016
QSAR MODELING ON SIGMA RECEPTOR LIGANDS

Yeditepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34755 stanbul, Trkiye
Sigma () receptors are functional, membrane-bound proteins distributed throughout the brain and peripheral organs. 1 and 2 receptor types are clearly established, and further pharmacological differentiation may be possible. 1 receptors are implicated in central nervous system (CNS) disorders such as depression, schizophrenia and dementia. Further, 1 receptor agonists have value as neuroactive agents, while antagonists may help alleviate cocaine addiction. The significance of the receptor system to human health is augmented through the overexpression of sites by a number of cancers. Thus, receptor ligands might be used for the detection and treatment of malignant neoplastic diseases. 2 receptors may be of particular prognostic relevance, as the extent of their expression seems indicative of the proliferative status of tumors [1].
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-stranded DNA via hydrogen atom abstraction from the sugar moiety. However, 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 pBluescript M13+ DNA was prepared and isolated according to standard protocols using Qiagene plasmid mini preparation kit. Gel was scanned on Gel documentation system (Gel-Doc-XR, BioRad, Hercules, 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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May 17-20, 2007
P 008
Ref: 0020
ANTIDEPRESSANT-LIKE EFFECT OF SOME NEW ARYL PROPANE DERIVATIVE COMPOUNDS

1
Fulya MORAL, 1Meri KKSAL, 1Mine YARIM, 2S. Srr BLGE, 2S. Elif AKSZ
Yeditepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34755 stanbul, Turkey 2 Ondokuz Mays University, Ondokuz Mays University, Faculty of Medicine, Department of Pharmacology, 55139, Kurupelit, Samsun, Turkey
1
In the early 1970s, evidence of the role of serotonin (5-hydroxytrytamine or 5-HT) in depression began to emerge and the hypothesis that enhancing 5-HT neurotransmission would be available mechanism to mediate antidepressant response was put forward. On the basis of this hypothesis, efforts to develop agents that inhibit the uptake 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 fewer side effects than the other classes of antidepressants. Fluoxetine, ()-N-methy-3-phenyl-3(,,-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 intolerance and activating effects such as nervousness, anxiety and imsonia 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 benzylpiperidine group to reach new antidepressant compounds with a faster onset of action, a better efficacy and less side-effects. The antidepressant-like effect of this synthesized compounds was studied in comparison with other antidepressants (Fluoxetine, Sertraline, Imipramine) 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, Atkinson ST, Hertel W, Nelson DL, Wainscott DB, Ahmad LJ, Shaw J, Threlkeld PG, Wong DT. Bioorg. Med. Chem. Lett., 13, 3939-3942, 2003.
problem in many regions of the world. The need for new antimalarials comes from the widespread resistance to those in current use. In this study, the use of parasites lactate dehydrogenase as an antimalarial drug target is evaluated. The cofactor binding site of Plasmodium vivax lactate dehydrogenase was compared to some other apicomplexan 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 feature that distinguishes Plasmodium and other apicomplexan lactate dehydrogenase enzymes from their human lactate dehydrogenase, supporting their suitability as targets in common drug design studies.
P 010
Ref: 0024
MUTAGENIC ANALYSIS OF ACTIVE SITE LOOP OF LACTATE DEHYDROGENASE BY MIMICKING EIMERIA TENELLA IN PLASMODIUM VIVAX
1
1
Venhar ELK, 2Dilek TURGUT-BALIK
University of Frat, Faculty of Arts and Sciences, Department of Biology, 23169 Elaz, Turkey 2 Yldz Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Davutpaa Campus, 34210 Esenler-stanbul, 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 endemic 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 enzymes 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 counterpart. 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 009
Ref: 0023
P 011
Ref: 0025
COMPARATIVE ANALYSIS OF THE COFACTOR BINDING SITE OF PLASMODIUM VIVAX LACTATE DEHYDROGENASE WITH SOME OTHER KNOWN APICOMPLEXAN COUNTERPARTS
1
1
SYNTHESIS OF AMINO ALCOHOL BASED CHIRAL LIGANDS AND THEIR APPLICATIONS IN ENANTIOSELECTIVE DIETHYLZINC ADDITION TO BENZALDEHYDES
Dilek Ik TAGIN, Canan NALEROLU
Hacettepe University, Faculty of Science, Department of Chemistry, 06800 BeytepeAnkara, Turkey
University of Frat, Faculty of Arts And Sciences, Department of Biology, 23169 Elaz, Turkey 2 Yldz Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Davutpaa Campus, 34210 Esenler-stanbul, 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. Malaria is one of the greatest causes of human misery and death. Despite continued efforts to control the disease, it remains a major health
Among asymmetric catalysis of C-C bond-formation, the enantioselective addition of diorganozinc reagents to aldehydes in the presence 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 catalysts, i.e. amino alcohols, amino thiols, diamines, disulfonamides,
53
and diols have been synthesized [3]. Among the chiral ligands reported, -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 synthesized (1S,2R)-2-((1H-pyrrol-2-yl)methylamino)-1-phenylpropan1-ol (1) and (1R, 2S)-2-((1H-pyrrol-2-yl)methyl amino)-1-phenylpropan-1-ol (2) were used as chiral ligands in the enantioselective addition of diethylzinc to benzaldehyde.
Table 1. The addition of diethylzinc to benzaldehyde with ligands 1 and 2. entry ligand 1 2 3 4 5 6 7 1 1 1 2 2 2 2 solvent Toluene Hexane Tol.+Hex. Toluene Hexane Tol.+Hex. Dichloromethane yield (%) 36 85 67 35 77 70 17 ee (%) 71 58 66 74 64 78 27 conf. S S S R R R R
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 Addition 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. After 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 extracted with ether and dried over anhydrous MgSO4 and the solvent was evaporated under reduced pressure. The crude product was purified 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 activity 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.
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

1
1 2
Grkem YALIN, 2Murat ZMECOLU, 1zlem ST, 1Mehmet ZSZ
Ege University, Faculty of Pharmacy, Department of Analytical Chemistry, zmir, Turkey Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, zmir, Turkey
Determinations of interactions between DNA and drugs are important aspects of biological studies in drug discovery and pharmaceutical development processes. The interactions of some molecules with DNA have been investigated 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 development studies.
P 013
Ref: 0031
GLYCOSYLATION IN ROOM TEMPERATURE IONIC LIQUID (RTIL) USING UNPROTECTED AND UNACTIVATED DONORS
1
1
Sultan N. BAYTA, 2Tae-joon PARK, 1Robert J. LINHARDT
Rensselaer Polytechnic Institute, Department of Chemistry and Chemical Biology, Troy, NY, USA 2 Rensselaer Polytechnic Institute, Department of Chemical and Biological Engineering, Troy, NY, USA
Glycosylation occurs between a donor and an acceptor in the presence of a promoter, which activates the donor. There are various factors 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 conventional organic solvents. Only very polar organic solvents, such as formamide, dimethylformamide, dimethylsulfoxide, and pyridine, easily dissolve significant amounts of sugars. It is important to investigate 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 reactions [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 benzyl glycosides, disaccharides and oligosaccharides. RTILs facilitate the use of unactivated and unprotected donors in these reactions, resulting 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 glycosidation 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 - current trends and future directions, Curr. Org. Synth., 2, 437-451, 2005.
P 015
Ref: 0034
AMINO TERMINAL ANALYSIS OF THE ACTIVE SITE LOOP OF LACTATE DEHYDROGENASE FROM THE MALARIA PARASITE PLASMODIUM VIVAX
1
1
Bnyamin ATMI, 1Dilek SADAK, 1Venhar ELK, 2Dilek TURGUT-BALIK
University of Frat, Faculty of Arts And Sciences, Department of Biology, 23169 ElazTurkey 2 Yldz Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Davutpaa Campus, 34210 Esenler-stanbul,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 LDHs 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 P 014 Ref: 0033 DEVELOPMENT OF VALIDATED METHOD FOR RISEDRONATE BY HPLC-MS MS FROM BIOLOGICAL MATERIAL
2 1
1
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
1
1
Zeynep rem DLER, 2Glay AHN-KO, 2Hseyin YALINKAYA, Duriehvar ZER-NAL, 1Dilek EROL
Yeditepe University, Faculty of Pharmacy, Department of Analytical Chemistry, stanbul, Turkey 2 Yeditepe University, Yeditepe Salk Hizmetleri A.., GLP Laboratory, Acbadem-stanbul, Turkey
University of Frat, Faculty of Arts and Sciences, Department of Biology, 23169 Elaz, Turkey 2 Yldz Technical University, Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering , Davutpaa Campus, 34210 Esenler-stanbul, 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 difficulties 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 biological 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 resolution was obtained by using Agilent Zorbax Eclipse XDB reversed phase C18 (150x4.6mm, 5m) and alendronate was used as an internal standart. The developed method was applied succesfully to biological material. 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 analysis 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.
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 different 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 active 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 target 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 making 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 possible without losing enzymatic activity. This observation emphasizes that the active site loop is a crucial region accross the apicomplexan LDHs.
55
P 017
Ref: 0036
INTERACTION OF SOME 3,4-DIHYDROQUINOLIN-(1H)2-ONE DERIVATIVES WITH RAT LUNG SEMICARBAZIDESENSITIVE AMINE OXIDASE (SSAO)
1 1
1
Samiye YABANOLU, 2Sevil Grkem SUNAL, 2Akgl YELADA, Glberk UAR
Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 16100 Ankara, Turkey 2 Hacettepe 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, Alzheimers 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 diazoheterocyclic compounds have been introduced as promising class of reversible amine oxidase inhibitors, three compounds with 3,4-dihydroquinoline-(1H)-2-one structure and their open ring derivatives were synthesized and the interaction of these compounds with SSAO purified from rat lung were evaluated. The compounds of Q (N-amino-3,4-dihydroquinoline-(1H)-2one) and QB (1-(Benzyliden-amino)-3,4-dihiydroquinoline-(1H)-2one) were synthesized by the reduction and the ring closure reaction of o-nitro-cinnamic acid whereas MBK (Tert-butyl-N-[cyclohexylcarbamoyl-(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-phenyl-2H-quinolin-1-yl-N-cyclohexyl-2-(4-chlorophenyl)acetamide) were synthesized by one pot Ugi-Knovenagel reaction. The structures 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 enzyme non-competitively and irreversibly suggesting that these compounds 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 semicarbazide-sensitive amine oxidase (SSAO), Ugi-Knovenagel condensation, substrate, inhibition
(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) produced form superoxide radical by dismutases. In this study, effects of one water soluable antioxidant, ascorbic acid (Vit C), and one lipid soluable antioxidant, -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), diabetic (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. Moreover, combined antioxidant given groups catalase activities were also higher than those of the diabetics and this increament was as effective 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 induced diabetes.
P 019
Ref: 0038
SYNTHESIS AND ANALGESIC ACTIVITY OF SOME BENZIMIDAZOLE DERIVATIVES

1 2
1
lhan IIKDA, 2mide DEMR, 2zgr Devrim CAN, 1Yusuf ZKAY, Yusuf ZTRK
Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskiehir, Turkey 2 Anadolu University, Faculty of Pharmacy, Department of Pharmacology, 26470 Eskiehir, Turkey
P 018
Ref: 0037
EFFECTS OF LIPOIC ACID AND VITAMIN C ADMINISTRATION ON STREPTOZOTOCIN INDUCED DIABETIC RAT LIVER CATALASE ACTIVITIES
Gkhan SAD, Tlin GRAY
Middle East Technical University, Department of Biochemistry, Ankara, Turkey
Diabetes mellitus which is a glucose metabolism disorder is associated 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 enzymatic 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
Heterocyclic compounds having two nitrogen atoms oriented in 1,3-positions in ring are endowed with broad spectrum of pharmaceutical properties. Imidazole and benzimidazole, shown as the instances for these heterocyclics, drugs have broaden scope in remedying various dispositions in clinical medicine [1]. Pharmaceutical properties concern antifungal and antimycotic, antiprotozoal, antineoplastic, antiulcer, antihistaminic and antiallergic, antihypertensive, anthelmintic, antioxidant, antiviral, antibacterial and antiparasitic 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 various 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 antipsychotic [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 corresponding 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 positions 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 ortho position of phenyl ring (compound 3) causes decrease in the analgesic activity. These findings indicate the importance of chemical sturucture and pharmacological activity relationships of the synthesized 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-hydroxybenzaldehyde 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.
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, antiproliferative 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, Ichkawa 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, Arpac, T, Ak, E, Yaln , Farmaco, 58, 497, 2003.
P 021 SYNTHESIS OF 3-PHENETHYLAMINO-1PHENYL/SUBSTITUTED PHENYL-1-PROPANONE HYDROCHLORIDES

1
1
Ref: 0041
Ebru METE, 2H. nci GL, 1Cavit KAZAZ
Atatrk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey 2 Atatrk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey
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]-BISBENZIMIDAZOLES
1
1
It has been reported that acetophenone derived several mono Mannich bases have cytotoxic, antifungal and anticonvulsant activities [1-5]. In this study, synthesis of 1-aryl-3-phenethylamino-1-propanone hydrochlorides has been realised to investigate their biological 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. Gl H, Vepsalainen J, Gl M, Erciyas E, Hanninen O. Cytotoxic activities of mono and bis Mannich bases derived from acetophenone against Renca and Jurkat cells, Pharm. Acta Helv., 74, 393-8, 2000. 2. Gl H, Gl M, Erciyas E. Syntheses and stability studies of some Mannich bases of acetophenones and evaluation of their cytotoxicity against Jurkat cells, Arzneimittel Forschung, 52, 628-35, 2002. 3. Gl H, al , Vepsalainen J. Synthesis of some mono-Mannich bases and corresponding azine derivatives and evaluation of their anticonvulsant activity, Arzneimittel Forschung, 54, 359-64, 2004. 4. Gl H, ahin F, Gl M, ztrk S, Yerdelen KO. Evaluation of antimicrobial activities of several Mannich bases and their derivatives, Arch. Pharm., 338, 335-8, 2005. 5. Gl H, 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.
lhan IIKDA, 1Yusuf ZKAY, 2Zerrin NCESU
Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskiehir, Turkey 2 Anadolu University, Faculty of Pharmacy, Department of Biochemistry, 26470 Eskiehir, 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 owing 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 encouraged the medicinal chemists to synthesize the large number of novel chemotherapeutic agents. Antitumoral activities of benzimidazole and bisbenzimidazole compounds were reported in several studies. Furthermore, there are clinical anticancer drugs, known as Hoechst-33258 and Hoechst-33342 dyes including bisbenzimidazole 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
P 022
Ref: 0042
SYNTHESIS OF 1-PHENETHYL-3-AROYL-4-ARYL-4PIPERIDINOLS
1
1
Ebru METE, 2H. nci GL, 1Cavit KAZAZ
Atatrk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey 2 Atatrk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey
It has been reported that piperidinol type of compounds have cytotoxic, antifungal and anticonvulsant activities [1-4]. In this study, synthesis of 1-phenethyl-3-aroyl-4-aryl-4-piperidinols has been realised to investigate their biological activities in future.
57
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. Gl H, al , Vepsalainen J. Synthesis and evaluation of anticonvulsant activities of some bis Mannich bases and corresponding piperidinols, Arzneimittel Forschung, 52, 863-9, 2002. 2. Gl M, Gl H, Das U, Hanninen O. Biological evaluation and structureactivity relationships of bis-(3-aryl-3-oxo-propyl)-methylamine hydrochlorides and 4-aryl-3-arylcarbonyl-1-methyl-4-piperidinol hydrochlorides as potential cytotoxic agents and their alkylating ability towards cellular glutathione in human leukemic T cells, Arzneimittel Forschung, 55, 332-7, 2005. 3. Gl H, ahin F, Gl M, ztrk S, Yerdelen KO. Evaluation of antimicrobial activities of several mannich bases and their derivatives, Arch. Pharm., 338, 335-8, 2005. 4. Gl M, Atalay M, Gl H, Nakao C, Lappalainen J, Hanninen O. The effects 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.
1. Stolfi RL, Martin DS. Therapeutic activity of maleic anhydride-vinyl ether copolymers against spontaneous, autochthonous murine mammary 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 Series 186, Washington DC, 1982. 3. Kaplan Can, H., Doan, AL., Rzaev, ZMO, ner, AH, Gner, A. Synthesis and Antitumor Activity of Poly(3,4-dihydro-2Hpyran-co-maleic anhydride-co-vinyl acetate), Journal Applied Polymer Science, 96, 23522359, 2005.
P 024
Ref: 0044
ANTIFUNGAL ACTIVITIES OF 3-PHENETHYLAMINO-1ARYL-1-PROPANONE HYDROCHLORIDES AND 3-AROYL4-ARYL-1-PHENETHYL-4-PIPERIDINOLS AGAINST SOME PLANT AND HUMAN PATHOGENIC FUNGI
1 3
1
H. nci GL, 1Canan ZELGL, 2Ebru METE, 3Fikrettin AHN, Dilat YURDAKUL
P 023
Ref: 0043
SYNTHESIS AND ANTITUMOR ACTIVITY OF POLY(2,3DIHYDROPYRAN-CO-MALEIC ANHYDRIDE-CO-VINYL ACETATE)

Hatice KAPLAN-CAN, 2A. Lale DOAN, 3Zakir M. O. RZAEV, 2 Ayegl HASEGEL-NER, 1Ali GNER
1
Atatrk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey 2 Atatrk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey 3 Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering, stanbul, Turkey
Hacettepe University, Faculty of Science, Department of Chemistry, Ankara, Turkey Hacettepe University, Institute of Oncology, Department of Basic Oncology, Ankara, Turkey 3 Hacettepe University, Faculty of Engineering, Department of Chemical Engineering, Ankara, Turkey
1 2
It is known that the water soluble anhydride-containing copolymers as polyanions and their functional derivatives have high biological 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 donoracceptor-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 thermalcopolymerization of complexed comonomers, terpolymer composition behavior relationships 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) testing method using calorimetric measurements of chemotherapic effect 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 carboxylic groups and on their regular distribution in side chain of the functional macromolecules [3].
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 pathogenic fungi (Rhizoctonia soloni-EB-ML, Fusarium oxysporium CE1, Sclerotinia sclerotiorum FD3, Aspergillus niger FS2, Aspergillus flavus Hak23, Alternaria alternata FS2002, Macrophamina phaseoli CE4, Botrytis cinerea MFD3) and 3 human pathogenic fungi (Microsporum canis-AO5, Candida albicans EA-07, Candida parapsilosis EA-08) using agar dilution assay in the concentration range of 6,25 to 200 g/ml [1]. Minimal Inhibition Concetrations (MIC) of the compounds were determined. Amphotericin-B was used as a reference compounds. Amphotericin-B was only effective against Macrophamina phaseoli CE4, Aspergillus niger FS2 and Aspergillus flavus Hak23 at 100 mg/ml. None of the compounds showed antifungal activity at the concentration 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 (g/ml) were as follows: Compound (g/ml): 13(25), 14(50) against Rhizoctonia soloni2001; 10, 13, 15 (12.5) , 2, 3, 8, 9, 11, 19, 20 (25), 1 (50), 7 (200) against Microsporum canis-A5, 11 (100) against Sclerotinia sclerotiorum-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. Gl H, ahin F, Gl M, ztrk S, Yerdelen KO. Evaluation of antimicrobial activities of several mannich bases and their derivatives, Arch. Pharm., 338, 335-8, 2005.
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May 17-20, 2007
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Ref: 0045
EVALUATION OF CYTOTOXICITY OF 1-ARYL-3PHENETHYLAMINO-1-PROPANONE HYDROCHLORIDES AND 1-PHENETHYL-3-BENZOYL-4-ARYL-4-PIPERIDINOLS BY BRINE SHRIMP BIOASSAY

1
1
Murat ZMECOLU, 2H. nci GL, 3Ebru METE
Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 35100 zmir, Turkey 2 Atatrk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey 3 Atatrk 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-piperidinols (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 bioassay (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 g/ml while reference compound 5-fluorouracil exhibited 2.99 g/ml of cytotoxicity.
Table 1. Cytotoxicity of the compounds in brine shrimp bioassay. Code Series 1 13 15 17 Series 2 4 6 8 4-CH3C6H4 4-CH3OC6H4 4-ClC6H4 165.70 0.186 13.85 0.191 573.33 0.156 2,4-(Cl)2C6H3 4-NO2C6H4 4-HOC6H4 65.79 0.083 3467.52 0.469 316.82 0.122 Aryl Cytotoxicity (g/ml)
been elucidated on the basis of 1H and 13C NMR spectral data (DEPT, COSY, HMQC, HMBC and NOE). 1H NMR spectra of the compound was well resolved and the unambiguous proton chemical-shift assignments were based on the multiplicity pattern of proton resonances 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)-1phenethyl-4-piperidinol was used for data collection on a fourcircle Rigaku RAXIS RAPIDS diffractometer equipped with a twodimensional area IP detector. The graphitemonochromatized Mo K radiation (=0.71073 ) and oscillation scans technique with =5 for one image were used for data collection. The lattice parameters were determined by the leastsquares methods on the basis of all reflections with F2>2(F2). Integration of the intensities, correction for Lorentz and polarization effects and cell refinement was performed using CrystalClear software [1]. The structures were solved by direct methods (SHELXS97) [2] and nonH atoms were refined by fullmatrix leastsquares method with anisotropic temperature factors (SHELXL-97) [2]. Compound 1, 3-(p-chlorobenzoyl)-4-(p-chlorophenyl)-1-phenethyl-4-piperidinol, crystallizes in the monoclinic crystal system with space group P21/a and the following unit cell parameters: a= 10.792.(4) , b= 12.863 (5), c= 16.950 (6) ; =90, = 97.77(4), = 90; V= 2331.3(16) 3 , and Z=4. The compound 1 has intermolecular O1-HN1 [O1-H; 0.820, HN1; 2.11, O1N1; 2.878(5) , O1H-N1 156], C13H-O2 [C13-H; 0.970, HO2; 2.54, C13O2; 3.373(6) , C13H-O2 144] hydrogen bonds.
1. Rigaku (2005), CrystalClear, Version 1.3.6. Rigaku American Corporation, 9009 New Trails Drive, The woodlands, TX 773815209, USA. 2. Sheldrick GM. SHELXS97 and SHELXL97, University of Gttingen, Germany, 1997.
P 027
Ref: 0047
STRUCTURE ELUCIDATION OF MONO MANNICH BASE 1-(p-METHOXYPHENYL)-3-PHENETHYLAMINO-1PROPANONE HYDROCHLORIDE AND SEMICYCLIC MONO MANNICH BASE 3-(p-METHOXYBENZOYL)-4-(pMETHOXYPHENYL)-1-PHENETHYL-4-PIPERIDINOL USING 1D AND 2D NMR SPECTROSCOPY
1
1
Cavit KAZAZ, 1Ebru METE, 2H. nci GL
1. Gl H, Gl 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.
Atatrk University, Faculty of Science & Arts, Department of Chemistry, 25240 Erzurum, Turkey 2 Atatrk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey
P 026
Ref: 0046
CRYSTAL STRUCTURE OF 3-(p-CHLOROBENZOYL)-4-(pCHLOROPHENYL)-1-PHENETHYL-4-PIPERIDINOL

1
1
Ertan AHN, 1Cavit KAZAZ, 1Ebru METE, 2H. nci GL
Atatrk University, Faculty of Science & Arts, Department of Chemistry, 25240 Erzurum, Turkey 2 Atatrk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey
Mono Mannich base 1-(p-methoxyphenyl)-3-phenethylamino-1propanone hydrochloride and semicyclic mono Mannich base 3-(pmethoxybenzoyl)-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. Proton 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. Gl M, Gl H, Das U, Hanninen O, Arzneimittel Forschung, 55(6), 3327, 2005.
NMR Study: The characterisation of compound 1, 3-(p-chlorobenzoyl)-4-(p-chlorophenyl)-1-phenethyl-4-piperidinol, have
59
P 028
Ref: 0048
P 029
Ref: 0049
INVESTIGATION OF CASEIN KINASE 2 (CK2) ENZYME AND ITS BINDING PROPERTIES WITH P53 PROTEIN, POLYLYSINE AND HEPARIN BY MALDI-MS
1
1
CYTOTOXICITIES OF N,N-BIS(3-DIMETILAMINO-1-ARYLPROPYLIDENE)HYDRAZINE DIHYDROCHLORIDES

1 3
1
mr ELKBIAK, Cansel TAAIR, Wayne E. CRISS, Bekir SALH

1 2 1
Kaan KKOLU, 2Mustafa GL, 1H. nci GL, 3Osmo HANNINEN, Mustafa ATALAY
Hacettepe University, Faculty of Science, Department of Chemistry, 06532 BeytepeAnkara, Turkey 2 Hacettepe Unversity, Faculty of Medicine, Oncology Institute, Department of Biochemstry, 06100 Shhiye-Ankara, Turkey
Atatrk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Erzurum, Turkey 2 Atatrk University, Faculty of Medicine, Department of Physiology, Erzurum, Turkey 3 University of Kuopio, Department of Physiology, Finland
Matrix-assisted Laser Desorption/Ionization, along with electrospray ionization, is now among the most important ionization methods for nonvolatile, high molecular weight compounds, in particular peptides, proteins, oligonucleotides, oligosaccharides and synthetic polymers. MALDI has also been successfully used for the investigation 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 - and two catalytic - or - 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 variety 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 including 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 and 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 molecules and CK2 and/or between these molecules and CK2 substrates, which probably improve the susceptibility of substrates to be phosphorylated or not. In order to get information about the mechanism of noncovalent interactions between these biomolecules, MALDIMS 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 Spectrometry, Mass Spectrometry Reviews, 17, 337366, 1998. 2. Schuster N, Gtz C, Faust M, Schneider E, Prowald A, Jungbluth A, Montenarh 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.
Synthesis of N,N-bis (3-dimethylamino-1-aryl-propylidene) hydrazine 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 hydrochloride 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 Jurkat 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 9.75-13.27M. The most effective compound was 4 in five compounds studied.
1. Gl H, 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)-4HYDROXYPHENYL]-3-ARYL-2-PROPEN-1-ONES AND EVALUATION OF THEIR ANTICONVULSANT ACTIVITIES

1
1
H. nci GL, 1K. zden YERDELEN, 2nsal ALI
Atatrk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey 2 Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Shhiye-Ankara, Turkey
In this study, Mannich bases with piperidine, 1-[3-(piperidinomethyl)-4-hydroxyphenyl]-3-aryl-2-propen-1-one, B1-B5, were synthesized starting from the chalcones, 1,3-diaryl-2-propen-1-one, A1-A5. Chemical structures of the compounds have been confirmed by 1 H-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 epilepsia, B1, B2 and B3 against both types of epilepsia, can be choosen as candidate compounds to develop new anticonvulsant compounds for further studies.
1. Gl H, al , Vepsalainen J. Synthesis of some mono-Mannich bases and corresponding azine derivatives and evaluation of their anticonvulsant activity, Arzneimittel Forschung, 54, 359-64 2004. 2. Gl H, al , Vepsalainen J. Synthesis and evaluation of anticonvulsant activities of some bis Mannich bases and corresponding piperidinols, Arzneimittel Forschung, 52, 863-9 2002.
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May 17-20, 2007
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Ref: 0051
DEVELOPMENT AND OPTIMIZATION OF A SURFACE AIDED ENZYMATIC DIGESTION SYSTEM TO USE IN PROTEOMICS STUDIES
1
1
Basri GLBAKAN, 1Asl ZTRK-AL, 2Talat YALIN, 1Bekir SALH
Hacettepe Unversity, Faculty of Science, Department of Chemistry, 06532 BeytepeAnkara, Turkey 2 zmir Institute of Technology, Faculty of Science, Department of Chemistry, 35430 zmir, Turkey
Proteomics is the study of all methods covering isolation, separation, identification, characterization of all proteins in living organisms and finding their functional roles and expression of the sequences of proteins in all tissues. The anomalies in the synthesis 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 involved. After completion of the Human Genome Project, proteomics 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 information 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 addition 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 intrinsic difficulties of conventional in-solution and in-gel trypsin digestion 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 different MALDI matrices, sample preparation methods, effect of functional 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 proteomics, Electrophoresis, 21, 1145-1154, 2001.
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), Aspergillus 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 g/ml using amphotericinB as the reference compound (1). Of the compounds, A1 against plant pathogens Sclerotinia sclerotioruma and Rhizoctonia solani, while C5 against plant pathogen Fusarium oxysporum, have shown 2-4 times more powerful antifungal activity compared with amphotericin-B. Of the compounds synthesized, A1 and C5, against plant pathogenic fungi can be choosen as candidate compounds for further studies to develop new antifungal compounds.
1. Gl H, ahin F, Gl M, ztrk S, Yerdelen KO. Evaluation of antimicrobial 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)-4HYDROXYPHENYL]-3-ARYL-2-PROPEN-1-ONES AND EVALUATION OF THEIR CYTOTOXIC ACTIVITIES AGAINST HUMAN T LYMPHOCYTES (JURKAT) CELLS AND RAT SKELETAL MUSCLE DERVED MYOBLAST CELLS (L6)
1 3
1
H. nci GL, 2Mustafa GL, 1K. zden YERDELEN, 3Osmo HANNINEN, Mustafa ATALAY
Atatrk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey 2 Atatrk University, Faculty of Medicine, Department of Physiology, 25240 Erzurum, Turkey 3 University of Kuopio, Faculty of Medicine, Department of Physiology, 1627 Kuopio, Finland
P 032
Ref: 0052
SYNTHESIS OF 1-[3-(DIBENZYLAMINOMETHYL)-4HYDROXYPHENYL]-3-ARYL-2-PROPEN-1-ONES AND EVALUATION OF THEIR ANTIFUNGAL ACTIVITIES

1
1
K. zden YERDELEN, 1H. nci GL, 2Fikrettin AHN
Atatrk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey 2 Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering, 34755-Kayda, stanbul, Turkey
In this study, Mannich bases with dibenzylamine 1-[3-(dibenzylaminomethyl)-4-hydroxyphenyl]-3-aryl-2-propen-1-one, (C1C5) 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 compared with their precursor chalcones (A1-A5) against 3 fungi species pathogenic in humans [Trichophyton rubrum (Hak-8), Trichophyton
In this study, Mannich bases with piperidine, 1-[3-(piperidinomethyl)-4-hydroxyphenyl]-3-aryl-2-propen-1-one, B1-B5 were synthesized starting from the chalcones, 1,3-diaryl-2-propen-1-one, A1A5. Chemical structures of the compounds have been confirmed by 1 H-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 human 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 cytotoxicity 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 corresponding chalcones. While all compounds synthesized had 3.20-9.43 times more powerful cytotoxicity than 5-FU against Jurkat cells, except 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. Gl M, Gl H, Das U, Hanninen O, Arzneimittel Forschung, 55(6), 3327, 2005.
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Ref: 0056
DETERMINATION OF PHYSICOCHEMICAL PROPERTIES OF SEVERAL SULFONAMIDES BY LIQUID CHROMATOGRAPHY IN ACETONITRILE-WATER BINARY MIXTURES
1
1
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-activity relationships between antibacterial activities and physicochemical properties of sulfonamides, J. Vet. Pharmacol. Therap., 20, 276-283, 1997. 2. Botsoglou NA, Fletouris DJ, Simeonidou EJ, Psomas IE. Retention behavior of multiple sulfonamides in various liquid chromatographic systems, 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 compounds, Chromatographia, 59, 79-87, 2004.
Nurullah ANLI, 1Gleren ALSANCAK, 2Adil DENZL
Sleyman Demirel University, Faculty of Science and Literature, Department of Chemistry, Isparta, Turkey 2 Hacettepe 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 chromatographic determination of pKa values of sulfonamides and the effect of the organic modifier content on the pKa values of these compounds are scarce, but the investigation of Mengelers et al. should be mentioned [1]. In this study, the dissociation constants of related series of sulfonamides (sulfodiazine, sulfothiazole, sulfomerazine, sulfomethazine, sulfomonomethoxine, sulfodoxine, sulfomethoxazole) were determined 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 according 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 sulfonamides are shown in Figure 1.
P 035
Ref: 0057
CONTROLLED RELEASE OF NAPROXEN FROM POLY (VINYL ALCOHOL) / SODIUM ALGINATE MICROSPHERES
1
1 2
Oya ANLI, 2Ebru KONDOLOT-SOLAK
Gazi University, Arts and Sciences Faculty, Department of Chemistry, 06500 Ankara, Turkey Gazi University, Atatrk Vocational College, Department of Chemistry, Ankara, Turkey
In this study microspheres of poly(vinyl alcohol)/sodium alginate (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 microscopy (SEM). Microspheres were also characterized by measuring the particle diameter, equilibrium swelling values and determining release profiles. 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 considerably. 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

1
1
Nursabah E. BAI, 2Demet SALMAN
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, sulfomethazine, sulfomonomethoxine, sulfodoxine, sulfomethoxazole).
Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, Turkey 2 Turkish Republic Instutition of Social Security, Ankara, Turkey
Their pKa values in ACN water mixture (50% v/v) were also potentiometricaly determined [3] and compared with the results obtained by LC methodology. The retention data of sulfonamides obtained by Reversed Phase Liquid Chromatography (RPLC) were correlated with the molecu-
Cabergoline is a synthetic dopamine agonist having high affinity to D2 receptors and used for early and advanced Parkinsons patients and hyperprolactinemy disorders [1]. Chromatographic and radioimmunoassay methods have been reported for quantification of cabergoline in body fluids [2-5], but there was not any spectrophotometric 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 determination of cabergoline in pharmaceutical preparations.
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Determination of cabergoline was performed by UV-VIS spectrophotometry at 280 nm wavelength and by 2nd derivative spectrophotometry at a range of 227-232 nm. HPLC (Shimadzu SCL-10AVP) connected to an electrochemical detector (Decade) was used in comparison 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 applications. Developed methods were validated according to the regulations 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 methods were linear over the range of 1-125 g mL-1. The limit of detection for the methods was 0.5 g mL-1 (RSD = 0.75%, Bias = 0.30) and the limit of quantification was 1.0 g 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 pharmaceutical preparations and there was no statistically significant difference 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, precise 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 parkinsons disease, Annals of Pharmacotheraphy, 31(10), 1205-17, 1997. 2. Persiani S, Pianezzola E., Broutin F, Fonte G, Benedetti MS. Radioimmunoassay 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 cabergoline in plasma and urine by high-performance liquid chromatography 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 spectrometry, Rapid Communications in Mass Spectrometry, 12, 33-39, 1998. 5. Igarashi K, Hotta K, Kasuya F, Abe K, Sakoda S. Determination of cabergoline and L-dopa in human plasma using liquid chromatography-tandem mass spectrometry, Journal of Chromatography B, 792, 55-61, 2003.
Ethanesulfonic acid hydrazide, C2H5SO2NHNH2, (esh) ; 5-methyl2-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, Salmonella enteritidis ATCC 13076, Staphylococcus aureus ATCC 25923 by using MICs method. MICs 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 (MICs values g/mL). All compounds exhibited the most activity against to Staphylococcus aureus between 60-672g. The presence of the NH group in the sulfonamides contributes positively to the increase of the activity of compounds against bacteria. In addition 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, zdemir , Karacan N et al., Z. Naturforsch., 54 b , 1553, 1999. 4. Zanatta N, Alues SH, Coelho HS et al., Bioorganic & Medicinal Chemistry, 15, 1947, 2007.
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DETERMINATION OF DISSOCIATION CONSTANTS OF SEVERAL SULFONAMIDES BY POTENTIOMETRY IN METHANOL, 2-PROPANOL, TETRAHYDROFURAN-WATER BINARY MIXTURES
1 3
1
Senem ANLI, 1Ebru UBUK-DEMRALAY, 2Hale SELM, Jose Luis BELTRAN
Sleyman Demirel University, Science & Literature Faculty, Department of Chemistry, 32260 Isparta, Turkey 2 Sleyman Demirel University, Research Centre, 32260 Isparta, Turkey 3 Barcelona University, Department of Analytical Chemistry, 08028 Barcelona, Spain.
P 037
Ref: 0059
INVESTIGATION OF ANTIMICROBIAL ACTIVITES OF NEW SULFONYLHYDRAZONE DERVATVES ON SOME MICROORGANISMS

mmhan ZDEMR-ZMEN, Fatma HAMURCU
Gazi University, Arts and Sciences Faculty, Department of Chemistry, 06500 Ankara, Turkey
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.
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 activity [1]. Methane sulfon amide residue has appeared as a suitable pharmacophoric equivalent to replace functional groups in drug design . Having hydrophilic character, like the sulfonyl group is considered as a suitable pharmacophoric equivalent for replacing functional groups in drug design [2]. In previous paper, we reported the antibacterial and cytotoxic effect of methanesulfonic acid hydrazide (msh) [3].
Fig 1. Scheme of dissociation equilibrium of sulfonamides.
Literature studies of the potentiometric determination of pKa values 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 obtained 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 investigated 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 upward 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 determination [3].
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 samples by liquid-liquid extraction with diethyl ether. The assay exhibited 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 precision (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 tablets 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.
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Ref: 0062
THE EFFECT OF TAUROLIDINE ON THICKNESS OF SCAR TISSUE IN RABBIT MODEL

1
1
Ali HAYAT, 2Fsun TEMAMOULLARI, 3Fsun BABA
University of Harran, Faculty of Veterinary Medicine, Department of Surgery, anlurfa, Turkey 2 University of Harran, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, anlurfa, Turkey 3 University of Harran, Faculty of Medicine, Department of Pathology, anlurfa, Turkey
Fig 2. Potentiometric titration curve of sulfomethaxazole in MeOHH2O, 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-activity relationships between antibacterial activities and physicochemical 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 ATE, Sami EREN, Selma ZLHAN, Tuncel ZDEN
Novagenix Bioanalytical R&D Centre, Ankara, Turkey
Indapamide, 3-(aminosulfonyl)-4-chloro-N-(2,3-dihydro-2-methyl-1H-indol-1-yl)-benzamide, is an oral antihypertensive and diuretic agent [1]. Indapamide inhibits carbonic anhydrase enzyme, which reduces the vascular response to noradrenalin and angiotensin II by inhibiting the transportation of Ca+ into vascular smooth muscle. It shows diuretic effect by acting at the first parts of distal tubules [2].
Clinicans have used numerous strategies to combat wound infections, 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 taurinamide which reacts with cell wall constituents of microbial pathogens via methylene iminium ions preventing bacterial adhesion to biological surfaces. Taurolidine has a short half-life and is metabolised 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 solution 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: 2500200) were taken for the study. All rabbits were anesthetized with intramuscular 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 prepared 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 parameters, such as the thickness of scar tissue. Macroscopically, the wounds treated with 10% povidine iodine solution 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% taurolidine 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 positively 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 Medications, Clin. Tech. Equine Pract., 3, 164-172, 2004. 3. Kl, S, Timurkan, N, nsald, S, Gnay, C, stek, , Ylmaz, B. Comparison 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 patients, International Journal of Antimicrobial Agents, 24, 491-495, 2004.
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Ref: 0064
COMPARISON OF THE ROYAL JELLY AND POVIDONE IODINE ON WOUND HEALING IN RABBITS
1
1
Fsun TEMAMOULLARI, 2Ali HAYAT, 3Fsun BABA
University of Harran, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, anlurfa, Turkey 2 University of Harran, Faculty of Veterinary Medicine, Department of Surgery, anlurfa, Turkey 3 University of Harran, Faculty of Medicine, Department of Pathology, anlurfa, Turkey
P 041
Ref: 0063
THE QUANTITATIVE DETERMINATION OF FEXOFENADINE IN HUMAN PLASMA BY LC/MSD SYSTEM

Mberra EN, Evren LEYEN, Selma ZLHAN, Suna TOPTAN, Tuncel ZDEN
Novagenix Bioanalytical R&D Centre, Ankara, Turkey
Fexofenadine hydrochloride, ()-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-butyl]-, -dimethyl benzeneacetic acid hydrochloride has an empirical formula, C32H39NO4.HCl with a molecular weight of 538.1 which is the active metabolite of terfenadine 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 developed and validated for quantification of fexofenadine in human plasma. The LC-MS system was operated under the positive electrospray ionisation mode (ESI). After liquid-liquid extraction, fexofenadine analysis was performed through a C18 column with a mobile 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, coefficient correlation was 0.9993 0.9999. In quality control samples, 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 standard 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 fexofenadine 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.
Royal jelly (RJ) has been used worldwide for many years as medical products, health foods and cosmetics [1]. A number of biological and immuno-regulatory actions attributed to RJ have been reported. 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 hydrochloride (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 diameter were created using a template prepared from an X-ray film. Following incision different wounds of each animal were treated with RJ (83 mg/ml Royal Jelly-Arjel 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 beginning of healing, granulation of the tissue and the first day of epithelization 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 epithelization 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 gauzeapplied wounds, whereas it wasnt completed on 0.9 % sodium chloride 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 process (to POD 4) was followed by the contraction process (to PODs 6-8). The contraction in RJ gauze-applied wounds (to PODs 16) became more than other wounds. The thickness of scar tissue was significantly 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 conclusion, RJ application in full-thickness skin defects in rabbits accelerated wound healing.
1. Hidaka S, Okamoto Y, Uchiyama S, Nakatsuma A, Hashimoto K, Ohnishi 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 044
Ref: 0066
INVESTIGATION OF CYTOTOXIC EFFECTS OF ANHYDRIDE CONTAINING WATER-SOLUBLE COPOLYMERS ON L929 MOUSE FIBROBLASTS
1 2
1
TOPIC ADMINISTRATION OF YARROW EXTRACT ON WOUND IN RABBITS

1
1
Fsun TEMAMOULLARI, 2Ali HAYAT, 3Fsun BABA
Esin AKBAY, Handan SEVM, zer Aylin GRPINAR, Hatice KAPLAN-CAN, 1Mehmet Ali ONUR, 3Zakr M. O. RZAEV, 2Ali GNER
1 1
Hacettepe University, Faculty of Science, Department of Biology, 06800 BeytepeAnkara, Turkey 2 Hacettepe University, Faculty of Science, Department of Chemistry, 06800 BeytepeAnkara, Turkey 3 Hacettepe University, Department of Chemical Engineering, Faculty of Engineering, 06800 Beytepe-Ankara, Turkey
University of Harran, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, anlurfa, Turkey 2 University of Harran, Faculty of Veterinary Medicine, Department of Surgery, anlurfa, Turkey 3 University of Harran, Faculty of Medicine, Department of Pathology, anlurfa, Turkey
Synthetic polymers, water-soluble or in the form of hydrogels, nanoparticles, dendrimers or microspheres, are materials with which we are in daily contact or which are under development as materials 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 physicochemical characteristics of synthetic polymers exceeded those of materials available from natural sources [1]. In this study, complex-radical copolymerization of maleic anhydride (MA), and acrylic acid (AA) and ternary polymerization of maleic anhydride (MA), vinyl acceptordonoracetate (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 o C under a nitrogen atmosphere. The co- and terpolymer synthesized by the use of 1: 1 and 1: 2: 1 molar ratio of initial monomers, respectively. 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. Following a 24 hour of incubation, the cell culture medium was removed and fresh medium containing poly(MA-co-AA) and poly(MA-coVA-co-AA) was added. Poly(MA-co-AA) and poly(MA-co-VA-coAA) 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 apoptosis of treated cells and of the control group. The results showed that there were differences between poly(MAco-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 proliferation characteristics of cells. Cytotoxicity of results can be explained 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 biologically active materials, Annals of the New York Academy of Sciences, 446 (1), 160168, 1985. 2. Kaplan CH, Doan AL, Rzaev ZMO, Uner AH, Gner A. Synthesis, characterization and antitumor activity of poly(maleic anhydride-co-vinyl acetate-co-acrylic acid), Journal Applied Polymer Science, 100, 34253432, 2006.
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 effects 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 extract 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 epithelization 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 proliferation progression was significantly different on PODs 4 and 16 within yarrow extract group. Such a relation was not found on PODs 8-12 ((P> 0.05). The degree of inflammatory cell infiltration was significantly 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% povidoneiodine 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. Assesment of two medicinal plants Psidium guajava L. and Achillea millefolium L. in vitro and in vivo assays, Genetics and Moleculer Biology, 26(4), 551-555, 2003. 2. Baytop, T. Trkiyede Bitkiler ile Tedavi (Gemite ve Bugn), stanbul niversitesi Eczaclk Fakltesi, stanbul, s.166-167, 1999. 3. Frastvedt E., Stashak TD, Othic A. Update on Topical wound medications, Clinical Techinique Practice, 3, 164-172, 2004. 4. Candan F, nl M, Tepe B, Daferera D, Polissiu M, Skmen A, Akpulat HA. Antioxidant and antimicrobial activity of the essential oil and methanol 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

1 2
1
Handan SEVM, 1Esin AKBAY, 1zer Aylin GRPINAR, Zafer C. EHREL, 1Mehmet Ali ONUR, 1Akn TMER
Hacettepe University, Faculty of Science, Department of Biology, 06800 BeytepeAnkara, Turkey 2 Hacettepe University, Faculty of Dentistry, Department of Pediatric Dentistry, 06100 Shhiye-Ankara, Turkey
Although previous studies have suggested that topical use of dexamethasone 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 separately. 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 interferon-beta gene expression by dexamethasone in murine L929 cells, Molecular Endocrinology, 9, 199-207, 1995.
In this present study, poly(maleic anhydride-alt-acrylic acid) copolymer, 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 compatible blends were obtained due to this strong hydrogen bond formation 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-microbial activity of anhydride copolymers and their derivatives prepared by ionizing radiation, Eur. Polym. J., 40, 2203-2210, 2004.
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Ref: 0070
KINETIC EVALUATION OF THE SUBSTITUTIONS ON BISBENZIMIDAZOL DERIVATIVES ON THEIR INHIBITORY ACTIVITIES OF MAMMALIAN DNA TOPOISOMERASE I
1 2
1
Sevil ZENCR, 2A. Selcen ALPAN, 2Pnar ALCIL, 2Gne OBAN, H. Semih GNE, 1Zeki TOPU
Ege University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, 35100 zmir, Turkey 2 Ege University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 35100 zmir, Turkey
P 046
Ref: 0069
INVESTIGATION OF INTERACTIONS IN POLY(MA-ALT-AA)/ PVP BLENDS

1
1 2
Hatice KAPLAN-CAN, 1Serap KAVLAK, 1Ali GNER, 2Zakir M. O. RZAEV
Hacettepe University, Faculty of Science, Department of Chemistry, Ankara, Turkey Hacettepe 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 interaction 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].
1H-Benzimidazole derivatives are known to have antibacterial, antifungal, 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 catalyzing the concerted breakage and rejoining of DNA strands during many genetic processes including DNA replication, transcription, recombination and transposition [3]. Because of the increased awareness 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-2yl)ethane and showed a significant interference of this compound on the mammalian type I topoisomerase using in vitro plasmid supercoil relaxation assays [4-6]. A known topoisomerase I poision, Camptothecin, 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. Gne HS, Coar G. Arzneimittel-Forschung/ Drug Res., 42, 1045-1048, 1992. 2. Alpan AS, Gne HS, Topu Z. 1H-Benzimidazole Derivatives As Mammalian 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. Topu Z, Castora FJ. Biochim. Biophys. Acta, 1264, 377-387, 1995. 6. Topu Z. J. Clin. Pharm. Ther., 26, 405-416, 2001.
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LC-DAD METHOD FOR THE DETERMINATION OF pKA VALUES OF SULFONAMIDES AND OPTIMIZING CHROMATOGRAPHIC SEPARATION BY VARYING SOLVENT COMPOSITION
1 3
1
SYNTHESIS AND CHARACTERZATION OF NOVEL DENDRITIC POLYMERS FOR ANTI-CANCER DRUG ENCAPSULATION AND RELEASE
1
1 2
Esra G, 2Gngr GNDZ, 1Ufuk GNDZ
Nurullah ANLI, Gleren ALSANCAK, Zerrin ERDEMGL, Jose Luis BELTRAN, 3Jose BARBOSA
1 2
Middle East Technical University, Department of Biological Sciences, Ankara, Turkey Middle East Technical University, Department of Chemical Engineering, Ankara, Turkey
Sleyman Demirel University, Science & Literature Faculty, Departament of Chemistry, 32260 Isparta, Turkey 2 Anadolu Unversity, BBAM, 32260 Eskiehir, Turkey 3 Barcelona University, Department of Analytical Chemistry, 08028 Barcelona, Spain
Systematic optimization of liquid chromatographic (LC) methods requires an accurate knowledgement of the parameters that influence the separation of the compounds. The linear correlation between the logarithm of retention factor and Reichardts 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 dissociation behavior of the compounds and the pH of the acetonitrilewater mobile phases [1]. The aim of this work is to analyze sulfodiazine, sulfothiazole, sulfomerazine, sulfomethazine, sulfodoxine, sulfomonometoxine, sulfomethoxazole like sulfonamides efficiently using HPLC method. The separation conditions have been optimized using the relationships between Reichardts 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 modifier 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 retention of sulfonamides is expected to pass through a maximum at an intermediate mobile phase pH. The pH dependent retention profiles 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 values 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 suggests that the R side chain plays an important role in the hydrophobic 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, Chromatographia, 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. Jimnez-Lozano E, Marqus I, Barrn D, Beltrn JL, Barbosa J. Determination 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.
Controlled drug delivery by biocompatible artificial systems has become one of the most interested research areas because of the ability to reduce the problems of conventional chemotherapy. By dendritic polymer 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 systems with their encapsulation stability for hydrophobic drugs and with their biosafety properties [2]. The aim of this study is to design hyperbranched 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 conjugated 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 wavelenght 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 dendritic 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 polymer have indicated the expected hydroxyl end groups. SEC studies have shown the molecular weight distribution of the dendritic polymer. According to the molecular characterization results, various polymer to drug ratios were tested for encapsulation studies.The encapsulation efficieny variations with respect to hyperbranched polymer (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 decreased with increasing drug ratio. Sustained release profiles of dendritic 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, Bioconjugate 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, Gndz G, Ksakrek D, Akhmedov . M. Synthesis and characterization of hyperbranched and air drying fatty acid based resins, Prog. Org. Coat., 55, 330-336, 2006.
when PANI and PVF+ClO4- were codeposited. The peaks belonging to both PVF and PANI were observed clearly from their cyclic voltammograms. 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 enzyme. 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. Optimum conditions for the determination of the relationship between the response current and the concentration of catechol are that the potential 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 M and from 3.91 to 64 000 M, respectively. 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.
P 050 DETERMINATION OF CATECHOL USING MODIFIED ELECTRODE WITH A COMPOSITE OF POLY(VINYLFERROCENE) AND POLYANILINE
Ref: 0073
Muammer KAVANOZ, Nuran ZEK-PEKMEZ, Kadir PEKMEZ, Attila YILDIZ

Hacettepe 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 clinical 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, isoprenalin, 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 development of new methods, that makes possible the minimal use of reagent 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 polymers due to its high conductivity, good redox reversibility and good stability in aqueous solutions and air. These properties provide favorable 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 methylene chloride medium. Thin and more adhesive films were obtained
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 M to 500 M, d) from 3.91 M to 64000 M. 1. Rita de Cassia Silva Luz, Flavio Santos Damos, Adriano Bof de Oliveira, Johannes 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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THE UV-SPECTROSCOPIC METHOD FOR DETERMINATION OF DISSOCIATION CONSTANTS OF SEVERAL SULFONAMIDES IN WATER AND ACETONITRILE-WATER BINARY MIXTURES
1
1
DETERMINATION OF PHYSICOCHEMICAL PARAMETERS AND SAR STUDY ON THE SOME BENZIMIDAZO[1,2a]PYRIMIDINE DERIVATIVES
Asiye MER
Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskiehir, Turkey
Senem ANLI, 1Gleren ALSANCAK, 2Jose Luis BELTRAN, 2Jose BARBOSA
Sleyman Demirel University, Science and Literature Faculty, Department of Chemistry, 32260 Isparta, Turkey 2 Barcelona University, Department of Analytical Chemistry, 08028 Barcelona, Span.
Sulfonamides are anti-bacterial and anti-infective drugs commonly used to treat in medicine and veterinary practice. The physicochemical profiling of drugs includes the determination of their dissociation constants. Data on proton dissociation of pharmacologically active substances are of utmost interest for the understanding of stability and solubility of drugs. A major factor of drug permeation 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 concentration and allows suitable absorbance measurement in aqueous solution even for products with low aqueous solubility. Acetonitrile (ACN)-water mixtures are usually employed for pKa determination 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, sulfamonomethoxine, 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].
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 revealed 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), molecular refraction (MR) [2], molecular volume (MV) [3], molecular connectivity index (MCI) [4], paracor (Par) [5-7]}, hydrophobic {partition coefficient [8], hydrophobic substituent constant () [9]} and electronic{electronic substituent constant [10]} parameters of 2,4-di- and 2,3,4-trisubstituted benzimidazo[1,2-a]pyrimidine derivatives 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 1 2 3 4 5 R1 OH OH OH OH OH R2 H H H Ph Me R3 Me Ph n-Pr Me Me Compound 6 7 8 9 10 R1 Ph Ph Me Me Me R2 H H H Me Et R3 Me Ph Me Me Me
Fig 1. UV-spectrum of sulfadiazine obtained at various pH in ACNwater 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. Jimnez-Lozano E, Marqus I, Barrn D, Beltrn JL, Barbosa J. Determination 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 sulfonamides in capillary electrophoresis, J. Chromatogr. A, 792, 37-47, 1997.
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 antineoplastic activity.
1. Meri A, ncesu Z, Karayel A, zbey 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 quantitative structure-activity studies, Eur. J. Med. Chem.-Chim. Ther., 12, 109112, 1977. 3. Berkem AR, Baykut S., Fizikokimya Kitab, stanbul niversitesi Yaynlar, Say: 2735, Kimya Fakltesi, No. 42, 1980. 4. Kier LB, Hall LH. Derivation and significance of valence molecular connectivity, J. Pharm. Sci. 70, 583-9, 1981. 5. Quayle QR. The parachors of organic compounds, Chem. Rev., 53, 43989, 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. Aliphatic hydrocarbons, J. Chem. Soc., 133, 1946. 8. Rekker RF, De Kort HM. The hydrophobic fragmental constant, an extension 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, Nikaitan D, Lien EJ. Aromatic substituent constant for structure activity correlations, J. Med. Chem., 16, 1207-26, 1973. 10. Ak-ener E, Yaln . Kantitatif Yap-Etki likileri Analizleri (QSAR), Ankara niversitesi Eczaclk Fakltesi Yaynlar No: 86, 2003. (ISBN 975-482-585-8)
P 054
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EFFECT OF USNIC ACID ON TISSUE NITRIC OXIDE SYNTHASE ACTIVITY AND GLUTATHIONE LEVEL IN TITANIUM-IMPLANTED SUBJECTS
Fehmi ODABAOLU, 2Hayati AYGN, 2mer Selim YILDIRIM, Zekai HALICI, 1Mesut HALICI, 4Zafer OKUMU, 5Ali ASLAN, 6 Ahmet AKIR, 7Cavit KAZAZ
1 3
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ENHANCED ENZYME ACTIVITY OF IMMOBILIZED LIPASE AS BIOCATALYST FOR SYNTHESIS OF ESTERS IN ORGANIC MEDIA
Taylan K. ZTRK, Funda KARTAL, Ali KILIN
Ege University, Faculty of Science, Department of Biochemistry, zmir, Turkey
Atatrk University, Faculty of Pharmacy, Department of Biochemistry, 25240 Erzurum, Turkey 2 Atatrk University, Faculty of Medicine, Department of Orthopedics and Traumatology, 25240 Erzurum, Turkey 3 Atatrk University, Faculty of Medicine, Department of Pharmacology, 25240 Erzurum, Turkey 4 Atatrk University, Faculty of Veterinary Medicine, Department of Surgery, 25240 Erzurum, Turkey 5 Atatrk University, Kazm Karabekir Education Faculty, Department of Biology, 25240 Erzurum, Turkey 6 Atatrk University, Kazm Karabekir Education Faculty, Department of Chemistry, 25240 Erzurum, Turkey 7 Atatrk University, Faculty of Science, Department of Chemistry, 25240 Erzurum, Turkey
1
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 enhancement 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 transesterification (include alcoholysis, acidolysis and interesterification). Lipase catalized esterification reactions have been actively pursued 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 polyhydric alcohols like glycerol, sorbitol and other carbohydrates (calledemulsifiers/surfactants) find immense application in food and pharmaceutical 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 effects of organic solvent type on esterification activity were studied. The effects of reaction time and reaction temperature were also studied. 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. Kln A, nal S, Telefoncu A. Chemical attachment of porcine pancreatic lipase to crosslinked poly(vinyl alcohol) by means of adipoyldichloride, Process Biochemistry, 38, 641-647, 2002.
Debris due to the frictions as well as biochemical and magnetic reactions 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 cytotoxicity [1, 2]. Usnic acid (UA) (Fig. 1) is a dibenzofuran derivative biosynthesised 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]. Eighteen 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 determined in the activities of iNOS (inducible), cNOS (constitutive), and tNOS (total) respectively in Ti-implanted rabbits compared to control 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. Surgical 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 administration may affect cytotoxicity via suppressing iNOS activity and increasing GSH level.
Keywords: Titanium implant, usnic acid, nitric oxide synthase, glutathione
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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. Treatments NITRIC OXIDE SYNTHASE (NOS) ACTIVITY (mol/min/mg tissue)a N tNOS cNOS iNOS Amount of GSH (nmol/mg tissue)a
TIT+UA (local) TIT+UA (oral) TIT+OO (local) TIT+OO (oral) TIT (control) Healthy tissue
a
3 6.840.27** 3.070.17** 3.770.19** 3.440.02* 3 2.580.19** 2.470.18** 0.120.07** 3.960.04** 3 3.650.27** 3.520.25* 0.130.02** 3.520.12* 3 3.400.37** 2.850.40** 0.550.05** 2.510.13* 3 5.430.21** 4.390.22** 1.040.03** 2.910.08** 3 3.190.08 2.720.06 0.480.02 4.210.01
MeansSEM of tissues of six legs in each group. N: The number of rabbits. *Significant at p<0.05; **Significant at p<0.01.
The basic structures of these compounds were confirmed by IR, H-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 cm1 in their IR spectra assignable to C=O groups. Also, compounds 3 and 4 have a one stretching band at 1783-1779 cm1. 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 -NCH2-CH2-. Characteristic singlet peak of NH for compounds 3 and 4 were observed at 12.4 ppm.
1
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 apoptosis, Am. J. Med. Gen., 106, 62-70, 2001. 3. Odabaolu F, akr A, Sleyman H, Aslan A, Bayr Y, Halc M, Kazaz, C. Gastroprotective and antioxidant effects of usnic acid on indomethacine-induced gastric ulcer in rats, J. Ethnopharmacology, 103 (1), 59-65, 2006.
1. Malawska B. New Anticonvulsant Agents, Curr Top Med Chem 5(1), 6985, 2005. 2. Popp FD. Potential anticonvulsants. IX. Some isatin hydrazones and related 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 (Weinheim), 335(4), 129-134, 2002. 5. Tacconi G, Righetti PP, Desimoni G. Einfache Darstellung von N-substituierten Isatinen, J. Prakt. Chem., 315(2), 339-344, 1973.
P 055
Ref: 0078 P 056 Ref: 0079
SYNTHESIS AND CHARACTERIZATION OF SOME NOVEL ISATIN DERIVATIVES IN WHICH ANTICONVULSANT ACTIVITY IS PREDICTED
Ebubekir SEPTOLU, Mutlu DLSZ-AYTEMR, nsal ALI
Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100-Shhiye, Ankara, Turkey
PHENOLIC COMPOUNDS OF SIDERITIS OZTURKII AND THEIR IN VIVO ANTI-INFLAMMATORY AND ANTINOCICEPTIVE ACTIVITIES
1
1
Pnar AHN, 2Esra KPEL, 1hsan ALI, 1Nurten EZER, 3Erdem YELADA
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 future work. The synthesized compounds were prepared by Tacconi and colleagues 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-bromoethyl)-1H-indole-2,3-dione with benzoxazol-2-one derivatives gave compounds 1 and 2. Final compounds 3 and 4 which have phenylhydrazone group were prepared by heating compounds 1 or 2 with phenylhydrazine in ethanol.
Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Ankara, Turkey 2 Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, Ankara, Turkey 3 Yeditepe University, Faculty of Pharmacy, Department of Pharmacognosy, stanbul, 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 phytochemical studies on Turkish Sideritis species, we now report the isolation of phenolic compounds from S. ozturkii Ayta & 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 antinociceptive 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 spectroscopic 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. Although the activity of ozturkosides A and B were found insignificant 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-inflammatory activities together.
1. Ayta 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 Tp Publications, stanbul, p.375, 1999. 3. Ezer N, Vila R, Caigueral S, Adzet T. Essential oil composition of four Turkish species of Sideritis, Phytochem., 41, 203-205, 1996. 4. Akco Y, Ezer N, zelik B, Abbasolu U. Iridoid glycosides from Sideritis lycia Boiss & Heldr. and its antimicrobial activities, FABAD J. Pharm. Sci., 23, 99-103, 1998. 5. ahin FP, Tademir D, Redi P, Ezer N, al . Three new acylated flavon glycosides from Sideritis ozturkii Ayta & Aksoy, Phytochem., 65, 20952099, 2004; 66, 125, 2005. 6. Kpeli E, Harput U, Varel M, Yeilada E, Saracolu . Bioassay-guided isolation of iridoid glucosides with antinociceptive and anti-inflammatory activities from Veronica anagallis-aquatica L., J. Ethnopharmacol., 102, 170176, 2005.
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 2.57fold increase in activity of inducible nitric oxide synthase (iNOS) in CAR-induced rats compared to control rats. However, oral administration of ALA, IND and DIC significantly decreased iNOS activity. All doses of ALA were more effective than IND and DIC for decreasing iNOS activity. These results suggest that the anti-inflammatory 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. Dose mg/kg body wt Acute antiinflammatory effect (Inhibition %) Chronic antiinflammatory effect (Inhibition %) Number of animals
P 057
Ref: 0080
Treatment ALA DIC IND CONTROLS
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
Fehmi ODABAOLU, 2Zekai HALICI, 3Hayati AYGN, 1Mesut HALICI, 1 Yasin BAYIR, 4Ahmet AKIR, 5Elif ADIRCI, 1Fadime ATALAY
1
2x6 2x6 2x6 2x6 2x6 2x6
50 100 200 25 25 -
18.5 29.6 40.7 44.4 55.5 -
67.7 68.9 69.9 76.1 83.8 -
Atatrk University, Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey

1
Alpha-lipoic acid (ALA) is a dithiol that is found naturally in mitochondria as the coenzyme for pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. ALA has been shown to combat oxidative stress by quenching a variety of intracellular reactive oxygen 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, nitric 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 investigated 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 SpragueDawley 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/
Dose mg/kg body wt.
Atatrk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey Atatrk University, Faculty of Medicine, Department of Orthopedics and Traumatology, Erzurum, Turkey 4 Atatrk University, Kazm Karabekir Education Faculty, Department of Biology, Erzurum, Turkey 5 Atatrk University, Faculty of Pharmacy, Department of Pharmacology, Erzurum, Turkey
2 3
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. Number of animals
cNOS
Treatment ALA CAR+DIC CAR+IND CAR HEALTHY
6 6 6 6 6 6 6
50 100 200 25 25 -
2.700.53* 0.170.02** 2.860.55 3.100.25* 0.130.05** 3.230.29* 3.230.37* 0.130.03** 3.370.37* 2.280.07 2.040.05 2.220.25 2.510.37 0.180.03** 2.460.07 0.220.01** 2.250.05 0.360.06** 2.580.24 0.140.03 2.650.37
1. Bilska A, Wlodex L. Lipoic acid- the drug of future?, Pharmacol Report, 57, 570-577, 2005. 2. Odabaolu F. Alpha lipoic acid, Pharma ark, 1 (4), 12-15, 2006.
tNOS
iNOS
73
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. Sleyman H, Odabaolu F, Aslan A, akr A, Karagz Y, Ger F, Halc M, Bayr Y. Antiinflammatory and antiulcer effects of aqueous extract of Lobaria pulmonaria, Phytomedicine, 10 (6-7), 552-557, 2003.
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 IND+MLK IND+MLK N 6 6 6 6 6 6 6 6 Dose mg/kg body wt. 5 10 20 25 25 30 25 Ulcer index (UI) 2.980.88** 2.210.91** 2.030.49** 0.160.08*** 2.540.76** 0.00*** 7.290.43*** % Inhibition 59.1 69.7 72.2 97.8 65.2 100 -
P 058
Ref: 0081
IND+MLK IND+FAM IND+RAN IND+LAN IND Healthy
GASTROPROTECTIVE EFFECT OF MONTELUKAST (SINGULAIRE) ON INDOMETHACINE-INDUCED GASTRIC ULCER IN RATS AND ITS RELATION WITH SOME GLUTATHIONE METABOLISM PARAMETERS
1 4
1
Gnnur ZBAKI-DENGZ, 2Zekai HALICI, 3Fehmi ODABAOLU, Elif ADIRCI, 2Halis SLEYMAN
Karaelmas University, Faculty of Medicine, Department of Pharmacology, Zonguldak, Turkey 2 Atatrk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey 3 Atatrk University, Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey 4 Atatrk 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 lesions induced by stress, ethanol and NSAIDs is well known [1]. The glutathione (GSH), glutathione S-transferase (GST) and glutathione reductase (GR) play an important role in the prevention of the gastric damages [2]. On the other hand, montelukast (MLK), a selective reversible cysteinyl leukotriene-1 receptor (LTD4 receptor) antagonist is used in the treatment of asthma [3] and presently nothing known about its effects on the gastro-intestinal system. We have investigated alterations in the GSH level and the activities of antioxidative 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 180190 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 received 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 millimeter 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 administration. These results suggest that the gastroprotective effect of MLK on IND-induced ulceration can be attributed to its ameliorating effect on the oxidative damage.
Table 2. Effects of the MLK, RAN and LAN on GSH, GST and GR in rats IND-induced tissues. IND was compared with healthy and treated groups with IND. Treatment N Dose GST (mg/kg) ACTIVITY (EU) 5 10 20 30 25 25 26,290,5* 28,220,7* GR ACTIVITY (EU) 34.50.6* 29.60.8* GSH LEVEL 3.310.06* 3.760.04* 4.240.05** 4.850.03** 4.170.10** 3.050.04** 4.120.04
IND+MLK IND+MLK IND+MLK IND+LAN IND+RAN IND HEALTHY
6 6 6 6 6 6 6
33,080,8** 27.60.7** 33,650,4** 17,330,8* 23,530,3* 28,600,4 43.10.6* 25.20.2** 37.70.9** 25.60.5
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. Odabaolu F, akr A, Sleyman H, Aslan A, Bayr Y, Halc M, Kazaz, C. Gastroprotective and antioxidant effects of usnic acid on indomethacine-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
Gnnur ZBAKI-DENGZ, 2Fehmi ODABAOLU, 3Zekai HALICI, 4 Elif ADIRCI, 2Mesut HALICI, 3Halis SLEYMAN
1
tric ulcers. These results suggest that the gastroprotective effect of AMD on IND-induced ulceration can be attributed to its ameliorating 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
Karaelmas University., Faculty of Medicine, Department of Pharmacology, Zonguldak, Turkey 2 Atatrk University, Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey 3 Atatrk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey 4 Atatrk University, Faculty of Pharmacy, Department of Pharmacology, Erzurum, Turkey
1
Amiodarone (AMD){2-butyl-3-(3;5diiodo-4-diethyl-aminoethoxy-benzoyl) -benzofuran} is a multiple ion (Ca++, Na+, K+) channel blocker drug and is also a non competitive - and -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 developing numerous adverse side effects. Of greatest concern is AMD-induced 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 phospholipid metabolism [1-3]. 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 lesions induced by stress, ethanol and NSAIDs 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 ulcer. 42 male Sprague-Dawley rats, weighing 180190 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 millimeter paper. Then tissues grounded within liquid nitrogen for assays of SOD, CAT and MPx activities. In the present study, we found that 1) AMD, LAN and RAN reduced the development of IND-induced gastric damages, at a greater magnitude for AMD and LAN than for RAN; 2) AMD and RAN alleviated 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-
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. 25 50 100 25 30 25 Ulcer index (UI)a [Ulcerated area / Total stomach area] x 100 2.75 0.70 * 2.28 0.83 ** 0.89 0.46 *** 1.64 0.85 ** 0 0 *** 6.04 1.27 *** 0 0 % Inhibitionb
6 Amiodarone 6 6 Ranitidine Lansoprazole Indomethacin Healthy group

a b
54.5 62.3 85.3 72.8 100 -
6 6 6 6
Mean damage index SEM of six animals in each group. N: The number of rats. % 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 rats indomethacin (IND)-induced gastric tissue. Treatment N Dose CAT SOD MPx Activity mg/kg (mmol/min/ (mmol/min/mg (mol/min/mg body mg tissue) tissue) tissue) wt 25 50 100 25 25 115,991,20 94,200.98* 10,220.08 11,470.26* 13,510.14* 5,730.11* 9,750.07* 7,650.61
6 IND+AMD 6 6 IND+RAN IND 6 6
90,171,20* 107,450.81** 74,051,10* 125,920.28** 50,680,84* 116,080.06** 117,890,36* 74,570.52** 74,930,95 122,351.13
Healthy rats 6
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 function: A review of the physiology and clinical manifestations, J. Clin. Pharmacol., 30, 588-595, 1990.
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4. 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. 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, Canadian 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 peroxidase- and lactoperoxidase catalyzed reactions involving iodide, J. Biol. Chem., 259, 13783-13790, 1984.
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. nkol T, Doruer DS, Ito S, ahin MF. Synthesis and antinociceptive activity of (5-chloro-2-benzothiazolinon-3-yl)acetamide derivatives, Archiv der Pharmazie, 333(10), 337-340, 2000. 5. Kontogiorgis CA, Hadjipavlou-Litina DJ. Non steroidal anti-inflammatory 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 060
Ref: 0083
P 061 SYNTHESIS AND ANTIMICROBIAL EVALUATION OF 6-SUBSTITUTED-3(2H)-PYRIDAZINONE-2-YL ACETOHYDRAZDE DERIVATIVES

1 2
1
Ref: 0085
SYNTHESIS OF SOME 5-CHLORO-6-(THIAZOL4-YL)-2OXO-3H-BENZOTHIAZOLE DERIVATIVES AS POTENTIAL COX-2 INHIBITORS

Selcen ADAK, Deniz S. DORUER, Serdar NL
Mehtap GKE, 1Glay YELKEN, 2Serpil POLAT, 3Seda TEZCAN, Mehmet SERN
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 COX2 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 addition, potent selective COX-2 inhibitors which bear thiazole structure 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 structure may lead to compounds with increased analgesic and anti-inflammatory 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 inhibitory potency will be evaluated by using in vitro human whole blood assay.
Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey 2 Mersin University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Mersin, Turkey 3 Mersin 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 multidrug resistant Gram-positive bacteria, in particular, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) is causing a serious menace. Moreover, the emergence of vancomycin resistant MRSA can be anticipated in foreseeable 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 hyrazidehydrazones have been claimed to exhibit appreciable antimicrobial activity [5-9]. On the basis of these observations a new series of 6substituted-3(2H)-pyridazinone-2-yl acetohydrazide (I) was synthesized using an appropriate synthetic route. The structure elucidation 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 Staphylococcus 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.
1. Menozzi G, Merello L, Fossa P, Mosti L, Piana A, Mattioli F. 4-Substituted 1,5-diarylpyrazole, analogues of celecoxib: synthesis and preliminary evaluation of biological properties, IL Farmaco, 58, 795-808, 2003.
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1. Snmez M., Berber I., Akba 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. Akba E, Berber I, ener A, Hasanov B. Farmaco, 60(1), 23-6, 2005. 4. Takaya M. Yakugaku Zasshi., 113(9), 676-81, 1993. 5. Koyiit-Kaymakolu B, Orun E, nsalan 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. Kkgzel G, Kocatepe A, De Clercq E, ahin 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), 8768, 2006. 9. Grover G, Kini SG. Eur. J. Med. Chem., 41(2), 256-62, 2006.
P 063
Ref: 0087
SYNTHESIS OF 6-(SUBSTITUE THIAZOL4-YL)-2-OXO3H-BENZOXAZOLE DERIVATIVES AS POTENTIAL COX-2 INHIBITORS

eyma CANKARA, Serdar NL
P 062
Ref: 0086
SYNTHESIS AND ANTIMICROBIAL EVALUATION OF 5-CHLORO-2(3H)-BENZOXAZOLINONE-3-ACETYL-2-(p-SUBSTITUTED BENZAL)HYDRAZONE DERIVATIVES

1 2
1
Tijen NKOL, 1Mehtap GKE, 1Ali Ulvi TOSUN, 2Serpil POLAT, Mehmet SERN, 3Seda TEZCAN
Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey 2 Mersin University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Mersin, Turkey. 3 Mersin University, Faculty of Medicine, Department of Clinical Microbiology, Mersin, 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 incorporation 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 COX2 inhibition. Thus, we have designated a series of 6-(substitute thiazole4-yl)2-oxo-3H-benzoxazole derivatives and these compounds were prepared 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].
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-hydrazones have been demonstrated to possess antibacterial, and antifungal activities [1-4]. Furthermore, it is reported in the literature that 2(3H)-benzoxazolinone derivatives can exhibit diverse activities. Particularly antibacterial [3] and antifungal [4] activities have been scrutinized intensively. In addition, it has been published that chlorinated 2(3H)-benzoxazolinone 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 benzaldehyde moieties in the same molecule. Synthesized 5-chloro-2(3H)benzoxazolinone-3-acetyl-2-(p-substituted benzal)hydrazone I derivatives were evaluated for screening antibacterial and antifungal activities on microorganisms respectively on four bacteria and two Candida species.
1. nl S, nkol T, Dndar Y, kcelik B, Kpeli E, Yeilada E, Noyanalpan N, ahin 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, 353361, 2003. 2. Narender M, Reddy MS, Sridhar R, Nageswar YVD, Rama Rao K. Aqueous phase synthesis of thiazoles and aminothiazoles in the presence of -cyclodetrin, Tetrahedron Letters, 46, 5953-5955, 2005. 3. Patrignani P, Panara Mr, Greco A, Fusco O, Natoli C, Iacobelli S, Cipollone F, Ganci A, Creminon C, Maclouf J, Patrono C. Biochemical and Pharmacological Characterization of the Cyclooxygenase Activity of Human Blood Prostaglandin Endoperoxide Synthases, The Journal of Pharmacology and Experimental Therapeutics, 271, 1705-1712, 1994.
P 064 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. Koyiit-Kaymakolu B, Orun E, nsalan 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), 859860, 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.
Ref: 0088
INHIBITION EFFECT OF NEW SULFONAMIDE DERIVATIVES ON CARBONIC ANHYDRASE

mmhan ZDEMR-ZMEN, Fatma ARSLAN, Fatma HAMURCU
Gazi 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-
77
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 hydrazide (esh), 5-methyl-2-hydroxyacetophenone ethanesulfonylhydrazone (5mafesh) and its Ni(II) complex on CAII have been investigated. 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, Kfreviolu , Nalbantolu B., Bioorg. & Med. Chem., 5(3), 515, 1997. 2. Chakravarty S , Kannan KK., J. of Mol. Biol., 243, 298, 1994.
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
len URLU, Serdar NL
Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey
P 065
Ref: 0089
DESIGN AND SYNTHESIS OF NOVEL TIAZOLE DERIVATIVES AS POTENTIAL CYCLOOXYGENASE- 2 (COX-2) INHIBITORS
Leyla UZUN, Tijen NKOL, Serdar NL
Gazi 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 (COX2) 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 inhibitors 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-benzothiazoles were accomplished by the reaction of 6- bromoacetyl-2oxo-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.
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 inflammation and has emerged as the isoform that is primarily responsible 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-1,3thiazol-4-yl)-3H-benzoxazole derivatives were synthesized by condensation of 6-bromoacetyl-5-chloro-2-oxo-3H-benzoxazole and appropriate thiobenzamides derivatives in diethylenglicoldimethylether. 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, Tetrahedron Letters, 43, 3193-3196, 2002.
P 067 PHYSICOCHEMICAL CHARACTERIZATION OF BIODEGRADABLE CARDIOVASCULAR STENTS

Can SARISZEN, Betl ARICA, Sema CALI, A. Atilla HINCAL
Ref: 0091
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 cyclodextrin, Tetrahedron Letters, 46, 5953-55, 2005.
Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100 Ankara, Turkey
The aim of our study was to realize physicochemical characterization 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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May 17-20, 2007
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 added 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 including 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 (20C to 300C at a heating rate of 10C/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 136m0.05. Based on ease of handling for perivascular placement, this film thickness was assessed to be optimum. Thin films (100m) tended to fold and 200m films were not elastic and easy to crack. Incorporation of drug or drug loaded microspheres did not interfere during the formation 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 biodegradable 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 period 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 explained by the slow degradation of the PLGA polymer in the release medium. Nevertheless, approximately 5-62% of the PA from the PAloaded 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 Length Diameter Thickness Microspheres (cm) (mm) (m) None (Empty Stent) PA Incorporated Microsphere Incorporated None (Empty Stent) PA Incorporated Microsphere Incorporated 1.5 1.5 1.5 1.5 1.5 1.5 3 3 3 3 3 3 136.55 m 1403.6 m 1454 m 1098 m 119.34.2 m 118.73.2 m Specific Surface Area 1,32 m2/g 2,14 m2/g 0,44 m2/g -
PLGA 75:25 PLGA 75:25 PLGA 75:25 PLGA 50:50 PLGA 50:50 PLGA 50:50
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
1
1
Yusuf ERGN, 2Ufuk Gney ERGN, 3zlem ORHAN, 4Ekrem KK
Kahramanmara St mam University, School of Medicine, Department of Pharmacology, 46100 Kahramanmara, Turkey 2 Kahramanmara St mam University, School of Medicine, Department of Family Medicine, 46100 Kahramanmara, Turkey 3 Kahramanmara St mam University, School of Medicine, Department of Psychiatry, 46100 Kahramanmara, Turkey 4 Osmaniye High School of Science, Osmaniye, Turkey
Previous studies have shown that nitric oxide synthase inhibitors and melatonin are as efficacious as conventional antidepressant 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 melatonin 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 sufficient 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 significant 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.
79
of natural products such as carbohydrates and prostaglandins [1]. A series of key precursors to the IMDA reaction of furane diene (2ac) have been prepared via facile alkylation and protection. While the cycloaddition process for (3ac) was afforded in hot toluene, a commercial microwave (2450 MHz) was used for the synthesis of (5ab) (Figure 1).
Figure 1. The effects of saline, 1% alcohol, NG-nitro-L-arginine (LNNA: 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 1. i. Br2, Et3N, DCM, 0C, 98%; NaBH4, CeCl3.7H2O, MeOH, 0C, 97%; PBr3, Pyridine, PhH, 0C reflux, 68%; KOtBu, CHBr3, Pentane, 0C; Heat 155C, 1h, 78%; 2,3-dibromocyclopentene or 2,3-dibromocyclohexene, K2CO3, THF, reflux, 3d; (Boc)2O, DMAP, DCM, 0C, 2h.
Treatment of fused oxy- and thio-heterotricycles (5ab) with borontriluoride-etherate in dichloromethane at 78C cleaved EpoxyBridge and concomitant aromatisation gave the isobenzo-furanol and thiophenol (6ab) in 7276% yields respectively (Figure 2) [2].
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 active 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-Lemaitre B. Effects of melatonin receptor ligands on swim test immobility, Neuroreport, 9, 249-253, 1998. 5. Mantovani M, Prtile R, Calixto JB, Santos ARS., Rodrigues ALS. Melatonin 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. Figure 2. 1. Lipshutz BH. Chem. Rev., 86, 795, 1986. Wang Q, Padwa A, Rh(I)-Catalyzed 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. Heterocyclic Commun., 3&4, 233, 2006. Karaarslan M, Gktrk E, Demircan A. Thermal intramolecular DielsAlder reaction of furan; Synthesis of nitrogen 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 nc CAN, Gksel ALTIOKKA
Anadolu University, Faculty of Pharmacy, Department of Analytical Chemistry, Eskiehir, Turkey
P 069
Ref: 0093
A UTILITY OF FURAN CORED COMPOUNDS; SYNTHESIS OF NITROGEN TETRACYCLES, ISOBENZOFURANOL AND ISOBENZOTHIOPHENOL
Muhsin KARAARSLAN, Ersen GKTRK, Aydn DEMRCAN
University of Nide, Faculty of Sciences & Letters, Department of Chemistry, Kampus, 51100 Nide, Turkey
Intramolecular DielsAlder (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
Anthocyanins constitute one of the major groups of natural pigments 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 develop 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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May 17-20, 2007
to photodiode array detection. Two anthocyanins and cyanidin (internal 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 m PVDF filters, and injected into the system with no further extraction or concentrating steps.
thocyanins were resolved by reversed phase technique within about 16 minutes. Analytes were detected at 520 nm wavelength and mobile 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 m PVDF filters, without any extraction or concentration steps.
Figure 1. A typical chromatogram of grape juice sample (PG: Pelargonidin, MV: Malvidin, IS: Internal standard). 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 biological activities of anthocyanins, Phytochemistry, 64, 923-933, 2003. 2. DellAgli 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. Anthocyanins in medicine, Pol. J. Pharmacol., 55, 699-702, 2003. 4. Bravo L. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance, Nutr. Rev., 56, 317-333, 1998.
Anthocyanins were detected in ppb levels with adequate chromatographic resolution. Linearities were obtained between the ranges of 78.1 390.7 ppb for pelargonidin and 88.15 440.7 ppb for malvidin. 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 nutritional significance. Nutr. Rev., 56, 317-333, 1998. 2. DellAgli M, Busciala A, Bosisio E. Vascular effects of wine polyphenols, Cardiovasc. Res., 63, 593-602, 2004.
P 072
Ref: 0097
P 071
Ref: 0096
RAPID DETERMINATION OF ACRYLAMIDE IN POTATO CHIPS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE ARRAY DETECTION
Nafiz nc CAN, Gksel ALTIOKKA
SCREENING OF ANTHOCYANINS PELARGONIDIN AND MALVIDIN IN COMMERCIAL FRUIT JUICES USING LIQUID CHROMATOGRAPHY COUPLED TO DIODE ARRAY DETECTION
Gksel ALTIOKKA, Nafiz nc CAN
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 reproducible method for the determination of two anthocyanins, pelargonidin 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-
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 determination 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 array 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
81
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-concentration steps.
in many of the vegetables such as red cabbage and turnip [1]. Anthocyanins, 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 cardiovascular 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 Sciences, Inertsil ODS-3, 3 m, 150 x 4.6 mm) with excellent accuracy (recovery more than 99%) and precision (RSD% < 0.9). Both compounds were separated with adequate resolution factor which was higher than 3. Cyanidin and petunidin signals were found to be linear 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 homemade potato chip sample (AA: Acrylamide, MAA: Methacrylamide)
Acrylamide was detected in ppb levels with adequate chromatographic resolution. The developed method was validated according to the recommendations of International Conference of Harmonization Q2(R1) and system suitability parameters were tested according to United States Pharmacopeia. The method was found to be linear in the ranges of 2.9-14.2 ppb, giving LOD and LOQ concentrations of 0.93 and 2.82 ppb, respectively. As a result, it was observed 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 acrylamide, a carcinogen formed in heated foodstuffs, J. Agric. Food Chem., 50, 4998-5006, 2002. 3. JECFA - Joint FAO/WHO Expert Committee on Food Additives, Summary 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, Macdonald N, Clewell H, Allen B, van Landingham C. Acrylamide: Review of toxicity data and dose-response analyses for cancer and noncancer effects, 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 Agency for Research on Cancer (IARC),Geneva, Switzerland, 1997. Figure 1. A typical chromatogram of turnip sample (CY: Cyanidin, PT: Petunidin, IS: Internal standard)
Developed method was validated according to the recommendations of International Conference of Harmonization. As a result, it can be concluded that both anthocyanins exist at high levels especially in red cabbage and turnip, while there is no evidence was observed 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 nutritional significance, Nutr. Rev., 56, 317-333, 1998. 2. Kong JM, Chia LS, Goh NK, Chia TF, Brouillard R. Analysis and biological activities of anthocyanins, Phytochemistry, 64, 923-933, 2003. 3. Kowalczyk E, Krzesinski P, Kura M, Szmigiel B, Blaszczyk J. Anthocyanins in medicine, Pol. J. Pharmacol., 55, 699-702, 2003. 4. DellAgli M, Busciala A, Bosisio E. Vascular effects of wine polyphenols, Cardiovasc. Res., 63, 593-602, 2004.
P 073
Ref: 0098
IMPROVED HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF CYANIDIN AND PETUNIDIN IN FRESH VEGETABLES USING AN ODS-3 REVERSED-PHASE COLUMN
Gksel ALTIOKKA, Nafiz nc CAN, Zeki ATKOAR
As a subgroup of the polyphenols family, anthocyanins are known as one of the best natural antioxidant compounds that can be found
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May 17-20, 2007
P 074
Ref: 0100
MOLECULAR MODELLING STUDIES OF BENZO[]CARBAZOLE DERIVATIVES AS ESTROGEN RECEPTOR INHIBITORS

Tuba TAKIN, Fatma SEVN
Hacettepe University, Faculty of Science, Department of Cemistry, Ankara, Turkey
There have been some researches about a number of 5,6-dihydro11-alkylbenzo[]carbazoles derivatives with one or two hydroxy groups in the aromatic rings for their binding affinities for the estrogen 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 molecule. The most statistically significant descriptors like substituent hydrophobicity constant (), electrophilicity index () 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-11alkylbenzo[]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, Srvastava HK, Singh PP. International Journal of Quantum Chemistry, 104, 87100, 2005.
maintain normal concentrations of about 60 M in plasma It is the most common electroactive biological compound [1-4]. So, the electrochemical oxidation of ascorbic acid has been carried out at glassy carbon electrode in various aqueous solution in the pH range of 0.6410.15 (Britton-Robinson, acetat, phosphate buffers and 0.5 M sulfuric acide solution) by cyclic (CV) and differential pulse voltammetry (DPV). The best results were obtained for the quantitative determination 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 regression equation was obtained as Ip (A) = 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 obtained in tablet dosage forms and some fruit juices.
1. Erdurak-Kl CS, Uslu B, Doan B, zgen U, zkan SA, Cokun M. Anodic voltammetric behaviour of ascorbic acid and its selective determination in pharmaceutical dosage forms and some rosa species of Turkey, 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 Gilmanns the pharmacological 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

nci Selin DOAN, Selma SARA
Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Ankara, Turkey
P 075
Ref: 0101
RAPID AND SENSITIVE VOLTAMMETRIC DETERMINATION OF ASCORBIC ACID IN TABLET DOSAGE FORMS AND SOME FRUIT JUICES
1 1
1
Selehattin YILMAZ, 1Gke AKIN, 1Sultan YAMUR, 1Glen TRKER, Glen SALIKOLU, 1Hseyin ERDUAN
anakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of Chemistry, 17020 anakkale, Turkey 2 anakkale Onsekiz Mart University, Faculty of Arts and Sciences, Department of Biology, 17020 anakkale, Turkey
3,4-Dihydropyrimidin-2(1H)-ones(thiones) have been reported to possess diverse pharmacological properties, such as antibacterial, antiviral, antitumor, antiinflammatory, antihypertensive, calcium 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 interest. Earlier, we have described the synthesis and calcium antagonistic 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-acetyl6-methyl-4-(substituted phenyl)-3,4-dihydropyrimidin-2(1H)-thiones which are expected to have calcium channel blocker and antibacterial activities.
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 playing 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 infused ascorbic acid in the urine, daily doses of 200 mg are needed to
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The title compounds were prepared by using the Biginelli threecomponent 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 compounds 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 S, Sara S, elebi S, Erol K. Synthesis of 4-aryl-3,4-dihydropyrimidine-2(1H)-thione derivatives as potential calcium channel blockers, Bioorg. Med.Chem., 14, 8582-8589, 2006. 3. Yarm M, Sara S, Ertan M, Batu S, Erol K. Synthesis, structural elucidation and pharmacological properties of some 5-acetyl-3,4-dihydro6-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 078 STUDIES ON NEW OXIME ESTER DERIVATIVES OF NAFIMIDONE ALCOHOL AS POTENTIAL ANTICONVULSANT COMPOUNDS
1
1
Ref: 0104
Burcu SELMOLU, 2Arzu KARAKURT, 1Sevim DALKARA
Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Ankara, Turkey 2 nn 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:
P 077
Ref: 0103
AN EXPERIMENTAL DESIGN APPROACH TO SELECTING THE OPTIMUM LIQUID CHROMATOGRAPHIC CONDITIONS FOR THE DETERMINATION OF LOCAL ANESTHETICS
1
1
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-imidazole Derivatives, a New Class of Anticonvulsant Agents. J. Med. Chem., 24, 67-74, 1981. 2. Karakurt A, Dalkara S, zalp M, zbey 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 Activities, Eur. J. Med. Chem., 36, 421-433, 2001.
Aysun DNEL, 2Nursabah E. BAI
Hacettepe University, Faculty of Medicine, Department of Pharmacology, Ankara, Turkey 2 Hacettepe 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 m) analytical column. An optimised mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (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 oxybuprocaine. 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 lidocaine, proparacaine and 100 ng ml-1 for bupivacine, oxybuprocaine (RSD 15 %, n=6).
Keywords: Local anesthetics, validation, experimental design and method optimization
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
1
1
Emirhan NEMUTLU, 1Sedef KIR, 2Sinan BEKSA
Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 Ankara, Turkey 2 Hacettepe Unversity, 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 connection to a Phenomenex C18 column (4 mm x 3.0 mm i.d.). The optimum assay conditions were found with experimental designs. Optimum 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. Marbofloxacin (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 moxifloxacin 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 g mL-1. The LOD and LOQ were 0.010 and 0.035 g mL-1, respectively. The absolute recoveries 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.
cies. The chloroform-soluble compounds extracted from Scorzonera 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 inhibit QS-regulated carbapenem antibiotic production in an important 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 infections 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., 42:569571, 1998.
P 080
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INVESTIGATION FOR NATURAL QUORUM SENSING INHIBITORS: ALTERNATIVE TO ANTIBIOTICS?

1 1
1
P 081
Ref: 0107
Glgn BOGELMEZ-TINAZ, Seyhan ULUSOY, Fatma Filiz COKUN-ARI, 2Aysel UUR, 2zgr CEYLAN
1
Sleyman Demirel University, Faculty of Arts and Sciences, Department of Biology, Isparta, Turkey 2 Mula University, Faculty of Arts and Sciences, Department of Biology, 480000 Mula, Turkey
DEVELOPMENT OF A CAPILLARY ZONE ELECTROPHORESIS METHOD FOR THE SIMULTANEOUS ANALYSIS OF OLMESARTAN MEDOXOMIL AND HYDROCHLOROTHIAZIDE
Mustafa ELEBER, Sacide ALTINZ
Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, Ankara, 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, pharmaceutical companies are now focusing on finding novel bacterial targets and new ways to control and eradicate bacterial infections [1]. To infect a host and cause a disease, bacteria produce an array of virulence 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 physiological 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-
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 effective option for antihypertensive therapy, demonstrating greater blood pressure reduction than monotherapy [2]. In the literature, there hasnt been any method described for the simultaneous analysis of olmesartan medoxomil and hydrochlorothiazide. A capillary zone electrophoresis method with ultraviolet detection for the simultaneous analysis of olmesartan medoxomil and hydrochlorothiazide in synthetic tablets was developed. A fused silica capillary (50 m 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 functional features of olmesartan medoxomil, an angiotensin receptor blocker, Journal of Cardiovascular Pharmacology, 46, 585-593, 2005. 2. Steven GC, Michael AW, Antonia CW, Donald JH. Evaluation of antihypertensive therapy with the combination of olmesartan medoxomil and hydrochlorothiazide, AJH, 17, 252259, 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

1
1
Aysun DNEL, 2Nursabah E. BAI, 1Atila BOZKURT
Hacettepe University, Faculty of Medicine, Department of Pharmacology, 06100 Ankara, Turkey 2 Hacettepe 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 (NAT2) activities in 101 Turkish volunteers (47 males and 54 females). Spot urine samples were analyzed 5 h after 100 mg caffeine consumption. 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-3methyluracil (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 chloroform/isopropanol (85:15, v/v) and separated on a reversed phase C18 (4.6 x 2.5 mm i.d., particle size 5 m) 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 enzyme activities of CYP1A2, CYP2A6, NAT2 and XO were found as 5.286.12, 0.220.11, 0.330.17, and 0.650.16 (meanSD), respectively. Smoking and gender were not affected CYP1A2, and CYP2A6 activities. The developed RP-HPLC method was validated and successfully 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
logical studies have been conducted on the roots of Paeonia species. Monoterpen glycosides are the active constituents for this plant species. Among the terpen glycosides, paeoniflorin (1) and oxypaeoniflorin (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 methods. The structure elucidations of 1 and 2 were achieved by combination of 1D and 2D-NMR experiments and mass spectrometry. The HPLC separation was achieved on a Nucleosil 100-5 C18 (5 m, 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 tailing 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 g/ml for 1 and 0.25-60 g/ml for 2. The developed method was found to be useful for determination 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. bodurii collected from the flora of Turkey.
P 084 STABILITY OF LIQUID AND SOLID FORMS OF RECOMBINANT HUMAN INTERFERON

Bilgen ZBATIR, Filiz NER
Ref: 0114
Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, 06100 Ankara, Turkey
P 083 DETERMINATION OF PAEONIFLORIN AND OXYPAEONIFLORIN IN PAEONIA SPECIES USING VALIDATED HPLC METHOD
1
1
Ref: 0112
Semra KOYUNOLU, 2Sedef KIR, 3Ali A. DNMEZ, 4hsan ALI
Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, 06100 Shhiye-Ankara, Turkey 2 Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 Shhiye-Ankara, Turkey 3 Hacettepe University, Faculty of Science, Department of Biology, 06100 Shhiye-Ankara, Turkey 4 Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, 06100 Shhiye-Ankara, Turkey
Paeonia L., the largest genus in the family Paeoniacea, is represented 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-
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 administration 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-2b 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 formulations of interferon present promising results in some references [2]. In this study we prepared a solid lozenge form and liquid form of rHuIFN-2b (recombinant human interferon alfa 2b) and examined 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-2b 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 rHuIFN-2b Sodium phosphate dibasic anhydrous Sodium phosphate monobasic monohydrate Disodium EDTA Polysorbate 80 (Tween 80) Phenol Sodium chloride Water for injection. 1. Shojaei AH. Buccal mucosa as a route for systemic drug delivery: A review, J. Pharm. Pharmaceut. Sci.,1(1), 15-30, 1998. 2. Ship JA., Fox PC, Michalek JE, Cummins MJ, Richards AB. Treatment of pimary Sjgrens syndrome with low-dose natural human interferon- administered by the oral mucosal route: a phase II clinical trial, Journal of Interferon and Cytokine Research, 19, 943-951, 1999. Solid formulation rHuIFN-2b Gelatin Glycerol Sucrose Water for injection
P 086
Ref: 0118
ANTIMICROBIAL ACTIVITY OF 3-HYDROXY-6-METHYL-2SUBSTITUTED 4H-PYRAN-4-ONE DERIVATIVES

1
1
Ekrem KILI, 2Mutlu DLSZ-AYTEMR
Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, 06100 Shhiye-Ankara, Turkey 2 Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Shhiye-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 antimicrobial agents with novel modes of action represents a main target in chemotherapy. It has been shown that hydroxypyran-4H-one derivatives have antimicrobial activity [1, 2]. In our previous studies, we synthesized some Mannich bases of 3-hydroxy-6-methyl-4H-pyran4-one derivatives and investigated their antimicrobial activity [3].
P 085
Ref: 0116
HPLC-ECD DETERMINATION OF EPINEPHRINE PLASMA CONCENTRATIONS IN VARIOUS DENTAL PATIENTS

1 2
1
eyda DEMRCAN, 2Aye Gl AKALIN, 1Filiz SAYIN, 2Gke MERAL, Ferda TAAR, 1Sedef KIR, 1Nursabah E. BAI
Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100, Shhiye-Ankara, Turkey 2 Hacettepe University, Faculty of Dentistry, Department of Oral Surgery, 06100-ShhiyeAnkara, 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 m, 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 validated for the parameters such as specificity, accuracy and extraction recovery and was applied to three different patient groups. The recovery of epinephrine after extraction from spiked plasma was 97 0.03 % (meanSD). 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 diseased older dental patients after the administration of local anesthesia with epinephrine were obtained.
Keywords: Epinephrine; HPLC; Electrochemical Detector (ECD); Validation; Plasma; Dental Patients
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 bacteria 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 g/ml), which was carrying (4-c hlorophenyl)phenylmethyl substituent showed antibacterial activity against S. aureus. The other compounds had no valuable inhibitory activity. Compound 5 (MIC: 16 g/ml) was also the most effective compound towards C. albicans than the others (MIC: 32-64 g/ml).
1. Aytemir MD, Erol DD, Hider RC, zalp M. Synthesis and evaluation of antimicrobial activity of new 3-hydroxy-6-methyl-4-oxo-4H-pyran-2carboxamide derivatives, Turk J. Chem., 27(6), 757-764, 2003. 2. Kayahara H, Shibata N, Tadasa K, Kotani T. Amino acid and peptide derivatives of kojic acid and their antifungal properties Agric. Biol. Chem., 54(9), 2441-2442, 1990. 3. Aytemir MD, al , zalp M. Synthesis and evaluation of anticonvulsant and antimicrobial activities of 3-Hydroxy-6-methyl-2-substituted 4H-pyran-4-one derivatives. Arch. Pharm. Pharm. Med. Chem. 337, 281288, 2004. 4. National Committee for Clinical Laboratory Standards (NCCLS), Methods 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), Reference method for broth dilution susceptibility testing of yeast, Approved Standard M27-A, 17(9) Villanova, PA. (1997).
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INHIBITION OF RAT LIVER MONOAMINE OXIDASE (MAO)A AND B BY GENTIANELLA CAUCASEA (Loddiges ex Sms) HOLUB EXTRACTS
1 1,3
1
Tayfun ERSZ, 1Zeliha . AKDEMR, 2Samiye YABANOLU, Funda Nuray YALIN, 1Duygu KAYA, 4. rem ANKAYA, 2Glberk UAR
Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, 06100 Ankara, Turkey 2 Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, 06100 Ankara, Turkey 3 Hacettepe University, Faculty of Pharmacy, Department of Pharmacy Management, 06100 Ankara, Turkey 4 Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Botany, 06100 Ankara, Turkey
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. Monoamine oxidase inhibitors from Gentiana lutea. Phytochemistry 65: 22552260, 2004. 6. Pritchard NM, Gentianella in Flora of Turkey and the East Aegean Islands (Ed. Davis PH) University Press, Vol 6 Edinburg, 1978. 7. Suzuki O, Katsumata Y, Oya M, Chari VM, Vermes B, Wagner H, Hostettmann 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 spectrophotometric assay for monoamine oxidase and related enzymes in tissue homogenates. Anal Biochem 244: 384-392, 1997.
Monoamine oxidase (MAO), which is found in two forms designated 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 Parkinsons and Alzheimers 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 neuroprotectans in the treatment or prevention of depression, psychosis, schizophrenia, Alzheimers and Parkinsons diseases [5]. Gentianella caucasea is a biennial stem to 30 cm grown in Northern and Northeastern Anatolia [6]. Preliminary works on Gentianella 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 methanol, 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 using 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 without extracts, versus extract concentration. All Gentianella extracts tested inhibited rat liver MAO isoforms in a dose-and time-dependent manner. Incubation of the enzyme with the extracts at 37 0C for 60 min. increased the inhibitory action of the extracts. Methanol extract inhibited MAO-B isoform selectively with IC50 value of 38.22.26 g dry weight/mL whereas chloroform, petrolum ether, and water extracts inhibited MAO-A selectively with IC50 values of 58.74.31, 64.335.06 and 102.137.55 g dry weight/mL, respectively. Xanthones of Gentianella caucasea may be a possible source of pharmaceuticals for the treatment and prevention of depression, Parkinsons and Alzheimers 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 inducing anticonvulsant activity in the kindling model of epilepsy. J Pharmacol Exp Ther 288: 984-992, 1999. 2. Uar G, Gkhan N, Yeilada A, Bilgin A. 1-N-substituted thiocarbamoyl3-phenyl-5-thienyl-2-pyrazolines: A novel cholinesterase and selective monoamine oxidase-B inhibitors for the treatment of Parkinsons and Alzheimers diseases. Neurosci Lett 382: 327-331, 2005. 3. Erturul A, Uar G, Baar K, Demir B, Yabanoglu S, Ulu B. Influence of clozapine on platelet serotonin, monoamine oxidase and plasma serotonin levels. Pscyhiatr Res 149: 49-57, 2007.
P 088
Ref: 0120
NEW MOLECULARLY IMPRINTED MICROSPHERES FOR COLONIC DELIVERY OF 5-AMINOSALICYLIC ACID

Yeliz TUN, Ersin BAYKARA, Kezban ULUBAYRAM
Hacettepe 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 imprinted 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 microspheres (MIPs) has been investigated for colonic delivery of 5aminosalicylic acid (5-ASA), an active agent used in treatment of ulcerative colitis and Crohns disease. For the first time, 5-ASA imprinted 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 diameter 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 polymerization 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 microspheres 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. Tun Y, Hasrc N, Yeilada 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 nanotechnology in drug delivery, Adv. Drug Del. Rev., 56, 1529-1531, 2004.
88
May 17-20, 2007
P 089
Ref: 0121
BIOLOGICALLY ACTIVE COMPOSITE SCAFFOLD FOR TISSUE ENGINEERING APPLICATIONS

1 1
1
Sinan GVEN, 2Nesrin HASIRCI, 3Sevda MFTOLU, Kezban ULUBAYRAM
Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, Ankara, Turkey 2 Middle East Technical University, Faculty of Arts and Sciences, Department of Chemistry, Ankara, Turkey 3 Hacettepe 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 transplantation [1]. In scaffold oriented tissue engineering, the scaffolds should mimic the structure and biological function of native extracellular 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, freezing temperatures and molding. Among the prepared scaffolds at different conditions, scaffolds (SC) prepared at 500 rpm and frozen at 80C 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 degradation 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, (SCRGD-GF), and incubated in media either free of serum or containing serum. Scaffolds immobilized with RGDS and PDGF-BB had the highest attached cell number by the day 15. According to scanning 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 matrices for tissue regeneration, Advanced Drug Delivery Reviews, 33, 65182, 1998.
tions on platelets aggregation were investigated in vitro using adenosine-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 platelet 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 aggregation 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. t was found that the combination of aspirin and coriander seeds 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 investigated by determination of their effects on activated partial thromoplastine time (APTT). None of tested extracts affected prothrombine time (PT) values. As a result, the corianders seeds volatile oil and parsley leaves extracts are of the interest for open new approaches towards explanation of the beneficial effects on several manifestations of cardiovascular diseases like hypertension, arteriosclerosis and ischemic diseases and it could be assessed as useful natural source of pharmaceutical agents.
P 090
Ref: 0122
EFFECT OF CORIANDER (Coriander sativum L) AND PARSLEY (Petroseleinum sativum Hoffm.) EXTRACTS ON PLATELET AGGREGATION AND BLOOD COAGULATION
Muhammad Jamal SHAMMOUT
University of Jordan, Analytical Toxicology, Amman, Jordan
Certain herbs may cause pharmacological effects on some cardiovascular aspects such as platelet aggregation and blood coagulation during homeostasis processes. In this study, the volatile oils and crude extracts of Coriander (Coriander sativum L) and Parsley (Petroseleinum sativum Hoffm.) were isolated using steam distillation and heating under reflux techniques. The effects of herbs prepara-
89
Author Index
A
AKALIN, Aye Gl 87 AKDEMR, Zeliha . 88 YELADA, Akgl 56 EN, Alaattin 51 ASLAN, Ali 71 GNER, Ali 58, 66, 67 HAYAT, Ali 64, 65, 66 KILIN, Ali 71 ONUR, Mehmet Ali 66 KARAKURT, Arzu 84 MER, Asiye 70 TMER, Akn 67 ZTRK-AL, Asl 61 BOZKURT, Atila 86 HINCAL, A. Atilla 78 YILDIZ, Attila 69 DEMRCAN, Aydn 80 DEMR, Ayhan 48 GRPINAR, zer Aylin 66, 67 HASEGEL-NER, Ayegl 58 UUR, Aysel 85 DNEL, Aysun 84, 86
SARISZEN Can 78 KAZAZ Cavit 57, 59, 71 CROOKS, Peter A. 35 AKIR, Ahmet 71, 73
GEYKOLU, Fatime 51, 52 ARSLAN, Fatma 77 HAMURCU Fatma 63, 77 SEVN Fatma 83 ODABAOLU Fehmi 71, 73, 74, 75 AHN, Fikrettin 58, 61 COKUN-ARI, Fatma Filiz 85 FILLET, M. 14 ALAR, Fulya 43, 44 MORAL, Fulya 53 KARTAL, Funda 71 BABA, Fsun 64, 65, 66 TEMAMOULLARI, Fsun 64, 65, 66
HASIRCI, Nesrin 89 ARDA-AKDOAN, Hatice 51 KAPLAN-CAN, Hatice 58, 66, 67 AYGN, Hayati 71, 73 ERDEM-YURTER, Hayat 48 ERDUAN, Hseyin 83 YALINKAYA, Hseyin 55
D
SALMAN, Demet 62 DEMR, Ayhan S. 36 DEMRCAN, eyda 87 DENZL, Adil 62 CAN, zgr Devrim 56 DAYANGA-ERDEN Didem 48 EROL, Dilek 52, 55 SADAK, Dilek 55 TURGUT-BALIK, Dilek 53, 55 YURDAKUL, Dilat 58 DLSZ-AYTEMR, Mutlu 72, 87 DNMEZ, Ali A. 86 ZER-NAL Duriehvar 55
I
ALI, hsan 72, 86 IIKDA, lhan 56, 57 GL, H. nci 47, 57, 58, 59, 60, 61 DLER, Zeynep rem 55 , Hseyin 46 GR, S. Belgin 46
G
BORA, Gamze 48 GEURTS, P. 14 GIMNEZ, E. 8 AKIN, Gke 83 SAD, Gkhan 56 ALTIOKKA, Gksel 80, 81, 82 SUNAL, Sevil Grkem 56 YALIN, Grkem 54 GREF, Ruxandra 18 AHN-KO, Glay 55 YELKEN, Glay 76 UAR, Glberk 47, 56 TRKER, Glen 83 ALSANCAK, Gleren 62, 68, 70 BOGELMEZ-TINAZ, Glgn 85 SALIKOLU, Glen 83 OBAN, Gne 67 ERGN, Ufuk Gney 79 GNDZ, Gngr 68 ZBAKI-DENGZ, Gnnur 74, 75 GVEN, Sinan 89
J
J. LINHARDT, Robert 54 BARBOSA , Jose 68, 70
E
BAI, Nursabah E. 62, 84 CRISS, Wayne E. 60 UBUK-DEMRALAY, Ebru 63 KONDOLOT-SOLAK, Ebru 62 METE Ebru, 57, 58, 59 GRDAL, Ece 52 KK, Ekrem 79 AKSZ, S. Elif 53 ADIRCI, Elif 73, 74, 75 NEMUTLU, Emirhan 84 ERCAN, Aye 47 YELADA, Erdem 72 ERLHAN, smail 46 GKTRK, Ersen 80 ERSZ, Tayfun 88 AHN, Ertan 59 AKBAY, Esin 66, 67 G, Esra 68 KPEL, Esra 72 LEYEN, Evren 65
K
ZTRK, Taylan K. 71 KKOLU, Kaan 60 KABANOV, A. V. 39 PEKMEZ, Kadir 69 KANSU, Emin 3 KAYA, Duygu 88 YELEK, Kemal 48 KILI, Ekrem 87 KIR, Sedef 87
B
BARBOSA, J. 8 BAI, Nursabah E. 86, 87 GLBAKAN, Basri 43, 61 BAYKARA, Ersin 88 BAYRAM, Hakan 25 SALH, Bekir 43, 60, 61 BENAVENTE, F. 8 ARICA, Betl 78 EKEN, Bircan 52 BRASH, Douglas 47 ATMI, Bnyamin 55 SELMOLU, Burcu 84
L
DOAN, A. Lale 58 UZUN, Leyla 78 LOUIS, Ed. 14 BELTRAN, Jose Luis 63, 68, 70
C
C. EHREL, Zafer 67 ALI, nsal 72 ZELGL, Canan 58 NALEROLU, Canan 53 ANKAYA, . rem 88 TAAIR, Cansel 60
M
MALAISE, M. 14 MARCO, Jos Luis 4 ZSZ, Mehmet 54 SERN, Mehmet 76, 77 GKE, Mehtap 76, 77 MERAL, Gke 87
H
SELM, Hale 63 SLEYMAN, Halis 74, 75 SEVM, Handan 66, 67 TRKEZ, Hasan 51, 52
F
ATALAY, Fadime 73
93
AUTHOR INDEX
AUTHOR INDEX
KKSAL, Meri 53 MERVILLE, M-P. 14 HALICI, Mesut 71, 73, 75 MEUWIS, M-A. 14 YARIM Mine 52, 53 KAVANOZ, Muammer 69 EN, Mberra 65 MFTOLU, Sevda 89 KARAARSLAN, Muhsin 80 ZMECOLU, Murat 54, 59 KIZIL, Murat 52 ATALAY, Mustafa 60, 61 ELEBER, Mustafa 85 GL, Mustafa 60, 61
AHN, Pnar 72 PKN, Erhan 38
YALIN, Talat 61 TAAR, Ferda 87 TAGIN, Dilek Ik 53 NKOL, Tijen 77, 78 YALINKAYA, Timuin 43 TAKIN, Tuba 83 GRAY, Tlin 56 TUN, Yeliz 88 ZDEN, Tuncel 64, 65
ATKOAR, Zeki 82 TOPU, Zeki 67 ATE, Zeliha 64 ERDEMGL, Zerrin 68 NCESU, Zerrin 57
S
DORUER, Deniz S. 76 ALTINZ, Sacide 85 AHN, Fethi 28 YABANOLU, 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, mer Selim 71 DOAN, nci Selin 83 ZLHAN, Selma 64, 65 SARA, Selma 83 CALI, Sema 78 GNE, H. Semih 67 KOYUNOLU, Semra 86 ANLI, Senem 63, 70 SENY, D. de 14 SEPTOLU, Ebubekir 72 KAVLAK, Serap 67 NL, Serdar 76, 77, 78 POLAT, Serpil 76, 77 ZENCR, Sevil 67 DALKARA, Sevim 48, 84 ULUSOY, Seyhan 85 CANKARA, eyma 77 SHAMMOUT, Muhammad Jamal 89 BEKSA, Sinan 84 SIPPL, Wolfgang 17 BLGE, S. Srr 53 URLU, len 78 YAMUR, Sultan 83 TOPTAN, Suna 65
U
UAR, Glberk 88 GNDZ, Ufuk 68 BYKDEMRC, Uur 44 ULUBAYRAM, Kezban 88, 89 TOSUN, Ali Ulvi 77 DEMR, mide 56 ZDEMR-ZMEN, mmhan 63, 77 AKKAYA, Engin Umut 45 SALGIN-GKEN, Umut 47 NAL, Serhat 20 ALI, nsal 60 UNZETA, Mercedes 4 UZUN, mrm 24
N
BAYTA, Sultan N. 54 GKHAN-KELEK, Nesrin 47 ZEK-PEKMEZ, Nuran 69 EZER, Nurten 72 ANLI, Nurullah 62, 68
O
RZAEV, Zakir M. O. 58, 67, 66 , . Hamdi 47 ELKBIAK, mr 60 CAN, Nafiz nc 80, 81, 82 NER, Filiz 86 ONUR, Mehmet Ali 67 HANNINEN, Osmo 60, 61 ANLI, Oya 62 ZALP-YAMAN, eniz 46 ZBATIR, Bilgen 86 YERDELEN, K. zden 60, 61 KL, zge 43, 44 CEYLAN, zgr 85 ZKAN, Serkan 27 DEMREL, zlem 43 ORHAN, zlem 79 ST, zlem 54 ZTRK, Recep 22
V
ELK, Venhar 53, 55 ADAR, Vildan 43, 44
W
WEHENKEL, L. 14
Y
YABANOLU, Samiye 88 YALIN, Funda Nuray 88 BAYIR, Yasin 73 YELADA, Erdem 29 APAN, Yilmaz 45 ERGN, Yusuf 79 ZKAY, Yusuf 56, 57 ZTRK, Yusuf 56
P
PARINI, Angelo 7 AYHAN, Peruze 48 ALCIL, Pnar 67
Z
OKUMU, Zafer 71 HALICI, Zekai 71, 73, 74, 75
T
PARK, Tae-joon 54
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International Symposium on Drug Research and Development

May 17-20, 2007

From Chemistry to Medicine DRD 2007

Kervansaray Convention Center & Hotel, LARA, ANTALYA / TRKYE

International Symposium on Drug Research and Development

May 17-20, 2007

From Chemistry to Medicine DRD 2007

10.20-10.50 Hall A 10.50-12.10 Chairpersons: 10.50-11.30 11.3012.10 12.1012.35