DPhG Jahrestagung 2012 - Ernst-Moritz-Arndt-Universität Greifswald
DPhG Jahrestagung 2012 - Ernst-Moritz-Arndt-Universität Greifswald
DPhG Jahrestagung 2012 - Ernst-Moritz-Arndt-Universität Greifswald
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<strong>DPhG</strong>-<strong>Jahrestagung</strong> <strong>2012</strong><br />
Moleküle, Targets und Tabletten -<br />
Translationale Forschung für Arzneimittel der Zukunft<br />
11. – 13. Oktober in <strong>Greifswald</strong><br />
Tagungsband<br />
www.dphg.de<br />
ISBN: 978-3-00-039318-1<br />
Verlag: <strong>Universität</strong>sbibliothek <strong>Greifswald</strong>
Sponsoren der <strong>DPhG</strong>-<strong>Jahrestagung</strong> <strong>2012</strong>
Förderer der <strong>DPhG</strong>-<strong>Jahrestagung</strong> <strong>2012</strong>
<strong>DPhG</strong><br />
Deutsche Pharmazeutische Gesellschaft e. V.<br />
<strong>Jahrestagung</strong> <strong>2012</strong><br />
Programm<br />
&<br />
Tagungsband<br />
Institut für Pharmazie<br />
der<br />
Mathematisch-Naturwissenschaftlichen Fakultät<br />
<strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-<strong>Universität</strong><br />
<strong>Greifswald</strong><br />
11.–13. Oktober
4<br />
Moleküle, Targets und Tabletten –<br />
Translationale Forschung für Arzneimittel der Zukunft<br />
Wissenschaftliches Komitee Organisationskomitee<br />
Prof. Dr. Patrick J. Bednarski Prof. Dr. Werner Weitschies<br />
Prof. Dr. Thomas Jira Prof. Dr. Christoph Ritter<br />
Prof. Dr. Heyo K. Kroemer Prof. Dr. Andreas Link<br />
Prof. Dr. Ulrike Lindequist PD Dr. Gregor Radau<br />
Prof. Dr. Sandra Klein Dr. Winfried Stahlkopf<br />
Prof. Dr. Andreas Link Dr. Thomas Emmrich<br />
Prof. Dr. Bernhard H. Rauch Dipl.-Pharm. Lisa Wilde<br />
Prof. Dr. Christoph Ritter Dipl.-Pharm. Felix Wilde<br />
Prof. Dr. Thomas Schweder<br />
Prof. Dr. Werner Siegmund<br />
Prof. Dr. Werner Weitschies<br />
Dipl.-Pharm. Heidi Lemmerhirt
Inhaltsverzeichnis<br />
Allgemeine Informationen 6<br />
Programm 7<br />
Vorprogramm – Mittwoch, 10. Oktober <strong>2012</strong> 8<br />
Pharmaziehistorische Veranstaltung Pharmazie in <strong>Greifswald</strong> 8<br />
Hauptsymposium Arzneimittelkontrolle 8<br />
Hauptprogramm – Donnerstag, 11. Oktober <strong>2012</strong> 9<br />
Diskussionsveranstaltungen Pharmazie 2020 – Perspektiven in Forschung und Lehre 9<br />
Eröffnung der <strong>Jahrestagung</strong> <strong>2012</strong> 9<br />
Hauptprogramm – Freitag, 12. Oktober <strong>2012</strong> 11<br />
Hauptprogramm – Sonnabend, 13. Oktober <strong>2012</strong> 15<br />
Vorträge 17<br />
Plenarvortrag Moleküle 18<br />
Plenarvortrag Targets 19<br />
Plenarvortrag Tabletten 20<br />
Sessionvorträge Moleküle 21<br />
Session Struktur-basiertes Design 21<br />
Session Molekulare Werkzeuge 25<br />
Session IVIVC: Modelle und Modellierungen 29<br />
Session Moderne Trenntechniken 33<br />
Session Naturstoffe 36<br />
Session Klinische Wirkstoffentwicklung 40<br />
Sessionvorträge Targets 44<br />
Session Proteomics und Metabolomics 44<br />
Session Transporter und Ionenkanäle 48<br />
Session Nukleäre Rezeptoren und Transkriptionsfaktoren als Wirkstofftargets 52<br />
Session Thrombozytenfunktion und Gerinnung 58<br />
Session Neue Targets und Testsysteme 61<br />
Session Tumortherapie und Resistenzmechanismen 65<br />
Sessionvorträge Tabletten 69<br />
Session Nanomedizin 69<br />
Session Pharmaverfahrenstechnik 75<br />
Session Bioanalytik: Testsysteme und Datenqualität 79<br />
Session Spezielle Darreichungsformen 83<br />
Session Optimierte Patientenversorgung 87<br />
Session Arzneimittelüberwachung/Regulatory Affairs 90<br />
Poster 93<br />
Autorenverzeichnis 141<br />
5
Allgemeine Informationen<br />
Lagepläne der Innenstadt <strong>Greifswald</strong>s sowie vom Campus finden sich am Ende des Tagungsbandes.<br />
Internetzugang<br />
Die <strong>Universität</strong> <strong>Greifswald</strong> nimmt am eduroam-Projekt (education roaming) teil. eduroam ermöglicht es Ihnen, sich<br />
mit Ihrer persönlichen Nutzerkennung Ihres Rechenzentrums in das Internet einzuloggen und einen gesicherten<br />
WLAN-Zugang an allen teilnehmenden Forschungs- und Ausbildungseinrichtungen, also auch hier während der<br />
Tagung in <strong>Greifswald</strong> nutzen zu können. Die allgemeinen, von Ihrer Hardware (Laptop, PC, Mobiltelefone)<br />
unabhängigen Einstellungen zur Nutzung dieses Dienstes sind im Folgenden aufgeführt:<br />
SSID eduroam<br />
Sicherheitstyp Firmenweiter WPA2 (WPA2 Enterprise)<br />
äußeres Identifizierungsverfahren TTLS<br />
inneres Identifizierungsverfahren PAP<br />
externe Identität anonymous@uni-greifswald.de<br />
innere Identität Ihre persönliche Nutzerkennung am URZ<br />
Vertrauenswürdige Zertifikate Deutsche telekom Root CA 2<br />
Weitere Informationen unter: http://www.rz.uni-greifswald.de/dienste/zugang-zum-uni-netz/eduroam.html<br />
Alternativ dazu kann man sich über den Tagungsaccount in das Netzwerk der Uni <strong>Greifswald</strong> einloggen:<br />
Benutzer<br />
Passwort<br />
<strong>DPhG</strong>_<strong>2012</strong><br />
Die <strong>Universität</strong> übernimmt für Schäden, die aus der Anwendung des Funknetzes (Wireless-LAN) entstehen, keinerlei<br />
Haftung.<br />
Auf Seite 151 findet sich ein Linienplan des öffentlichen Nahverkehrs <strong>Greifswald</strong> (Bus).<br />
6
Programm
Vorprogramm – Mittwoch, 10. Oktober <strong>2012</strong><br />
► Die Veranstaltungen des Vorprogramms finden im Hörsaal des C_DAT statt.<br />
Pharmaziehistorische Veranstaltung<br />
Pharmazie in <strong>Greifswald</strong><br />
14:00–14:15 Begrüßung und Einführung<br />
Prof. Dr. Christoph Friedrich (Marburg)<br />
14:15–15:00<br />
15:00–15:45<br />
15:30–15:45 Kaffeepause<br />
15:45–16:30<br />
16:30–17:15<br />
Zur Entwicklung des Pharmaziestudiums an der <strong>Universität</strong> <strong>Greifswald</strong> vor 1945<br />
Prof. Dr. Christoph Friedrich (Marburg)<br />
Zur Entwicklung der Pharmazie an der <strong>Universität</strong> <strong>Greifswald</strong> nach 1945<br />
Prof. Dr. Thorsten Beyrich (<strong>Greifswald</strong>)<br />
Das Apothekenwesen der Stadt <strong>Greifswald</strong> von den Anfängen bis zum 20.<br />
Jahrhundert<br />
Dr. Hartmut Bettin (<strong>Greifswald</strong>)<br />
Die Homöopathie an der <strong>Universität</strong> <strong>Greifswald</strong><br />
Dr. Ulrich Meyer (Berlin)<br />
Vorprogramm<br />
Abend für Offizinapotheker<br />
18:00–18:45 Chair: Prof. Dr. Andreas Link<br />
Michael Jackson – DieSehnSUCHT nach Schlaf<br />
Prof. Dr. Dieter Steinhilber/Prof. Dr. Theo Dingermann (Frankfurt am Main)<br />
19:00–20:00<br />
Individualisierte Medizin<br />
Podiumsgespräch Dr. Hildegard Kaulen (FAZ), Prof. Dr. Theo Dingermann (Frankfurt am<br />
Main), Prof. Dr. Wolfgang Lieb (<strong>Greifswald</strong>), Dr. Christian Ude (Darmstadt), u.a.<br />
Ganztägiges<br />
Hauptsymposium Arzneimittelkontrolle<br />
Eigenverantwortlich organisiert und selbständig durchgeführt von der Fachgruppe Arzneimittelkontrolle –<br />
Pharmazeutische Analytik.<br />
► Ort: Biotechnikum, Raum 0.09<br />
Das Programm publiziert die Fachgruppe unter:<br />
http://www.pharmchem.tu-bs.de/forschung/waetzig/#veranstaltungen<br />
ab 17:00 Uhr<br />
Hörsaal<br />
Pharmazie<br />
Sitzung des Verbandes der Professoren an Pharmazeutischen Hochschulen der<br />
Bundesrepublik Deutschland e. V. (VdPPHI)<br />
8 Programm – 10.10.
Hauptprogramm – Donnerstag, 11. Oktober <strong>2012</strong><br />
Diskussionsveranstaltungen<br />
Pharmazie 2020 – Perspektiven in Forschung und Lehre<br />
08:30–10:00 5 Parallelveranstaltungen der einzelnen Prüfungsfächer<br />
Audimax HS 1 Audimax HS 2 Audimax HS 3 Audimax HS 4 Audimax HS 5<br />
09:45<br />
Uni Hauptgebäude<br />
Pressekonferenz<br />
im Beratungsraum des Rektorats, Domstr. 11, Eingang 2<br />
10:00–10:30 Pause (Fußweg in die Stadthalle)<br />
10:30–12:00<br />
Stadthalle, Kaisersaal<br />
13:00–13:30<br />
Stadthalle, Kaisersaal<br />
13:30–14:15<br />
Stadthalle, Kaisersaal<br />
14:15–15:00<br />
Stadthalle, Kaisersaal<br />
Abschlussdiskussion Plenarveranstaltung<br />
Chair: Prof. Dr. Angelika M. Vollmar /Prof. Dr. Dieter Steinhilber<br />
Eröffnung der <strong>Jahrestagung</strong> <strong>2012</strong><br />
Moleküle, Targets und Tabletten –<br />
Translationale Forschung für Arzneimittel der Zukunft<br />
Dipl.-Ing. Jörg Hochheim, Leiter des Dezernats für Bauwesen und Umwelt und<br />
1. Stellvertreter des Oberbürgermeisters der Hansestadt <strong>Greifswald</strong><br />
Prof. Dr. Rainer Westermann, Rektor der <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-<strong>Universität</strong> <strong>Greifswald</strong><br />
Plenarvortrag Moleküle<br />
Prof. Dr. Rolf Müller (Saarbrücken)<br />
Genom-basierte mikrobielle Naturstoff-Forschung für die<br />
Entwicklung von anti-Infektiva<br />
Plenarvortrag Targets<br />
Prof. Dr. Kathrin Mädler (Bremen)<br />
Anti-entzündliche Therapien für Diabetes – Rettung für die<br />
Beta-Zelle<br />
15:00–15:30 Fußweg zum neuen Campus<br />
15:30–16:00 Kaffeepause<br />
Programm – 11.10. 9
Moleküle ► Hörsaal Biochemie<br />
16:00–17:30 Struktur-basiertes Design<br />
Chair: Prof. Dr. Wolfgang Sippl & Prof. Dr. Christoph Sotriffer<br />
Prof. Dr. Gerhard Wolber (Berlin): „New algorithms for 3D pharmacophore-based virtual screening“<br />
Prof. Dr. Holger Gohlke (Düsseldorf): „From Determinants of RUNX1/ETO Tetramerization to Small-Molecule<br />
Protein-Protein Interaction Inhibitors Targeting Acute Myeloid Leukemia“<br />
Dr. Peter Kolb (Marburg): „Docking to GPCRs: ligand diversity, efficacy and selectivity“<br />
Dr. Andrea Straßer (Regensburg): „Molecular dynamic simulation of a symmetric active state hβ2R-Gαβγhomodimer<br />
complex - structural and energetical analysis“<br />
Targets ► Hörsaal Biotechnikum<br />
16:00–17:30 Proteomics und Metabolomics<br />
Chair: Prof. Dr. Irmgard Merfort & Prof. Dr. Thomas Schweder<br />
Prof. Dr. Uwe Völker (<strong>Greifswald</strong>): „OMICs-Analysen von Populations-basierten Kohorten – Mögliche Ansätze<br />
für das Biomarkerscreening“<br />
Prof. Dr. Thomas Efferth (Mainz): „Drug target fishing in silico and in vitro“<br />
Dr. Manuel Liebeke (London): „From whole-organism to sub-cellular localization of metabolites by mass<br />
spectrometry imaging“<br />
Rico Schwarz (Halle): „Incorporation of unnatural amino acids to monitor conformational changes in peroxisome<br />
proliferator-activated receptor alpha (PPARα)“<br />
Tabletten ► Hörsaal Physik<br />
16:00–17:30 Nanomedizin<br />
Chair: Prof. Dr. Udo Bakowsky & Prof. Dr. Claus-Michael Lehr<br />
Prof. Dr. Claus-Michael Lehr (Saarbrücken): „Nanomedicines for targeted drug delivery to the inflamed intestinal<br />
mucosa“<br />
Prof. Dr. Udo Bakowsky (Marburg): „Selective gene vehicles for enhanced DNA and siRNA delivery“<br />
Dr. Christian Wischke (Teltow): „Modulating immune responses of human cells by adjuvant loaded particulate<br />
carriers“<br />
Jun.-Prof. Dr. Peter R. Wich (Mainz): „Acid-Degradable Dextran Particles for the Delivery of Biotherapeutics“<br />
Astrid Müller (Jena): „Characterization of Bacterial Nanocellulose as Drug Delivery System for Low and High<br />
Molecular Weight Drugs“<br />
Dr. Mathias Könczöl (Freiburg): „Biomedical applications of magnetite in the respiratory system - Toxicological<br />
considerations and mechanistic studies in A549 lung cells“<br />
17:30–19:00 Posterpräsentationen (Institut für Biochemie)<br />
19:00–22:00 Begrüßungsabend, Posterpräsentationen (Institut für Biochemie)<br />
10 Programm – 11.10.
Hauptprogramm – Freitag, 12. Oktober <strong>2012</strong><br />
Moleküle ► Hörsaal Biochemie<br />
09:00–10:30 Molekulare Werkzeuge<br />
Chair: Prof. Dr. Ulrike Holzgrabe & Prof. Dr. Andrea Sinz<br />
Dr. Marcus Bantscheff (Cellzome AG): „Chemoproteomic target deconvolution of bioactive molecules“<br />
Christopher Lang (Erlangen): „Molecular tools for positron emission tomography (PET): Development of a highly<br />
selective radiotracer for the detection of neurotensin receptor 1 positive tumors“<br />
Prof. Dr. Wolfgang Maison (Hamburg): „Analogs of siderophores and mussel adhesion proteins for applications<br />
in clinical hygiene and implant medicine“<br />
Dr. Eberhard Heller (Würzburg): „Microwave-Enhanced Synthesis of Fluorescent Dyes for Pharmacological<br />
Purposes Using Real-Time Observation by UV/Vis Spectroscopy“<br />
Prof. Dr. Armin Buschauer (Regensburg): „Fluorescent and radiolabelled ligands as molecular tools for NPY<br />
receptors“<br />
10:30–11:00 Kaffeepause<br />
11:00–12:30 IVIVC: Modelle und Modellierungen<br />
Chair: Prof. Dr. Charlotte Kloft & Prof. Dr. Werner Weitschies<br />
Dr. Thorsten Lehr (Saarbrücken): „From Mice to Men: Application and Impact of in vivo Modeling and Simulation<br />
in Drug Research and Development“<br />
Dr. Grzegorz Garbacz (<strong>Greifswald</strong>): „Estimating the In Vivo Release Behaviour of Extended- and Immediate-<br />
Release Products using biorelevant bissolution stress tests“<br />
Dr. Steffi Hansen (Saarbrücken): „Improved input parameters for diffusion models of skin absorption“<br />
Arun Jain (<strong>Greifswald</strong>): „Interindividual gastrointestinal erosion and inter- as well as intraindividual<br />
gastrointestinal transit time variability of hydrophilic matrix tablets“<br />
12:30–13:30 Mittagspause (C_DAT Seminarraum)<br />
13:30–15:00 Moderne Trenntechniken<br />
Chair: Prof. Dr. Thomas Jira & Prof. Dr. Michael Lämmerhofer<br />
Prof. Dr. Hermann Wätzig (Braunschweig): „Continuous Improvement in Pharmazeutischer Analytik: schneller,<br />
präziser, selektiver“<br />
Prof. Dr. Andrea Sinz (Halle): „Untersuchung von Proteinen und Metaboliten -- Was können moderne<br />
Massenspektrometer leisten?<br />
Evamaria Falck (Münster): „Metabolism studies of Ifenprodil, a potent NR2B-selective NMDA receptor<br />
antagonist“<br />
Dr. Anette Thiessen (Anton Paar GmbH): „Macroscopic Questions and Molecular Answers: Measuring Optical<br />
Rotation in Latest Chiral Separation Techniques“<br />
15:00–15:30 Kaffeepause<br />
15:30–17:00 Naturstoffe<br />
Chair: Prof. Dr. Ulrike Lindequist & Prof. Dr. Angelika M. Vollmar<br />
Dr. Birgit Kraus (Regensburg): „Hepatic bioactivity of phenolic natural compounds“<br />
Dr. Karin von Schwarzenberg (München): „Mode of cell death induction by pharmacological V-ATPase<br />
inhibition“<br />
Dr. Timo Niedermeyer (Berlin/<strong>Greifswald</strong>): „Cyanobacteria in Anticancer Natural Products Research“<br />
Prof. Dr. Florian C. Stintzing (WALA Heilmittel GmbH): „Applied phytochemical research – an industry’s<br />
perspective“<br />
Programm – 12.10. 11
17:15–18:45 Klinische Wirkstoffentwicklung<br />
Chair: Prof. Dr. Klaus Mohr & Prof. Dr. Werner Siegmund<br />
Prof. Dr. Jörg König (Erlangen): „Single- and multiple-transfected cell lines as tools for the analysis of drug<br />
transport and of the interplay of drug transport and drug metabolism“<br />
Dr. Stefan Oswald (<strong>Greifswald</strong>): „Methods to investigate the clinical relevance of drug transporters“<br />
Dr. Dieter Runge (PRIMACYT GmbH): „The functionality of uptake transporters in primary hepatocytes of<br />
different species can be verified by estrone-3-sulfate”<br />
Anett Engel (<strong>Greifswald</strong>): „Solubilizing agents influence the pharmacokinetics of probe drugs by transporter<br />
interaction“<br />
20:00–23:00 Gesellschaftsabend (Stadthalle, Theatercafé)<br />
Targets ► Hörsaal Biotechnikum<br />
09:00–10:30 Transporter und Ionenkanäle<br />
Chair: Prof. Dr. Bernhard H. Rauch & Prof. Dr. Peter Ruth<br />
Prof. Dr. Marc Freichel (Heidelberg): „TRP channels as regulators of blood pressure and fertility“<br />
Dr. Robert Lukowski (Tübingen): „Role of Ca2+ -activated K + Channels in Breast Cancer Proliferation“<br />
PD Dr. Gabriele Jedlitschky (<strong>Greifswald</strong>): „Transporters in human platelets: physiological function and impact for<br />
pharmacotherapy“<br />
PD Dr. Stephan Reichl (Braunschweig): „Expression analysis of drug transporter proteins in RPMI 2650 cell line<br />
and excised human nasal mucosa“<br />
10:30–11:00 Kaffeepause<br />
11:00–12:30 Nukleäre Rezeptoren und Transkriptionsfaktoren als<br />
Wirkstofftargets<br />
Chair: Prof. Dr. Manfred Schubert-Zsilavecz & Prof. Dr. Dieter Steinhilber<br />
Prof. Dr. Ronald Gust (Innsbruck): „Investigation on the estrogen receptor alpha selectivity of 2,3,5-triaryl-1Hpyrroles“<br />
Prof. Dr. Manfred Jung (Freiburg): „Chemical epigenetics: histone modifying enzymes as targets to regulate<br />
transcription“<br />
Prof. Dr. Irmgard Merfort (Freiburg): „Helenalin derivatives and their impact on transcriptional control of<br />
mediators involved in rheumatic diseases“<br />
Prof. Dr. Frank Böckler (Tübingen): „Rescuing mutant p53 using halogen-enriched fragment libraries (HEFLibs)“<br />
Jun. Prof. Dr. Eugen Proschak (Frankfurt): „Novel leads for PPARs and FXR: a computer-aided drug design<br />
approach“<br />
Prof. Dr. Dieter Steinhilber (Frankfurt): „Inhibitors of class I histone deacetylases induce 5-lipoxygenase<br />
expression“<br />
12:30–13:30 Mittagspause (C_DAT Seminarraum)<br />
13:30–15:00 Thrombozytenfunktion und Gerinnung<br />
Chair: Prof. Dr. Susanne Alban & Prof. Dr. Andreas Greinacher<br />
Prof. Dr. Susanne Alban (Kiel): „Novel oral anticoagulants – State of the art and aspects of their application“<br />
Prof. Dr. Andreas Greinacher (<strong>Greifswald</strong>): „Novel platelet inhibitors – State of the art and aspects of their<br />
application”<br />
Dr. Krystin Krauel (<strong>Greifswald</strong>): „Naturally occurring PF4/polyanion complexes prime for heparin-induced<br />
thrombocytopenia as a misdirected host defense mechanism“<br />
12 Programm – 12.10.
15:00–15:30 Kaffeepause<br />
15:30–17:00 Neue Targets und Testsysteme<br />
Chair: Prof. Dr. Manfred Jung & Prof. Dr. Stefan Laufer<br />
Prof. Dr. Conrad Kunick (Braunschweig): „PfGSK-3: A target for new antimalarial drugs?“<br />
Prof. Dr. Daniel Rauh (Dortmund): „Chemical Oncology – Converging Cancer Genetics, Structural Biology and<br />
Medicinal Chemistry“<br />
Dr. Dante Rotili (Rom): „Development of lysine mimic quinazoline-based selective inhibitors of a Jmj-C histone<br />
demethylase family“<br />
Prof. Dr. Mike Schutkowski (Halle): „Profiling of epigenetic targets using peptide microarrays“<br />
17:15–18:45 Tumortherapie und Resistenzmechanismen<br />
Chair: Prof. Dr. Patrick J. Bednarski & Prof. Dr. Conrad Kunick<br />
Prof. Dr. Rolf W. Hartmann (Saarbrücken): „Anti-infectives research to combat drug resistant tumors?“<br />
Prof. Dr. Matthias Kassack (Düsseldorf): „Chemoresistance of ovarian cancers against platinum complexes“<br />
Prof. Dr. Ingo Ott (Braunschweig): „Medicinal Chemistry with Organometallics - From Catalysts to Anticancer<br />
Drugs?“<br />
Dr. Matthias Tacke (Dublin): „NHC-Silver(I) and NHC-gold(I) complexes as bioorganometallic anticancer and<br />
antibacterial drugs“<br />
Riad Schulz (<strong>Greifswald</strong>): „A new class of glutathione peroxidase inhibitors is able to reverse resistance to<br />
chemotherapeutics in human B-cell lymphoma cell lines“<br />
20:00–23:00 Gesellschaftsabend (Stadthalle, Theatercafé)<br />
Tabletten ► Hörsaal Physik<br />
09:00–10:30 Pharmaverfahrenstechnik<br />
Chair: Prof. Dr. Peter Kleinebudde & Prof. Dr. Christel Müller-Goymann<br />
Prof. Dr. Heike Bunjes (Braunschweig): „Formulierungs-Screening mit kolloidalen Wirkstoffträgersystemen“<br />
Dr. Markus Thommes (Düsseldorf): „Pharmaceutical Extrusion technology – Recent developments“<br />
PD Dr. Hubert Rein (Bonn): „Hot melt extruded dosage forms“<br />
André Bitterlich (Braunschweig): „Nanogrinding of naproxen: influence of process parameters on product<br />
quality“<br />
10:30–11:00 Kaffeepause<br />
11:00–12:30 Bioanalytik: Testsysteme und Datenqualität<br />
Chair: Prof. Dr. Hermann Wätzig & Prof. Dr. Gerhard Winter<br />
Prof. Dr. Knut Baumann (Braunschweig): „Bildverarbeitung für biomedizinisch eingesetzte Mikroskopie“<br />
Dr. Achmed Besheer (München): „The use of microscale thermophoresis (MST) for protein characterization and<br />
formulation“<br />
Tim Menzen (München): „High-Throughput Protein Melting Temperature Analysis by Differential Scanning<br />
Fluorimetry“<br />
Prof. Dr. Christiane Ritter (Braunschweig): „NMR Spektroskopie von hochmolekularen Proteinkomplexen“<br />
12:30–13:30 Mittagspause (C_DAT Seminarraum)<br />
Programm – 12.10. 13
13:30–15:00 Spezielle Darreichungsformen<br />
Chair: Prof. Dr. Jörg Breitkreutz & Prof. Dr. Sandra Klein<br />
Prof. Dr. Jörg Breitkreutz (Düsseldorf): „Orodispersible dosage forms – novel concepts, innovative products,<br />
new therapeutic options“<br />
Marco Neumann (<strong>Greifswald</strong>): „Challenges in developing gastroretentive dosage forms“<br />
Matthias Rischer (Losan Pharma GmbH): „Innovative nanoparticular dosage forms – advantages in<br />
pharmacokinetics and administration“<br />
Susanne Kirchhof (Regensburg): „Development and pharmaceutical technological applications of Diels-Alder<br />
hydrogels”<br />
15:00–15:30 Kaffeepause<br />
15:30–17:00 Optimierte Patientenversorgung<br />
Chair: Prof. Dr. Ulrich Jaehde & Prof. Dr. Christoph Ritter<br />
Prof. Dr. Ulrich Jaehde (Bonn): „Medication safety research as challenge for pharmaceutical scientists“<br />
Prof. Dr. Thilo Bertsche (Leipzig): „Klinische Pharmazeuten zur Verbesserung der Patientensicherheit in der<br />
Arzneimitteltherapie“<br />
Prof. Dr. Petra A. Thürmann (Witten/Herdecke): „Arzneimitteltherapiesicherheit in Alten- und Pflegeheimen“<br />
Dr. Thomas Fiß (<strong>Greifswald</strong>): „Identifikation von Risikofaktoren für das Auftreten von Arzneimittelbezogenen<br />
Problemen im Rahmen von häuslichen Medikationsreviews“<br />
17:15–18:45 Arzneimittelüberwachung/Regulatory Affairs<br />
Chair: Dr. Olaf Queckenberg & PD Dr. Klaus Raith<br />
Dr. Andreas Sutter (Bayer AG): „Analyzing the use of (Q)SAR models for the evaluation of potential genotoxic<br />
impurities: A cookbook in support of ICH M7“<br />
Dr. Adrian Funke (Bayer AG): „Control strategy for an active coating process based on systematic process<br />
development (DoE) and in-line Raman spectroscopy (PAT)“<br />
Dr. Isabel Astner (Braunschweig): „Gewebe und Zellen – Arzneimittel für neuartige Therapien“<br />
Dr. Nicholas Schramek (Oberschleißheim): „Analytik von illegalen Arzneimitteln“<br />
20:00–23:00 Gesellschaftsabend (Stadthalle, Theatercafé)<br />
14 Programm – 12.10.
09:00–09:45<br />
HS Nord Med.<br />
10:00–11:30<br />
HS Nord Med.<br />
11:30–11:45<br />
HS Nord Med.<br />
Hauptprogramm – Sonnabend, 13. Oktober <strong>2012</strong><br />
11:45–12:15 Kaffeepause<br />
12:15–13:00<br />
HS Nord Med.<br />
Plenarvortrag Tabletten<br />
Prof. Dr. Wolfgang Frieß (München)<br />
Analyse von Aggregaten und Partikeln in Proteinarzneimitteln<br />
– was ist eigentlich das Problem?<br />
Kurzvorstellung von Leuchtturmprojekten<br />
• Helmholtz Institute for Pharmaceutical Research (HIPS)<br />
Saarland<br />
• Center of Drug Absorption and Transport (C_DAT)<br />
<strong>Greifswald</strong><br />
• Zentrum für Pharmaverfahrenstechnik (PVZ)<br />
Braunschweig<br />
• Fraunhofer Projektgruppe – Translationale Medizin und Pharmakologie (TMP) Frankfurt<br />
am Main<br />
Pharmazie 2020 – Perspektiven in Forschung und Lehre<br />
Chair: Prof. Dr. Dieter Steinhilber<br />
Abschlussveranstaltung<br />
Verleihung von Ehrungen und Preisen<br />
Programm – 13.10. 15
Vorträge
Plenarvortrag Moleküle<br />
Genom-basierte mikrobielle Naturstoff-Forschung für die Entwicklung von anti-Infektiva<br />
Müller R<br />
Helmholtz-Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Pharmaceutical Biotechnology,<br />
Saarland University, Campus, Building C2 3, 66123 Saarbrücken, Germany<br />
Microorganisms continue to be one of the most important resources of bioactive molecules exhibiting potential for<br />
pharmaceutical application (1). This is especially true for urgently needed antibacterials which almost exclusively are<br />
derived from bacteria or fungi. Aiming at the identification of novel hit and lead structures ideally addressing new<br />
targets in pathogens, genome analysis of microbial producer organisms has become very useful for the in silico<br />
prediction of the hypothetical potential of a given organism for the production of secondary metabolites (2,3).<br />
Although revealing tremendous potential in terms of the presence of typical natural product biosynthetic genes, the<br />
translation of this knowledge into "real compounds in the flask" clearly represents the bottleneck in this approach. In<br />
the presentation I will discuss our recent efforts to develop and employ genome mining to a) identify (4,5), b)<br />
heterologously express (6,7) and c) modify secondary metabolite biosynthesis (8,9). In addition, I will present two<br />
success stories where novel natural product scaffolds were isolated and have been found to indeed address new<br />
targets in pseudomonads and staphylococci (10-12).<br />
Reference List:<br />
1. Newman, D. J., Cragg, G. M.: J.Nat.Prod. <strong>2012</strong>, 75(3): 311–335.<br />
2. Bode, H. B., Müller, R.: Angew.Chem.Int.Ed. 2005, 44(42): 6828–6846.<br />
3. Wenzel, S. C., Müller, R.: Natural Product Reports 2011,26(11):1385-407.<br />
4. Schneiker, S. et al.: Nat.Biotechnol. 2007, 25(11): 1281–1289.<br />
5. Cortina, N. S. et al.: Angew.Chem.Int.Ed. <strong>2012</strong>, 51(3): 811–816.<br />
6. Wenzel, S. C. et al.: Chem.Biol. 2005, 12(3): 349–356.<br />
7. Fu, J. et al.: Nat.Biotechnol. <strong>2012</strong>, 30 440–446.<br />
8. Rachid, S. et al.: ChemBioChem 2011, 12(6): 922–931.<br />
9. Quade, N. et al.: Nat Chem Biol Nat Chem Biol <strong>2012</strong>, 8(1): 117–124.<br />
10. Bielecki, P. et al.: ChemBioChem <strong>2012</strong>, in press.<br />
11. Steinmetz, H. et al.: Angew.Chem.Int.Ed. 2011, 50(2): 532–536.<br />
12. Dehn, R. et al.: Angew.Chem.Int.Ed. 2011, 50(17): 3882–3887.<br />
18 Plenarvorträge
Plenarvortrag Targets<br />
Anti-inflammatory compounds as novel tools to rescue the pancreatic β-cell in diabetes<br />
Maedler K<br />
Islet Biology Laboratory, Centre for Biomolecular Interactions Bremen, University of Bremen, Germany<br />
In both, type 1 and type 2 diabetes mellitus the main processes leading to β-cell failure are apoptosis and loss of<br />
function. Although the β-cell has an enormous capacity to adapt to conditions of higher insulin demand, e.g. in<br />
obesity and pregnancy, its continuous overstimulation leads to a β-cell overwork, subsequent loss of function and<br />
apoptosis and diabetes progression. This is a result from a complex interplay of environmental factors together with<br />
genetic predisposition.<br />
Many studies demonstrate how cytokines and chemokines have an active role in triggering the immune-response<br />
against the β-cell population. In a recent study we have identified 2 classical inflammatory mediators, the cytokine IL-<br />
1β and the chemokine CXCL10, which both play an active role in triggering β-cell destruction.<br />
We have identified the Toll like receptor 4 as the receptor for CXCL10 and as new pathway for the induction of β-cell<br />
apoptosis.<br />
In the lecture, I will give an overview on mechanisms of destruction of the β-cell in diabetes, the influence of obesity,<br />
the crosstalk between fat and pancreas and resulting new therapeutic approaches to fight onset and progression of<br />
diabetes.<br />
Key words: diabetes, β-cells, apoptosis, proliferation, inflammation, IL-1β, CXCL10<br />
Plenarvorträge 19
Plenarvortrag Tabletten<br />
Analysis of aggregates and particles in protein drug products – what is the challenge?<br />
Frieß W<br />
Department of Pharmacy; Pharmaceutical Technology and Biopharmaceutics; Ludwig-Maximilians Universitaet Muenchen, Butenandtstr. 5,<br />
D-81377 Muenchen<br />
Protein aggregates and particles are currently a hot topic in pharmaceutical industry as well as for the regulatory<br />
agencies. FDA sees a strong scientific basis for aggregates, including sub-visible particulates, to induce immune<br />
responses but insufficient data to determine the likelihood of immune response induction given the levels/types of<br />
aggregates in any given product. Therefore, aggregates and sub-visible particulates should be assumed critical to<br />
the observed safety and efficacy profile and controlled commensurate with risk [1]. In order to adequately define the<br />
actual risk, on the one hand more detailed studies on the causes and correlation of protein aggregates and particles<br />
with immunogenicity are deemed necessary. On the other hand the detailed characterization of protein particles is<br />
mandatory. The latter includes both quantity and quality such as size, shape, morphology, bond (covalent or<br />
noncovalent) or protein conformation. This implies that no single method can cover the entire range but instead a<br />
multitude of orthogonal methods is applied for characterization [2,3]. All methods come with limitations which need to<br />
be considered and new methods are developed. For example classical size exclusion chromatography (SEC) may<br />
provide inaccurate results in the analysis of protein aggregates due to protein adsorption to column media as well as<br />
dissociation or formation of aggregate species at the higher salt concentration typically applied in the eluent.<br />
Orthogonal methods like analytical ultracentrifugation or asymmetrical flow field flow fractionation should always be<br />
used in the development of SEC methods and to corroborate the accuracy of SEC results [4]. In order to obtain<br />
additional information, methods can be combined, for example a combination of SEC with addition of fluorescent dye<br />
enables to trace unfolding of the individual separate protein species [5]. Another option is to combine SEC with the<br />
determination of individual second virial coefficients of monomer and oligomers in protein samples [6]. For<br />
characterization of subvisible particles several new methods have been established. Nanoparticle tracking analysis<br />
(NTA) is an innovative system for sizing particles from about 30 to 1,000 nm based on laser light scattering<br />
microscopy to track individual nanoparticles moving under Brownian motion [7]. Another system called Archimedes is<br />
based on a fluidic microchannel embedded inside a resonator through which the sample is transported and as<br />
particles flow through the resonators the change in resonant frequency gives the individual particle mass and size<br />
with high resolution [8]. But also established methods like the classic Coulter Counter experience a renaissance as<br />
the results of the standard optic based methods to analyse subvisible particles like light obscuration of micro flow<br />
imaging depend on refractive index differences between particle and formulation [9]. Ultimately these developments<br />
also give rise to new guidelines to direct the industry and USP has just published a chapter “Subvisible Particulate<br />
Matter in Therapeutic Protein Injections” for comment in the Pharmacopia Forum. The critical evaluation of<br />
protein aggregates and particles is not only challenging for standard formulations but also presents a major<br />
roadblock for drug delivery systems [10].<br />
References:<br />
1. Rosenberg A.: Workshop on protein aggregation and immunogenicity, July <strong>2012</strong> Breckenridge, CO.<br />
2. Mahler H.-E. et al.: J. Pharm. Sci. 2009, 98(9): 2909–2934.<br />
3. Zölls S. et al.: J. Pharm. Sci. <strong>2012</strong>, 101(3): 914-935.<br />
4. Carpenter J. et al.: J. Pharm. Sci. 2010, 99(5), 2200–2208.<br />
5. Printz M., Friess W.: J. Pharm. Sci. <strong>2012</strong>, 101(2): 826-837.<br />
6. Printz M., Kalonia D., Friess W.: J. Pharm. Sci. <strong>2012</strong>, 101(1): 363-372.<br />
7. Filipe V., Hawe A., Jiskoot W.: Pharm. Res. 2010, 27(5): 796-810.<br />
8. Burg T.P. et al.: Nature 2007, 446(7139), 1066-1069.<br />
9. Hawe A.: Workshop on protein aggregation and immunogenicity, July <strong>2012</strong> Breckenridge, CO.<br />
10. Jiskoot W. et al.: J. Pharm. Sci. <strong>2012</strong>, 101(3): 946-954.<br />
20 Plenarvorträge
Sessionvorträge Moleküle<br />
Session Struktur-basiertes Design<br />
New algorithms for 3D pharmacophore-based virtual screening<br />
Spitzer G1 , Liedl K1 , Wolber G2 1Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria<br />
2Institute of Pharmacy/Pharmaceutical Chemistry, Computer-Aided Drug Design, Freie <strong>Universität</strong> Berlin, Königin-Luisestr. 2+4, 14195 Berlin,<br />
Germany<br />
Virtual screening using three-dimensional arrangements of chemical features (3D pharmacophores) has become a<br />
highly relevant method for virtual hit identification and lead optimization. Although frequently used, considerable<br />
differences exist in the interpretation of chemical features and the implementation of their corresponding 3D overlay<br />
algorithms [1].<br />
We have recently continued the development of our 3D alignment algorithm relying on pattern recognition. Based on<br />
this method, a workflow for high-performance multi-conformational database screening has been created using new<br />
fingerprint filtering techniques that delivers a higher degree of geometric accuracy than previously published or<br />
commercially available approaches. Despite the considerable gain in geometric subsampling and the resulting<br />
increased alignment accuracy, this technique allows for computationally highly efficient virtual pharmacophore<br />
screening allowing to process millions of molecules in hours on a desktop computer. The impact of increased<br />
geometric accuracy on virtual screening will be presented and discussed by means of benchmarks and prospective<br />
screening studies.<br />
References:<br />
1. Spitzer G et al., J. Chem Inf. Model 2010, 50(7): 1241-1247.<br />
Sessionvorträge Moleküle 21
From Determinants of RUNX1/ETO Tetramerization to Small-Molecule Protein-Protein Interaction<br />
Inhibitors Targeting Acute Myeloid Leukemia<br />
Metz A1 ; Schanda J2 ; Wichmann C2 ; Grez M2 ; Gohlke H1 1 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University, <strong>Universität</strong>sstr. 1, 40591 Düsseldorf, Germany<br />
2 Georg-Speyer-Haus, Institute for Biomedical Research, Paul-Ehrlich-Str. 42-44, 60596 Frankfurt, Germany<br />
A promising way to interfere with biological processes is through the control of protein-protein interactions by means<br />
of small molecules. Many of these small-molecule modulators known to date have not been found by rational design<br />
approaches. In large part this is due to the challenges that one faces in dealing with protein binding epitopes.<br />
However, recent advances in the understanding of the energetics and dynamics of protein binding interfaces open up<br />
a way to apply rational design approaches also for finding protein-protein interaction modulators. Here, we target the<br />
homotetramerization of the NHR2 domain within the RUNX1/ETO fusion protein using computational methods<br />
developed in our group [1, 2, 3]. Homotetramerization of the NHR2 domain leads to the onset of acute myeloid<br />
leukemia.<br />
Initially, energetic contributions of individual amino acids within the NHR2 homotetramer were analyzed. Ala<br />
substitution of amino acids with strong energetic contributions abolished tetramer formation. The resulting NHR2<br />
dimers fail to induce leukemia in mice. These studies revealed the existence of a hot spot at the NHR2 dimertetramer<br />
interface, suitable for a molecular intervention.[4] Based on the hot spot information, an 18-mer peptide was<br />
designed that inhibits NHR2 tetramerization. This peptide was used as a model compound to establish in vitro<br />
assays. Using the hot spot and peptide information, small-molecule inhibitors of the NHR2 tetramerization were<br />
identified by virtual screening and validated by several in vitro assays. Some of these compounds led to the induction<br />
of differentiation markers and apoptosis in NHR2-dependent cell lines. These compounds may thus be potential<br />
chemotherapeutics in the case of acute myeloid leukemia.<br />
References:<br />
1. Kruger DM, Gohlke H, Nucl. Acids Res. 2010, 38: W480-W486.<br />
2. Gohlke H, Kiel C, Case DA, J. Mol. Biol. 2003, 330: 891-913.<br />
3. Metz A et al., J. Chem. Inf. Model. <strong>2012</strong>, 52, 120-133.<br />
4. Wichmann C et al., Blood 2010, 116: 603-613.<br />
22 Sessionvorträge Moleküle
Docking to GPCRs: ligand diversity, efficacy and selectivity<br />
Kolb P 1 ; Rosenbaum D M 2 ; Schmidt D 1 ; Bernat V 3 ; Tschammer N 3 ; Kobilka B K 2 ; Shoichet B K 4<br />
1 Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35039 Marburg, Germany<br />
2 Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305, USA<br />
3 Chemistry and Pharmacy, Friedrich Alexander University Erlangen, Schuhstraße 19, 91052 Erlangen, Germany<br />
4 Pharmaceutical Chemistry, University of California, 1700 4th Street, Box 2550, San Francisco, CA 94158, USA<br />
With GPCRs becoming amenable to crystallization in the recent past [1], structure-based computational approaches<br />
to ligand discovery have become possible. As only about 1% of the GPCRome has been solved, however, receptor<br />
structure prediction is equally important. I will discuss the usage of x-ray structures and homology models for docking<br />
and highlight some of the conclusions that can be drawn from three recent docking campaigns.<br />
Campaign 1 used the recently determined X-ray structure of the β2-adrenergic receptor [2] to investigate the<br />
advantages and limitations inherent in a structure-based approach to ligand discovery against this and related GPCR<br />
targets. Approximately 1 million commercially available, “lead-like” molecules were docked against the β2-adrenergic receptor structure. On testing of 25 high-ranking molecules, 6 were active with binding affinities
Molecular dynamic simulation of a symmetric active state hβ2R-Gαβγ-homodimer complex –<br />
structural and energetical analysis<br />
Straßer A1 ; Wittmann H-J2 1 Department of Pharmaceutical/Medicinal Chemistry II, University of Regensburg, 93040 Regensburg, Germany<br />
2 Faculty of Chemistry and Pharmacy, University of Regensburg, 93040 Regensburg, Germany<br />
Homo- and heterodimers of several GPCRs are suggested by a large number of experimental studies [1]. Most<br />
experimental studies give hint for asymmetric dimers, consisting of two receptor protomers, coupling to only one<br />
heterotrimeric G protein [1]. However, there is only hint, but no clear experimental evidence for a symmetric<br />
homodimer, consisting of two receptor protomers, each coupling to one heterotrimeric G protein [1].<br />
Based on the crystal structure of the κ-opiod receptor [2] and bovine rhodopsin [3], we manually aligned two active<br />
hβ2R-Gαβγ-complexes [4] in a symmetric manner. For refinement of the resulting hβ2R-Gαβγ-homodimer, we<br />
performed an extensive scan of the potential energy surface in an analogous manner, as already described [5].<br />
Thereby, one hβ2R-Gαβγ-complex was translated along and rotated around the x-, y- and z-axis. Subsequently, the<br />
resulting hβ2R-Gαβγ-homodimers were energetically minimized and the complex with smallest potential energy was<br />
embedded into a POPC lipid bilayer and molecular dynamic simulation of the hβ2R-Gαβγ-homodimer (Fig. 1A) was<br />
performed, including lipid, sodium and chloride ions, extra- and intracellular water.<br />
The MD simulation revealed strong van-der-Waals interactions between both hβ2 receptors. POPC molecules placed<br />
between both hβ2 receptors stabilize the hβ2R-hβ2R-interaction (Fig. 1B). Additionally, the symmetric active state<br />
hβ2R-Gαβγ-homodimer is strongly stabilized by electrostatic and hydrogen bond interaction between the Gα- and<br />
Gβ-subunits of complex 1 and 2 (Table 1). Furthermore, hydrogen bond and electrostatic interactions were detected<br />
between the Gβ subunits of different monomers (Table 1).<br />
Table 1: Interactions within the hβ2R-Gαβγ-homodimer<br />
hβ2R (1) –<br />
hβ2R (2)<br />
Gα (1) –<br />
Gβ (2)<br />
Gβ (1) –<br />
Gβ (2)<br />
hβ2R (1)–<br />
Gαβγ (2)<br />
no.<br />
of H<br />
bonds<br />
dist.<br />
don-acc<br />
< 0.35<br />
nm<br />
Eelec.(SR)<br />
[kJ/mol]<br />
EvdW<br />
[kJ/mol]<br />
3 3 -288 ± 36 -329 ± 15<br />
6 7 -543 ± 46 -142 ± 15<br />
3 2 -135 ± 44 -105 ± 12<br />
0 0 -5 ± 4 -4 ±1<br />
The molecular dynamic simulation revealed a stable symmetric active state hβ2R-Gαβγ-homodimer. Thus, the<br />
suggested homodimerization between two hβ2 receptors, based on experimental studies [6], could be also found in<br />
silico. The amino acids, determined in silico to be responsible for the interaction between both hβ2R-Gαβγcomplexes<br />
represent a good starting point for further experimental studies.<br />
References:<br />
1. Kamal, M.; Maurice, P.; Jockers, R.: Pharmaceuticals 2011, 4 273-284.<br />
2. Wu, H. et al.: Nature <strong>2012</strong>, doi:10.1038/nature10939.<br />
3. Park, J.H. et al.: Nature 2008, 454 183-187.<br />
4. Rasmussen, S.G.F. et al.: Nature 2011, doi:10.1038/nature10361.<br />
5. Strasser, A.; Wittmann, H.-J.: J Comput Aided Mol Des 2010, 24 759-769.<br />
6. Angers, S. et al.: PNAS 2000, 97(7)3684-3689.<br />
24 Sessionvorträge Moleküle
Session Molekulare Werkzeuge<br />
Chemoproteomic target deconvolution of bioactive molecules<br />
Bantscheff M<br />
Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany<br />
Chemoproteomics represents an emerging research discipline at the interface of medicinal chemistry, biochemistry,<br />
and cell biology focused on studying the molecular mechanisms of action of drugs and other bioactive small<br />
molecules. In this presentation, experimental strategies in current chemical proteomics research will be described<br />
and illustrated using recent examples of successful applications (e.g. [1,2]). Finally, areas in drug discovery where<br />
chemical proteomics-based assays using native endogenous proteins are expected to have substantial impact will be<br />
highlighted.<br />
References:<br />
1. Bantscheff et al. Nat Biotechnol. 2011, 29(3): 255-65.<br />
2. Dawson et al., Nature 2011, 478(7370): 529-33.<br />
Microwave-Enhanced Synthesis of Fluorescent Dyes for Pharmacological Purposes Using Real-<br />
Time Observation by UV/Vis Spectroscopy<br />
Heller E1 ; Lohse M J2 ; Hoffmann C2 ; Holzgrabe U1 1 Institute for Pharmacy and Food Chemistry 1, Am Hubland, D-97074 Würzburg, Germany<br />
2 Institute for Pharmacology and Toxicology 2, Versbacher Straße 9, D-97078 Würzburg, Germany<br />
Fluorescent dyes play an important role as markers to investigate biological signalling pathways or to study binding<br />
of active agents to receptors. It is possible to label proteins with 2 dyes (FRET technology, e.g. FlAsH and ReAsH<br />
[1,2]) or to mark the active agent with one dye [3]. Tagging of active agents with fluorescent dyes is an alternative<br />
method for radioactive marking.<br />
Here we aimed to synthesize several dyes of different structural classes, e.g. substituted fluoresceins, pyrilium and<br />
pyridine dyes, by means of microwave irradiation. Microwave-enhanced reactions are rather fast in comparison to<br />
conventional heating and hence, the reaction time can be shortened from hours or days to minutes. Real time<br />
monitoring of the reaction progress is often difficult, because classical monitoring (e.g. HPLC, TLC and NMR) can<br />
take up to 30 minutes analysis time. This can cause overreaction or decomposition of the product. We created a<br />
novel method which allows a real-time observation of the reaction progress in the microwave cavity. This allows<br />
detecting the exact end-point of the reaction [4] and avoids the formation of side products. The knowledge was used<br />
to speed up the synthesis of FlAsH and analogues. Additionally the dyes were obtained with excellent purity and high<br />
yields.<br />
References:<br />
1. Hoffmann, C. Lohse M. J.: BIOspektrum 2006, 12(5): 495-497.<br />
2. Lohse, M. J. et al.: Curr. Opin. Pharm. 2007, 7, 547-553.<br />
3. Schneider, E. et al.: Chembiochem. 2007, 8(16), 1981-1988.<br />
4. Heller, E. et al.: Eur. J. Org. Chem. 2010, 19, 3569-3573.<br />
Sessionvorträge Moleküle 25
Molecular tools for positron emission tomography (PET): Development of a highly selective<br />
radiotracer for the detection of neurotensin receptor 1 positive tumors<br />
Lang C<br />
Department of Chemistry and Pharmacy, Friedrich Alexander University Erlangen-Nürnberg (FAU), Schuhstr.19, D-91052 Erlangen, Germany.<br />
Neurotensin (NT) is an endogenous, 13 amino-acid peptide that mediates a number of pharmacological effects<br />
involving dopamine transmission, analgesia, and hypothermia [1]. In addition to these attributes NT acts as a key<br />
player in multiple steps of cancer progression in numerous types of cancer cells.[2] Those carcinogenic effects are<br />
presumably evoked by an abnormal expression of the neurotensin receptor 1 (NTS 1). Based on this overexpression<br />
we aimed for a specific labeling of tumors by molecular imaging techniques such as positron emission tomography<br />
(PET).<br />
In collaboration with the Clinic of Nuclear Medicine of FAU, we have reported on the design and synthesis of a<br />
radiolabeled derivative of the hexapeptide NT 8-13, the active fragment of the endogenous agonist neurotensin [3]. As<br />
an extension of these investigations, we planned to construct a 18F-labeled non-peptidic NTS 1 selective ligand based<br />
on the potent NTS antagonist SR142948A [4]. We herein report on the novel PET-Tracer 18F-LC40, which could be<br />
synthesized by taking advantage of our click chemistry based ligation of 2-deoxy-2-[ 18F]fluoroglucosyl azide (azido-<br />
[ 18F]FDG) to the alkyne functionalized analog of the lead structure. Receptor binding experiments using NTS 1<br />
expressing CHO cells indicated a Ki value for F-LC40 of 0.5 nM with 70 fold selectivity towards NTS 2. The precursor for<br />
18F-labeling was obtained by a palladium-catalyzed aminocarbonylation [5] of the respective bromoarene derivative with<br />
an alkynylamine using Mo(CO)6 as carbon monoxide source. The CuAAC using azido-[ 18F]FDG afforded the glycosyl<br />
ligand 18F-LC40 in a radiochemical yield of 20% in a total synthesis time of 75 min.<br />
The µPET studies using HT29 (human colon carcinoma cell line) xenografted nude mice demonstrated specific binding<br />
of 18F-LC40 in vivo. Furthermore, the tracer displayed a good tumor to blood ratio and excellent metabolic stability both<br />
in vitro and in vivo.<br />
O<br />
O<br />
O<br />
N N<br />
O<br />
NH COOH<br />
N<br />
18 F-LC40<br />
N<br />
N<br />
N<br />
N<br />
O OH<br />
OH<br />
References:<br />
1. for review, see: Myers, R et al., ACS Chem. Biol. 2009, 4(7): 503–525<br />
2. for review, see: Dupouy, S et al., Biochimie. 2011, 93: 1369–1378<br />
3. Maschauer, S et al., Angew. Chem. Int. Ed., 2010, 49(5): 976–979<br />
4. Labeeuw, B et. Al, 1996, Patent WO 96/3282.<br />
5. Appukkuttan, P et al., Tetrahedron Lett., 2008, 49 : 5625–5628<br />
18 F<br />
OH<br />
26 Sessionvorträge Moleküle
Analogs of siderophores and mussel adhesion proteins for applications in clinical hygiene and<br />
implant medicine<br />
Maison W1 ; Franzmann E2 ; Khalil F1 1 <strong>Universität</strong> Hamburg, Pharmaceutical and Medicinal Chemistry, Bundesstr. 45, 20146 Hamburg, Germany<br />
2 Justus-Liebig-<strong>Universität</strong> Giessen, Organic Chemistry, Heinrich-Buff-Ring 58, 35390 Giessen, Germany<br />
The chemical modification of material surfaces by molecular self-assembly of functionalized anchor molecules is an<br />
attractive concept for various applications in implant medicine, bone engineering and clinical hygiene. The major<br />
advantages of this principle for the generation of functional surfaces are a) the availability of efficient and scalable<br />
coating techniques (compatible with large surface areas and three dimensional objects), b) the generation of<br />
chemically well-defined surfaces and c) the need of relatively low amounts of anchor molecules.<br />
Recently, catecholates have received considerable interest as reagents for the mild modification of metal and metal<br />
oxide surfaces by convenient dip-and-rinse protocols.[1-3] This approach may be considered biomimetic, because<br />
catecholate moieties are key components of natural metal-binders such as mussel adhesion proteins. In addition,<br />
catecholates are abundant motifs in many siderophores like enterobactin.<br />
Inspired by the intriguing molecular symmetry of siderophores such as enterobactin, we present the design and<br />
synthesis of biomimetic triscatecholates with a tripodal binding motif based on adamantane scaffolds. These<br />
triscatecholates form extremely stable molecular monolayers on metal surfaces and may be covalently conjugated to<br />
any effector molecule of choice. We describe the immobilization properties of these multimeric catecholates on<br />
implant materials such as TiO2 and demonstrate antifouling properties and toxicity data for PEG (effector =<br />
polyethylene glycol) conjugates.<br />
References:<br />
1. J. H. Waite, J. H.; Tanzer, M. L.: Science 1981, 212(4498): 1038-1040.<br />
2. Brubaker, C. E., Messersmith, P. B.: Langmuir <strong>2012</strong>, 28(4): 2200-2205.<br />
3. E. Franzmann, F. et al.: Chem.-- Eur. J. 2011, 17(31): 8596-8603.<br />
Sessionvorträge Moleküle 27
Fluorescent and radiolabelled ligands as molecular tools for NPY receptors<br />
Buschauer A1 ; Keller M1 , Pluym N1 ; Kaske M1 , Erdmann D1 , Bernhardt G1 1 Lehrstuhl für Pharmazeutische/Medizinische Chemie II, <strong>Universität</strong> Regensburg, 93040 Regensburg, Germany<br />
In search for pharmacological tools for various G-protein coupled receptors (GPCRs) we explored the synthesis of<br />
fluorescent ligands and radioligands as and the applicability of the labelled compounds to cellular studies of<br />
neuropeptide Y (NPY) receptors as examples of peptidergic GPCRs. Four NPY receptor subtypes (Y1R, Y2R, Y4R,<br />
Y5R) are functionally expressed in humans. Whereas Y1R, Y2R and Y5R are more sensitive to NPY and peptide YY<br />
(PYY), the Y4R prefers the pancreatic polypeptide (PP) as the endogenous agonist.<br />
Two complementary approaches to labelled NPY receptor ligands were considered:<br />
(1) Preparation of “universal” ligands, addressing more than one receptor subtype. For instance, fluorescent<br />
derivatives of the endogenous peptides are useful molecular tools for confocal microscopy or flow cytometry [1]. The<br />
latter enables the simultaneous multiparametric determination of receptor subtype selectivities of investigational<br />
compounds under equilibrium conditions, using mixtures of cells expressing the respective receptors [1].<br />
(2) Synthesis of selective radiolabelled or fluorescent tracers, preferably nonpeptides, for the identification of NPY<br />
receptor subtypes on cells and in tissues, and for the determination of binding data. Special attention was paid to<br />
labelling strategies based on side chain modifications at the ε-amino group of lysine in peptides such as NPY (Y1,2,5R<br />
agonist) or [K4 ]hPP (Y4R agonist), and, in particular, at the guanidino group of arginine, both in peptides and<br />
nonpeptides [1-7].<br />
So far, the functionalisation of arginine at NG has been largely neglected in the design of GPCR ligands. However,<br />
previous studies from our laboratory demonstrated that the acylation of strongly basic guanidine residues, which are<br />
characteristic of numerous biologically active compounds, is a general, very effective biosisosteric approach to retain<br />
(or even increase) the pharmacological activities and to substantially improve the drug-like properties by lowering the<br />
basicity of GPCR ligands. This also paved the way to radioactive ( 3H, 18F) tracers and fluorescent ligands.<br />
Tritiated high affinity Y1R [5, 6] and Y2R antagonists were accessible by coupling of amino-functionalized guanidine<br />
derivatives with succinimidyl [2,3-3H]propanoate. By analogy, prototypical [ 18F]-PET ligands for the detection of Y1Rs,<br />
e.g. on breast cancer cells, were obtained. Interestingly, space-filling fluorophores were tolerated as well. To improve<br />
the signal-to-noise ratio, red-emitting fluorophores, e. g. cyanine or pyrylium dyes, or dyes emitting in the near<br />
infrared were preferred for coupling with argininamide-type Y1R [2, 7] and Y2R antagonist core structure by linkers<br />
attached to the guanidine group. This approach resulted in receptor affinities in the one- to two-digit nanomolar<br />
range. Labelling of short C-terminal peptides, modified, e.g., with cyclic β-amino acids, gave selective fluorescent tool<br />
for the Y4R. Green emitting ligands were designed with respect to colocalization studies.<br />
The presented strategies resulted in valuable pharmacological tools for the detection of the receptor of interest in<br />
vitro and/or in vivo as well as for the characterisation of receptors and ligands in radiochemical and fluorescencebased<br />
assays, including flow cytometry and confocal microscopy.<br />
References:<br />
1. Schneider, E. et al.: Chembiochem 2006, 7: 1400-1409.<br />
2. Schneider, E. et al.: Chembiochem 2007, 8: 1981-1988.<br />
3. Pluym, N. et al.: ChemMedChem 2011, 6: 1727–1738.<br />
4. Ziemek et al.: J. Recept. Signal Transduct. 2007, 27: 217.<br />
5. Keller, M. et al.: J. Med. Chem. 2008, 51: 8168-8172.<br />
6. Keller, M. et al.: ChemMedChem 2011, 6: 1566-1571.<br />
7. Keller, M. et al.: Bioorg. Med. Chem. 2011, 19: 2859-2878.<br />
28 Sessionvorträge Moleküle
Session IVIVC: Modelle und Modellierungen<br />
From Mice to Men: Application and Impact of in vivo Modeling and Simulation in Drug Research and<br />
Development<br />
Lehr T<br />
1 Clinical Pharmacy, Saarland University, Campus Building C2 3, Room 2.33, 66123 Saarbrücken, Germany<br />
The application and implementation of modeling and simulation in drug research and development has significantly<br />
changed in the past decades. The enhancements were and are mainly triggered by improved computational<br />
resources, the increased implementation of conceptual biological understanding, and the need to optimize the drug<br />
research and development process. Nowadays, modeling and simulation techniques are frequently applied, whereas<br />
the level of detail of these models can differentiate between empirical and mechanistic. However, the level of detail<br />
and the model type applied is highly variable and depending on the stage of the drug research/development process,<br />
the a priori knowledge available (i.e. in vitro or preclinical data) and the question that needs to be answered. Overall,<br />
there is still an urgent need for a more efficient drug research and development process. The application of modeling<br />
and simulation might be a cornerstone to meet this need.<br />
The objective of this presentation is to provide a brief overview about the application of modeling and simulation in<br />
the drug research and development process. The concept and impact of modeling and simulation shall be illustrated<br />
by selected examples.<br />
Sessionvorträge Moleküle 29
Estimating the In Vivo Release Behaviour of Extended- and Immediate-Release Products using the<br />
Biorelevant Dissolution Stress Testers<br />
Garbacz G 1,2 , Koziolek M 1 , Klein S 1 , Weitschies W 1<br />
1 University of <strong>Greifswald</strong>, Department of Pharmaceutics and Biopharmaceutics, Felix-Hausdorf-Straße 3, 17489 <strong>Greifswald</strong>, Germany<br />
2 Physiolution GmbH, Walther-Rathenau-Strasse 49a, 17489 <strong>Greifswald</strong>, Germany<br />
Introduction<br />
The estimation of the in vivo drug delivery behavior oral dosage forms by in vitro dissolution tests is a prerequisite for<br />
successful development of immediate release (IR) and modified release (MR) formulations. Unfortunately, the<br />
relevant aspects of the physiology of the gastro-intestinal (GI) tract cannot be simulated realistically using<br />
compendial dissolution tests. Thus, the development of predictive in vitro test systems capable of biorelevant<br />
simulation of GI transit conditions appears mandatory for rational dosage form development and characterization.<br />
Materials and methods<br />
In order to improve the predictive power of dissolution testing, we developed a novel dissolution test apparatuses<br />
mimicking the physical conditions of the fasting gastrointestinal passage of IR and MR dosage forms which is<br />
enabled by application of different dissolution vessels [1]. Parameters that can be depicted by our devices include GI<br />
pressure exerted by gastrointestinal (GI) motility, shear stresses generated during phases of GI-transport,<br />
temperature changes, media flow and emptying patterns and intermittent contact with intestinal fluids.<br />
Results<br />
The test results obtained for MR formulations indicate the susceptibility of modified release dosage forms towards<br />
mechanical stresses of biorelevant intensity [2, 3]. Moreover, the relationship between the mechanical resistance of<br />
MR tablets and the physico-chemical properties of the dissolution media was demonstrated. Our test results for IR<br />
formulations clearly indicated that both the dosage form disintegration and the dissolution are impacted by<br />
mechanical stress events of biorelevant intensity, temperature changes and different media flow conditions as they<br />
may occur in the fasting stomach.<br />
Conclusion<br />
The study results confirmed the usability of this novel method and the corresponding test protocols for the estimation<br />
of the in vivo drug delivery characteristics of solid oral dosage forms. Moreover, the study demonstrated the<br />
applicability of the novel methods for the identification of undesired drug release behavior in vivo.<br />
Acknowledgments: Federal Ministry of Education and Research (GASTROMAX, FKZ 13 N11368-370) is kindly acknowledged for the financial<br />
support.<br />
References:<br />
1. Garbacz, G., et al., Eur. J. Pharm. Biopharm. 2008, 70(2): 421-482.<br />
2. Garbacz, G., et al., Expert Opin. Drug. Deliv. 2010, 7(11): 1251-1261.<br />
3. Garbacz, G., et al., J. Pharm. Pharmacol. <strong>2012</strong>, 64(7): 944-968.<br />
30 Sessionvorträge Moleküle
Improved input parameters for diffusion models of skin absorption<br />
Hansen S1 ; Schaefer U2 1 Helmholtz Institute for Pharmaceutical Research Saarland, Saarland University, 66123 Saarbruecken, Germany<br />
2 Biopharmaceutics and Pharmaceutical Technology, Saarland University, 66123 Saarbruecken, Germany<br />
To use a diffusion model for predicting skin absorption requires accurate estimates of input parameters on model<br />
geometry, affinity and transport characteristics [1]. We determined input parameters for diffusion models of skin<br />
absorption focusing on partition and diffusion coefficients. These include experimental methods, extrapolation<br />
approaches, and correlations that relate partition and diffusion coefficients to tabulated physico-chemical solute<br />
properties.<br />
Exhaustive databases on lipid-water and corneocyte protein-water partition coefficients were collected and analyzed.<br />
Based on these improved equations were deduced which allow the prediction of solute partitioning into corneocytes<br />
and lipids from the octanol water partition coefficient logKow and improve predictions of the macroscopic SC-water<br />
partition coefficient.<br />
With diffusion coefficients the situation is more complex. Due to the still ongoing discussion of lipophilic and polar<br />
absorption routes across the SC the representation of these pathways varies greatly between individual models<br />
which therefore require very different diffusion coefficients or mass transfer coefficients. A plethora of theories has<br />
been used to obtain estimates. As a common principle all diffusion and mass transfer coefficients are inversely<br />
related to a measure of molecular size. The strength of this size dependence may be different (less pronounced in an<br />
aqueous environment than in the highly organized SC lipid bilayers, more pronounced in bilayer crossing than in<br />
lateral diffusion).<br />
In order to improve modeling of skin absorption from realistic (i.e. finite) doses it is essential to consider slow<br />
equilibrating processes such as reversible protein binding [2, 3]. High keratin binding and slow desorption kinetics are<br />
responsible (among other factors) for the formation of the stratum corneum reservoir reported especially in the<br />
context of corticosteroid absorption. We can demonstrate that equilibrium binding and the rate of desorption are both<br />
functions of solute lipophilicity. Our results prove that slow desorption from keratin may be a major contributor to the<br />
stratum corneum reservoir. Also they prove that reservoir formation is relevant for lipophilic solutes independent of<br />
drug class, thus allowing new options for topical pharmacotherapy.<br />
From these studies we can conclude that in order to improve modeling of skin absorption in the future diffusion<br />
models should include the vertical stratum corneum heterogeneity, slow equilibration processes, the absorption from<br />
complex non-aqueous formulations, and an improved representation of dermal absorption processes. This will<br />
require input parameters for which no suitable estimates are yet available.<br />
References:<br />
[1] S. Hansen, C.M. Lehr, U.F. Schaefer, Improved input parameters for diffusion models of skin absorption, Advanced Drug Delivery<br />
Reviews, (<strong>2012</strong>) accepted.<br />
[2] S. Hansen, D. Selzer, U.F. Schaefer, G.B. Kasting, An extended database of keratin binding, Journal of Pharmaceutical Sciences, 100<br />
(2011) 1712-1726.<br />
[3] S. Seif, S. Hansen, Measuring the stratum corneum reservoir: desorption kinetics from keratin, Journal of Pharmaceutical Sciences,<br />
accepted (<strong>2012</strong>).<br />
Sessionvorträge Moleküle 31
Interindividual gastrointestinal erosion and inter- as well as intraindividual gastrointestinal transit<br />
time variability of hydrophilic matrix tablets<br />
Jain A K1 ; Abrahmsson B3 ; Abrahmsén-Alami S3 ; Anschütz M2 ; Blume H2 ; Donath F2 ; Knopke C2 ;Schug B3 ;<br />
Söderlind E3 ; Tajarobi F3 , Viridé A3 , Weitschies W1 ;Welinder A3 1 Department of Biopharmaceutics and Pharmaceutical Technology, University of <strong>Greifswald</strong>, <strong>Greifswald</strong>, Germany<br />
2SocraTec R&D GmbH, Oberursel, Germany<br />
3AstraZeneca R&D Mölndal, Sweden<br />
Purpose<br />
The aim of this study was to investigate the interindividual gastrointestinal erosion rate (%/hr.) as well as variability in<br />
gastric emptying and colon arrival time of magnetically marked hydroxpropylmethylcellulose based matrix tablets.<br />
Materials and methods<br />
Table 1 Formulation composition of the hydroxypropylmethylcellulose matrix tablets<br />
Formulation 1* Formulation 2* Formulation 3* Formulation 5*<br />
Methocel K4M % (w/w) 23 10.1 - -<br />
Methocel K100 % (w/w) 17 30 40 20<br />
Dicalcium phosphate % (w/w) 57.6 57.5 57.6 77.6<br />
* Additionally all formulations contain 1.4% (w/w) Iron oxide (E172) and 1 % (w/w) Sodium Stearyl Fumarate<br />
Circular biconvex tablets of 350 mg with a diameter of 10 mm were prepared by direct compression at AstraZeneca<br />
R&D Lund, Sweden. In vivo gastrointestinal performance of tablets was investigated by magnetic marker monitoring<br />
(MMM) [1, 2] in a single centre, open label, cross over clinical study which was carried out in 5 healthy male<br />
volunteers. Formulation 1 and Formulation 2 were administered in fasted state while Formulation 3 and Formulation<br />
5 were administered under fasting as well as fed (30 min. after standard FDA breakfast) conditions. Mean, standard<br />
deviation (SD) and percentage relative standard deviation (%RSD) for gastrointestinal erosion rate, gastric emptying<br />
time and colon arrival time were calculated for each formulation as well as in each volunteer.<br />
Results<br />
Low variability in between-subject gastrointestinal erosion rate (9-29 %RSD) was observed between the subjects.<br />
However, high between-subject variability was observed for gastric emptying time (56-110% RSD) while<br />
interindividual variability was low for colon arrival time (0-39 %RSD). In only one volunteer, the Formulation 3, when<br />
administered under fed condition reached the colon at 260 min. Formulation 5 when administered under fed condition<br />
did not reach the colon in any volunteer. High intraindividual variability in gastric emptying time (59-110% RSD) was<br />
observed for all the formulations when administered in fasted state. Moderate intraindividual variability in colon arrival<br />
time (19-52 %RSD) was observed for all the formulations when administered in fasted state.<br />
Conclusion<br />
Erosion rate was found to be dependent on the formulation composition and exhibits low interindividual variability.<br />
Gastric emptying time didn’t exhibit dependency on the formulation composition and/or on the volunteers and shows<br />
high inter- as well as intra individual variability.<br />
References:<br />
1. Weitschies, W. et al.: J. Control Release. 2005, 108(2-3): 375–385<br />
2. Weitschies, W. et al.: Adv. Drug Deliv. Rev. 2005, 57(8): 1210–1222<br />
32 Sessionvorträge Moleküle
Session Moderne Trenntechniken<br />
Continuous Improvement in pharmaceutical analytics: faster, more precise and more selective<br />
Cianciulli C1 ; Redweik S1 ; Hahne T1 ; Alhazmi H1 , Xu Y2 ; Wätzig H1 1 TU Braunschweig, Institut für Medizinische und Pharmazeutische Chemie, Beethovenstr. 55, D-38106 Braunschweig, Germany<br />
2 Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, China<br />
The increasing need to properly answer to pharmaceutical and bioanalytical challenges induced tremendous<br />
progresses in electrophoresis and chromatography. Meanwhile Capillary Gel Electrophoresis (CGE), also known as<br />
Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS), is established in the pharmaceutical industry replacing<br />
SDS-PAGE gel electrophoresis, for example for the purity control of monoclonal antibodies. In order to quantify these<br />
components with sufficient precision using the same quality control method, a relative standard deviation (RSD [%])<br />
for the quantitative analysis under 2% is required. A reliable and highly precise CGE method could be obtained after<br />
thorough optimisation. It was crucial to increase the sample concentration and the injection volume in order to<br />
achieve sufficiently high signal-to-noise ratios (>70). The application of hydrodynamic injection is beneficial for the<br />
precision of the method compared to the traditionally used electrokinetic one. Linearity was demonstrated and limits<br />
of detection and quantitation were estimated. Both injection modes were compared in long series runs (n=48).<br />
Furthermore, the use of an internal standard was investigated. Thus, the RSD [%] of the migration time was reduced<br />
from 0.9% to 0.2% and the RSD [%] of peak areas was greatly improved. However, the normalisation to the total<br />
area further reduced the influence of the injection error. RSDs [%] for the peak area ratios of typically between 1 and<br />
2% were provided.<br />
Affinity Capillary Electrophoresis (ACE) can nicely detect interactions of proteins by the mobility changes which occur<br />
in the presence of various ligands [1]. ACE is able to explore changes in the conformation as well as charge changes<br />
of proteins. Six repeats were done with a very good precision due to the use of a special rinsing procedure [2]. The<br />
RSDs [%] of the migration times and the mobility ratios were typically below 2%, very often below 0.2%. The<br />
influence of various charged species e.g. metal ions, such as Cu2+ , Mn2+ and Mg2+ , pharmaceutical cations as<br />
ephedrine hydrochloride and pirenzepine dihydrochloride or anions such as glutamic acid and succinic acid was<br />
tested on the migration behavior of BSA, ß-lactoglobulin and ovalbumin. Furthermore the influence of<br />
phosphorylation on protein interactions was investigated at the activation site of the extracellular signal-regulated<br />
kinase 1 (ERK1). Organic cations and metal ions showed clearly different interactions with the proteins. Together<br />
with the advancements in mass spectrometry, NMR, and in other techniques such as THz spectrometry, deeper<br />
understanding of the behaviour of biomolecules will become possible, which in turn will strongly support the<br />
discovery of new pharmaceuticals.<br />
Y. Xu greatly acknowledges the funding from the Alexander von Humboldt Foundation<br />
References:<br />
1. Busch, M. H. A. et al.: J. Chromatogr. A 1997, 777(2): 311-328.<br />
2. El-Hady, D. et al.: J. Pharm. Biomed. Anal. 2010, 52(2): 232–241.<br />
Sessionvorträge Moleküle 33
Untersuchung von Proteinen und Metaboliten - Was können moderne Massenspektrometer leisten?<br />
Sinz A1 1 Abteilung für Pharmazeutische Chemie & Bioanalytik, Institut für Pharmazie, Martin-Luther-<strong>Universität</strong> Halle-Wittenberg, Wolfgang-<br />
Langenbeck-Str.4, D-06120 Halle/Saale<br />
Die Massenspektrometrie hat sich in den vergangenen Jahren zu einer immer bedeutenderen Methode für die<br />
Analyse klinischer Proben im Hochdurchsatz entwickelt. So findet man Massenspektrometer vor allem in den<br />
Bereichen, in denen die klassischen Verfahren, wie Immunoassays, limitiert sind, wie beispielsweise in der<br />
toxikologischen Analytik oder im Therapeutischen Drug Monitoring. Ebenso besitzt die Massenspektrometrie ein<br />
großes Potential für die qualitative und quantitative Untersuchung des Proteoms und Metaboloms in<br />
Körperflüssigkeiten, worauf in diesem Beitrag näher eingegangen wird.<br />
Biotransformation of the NR2B-selective NMDA receptor antagonist ifenprodil<br />
Falck E; Wünsch B<br />
Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-<strong>Universität</strong>, Hittorfstr. 58-62, 48153 Münster, Germany<br />
The NR2B-subunit of the NMDA receptor has a particular binding site which is named after ifenprodil (1) [1]. This<br />
compound shows a high affinity to the NMDA receptor. Its antagonistic affect can be used for the therapy of<br />
neurodegenerative diseases such as schizophrenia, epilepsy or parkinson’s disease.<br />
1<br />
The presentation will show the metabolism of ifenprodil, regarding the phase I and phase II metabolites resulting from<br />
hydroxylation and conjugation reactions.<br />
The metabolites were obtained by biotransformation with rat liver microsomes. Identification was possible using<br />
HPLC-ESI-MSn . The structures of the resulting fragments were confirmed by recording the exact mass yielding from<br />
time-of-flight detection.<br />
References:<br />
1. Williams, K.: Curr. Drug Targets 2001, 2: 285-298<br />
34 Sessionvorträge Moleküle
Macroscopic Questions and Molecular Answers: Measuring Optical Rotation in Latest Chiral<br />
Separation Techniques<br />
Thiessen A; Bertram N<br />
Anton Paar OptoTec GmbH, Albert-Einstein-Str. 5, 30926 Seelze-Letter, Germany, www.anton-paar.com<br />
Optical instruments are used in bio analytics to determine physical properties such as optical rotation or refractive<br />
index of chemical as well as biological compounds. The optical methods are among the oldest methods known, yet<br />
do the macroscopic optical properties tell a lot about the behaviour on the nano and molecular level. Technical<br />
innovations have dramatically improved the data quality of optical measurements in general and polarimeters<br />
especially within the last years, making the determination of optical rotation of chiral substances as accurate and<br />
traceable as never before.<br />
Pharmaceutical drugs often come as chiral substances. Their enantiomers may differ in their pharmacodynamic and<br />
pharmacokinetic interactions with receptors, enzymes and proteins in general. Therefore, one enantiomer – the<br />
eutomer – may have the desired pharmaceutical activity, whereas the other enantiomer – the distomer – may be<br />
inactive or even cause unwanted side effects. To ensure the desired efficacy and to avoid unwanted adverse effects<br />
of a drug, chiral substances may reliably be separated by the use of chiral HPLC columns.<br />
Polarimeters are used as chiral detectors in HPLC setups. But also after the separation process, a traceable and<br />
accurate polarimetric analysis is mandatory to verify the qualitative and quantitative separation of those enantiomers.<br />
Spectral polarimetry can be used to differentiate optically active substances by their rotational dispersion and may<br />
shed light onto molecular structures.<br />
We will present the possibilities of using polarimeters as rotation sensitive HPLC detectors and discuss principles<br />
and instrumentation for spectral polarimetry. We will then introduce novel developments in polarimetry to minimise<br />
the risk of measuring errors. This includes a live and recordable visualisation of the sample conditions inside the<br />
sample cell, avoiding errors from bubbles or particles. Saving a picture of the filling condition greatly improves the<br />
data traceability for pharmaceutical applications. The technology also indicates proper temperature distributions<br />
within the sample, raising the bar for high quality data generation in polarimetric measurements like never before.<br />
Another innovation automatically identifies the sample cell and checks its suitability for the selected method in order<br />
to avoid human error.<br />
We will briefly discuss how the enhanced traceability and data security improves compliance with pharmaceutical<br />
industry standards.<br />
Sessionvorträge Moleküle 35
Session Naturstoffe<br />
Hepatic bioactivity of phenolic natural compounds<br />
Kraus B 1 ; Decker M 1 ; Wolff H2 1 Lehrstuhl Pharmazeutische Biologie, <strong>Universität</strong> Regensburg, <strong>Universität</strong>sstrasse 31, 93053 Regensburg, Germany<br />
2 Carl Zeiss Microscopy, Kistlerhofstrasse 75, 81379 München, Germany<br />
The liver is the largest organ in the human body and manages a great number of functions. Its role as clearance<br />
organ bears the risk that substances that should be degraded or eliminated are hepatotoxic and lead to tissue<br />
damage [1]. The capacity of a substance to produce liver damage often results from the interaction of a series of<br />
complex cellular processes, involving uptake, biotransformation and elimination of the substance [2].<br />
There are a number of medicinal plants and natural compounds, e.g. silymarin/silibinin from milk thistle (Silybum<br />
marianum (L.) GAERTN.), which posess hepatoprotective properties and are used for the therapy of liver diseases [3].<br />
The mechanism of hepatoprotection by these compounds is generally by exerting multiple effects, including e.g.<br />
antioxidant, immunomodulatory, and antiinflammatory activities. In recent years Xanthohumol (XAN), a prenylated<br />
chalkone found in hop (Humulus lupulus L.) cones, has been reported to show hepatoprotective activity and to inhibit<br />
hepatic inflammation and fibrosis [4].<br />
We are interested in the hepatic bioactivity of prenylated and not prenylated chalcones from Humulus lupulus and<br />
(the putative hepatotoxic) Piper methysticum G. FORST. as well as metabolites and structural derivatives thereof,<br />
looking for structure activity-relationships. For this, we turned our attention to cellular bioavailability of XAN,<br />
visualizing and tracing its uptake, intracellular accumulation and distribution [5].<br />
In addition, we studied the hepatic bioactivity of the chalcones and metabolites. The influence of the substances on<br />
cell viability and mitotoxicity was determined and antiinflammatory mechanisms were adressed.<br />
In a further project, we analysed the potential of the flavonolignan silibinin to act hepatoprotective against tacrineinduced<br />
liver cell damage in hepatic stellate cells. We determined the capability of silibinin to reduce tacrinemediated<br />
cyto- and mito-toxicity applying either an equimolar mixture of silibinin and tacrine or a stable hybrid<br />
structure with silibinin covalently connected to tacrine [6].<br />
Acknowledgments: We like to thank Katharina Zenger and Magdalena Motyl for carrying out the cellular assays, Xinyu Chen for synthesis of<br />
the silibinin-tacrin-hybrid, Petr Jirásek and Susanne Vogel for synthesis of chalcones and metabolites, and Jörg Heilmann for fruitful discussion<br />
and support (all from Lehrstuhl Pharmazeutische Biologie, University Regensburg).<br />
References:<br />
1. Ramadori, G. et al.: J. Physiol. Pharmacol. 2008, 59 Suppl 1, 107-117.<br />
2. Guillouzo, A.: Environ. Health. Perspect. 1998, 106 Suppl 2, 511-532.<br />
3. Muriel, P., Rivera-Espinoza, Y.: J. Appl. Toxicol. 2008, 28, 93-103.<br />
4. Dorn, C., et al.: Mol. Nutr. Food Res. 2009, 54 Suppl 2:S205-13.<br />
5. Wolff, H., et al.: J. Agric. Food Chem. 2011, 59(24):12893–12901.<br />
6. Chen, X., et al.: J. Med. Chem. <strong>2012</strong>, DOI: 10.1021/jm300246n.<br />
36 Sessionvorträge Moleküle
Mode of cell death induction by pharmacological V-ATPase inhibition<br />
von Schwarzenberg K1 ; Wiedmann R M1 ; Oak P2 ; Schulz S3 ; Zischka H3 ; Wanner G4 ; Efferth T5 ; Trauner D6 ;<br />
Vollmar A M1 1Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University, 81377 Munich, Germany<br />
2Department of Pharmacy, Pharmaceutical Biology-Biotechnology, Ludwig-Maximilians-University, 81377 Munich, Germany<br />
3Institute of Toxicology, Helmholtz Center Munich, German Research Center for Environmental Health, 85764 Neuherberg, Germany<br />
4Department of Biology I, Ludwig-Maximilians-University, 82152 Martinsried, Germany<br />
5Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, 55099 Mainz, Germany<br />
6Department of Chemistry, Ludwig-Maximilians-University, 81377 Munich, Germany<br />
Recently V-ATPase, a multisubunit proton pump, has come into focus as an attractive target in cancer cell invasion.<br />
However little is known about the role of V-ATPase in cell death and especially the underlying mechanisms remain<br />
mostly unknown. We used archazolid B, a macrolide of myxobacterial origin being a novel very potent inhibitor of the<br />
V-ATPase as an experimental drug as well as a chemical tool to decipher V-ATPase related cell death signaling. We<br />
found that archazolid induced apoptosis in highly invasive tumor cells at nanomolar concentrations while showing<br />
almost no cytotoxicity in non tumorus cells. Apoptosis was caspase-dependant and executed by the mitochondrial<br />
pathway shown by breakdown of mitochondrial potential, leakage of cytochrom c from the mitochondria and<br />
activation of caspase-9. Prior to apoptosis induction archazolid lead to the activation of stress responses such as<br />
autophagy and glycolysis. Autophagy was induced at concentrations that do not alkalize lysosomes and was shown<br />
by degradation of p62 or fusion of autophagosomes with lysosomes. Autophagy was found to be a survival<br />
mechanism at low concentrations but could not be accomplished at higher concentrations due to lysosome<br />
alkalization by V-ATPase inhibition. Archazolid also lead to glycolysis induction which is mediated by the hypoxiainducible-factor-1<br />
alpha and induced due to energy stress. This was shown by a decline of the ATP level and arrest<br />
of energy consuming processes (AMPK, mTOR, eIF2alpha). As silencing HIF1alpha increases apoptosis, glycolysis<br />
was suggested to be an adaptive mechanism. We conclude that archazolid leads to energy stress which activates<br />
autophagy and glycolysis and finally leads to apoptosis. We propose V-ATPase as a promising drugable target in<br />
cancer therapy caught up at the interplay of apoptosis, autophagy and cellular/metabolic stress.<br />
Sessionvorträge Moleküle 37
Cyanobacteria in Anticancer Natural Products Research<br />
Niedermeyer T H J1 ; Daily A2 ; Swiatecka-Hagenbruch M1 , Moscow J A2 1 Cyano Biotech GmbH, Magnusstr. 11, 12355 Berlin, Germany<br />
2 Hematology-Oncology Department of Pediatrics of the University of Kentucky, 740 South Limestone, Lexington, Kentucky 40536, USA<br />
Cyanobacteria are a prolific source of natural products. Many secondary metabolites isolated from cyanobacteria<br />
show remarkable cytotoxicity. In the first part of this talk, cyanobacteria and some of the most interesting compounds<br />
that have potential for the exploitation as lead compounds or have already been advanced into clinical studies (e.g.<br />
the cryptophycins and dolastatins) are highlighted. In the second part, microcystins are presented as novel leads.<br />
Microcystins, rather known as hepatotoxins produced by cyanobacteria, are currently also studied for their potential<br />
as leads for anticancer drug development. They potently inhibit the eukaryotic protein phosphatase families PP1 and<br />
PP2A [1]. Microcystins display their toxicity after transporter-mediated uptake by the cell. Three human proteins are<br />
thought to be able to mediate this uptake, the organic anion transporting polypeptides OATP1B1, OATP1B3, and<br />
OATP1A2. Liver cells express OATP1B1 and OATP1B3 transporters and are thus able to absorb microcystins from<br />
the blood, resulting in a pronounced liver toxicity of microcystins. OATP1B3 is also expressed in hepatocellular<br />
carcinoma cells (HCC) as well as gastrointestinal and lung tumors. Recent data offer support that the OATP1B3<br />
protein is also expressed in a significant percent of breast and colon tumors [2, 3]. Interestingly, compared to<br />
OATP1B1, OATP1B3 is found only in low abundance in liver cells. Thus selectivity that favors OATP1B3 over<br />
OATP1B1 should lead to a decreased hepatic clearance / toxicity and an increased uptake in OATP1B3-expressing<br />
tumors, creating a therapeutic window for the respective compound.<br />
Since about 90 naturally occurring microcystins with variations in the seven constituting amino acids are known and<br />
many more structures are not described yet, there is high potential for isolating variants with unique properties. In<br />
fact, in our initial screening of 20 microcystins, we have found four microcystin structural variants with an IC50 in the<br />
low nanomolecular range and with transporter selectivity that favors OATP1B3 over OATP1B1 by a factor of more<br />
than ten. Some of these microcystin variants have not yet been described in the literature.<br />
Testing of further microcystin variants, the elucidation of their structures and the deduction of structure-activity<br />
relationships will enable us to find and create more selective and potent compounds.<br />
OH<br />
HN<br />
References:<br />
1. Watanabe, M. et al., Toxic Microcystis (CRC Press) 1995.<br />
2. Muto, M. et al., Cancer Sci. 2007, 98(10): 1570–1576.<br />
3. Lee, W. et al., Cancer Res. 2008, 68(24): 10315–10323.<br />
O<br />
NH 2<br />
NH<br />
N<br />
NH<br />
38 Sessionvorträge Moleküle<br />
COOH<br />
O<br />
H<br />
N<br />
O<br />
O<br />
N<br />
H<br />
N<br />
O<br />
O<br />
COOH<br />
Structure of a typical microcystin (Microcystin LR)<br />
NH<br />
HN<br />
O
Applied phytochemical research – an industry’s perspective<br />
Stintzing F C<br />
WALA Heilmittel GmbH, Dorfstrasse 1, 73087 Bad Boll/Eckwälden, Germany<br />
An overview is given on research conducted on selected European plants used for cosmetics and complementary<br />
medicine. A first key aspect will deal with the composition of a lipophilic extract from Cydonia oblonga and its<br />
ramifications for cosmetic purposes [1]. The impact of location and harvest date on the phenolic composition at the<br />
example of Salvia officinalis [2], Alchemilla mollis and A. vulgaris [3] will be a second focal point. A third topic will<br />
cover the biotransformation of hydrophilic compounds from Betula pendula [4], Hamamelis virginiana [5,6], Nicotiana<br />
tabacum [7] and Atropa belladonna [8] upon lactic acid fermentation. Fourth, the findings obtained by an in-depth<br />
screening including lipophilic and hydrophilic compounds of Mercurialis perennis will be presented [9,10,11]. Finally,<br />
most recent data on the stability evaluation of selected essential oils during exposure to light and elevated<br />
temperatures will be shown also covering new approaches for quality control measures [12,13,14,15].<br />
The data presented demonstrate that phytochemical research on the European flora is most rewarding and deserves<br />
a more attentive dedication even on plants that are seemingly studied well. The impact of processing resulting in<br />
specific compound patterns but also differing bioactivities should encourage both industry and academia to join<br />
forces in discovering and making use of the plethora of phytochemicals in local plants.<br />
Acknowledgments: The author is grateful to all co-authors and collaborators for their common interest and enthusiasm in conducting the<br />
investigations presented.<br />
References:<br />
1. Lorenz, P. et al.: Anal. Bioanal. Chem. 2008, 391(2): 633-646.<br />
2. Schnitzler, P. et al.: Phytomed 2008, 15(1/2): 62-70.<br />
3. Duckstein, S.M. et al., Ztschr. Naturforsch C/ J. Biosci. <strong>2012</strong>: submitted.<br />
4. Millet, A., Stintzing, F., Merfort, I.: J. Pharm. Biomed. Anal. 2009, 49(5): 1166-1171.<br />
5. Duckstein, S.M.., Stintzing, F.C.: Anal. Bioanal. Chem. 2011, 401(2): 677-688.<br />
6. Duckstein, S.M. et al.: Phytochem. Anal. <strong>2012</strong>: doi.10.1002/pca.2359.<br />
7. Millet, A., Stintzing, F., Merfort, I.: J. Pharm. Biomed. Anal. 2010, 53(2): 137-144.<br />
8. Schwarzenberger, M. et al.: Pharmazie <strong>2012</strong>, 67(5): 460-466.<br />
9. Lorenz, P. et al.:, Phytochem. Anal. 2009, 21(3): 234-245.<br />
10. Lorenz, P. et al., Ztschr. Naturforsch C/J. Biosci. 2010, 65(3/4): 174-179.<br />
11. Lorenz, P. et al., Phytochem. Anal. <strong>2012</strong>, 23(1): 60-71.<br />
12. Turek, C., Stintzing, F.C.: Anal. Bioanal. Chem. 2011, 400(9): 3109-3123.<br />
13. Turek, C., Stintzing, F.C.: J. Food Sci. 2011, 76(9): C1365-C1375.<br />
14. Turek, C., Stintzing, F.C.: Food Res. Int. <strong>2012</strong>, 46(1): 341-353.<br />
15. Turek, C., Kirschmann, N., Stintzing, F.C.: Ztschr. Arznei Gewürzpfla/J. Med. Spice Plants <strong>2012</strong>, 17(2): 73-79.<br />
Sessionvorträge Moleküle 39
Session Klinische Wirkstoffentwicklung<br />
Single- and multiple-transfected cell lines as tools for the analysis of drug transport and of the<br />
interplay of drug transport and drug metabolism<br />
König J, Fahrmayr C, Fromm M F<br />
Institute of Experimental and Clinical Pharmacology and Toxicology, Department of Clinical Pharmacology and Clinical Toxicology, Friedrich-<br />
Alexander-<strong>Universität</strong> Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany<br />
In liver drug disposition and effects are determined by the coordinated processes of drug uptake, drug metabolism<br />
and drug export. Following transporter-mediated drug uptake (e.g. by the organic anion transporting polypeptide<br />
OATP1B1, gene symbol SLCO1B1) and subsequent phase I (e.g. by the cytochrom P450 enzyme CYP3A4) and / or<br />
phase II metabolism (e.g. by UDP-glucuronosyltransferase 1A1) parent compounds or their metabolites are<br />
frequently exported by members of the ABC transporter superfamily (e.g. by the apically localized export pump<br />
MRP2, gene symbol ABCC2) into bile. So far, mostly single-transfected cells recombinantly overexpressing the<br />
protein of interest were used to investigate its role in drug disposition. This setup may be useful for determining the<br />
substrate spectrum of transporters or metabolizing enzymes or for the analysis of transporter-mediated drug-drug<br />
interactions but results in limited data regarding the interplay of drug transport and drug metabolism. Therefore,<br />
double- and multiple-transfected cell lines have been established expressing an uptake transporter together with an<br />
export pump [1, 2, 3] or expressing an uptake transporter simultaneously with the phase II enzyme UGT1A1 and the<br />
export pump MRP2 [4]. The latest development was the establishment of a quadruple-transfected cell line<br />
recombinantly overexpressing the uptake transporter OATP1B1, the phase I enzyme CYP3A4, the phase II enzyme<br />
UGT1A1 and the apically localized export pump MRP2. This presentation summarizes data regarding the usage of<br />
single- and multiple-transfected cell lines for the analysis of drug transport, transporter-mediated drug-drug<br />
interactions and the interplay of drug transport and drug metabolism.<br />
References:<br />
1. Cui et al.: Mol Pharmacol 2001, 60: 934-943.<br />
2. Letschert et al.: J Pharmacol Exp Ther 2005, 313: 549-556.<br />
3. Nies et al.: Naunyn Schmiedebergs Arch Pharmacol 2007, 376: 449-461.<br />
4. Fahrmayr et al.: Br J Pharmacol <strong>2012</strong>, 165: 400-406.<br />
40 Sessionvorträge Moleküle
Methods to investigate the clinical relevance of drug transporters<br />
Oswald S<br />
University Medicine of <strong>Greifswald</strong>, Center of Drug Absorption and Transport, Department of Clinical Pharmacology, Felix-Hausdorff-Str. 3,<br />
17487 <strong>Greifswald</strong>, Germany<br />
Transporter proteins are crucial determinates in the pharmacokinetics (absorption, distribution and excretion) of<br />
many drugs. Many of these transporters are highly expressed in tissues that are of outstanding relevance in drug<br />
disposition such as the intestine, the liver, the kidneys and at physiologically relevant blood-tissue barriers such as<br />
the blood-brain-barrier or the placenta.<br />
In general, one can distinguish between SLC (solute carrier) transporters which mostly facilitate the cellular uptake of<br />
their substrates, and ABC (ATP-binding cassette) transporters such as P-glycoprotein (P-gp, ABCB1) which pump<br />
their substrates out of the cell in an energy-dependent manner.<br />
So far hundreds of these transporters have been described, whereas the clinical impact of only few candidates (e.g.<br />
P-gp, OATP1B1) is well established. On the other side, there is an urgent need to know the distinct cellular transport<br />
properties of a certain drug to avoid unwanted transporter-based drug-drug interactions (by induction or inhibition),<br />
high inter-subject variability in drug disposition (e.g. by genetic variants) and safety issues.<br />
Consequently, current guidelines from regulatory agencies (e.g. FDA, EMA) recommend comprehensive in vitro<br />
characterization of new molecular entities with regards to their transporter affinity. After verification that a certain drug<br />
may be a substrate for a certain transporter protein clinical studies in humans are recommended to conclude on the<br />
potential clinical impact.<br />
The presentation will provide a brief overview about clinical state-of-the-art approaches as well as experimental<br />
studies in humans to conclude on the clinical relevance of the most important ABC and SLC transporters.<br />
Sessionvorträge Moleküle 41
The functionality of uptake transporters in primary hepatocytes of different species can be verified<br />
by estrone-3-sulfate<br />
Runge D1 ; Keiser M2 ; Ullrich A1 ; Pohl J2 ; Radebold J2 ; Damm G3 ; Nüssler A4 ; Siegmund W2 1PRIMACYT Cell Culture Technology GmbH, Schwerin, 19061, Federal Republic of Germany<br />
2Department of Clinical Pharmacology, University Medicine of <strong>Greifswald</strong>, <strong>Greifswald</strong>, 17487, Federal Republic of Germany<br />
3Charité Virchow Klinik, Berlin, 13353, Federal Republic of Germany<br />
4BG Trauma Hospital, Tübingen, 72076, Federal Republic of Germany<br />
Introduction: Primary mammalian hepatocytes are used for several in vitro applications like testing of drug<br />
metabolism, toxicity and transporter assays. However, little is known about species specific differences or similarities<br />
in the activity of uptake and efflux transporter. Therefore, we started a species specific characterization and<br />
compared the uptake of different substrates in hepatocytes from pharmacological relevant species which are<br />
commonly used for drug testing.<br />
Materials & Methods: Human, rat, dog and monkey hepatocytes were incubated in serum free media. 2-4 days after<br />
cell isolation a time and concentration dependent uptake of [3H]- estrone-3-sulfate (E3S), -bromosulfophthaleine<br />
(BSP), -digoxine (Dig) and -taurocholic acid (TA) at 4°C and 37°C was measured by liquid scintillation counting.<br />
Competition assays were performed using E3S as substrate and rifampicin, BSP, Dig or TA as competitors.<br />
Results: All hepatocytes showed a time-dependent and saturable increase in E3S and BSP uptake at 37°C<br />
compared to the uptake at 4°C. By contrast, Dig and TA showed a time-dependent and saturable transport only in<br />
hepatocytes of humanoids and rats, but not in dog. Moreover Dig uptake was about 10 fold higher in human than in<br />
rat and monkey hepatocytes. E3S revealed a high affinity to human (Km = 12.9 ± 10.1 µmol/l; Vmax = 84.2 ± 30.3<br />
pmol/mg × min), monkey (Km = 9.2 ± 2.2 µmol/l; Vmax = 24.8 ± 3.3 pmol/mg × min) and dog (Km = 3.3 ± 2.3 µmol/l;<br />
Vmax = 10.0 ± 2.1 pmol/mg × min). In rat hepatocytes E3S showed an increased uptake with increasing<br />
concentrations but no kinetic data could be calculated. For BSP and Dig no pharmacokinetic data could be calculated<br />
in any species and TA was transported only into hepatocytes of monkey (Km = 17.5 ± 12.5 µmol/l; Vmax = 5.8 ± 2.1<br />
pmol/mg × min). Rifampicin clearly inhibited the E3S uptake in monkey (IC50 = 7.9 (2.4; 25.9) µmol/l) and dog<br />
hepatocytes while uptake of E3S in rat hepatocytes was not influenced. E3S uptake was also inhibited by increasing<br />
concentration of BSP in dog (IC50 = 7.5 (5.1; 11.1) µmol/l), monkey (IC50 = 5.9 (3.1; 11.3) µmol/l) and rat (IC50 = 200<br />
(177; 226) µmol/l) hepatocytes. Dig and TA did not significantly influence the E3S uptake in primary hepatocytes in<br />
dog, rat and monkey hepatocytes.<br />
Discussion: Our data suggest that among all substrates tested E3S is the most suitable substrate to verify the<br />
functionality of uptake transporters in primary hepatocytes of humanoids, dogs, and rats. However, major differences<br />
were observed in the uptake of drugs within the species tested here. These species specific differences in<br />
hepatocellular uptake should receive attention when data obtained in drug transport, metabolism and toxicity assays<br />
are transferred from animal experiments to humans.<br />
This work was supported by the Ministry of Economy, Labour and Tourism of Mecklenburg-Vorpommern (Grant V630-F-108-2011/040 and<br />
V630-VB-196-2011/038).<br />
42 Sessionvorträge Moleküle
Solubilizing agents influence the pharmacokinetics of probe drugs by transporter interaction<br />
Engel A1 ; Keiser M1 ; Oswald S1 ; Scheuch E1 ; Berg S2 ; Freyse E-J2 ; Weitschies W3 ; Siegmund W1 1 Institute of Pharmacology, Dpt. of Clinical Pharmacology, Felix-Hausdorff-Str. 3, 17487 <strong>Greifswald</strong>, Germany<br />
2 Institute of Diabetes “Gerhardt Katsch” e.V., <strong>Greifswald</strong>er Str. 11e, 17495 Karlsburg, Germany<br />
3 Institute of Pharmacy, Dpt. of Biopharmaceutics and Pharmaceutical Technology, Felix-Hausdorff-Str. 3, 17487 <strong>Greifswald</strong>, Germany<br />
Background & Aims: Pharmaceutical excipients are supposed to be pharmacologically inactive by definition.<br />
However, there is ample evidence that solubilizing agents (e.g. Cremophor ® EL) might significantly influence CYP450<br />
dependent drug metabolism and efflux transport by ABCB1 and ABCC2 [1, 2, 3]. So far, little is known about the<br />
influence of pharmaceutical excipients on uptake transporters, which were shown to be involved in the disposition of<br />
many drugs. Therefore, we investigated the influence of polyethylene glycol 400 (PEG), hydroxypropyl-ß-cyclodextrin<br />
(HPCD), Solutol ® HS15 (SOL) and Cremophor ® EL (CrEL) on the organic anion-transporting polypeptides (OATP)<br />
1A2, 1B1, 1B3 and 2B1 and on the Na + /taurocholate cotransporting polypeptide (NTCP) in vitro. Furthermore, we<br />
studied the impact of PEG, HPCD and SOL on the pharmacokinetics of the probe drugs acetaminophen (AAP),<br />
talinolol (TAL) colchicine (COL) and cyclosporine A (CSA) in rats.<br />
Materials & Methods: In vitro inhibitory effects of the excipients were studied in competition assays using HEK293<br />
cells stably transfected with OATP1A2, 1B1, 1B3, 2B1 and NTCP, respectively, and in each case two established,<br />
radio-labelled reference substrates. Intracellular accumulation of taurocholic acid (TA; OATP1A2, NTCP), estrone-3sulfate<br />
(E3S; OATP1A2, 1B1, 2B1), bromosulfophthaleine (BSP; OATP1B1, 1B3, 2B1, NTCP) and estradiol-17ßglucuronide<br />
(E17ßGln; OATP1B3) was measured by liquid scintillation counting after cell lysis. For pharmacokinetic<br />
investigations, solutions of each probe drug with each excipient were administered intravenously as single or multiple<br />
doses to male Wistar rats (each N=6). Drug in saline served as control. Blood samples, urine, feces and organs were<br />
collected and drug concentrations determined by LC-MS/MS or HPLC-UV.<br />
Results: In vitro, PEG selectively inhibited the uptake of TA and E3S by OATP1A2 (IC50 0.05% and 0.14%) but not by<br />
1B1, 1B3, 2B1 and NTCP. HPCD strongly inhibited the TA, E3S and E17ßGln uptake by OATPs (IC50 0.001 –<br />
0.24%). SOL and CrEL were potent inhibitors of all investigated transporters with the strongest effect on the TA, BSP<br />
and E3S uptake by OATP1A2 and OATP2B1 (IC50 0.0003 – 0.01%). In vivo, all investigated excipients were able to<br />
significantly modulate the pharmacokinetics of at least one probe drug. Pharmacokinetics of CSA were most<br />
strikingly influenced by all three solubilizing agents, its single and multiple dose AUC was increased 2-5-fold and<br />
volume of distribution (VD) was reduced to a similar degree while the elimination half-life (t1/2) remained unchanged.<br />
SOL likewise affected AUC and VD of COL and AAP, but to a lesser extent. It also prolonged t1/2 of TAL and COL 2-3<br />
times. HPCD increased VD of TAL, supported by a dramatic accumulation of TAL in kidneys and duodenum.<br />
Furthermore t1/2 of TAL was doubled by HPCD. PEG only caused minor changes in the kinetics of TAL and COL but<br />
significantly increased renal clearance of unchanged AAP and reduced renal clearance of AAP conjugates.<br />
Conclusion: Frequently used solubilizing agents were shown to substantially influence intestinal and hepatic uptake<br />
transporters in vitro and to significantly modulate the pharmacokinetics of drugs in the rat. This might lead to<br />
unwanted drug interactions or adverse drug effects, which should be considered in drug development. However, the<br />
clinical relevance should be evaluated in further investigations.<br />
References:<br />
[1] Bravo González, R.C. et al.: Biopharm. Drug Dispos. 2004, 25(1): 37–49.<br />
[2] Hanke, U. et al.: Eur. J. Pharm. Biopharm. 2010, 76(2): 260–268.<br />
[3] ten Tije, A. J. et al.: Clin. Pharmacokinet. 2003, 42(7): 665–685.<br />
Sessionvorträge Moleküle 43
Sessionvorträge Targets<br />
Session Proteomics und Metabolomics<br />
OMICs approaches in the analysis of population-based cohorts – Potential avenues for biomarker<br />
screening<br />
Völker U, Ameling S1 , Endlich K2 , Felix S B3 , Gesell-Salazar M1 , Hammer E1 , Hoffmann W4 , Homuth G1 , Jehmlich N1 ,<br />
Kocher T5 , Lerch M M6 , Schurmann C1 , Nauck M7 , Schmidt F1 , Teumer A1 , Völzke H4 , Wallaschofski W7 1 Interfaculty Institute of Genetics and Functional Genomics<br />
2 Institute of Anatomy and Cell Biology<br />
3 Clinic for Internal Medicine B<br />
4 Institute for Community Medicine<br />
5 Department of Restorative Dentistry, Periodontology and Endodontology<br />
6 Clinic for Internal Medicine A<br />
7 Institute for Clinical Chemistry and Laboratory Medicine, University Medicine <strong>Greifswald</strong>, D-17475 <strong>Greifswald</strong>, Germany<br />
Different OMICs approaches are currently widely applied in the screening for diagnostic and prognostic biomarkers in<br />
case-control settings or for the exploration of the pathophysiology of different diseases. On the other side populationbased<br />
cohorts provide an extremely valuable resource particularly for the study of prevalent diseases. One such<br />
study, the Study of Health in Pomerania (SHIP), has been established in the last 15 years at the University of<br />
<strong>Greifswald</strong> and it is internationally acknowledged for its comprehensive high-quality phenotyping and quality control<br />
measures (1). The first wave (SHIP-0) was a cross-sectional epidemiological survey in West Pomerania, in the<br />
North-East of Germany, conducted from 1997-2001 and comprising 4310 participants. Two additional waves (SHIP-1<br />
and 2) with expanded phenotyping were conducted between 2001-2006 and 2008-<strong>2012</strong>. Finally, a separate stratified<br />
random sample of 8016 adults aged 20 to 79 years was drawn for SHIP-TREND for which examination started in<br />
2009 and until October <strong>2012</strong> more than 3700 subjects participated.<br />
Recently, we started to perform different OMICs analyzes with biomaterials of SHIP and SHIP-TREND, which started<br />
with genome-wide genotyping and metabolome analysis of urine. Joining epidemiological, clinical and functional<br />
genomics expertise we are now active partners of international consortia addressing the genetics of cardiovascular<br />
diseases (2, 3).<br />
The OMICs analyses are currently being expanded to include metabolomics measurements of plasma, expression<br />
profiling of whole blood and proteomics approaches for the characterization of biofluids such as plasma, urine and<br />
saliva. In the talk the progress in the analysis of the two population-based cohorts using OMICs technologies will be<br />
presented.<br />
The second part will be used to demonstrate, how such population-based cohorts can be used to provide reference<br />
values in case-control settings particularly in the field of cardiovascular research. The application of these<br />
technologies will be illustrated using dilated cardiomyopathy as an example (4).<br />
References:<br />
1. Völzke, H. et al.:Int J Epidemiol 2011, 40: 294-307.<br />
2. Newton-Cheh, C. et al.: Nat Genet 2009, 41: 666-676.<br />
3. Suhre, K. et al.: Nat Genet 2011, 43: 565-569.<br />
4. Ameling, S. et al.: Eur Heart J <strong>2012</strong>, in press.<br />
44 Sessionvorträge Targets
Drug target fishing in silico and in vitro<br />
Efferth T<br />
Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128<br />
Mainz, Germany<br />
Mechanisms of action of drugs are frequently difficult to unravel in a comprehensive fashion. Elegant techniques<br />
which appeared in recent years represent transcriptome-wide and proteome-wide expression profilings by<br />
microarrays to connect gene and protein expression signatures to drug, genes and diseases.<br />
Although clinical parameters are valuable for the determination of the prognosis of larger cohorts of patients, they are<br />
frequently less helpful to predict the treatment success for each individual patient due to inter-individual variability<br />
and heterogeneity. Therefore, personalized medicine aims to develop individualized treatment options adapted to<br />
factors relevant for the prognosis of each single patient. Molecular biomarkers identified by “-omics” technologies are<br />
expected to predict the likelihood of individual responsiveness (or toxicity) to treatment. This information can then be<br />
used to optimize treatment schedules in a custom-made fashion.<br />
However, microarray-based data do not allow conclusions on the protein targets of drugs. Considering that there is a<br />
huge amount of potentially drugable proteins, but that only a few hundred drug targets have been exploited so far,<br />
the development of drug target fishing techniques is urgently required. Identification of drug targets is mostly<br />
performed by fishing proteins with a modified drug from a protein solution isolated from cells or tissues. Activitybased<br />
probe profiling and compound-centric chemical proteomics are established methods for this purpose.<br />
Pointing to individualized cancer therapy, a current concept is to identify mutations in “driver” genes of cancer<br />
development and progression and to specifically develop small molecules or antibodies against patient-specific<br />
mutated proteins in cancer tissues. The hope is not only to improve individual response rates of tumors, but also to<br />
reduce side effects in normal tissues.<br />
In this presentation, we give an overview of current bioinformatical and wet-lab technologies to identify and verify<br />
drug targets and present selected examples.<br />
References:<br />
1. Volm, M.et al., Clin. Cancer Res. 2002, 8(6): 1843-8.<br />
2. Gillet, J.P./Efferth, T. et al., Cancer Res. 2004, 64(24): 8987-93.<br />
3. Efferth, T. et al., Mol. Cancer Ther. 2006, 5(8): 1986-94.<br />
4. Efferth, T. et al., Mol. Cancer Ther. 2008, 7(1): 152-61.<br />
5. Konkimalla, V.B., Efferth, T. Biochem. Pharmacol. 2010, 79(8): 1092-9.<br />
6. Youns, M. et al., Planta Med. 2010, 76(17): 2019-25.<br />
7. Sertel, S. et al., PLoS One <strong>2012</strong>, 7(5): e35584.<br />
8. Eichhorn, T. et al., Mol. Biosyst. <strong>2012</strong>, 8(4): 1311-8.<br />
Sessionvorträge Targets 45
From whole-organism to sub-cellular localization of metabolites by mass spectrometry imaging<br />
Liebeke M<br />
Department of Surgery and Cancer, Imperial College London, Sir Alexander Fleming Building, London, SW7 2AZ, UK<br />
(m.liebeke@imperial.ac.uk)<br />
Localization of metabolites in tissues or even within single cells is of emerging interest for comprehensive<br />
understanding of biological systems. Advances in mass spectrometry imaging (MSI) allow the mapping of individual<br />
molecules across a spatial distribution, but depending on the method used this distribution image is limited by the<br />
technical possibilities of the desired device. We report here the use of matrix assisted laser desorption ionization<br />
(MALDI) FTICR-MS to image tissue cross-sections (0.5cm 2 – 20µm resolution) and the application of secondary ion<br />
mass spectrometry (SIMS) to further elucidate the cellular distribution of selected small molecules (0.5mm 2 – 100nm<br />
resolution). Both MSI techniques gained comparable image results for the whole tissue cross-section but showed<br />
distinct differences for high spatial resolution imaging. The advantages of TOF-SIMS and MALDI MSI approaches<br />
will be pointed out, as well as problems and limitations of both techniques. Results of clinical interest (infected mouse<br />
lung) and environmental biology (earthworm) will be presented. We show the power of the whole-organism to subcellular<br />
metabolite localization, which may be able to reveal novel possible physiological implications in the analyzed<br />
organism.<br />
Figure 1 images from tissue sections generated by TOF-SIMS, observed ions indicated. Images for are presented in a gray scale from black to<br />
white. Higher brightness corresponds to higher signal intensities. Total counts (TC) correspond to the total ion signal from the specified ion.<br />
Maximum counts (MC) correspond to the maximal number of counts in a pixel. The field of view is 500 × 500 μm 2 .<br />
46 Sessionvorträge Targets
Incorporation of unnatural amino acids to monitor conformational changes in peroxisome<br />
proliferator-activated receptor alpha (PPARα)<br />
Schwarz R, Tänzler D, Ihling C H, Kölbel K, Sinz A<br />
Dept. Pharm.Chem./Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Halle/Saale, Germany<br />
Chemical cross-linking combined with an enzymatic digestion and mass spectrometric analysis of the reaction<br />
products has evolved into an alternative strategy to identify protein-protein and protein-ligand interactions. Here, we<br />
present the investigation of conformational changes in PPARα upon ligand binding by photo-affinity labeling<br />
combined with mass spectrometry. Peroxisome proliferator-activated receptors (PPARs) belong to the superfamily of<br />
nuclear receptors. PPARα plays a crucial role in lipid metabolism and poses an important target for designing<br />
antidyslipidemic drugs for the treatment of the metabolic syndrome.[1].<br />
Expression and purification of PPARα variants were optimized in respect to obtain high protein yields. The<br />
photoreactive amino acid para-benzoylphenylalanine (Bpa) was incorporated at position 258 of PPARα for<br />
conducting photo-affinity labeling studies (Figure 1). Bpa incorporation was confirmed by peptide mass fingerprint<br />
analyses. PPARα variant L258Bpa was employed for photo-cross-linking experiments in the absence and presence<br />
of the PPARα ligand GW6471. After the photo-cross-linking reaction, reaction mixtures were enzymatically digested<br />
and peptides were analyzed by high-resolution mass spectrometry (nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS).<br />
The distance constraints imposed by the photochemical cross-links served to monitor conformational changes in<br />
PPARα upon ligand binding.<br />
Conformational changes in PPARα that had already been suggested in previous studies [2] were verified. In the<br />
presence of the PPARα antagonist GW6471 a high number of cross-linked products were identified, which were<br />
mainly localized in the region comprising amino acids 215 to 226. These cross-links can only be accomodated if the<br />
flexible omega loop in PPARα, harboring the photo-amino acid Bpa258, exerts a huge conformational change and<br />
thus closes the ligand binding pocket according to a “mouse trap” principle. Our strategy demonstrates that photoaffinity<br />
labeling followed by a mass spectrometric analysis of the reaction products allows monitoring conformational<br />
changes in target proteins in a highly efficient manner.<br />
Figure 1: Three dimensional structure (pdb: 1KKQ) of GW6471-bound PPARα; Leu-258 that was replaced by Bpa is shown in green, Ω-loop<br />
(yellow), activation function helix 2 (orange), and GW6471 (magenta).<br />
References:<br />
[1] Plutzky et al.: Am J Cardiol 2003.<br />
[2] Müller et al.: J Med Chem 2009.<br />
Sessionvorträge Targets 47
Session Transporter und Ionenkanäle<br />
TRP channels as regulators of blood pressure and fertility<br />
Freichel M<br />
48 Sessionvorträge Targets
Role of Ca 2+ -activated K + Channels in Breast Cancer Proliferation<br />
Lukowski R<br />
Sessionvorträge Targets 49
Transporters in human platelets: physiological function and impact for pharmacotherapy<br />
Jedlitschky G 1 , Rauch B 1 , Greinacher A 2 , Kroemer H K 1<br />
1Department of Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine <strong>Greifswald</strong>, Felix-Hausdorff-Str. 3,<br />
17487 <strong>Greifswald</strong>, Germany<br />
2Department of Immunology and Transfusion Medicine, University Medicine <strong>Greifswald</strong>, Sauerbruchstrasse, 17487 <strong>Greifswald</strong>, Germany<br />
Platelets contain many biologically active molecules, such as factors triggering platelet aggregation, growth factors<br />
and many other compounds, which are secreted upon platelet activation. Transporters mediate accumulation of<br />
these molecules in platelet granules and –upon platelet activation- their translocation across the plasma membrane.<br />
From a more pharmacologic point of view, platelets represent pharmacokinetic micro-compartments, in which the<br />
combination of uptake and elimination transport, possibly together with intracellular metabolism determines<br />
pharmacokinetics of drugs. As a result of transporter-mediated uptake, drugs can act on the target structures inside<br />
the platelets and provoke therapeutically positive effects or negative side effects. On the other hand, elimination of<br />
drugs from the platelet by export pumps can limit the effect of the drug and lead to drug resistance.<br />
Several members of the two major transporter families, ATP-binding cassette (ABC) transporters and solute carriers<br />
(SLCs), have been identified in platelets. 1 An example of an ABC transporter is MRP4 (ABCC4), which has been<br />
shown to facilitate ADP accumulation in dense granules. 2 Several additional functions of MRP4 in platelets have<br />
been proposed, since MRP4 is a very versatile transporter and its localization can be shifted from granules to the<br />
plasma membrane upon activation of the platelets and under certain pathophysiological conditions. These functions<br />
include the release of lipid mediators as well as a role in aspirin resistance. Several other ABC proteins have been<br />
detected in platelets with functions in glutathione and lipid homeostasis including ABCC1 and ABCC3 as well as<br />
several members of the ABCA subfamily. 3 The serotonin transporter (SERT, SLC6A4) in the platelet plasma<br />
membrane represents a well characterized example of the SLC family. Furthermore, expression of the organic anion<br />
transporter OATP2B1 (SLCO2B1), a high affinity transporter for certain statins, has been demonstrated in platelets.<br />
Changes in transporter localization and expression can affect platelet function and drug sensitivity. Thus, due to their<br />
various functions, platelet transporters can either serve as drug targets or as drug delivery systems and these<br />
functions can be greatly altered in the setting of inherited or acquired platelet defects. Therefore, a detailed<br />
understanding of the expression, function and regulation of transporters in platelets will add to a better understanding<br />
of platelet biology and in many ways may result in improved therapeutic options.<br />
References:<br />
1. Jedlitschky, G. et al. Blood. <strong>2012</strong>, 119(15):3394-3402.<br />
2. Jedlitschky, G. et al. Am.J. Pathol. 2010, 176(3):1097-1103.<br />
3. Niessen, J. et al. Pharmacogenet. Genomics. 2010, 20(6):396-400.<br />
50 Sessionvorträge Targets
Expression analysis of drug transporter proteins in RPMI 2650 cell line and excised human nasal<br />
mucosa<br />
Reichl S<br />
Institut für Pharmazeutische Technologie, Technische <strong>Universität</strong> Carolo-Wilhelmina zu Braunschweig, Mendelssohnstraße 1, 38106<br />
Braunschweig, Germany<br />
The nasal mucosa is an interesting site for application of drugs with systemic action, since it has a number of<br />
advantages as a delivery route, including easy accessibility, extensive vascular supply and avoidance of<br />
gastrointestinal degradation as well as first pass metabolism. Therefore more and more applications for nasal<br />
systemic drug delivery have been developed during the last decades, and their number is still increasing [1].<br />
In the development of drugs for nasal application, identification and characterization of the different uptake and efflux<br />
systems are necessary. P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRP) are classified as<br />
ATP binding cassette (ABC) transporters based on their sequences, organisation of the ATP-binding domains and<br />
efflux function. ABC proteins represent a large family of integral membrane transporters that utilize the energy of ATP<br />
hydrolysis to carry specific substrates across membranes [2]. The solute carrier gene (SLC) superfamily encodes<br />
another large family of membrane-bound transporters, located in almost every cellular and organelle membranes.<br />
Proteins of the SLC family include passive transporters, ion-coupled transporters and exchangers [3].<br />
A comparison was performed between excised human nasal mucosa from turbinectomy surgeries and our in vitro<br />
model based on immortalized human nasal epithelial cells (RPMI 2650) concerning the mRNA expression of different<br />
efflux and uptake transporter proteins. First investigations to prove functionality of different ABC transporters in RPMI<br />
2650 cells was carried out using permeation studies and uptake assays.<br />
The mRNA expression of eight ABC transporters as well as 13 SLC transporters of excised human tissue and RPMI<br />
2650 epithelial model were investigated and compared. The results showed promising similarity in the possible<br />
presence or absence of these efflux and influx transporters in human tissue and the in vitro model. A similarity<br />
between RPMI 2650 models and human nasal mucosa has already been shown for barrier properties to evaluate<br />
passive drug permeation [4].<br />
To verify the functionality of the first transporter protein, P-gp, bidirectional transport studies using rhodamine 123<br />
with RPMI 2650 cells grown on polyethylene terephthalate filter were performed. Indirect immunofluorescence<br />
staining for P-gp was also carried out. The findings confirmed the assumption that P-gp is not functionally expressed<br />
in these epithelial cells.<br />
An uptake assay using 5(6)-carboxy-2′,7′-dichlorofluorescein as a substrate for MRP1 and MRP5 was carried out.<br />
The result indicated functional expression of these efflux transporters in RPMI 2650 cells. Identification and<br />
localization of the responsible transporter proteins have to be proved by immunohistochemistry.<br />
Further investigations will be focused on western blot, immunohistochemical analysis and permeation as well as<br />
uptake studies using specific substrates for active transport to verify the protein expression and functionality of<br />
further drug transporters in human nasal mucosa and in vitro model based on RPMI 2650 cell line.<br />
Acknowledgments:<br />
We are grateful to the German Federal Institute for Risk Assessment (BfR), which funded this work under grant no. 3-1329-469. Furthermore,<br />
the authors thank Prof. Dr. Schroeder, Dr. Schmidt, Städtisches Klinikum Braunschweig and Dr. Reintjes, HNO-Praxis Schlosscarree<br />
Braunschweig for the supply of human nasal mucosa specimens.<br />
References:<br />
1. Dimova, S. et al.: Toxicology In Vitro 2005, 19: 107–122.<br />
2. Jones, P.M., George, A.M.: Cell. Mol. Life Sci. 2004, 61: 682–699.<br />
3. He, L., Vasiliou, K., Nebert, D.W.: Human Genomics 2009, 3: 195–206.<br />
4. Wengst, A., Reichl, S.: Eur. J. Pharm. Biopharm. 2010, 74: 290–297.<br />
Sessionvorträge Targets 51
Session Nukleäre Rezeptoren und Transkriptionsfaktoren als Wirkstofftargets<br />
Investigation on the estrogen receptor alpha selectivity of 2,3,5-triaryl-1H-pyrroles<br />
Gust R<br />
Institute of Pharmacy, University of Innsbruck, 6020 Innsbruck, Austria<br />
The estrogen receptors ERα and ERβ are ligand-inducible transcription factors which belong to the nuclear hormone<br />
receptor superfamily. ER activation or inhibition influences growth, differentiation and physiological function of<br />
several organs. Selective ER modulators (SERM) like raloxifene or tamoxifen are used as partial ERα antagonists in<br />
the therapy of osteoporosis and hormone dependent ERα-positive breast cancer, respectively. Despite their obvious<br />
benefits, SERMs may cause some serious side effects, including thromboembolisms and endometrial cancer, which<br />
urgently require the search for new derivatives.<br />
In this study we wanted to design molecules that bind exclusively to ERα, which is over-expressed in hormonedependent<br />
breast cancer cells. If the compounds displayed cytotoxic activity too, we would have an adequate drug<br />
for the therapy of the ER-positive mammary carcinoma.<br />
We started our investigations from the observation that triarylpyrazoles (e.g. PPT) but not related 2,4,5triarylimidazoles<br />
showed high agonistic/antagonistic potency and ER subtype selectivity. This finding documents that<br />
the position of the N-atoms of the heterocycle in relation to the aromatic rings determines the ER binding and<br />
subtype selectivity.<br />
In continuation of this study we synthesized 1H-pyrroles whose N-position in relation to the aryl rings at the<br />
heterocycle was selected from the position in PPT resulting in Type A pyrroles or Type B pyrroles.<br />
While Type A pyrroles showed strong degradation under physiological conditions, Type A pyrroles were stable and<br />
activated the ER only in U2-OS/ERα cells. For all pyrroles no transactivation could be observed in ERß containing<br />
U2-OS cells, which documents that they are selective ERα agonists in cellular systems. Additionally, the most<br />
interesting drug 2,3,5-tris(2-fluoro-4-hydroxyphenyl)-1-propyl-1H-pyrrole inhibited the growth of hormone-dependent<br />
tumor cells comparable to the established drug 5-fluorouracil and therefore fulfills the above mentioned<br />
pharmacological profile.<br />
52 Sessionvorträge Targets
Chemical epigenetics: histone modifying enzymes as targets to regulate transcription<br />
Jung M<br />
Institute of Pharmaceutical Sciences, Albert-Ludwigs-<strong>Universität</strong>, Albertstr. 25, 79104 Freiburg,<br />
Background<br />
Epigenetics is defined as inheritable phenotypic traits that determined by mechanisms beyond the DNA sequence.<br />
Histone modifications are an important component of the epigenetic machinery and we are working on the drug<br />
discovery of epigenetic inhibitors as well as the use of small molecule substrates to test for cellular enzyme activity.<br />
Material and Methods<br />
We have used focussed library and virtual screening in collaboration with Wolfgang Sippl (University of Halle) to<br />
identify potential inhibitors of epigenetic targets. The candidates were then screened in in-vitro assays in our lab and<br />
in collaborating labs (M. Lübbert, R. Schüle, University Freiburg Medical Centre) for phenotypic and functional<br />
assays. We have used a fluorogenic substrate to screen cell culture, animal tissue samples and blood cells for<br />
histone deacetylase activity and have validate this using western blotting of acetyl-histones.<br />
Results<br />
A combination of virtual and focussed library screening has identified new inhibitors of histone deacetylases as well<br />
as the histone kinase PRK1. The PRK1 inhibitor lestaurtinib shows a suppression of androgen dependent gene<br />
transcription in prostate cancer cells1 . A carbamate has been identified as a prodrug for hydroxamic acids with<br />
increased intracellular activity2 and we currently work on testing different protecting groups and their effect on cellular<br />
permeability.<br />
We thank the Deutsche Forschungsgemeinschaft (Ju295/7-1 and Ju295/9-1, within SPP 1463) for funding.<br />
References:<br />
1. Köhler, J. et al.: PLoS ONE <strong>2012</strong>, 7: e34973.<br />
2. Schlimme, S. et al. ChemMedChem 2011, 6: 1193-8.<br />
Sessionvorträge Targets 53
Helenalin derivatives and their impact on transcriptional control of mediators involved in rheumatic<br />
diseases<br />
Jäger C1 , Patel H1 , Humar M2 , Günther S3 , Merfort I1 1 Depart. Pharmaceutical Biology and Biotechnology, Inst. Pharmaceutical Sciences, Albert-Ludwigs University, Stefan-Meierstr. 19, 79104<br />
Freiburg, Germany<br />
2 Depart. Anesthesiology and Critical Care Medicine, University Hospital Freiburg, Freiburg, Germany<br />
3 Inst. Pharmaceutical Scienecs, Pharmaceutical Bioinformatics, Albert-Ludwigs University, Hermann-Herderstr. 9, 79104 Freiburg, Germany<br />
Helenalin derivatives are sesquiterpene lactones from the pseudoguaianolide type. They are considered as the<br />
effective compounds in Arnica montana flowers which preparations have been proven to exert beneficial effects in<br />
rheumatic complaints, such as osteoarthritis and rheumatoid arthritis (1). We here report studies on the molecular<br />
mode of actions which contribute to a better understanding of these beneficial effects.<br />
Both rheumatic diseases are characterized by chronic joint destruction. Whereas IL-1β and TNF-α are well-known<br />
players in this process it has become more and more obvious that IL-17 is also involved (2). This cytokine stimulates<br />
fibroblasts, macrophages and chondrocytes which for their part release various mediators with effects on<br />
inflammation, cartilage damage and bone erosion. Interestingly, IL-17 does not only induce TNF-α and IL-1β, but<br />
was shown to have also additive or even synergistic effects with TNF-α and IL-1β during the induction of cytokine<br />
expression and joint damage in vitro and in vivo.<br />
We could show with helenalinbutyrate that mRNA levels of IL-17 are reduced in primary human T-cells.<br />
Consequently, pretreatment of human primary chondrocytes with helenalinisobutyrate or<br />
dihydrohelenalinmethacrylate drastically reduced gene and protein expression of matrix metalloproteinase 1 (MMP1)<br />
and 13 (MMP13) at low concentrations with helenalinbutyrate being mostly active. Additionally, this sesquiterpene<br />
lactone lowered synergistically upregulated mRNA and protein of COX-2 by IL-17 and IL-1β. Transcription of MMPs<br />
is tightly regulated by the transcription factors AP-1 and NF-κB which were demonstrated to be directly targeted by<br />
the helenalin derivatives. The effect on NF-κB has already been studied intensively (3) in contrast that one on AP-1.<br />
Impacts on these transcription factors were supported by molecular docking studies.<br />
Acknowledgments: We are grateful to Bioforce company for financial support.<br />
References:<br />
1. Merfort, I. Curr. Drug Targets. 2011, 12: 1560-73.<br />
2. Koenders, M.I., Joosten, L.A.B., van den Berg, W.B., Ann. Rheum. Dis. 2006, 65: 29-33.<br />
3. Garcia-Pineres, A. et al., J. Biol. Chem. 2001, 276: 391113-20.<br />
54 Sessionvorträge Targets
Rescuing mutant p53 using halogen-enriched fragment libraries (HEFLibs)<br />
Boeckler F M1 ; Wilcken R1,2 ; Fersht A R2 ; Joerger A C2 1 Laboratory for Molecular Design and Pharmaceutical Biophysics, Department of Pharmaceutical and Medicinal Chemistry, Institute of<br />
Pharmacy, Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany<br />
2 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom<br />
The destabilizing cancer mutation Y220C creates a druggable surface crevice in the DNA-binding core domain of the<br />
transcription factor and tumor suppressor p53 [1,2]. We developed a strategy exploiting halogen bonding for lead<br />
discovery to stabilize the mutant with small molecules. We designed halogen-enriched fragment libraries (HEFLibs)<br />
as starting points to complement classical approaches [3]. From screening of HEFLibs and subsequent structureguided<br />
design, we developed substituted 2-(aminomethyl)-4-ethynyl-6-iodophenols as p53-Y220C stabilizers. Crystal<br />
structures of their complexes highlight two key features: (i) a central scaffold with a robust binding mode anchored by<br />
halogen bonding of an iodine with a main chain carbonyl and (ii) an acetylene linker, enabling the targeting of an<br />
additional subsite in the crevice. The best binders showed induction of apoptosis in a human cancer cell line with<br />
homozygous Y220C mutation. Structure-activity relationships show correlations to QM-based interaction energies of<br />
halogen-bonding. Our structural and biophysical data suggest a more widespread applicability of HEFLibs in lead<br />
identification, yielding lead structures that feature binding modes hardly obtainable by other techniques.<br />
References:<br />
1. Joerger, A. C.; Ang, H. C.; Fersht, A. R.: Proc. Natl. Acad. Sci. U. S. A. 2006, 103(41): 15056-61.<br />
2. Boeckler, F. M. et al.: Proc. Natl. Acad. Sci. U. S. A. 2008, 105(30): 10360-10365.<br />
3. Wilcken, R. et al.: J. Am. Chem. Soc. <strong>2012</strong>, 134(15): 6810–6818.<br />
Sessionvorträge Targets 55
Novel leads for PPARs and FXR: a computer-aided drug design approach<br />
Proschak E1 , Steri R, Achenbach J, Lamers C, Rau O, Schneider G, Steinhilber D, Schubert-Zsilavecz M1 Goethe-University of Frankfurt, Institute of Pharmaceutical Chemistry, OSF/ZAFES, Max-von-Laue Str. 9, D-60438 Frankfurt, Germany<br />
Peroxisome proliferator-activated receptors (PPARs) and farnesoid X receptor are in the main focus of<br />
pharmaceutical research for a wide range of metabolic disorders. Novel findings in this area indicate that although<br />
these targets have been investigated intensively, there is still an unmet need for novel safe and efficient lead<br />
structures. This presentation demonstrates the impact of computer-aided drug design techniques applied to<br />
discovery and optimization of novel lead structures for PPARs and FXR. We present three different approaches to<br />
identify novel lead structures: virtual screening, de novo design and in silico drug repurposing.<br />
In our virtual screening study we were able to identify a potent and selective PPARa agonist using a combination of<br />
pharmacophore- and shape-based screening [1]. The software retrieved a potent hit (EC50 = 44 nM), with >100fold<br />
selectivity against PPARγ. In a second application, we designed a lead structure de novo, starting from the known<br />
PPARα agonist GW590735 [2]. In the design process, two moieties were replaced by bioisosteric groups suggested<br />
by SQUIRRELnovo, which are responsible for compound potency. The new lead structure yielded submicromolar<br />
activity on PPARα (EC50 = 0.51 µM) in a cell-based reporter-gene assay [3].<br />
Furthermore we were able to identify the molecular basis for the glucose-lowering effects of imatinib based on FXR<br />
activation using a machine-learning approach [4]. We applied self-organizing maps to self-organizing map (SOM) is a<br />
virtual screening method for correlation of molecular structure and potential biological activity [5]. We used a SOM<br />
trained with known FXR ligands to screen the DrugBank database [6] for potential ligands for FXR. We were able to<br />
identify six approved drugs out of the Drugbank as FXR modulators (ketoconazole, pentamidine, dobutamine,<br />
imatinib, papaverine and montelukast) by using a SOM for screening of the DrugBank database. We show FXR<br />
modulation by selected compounds in a full length FXR transactivation assay and modulation of a FXR target gene<br />
by imatinib.<br />
Acknowledgments: We gratefully acknowledge financial support from the Else-Kröner-Fresenius-Stiftung, Merz Pharmaceuticals, Lipid<br />
Signaling Forschungszentrum Frankfurt (LiFF), Oncogenic Signaling Frankfurt (OSF) and Fonds der Chemischen Industrie for financial<br />
support.<br />
References:<br />
1. Proschak, E., Zettl, H., Tanrikulu, Y., Weisel, M., Kriegl, J.M., Rau, O., Schubert-Zsilavecz, M., Schneider, G., ChemMedChem 2009, 4:<br />
41–44.<br />
2. Sierra ML, Beneton V, Boullay A-B, Boyer T, Brewster AG, Donche F, et al., J Med Chem 2007, 50: 685–95.<br />
3. Proschak, E., Sander, K., Zettl, H., Tanrikulu, Y., Rau, O., Schneider, P., Schubert-Zsilavecz, M., Stark, H., Schneider, G., ChemMedChem<br />
2009, 4: 45-48.<br />
4. Steri, R., Achenbach, J., Steinhilber, D., Schubert-Zsilavecz, M., Proschak, E., Biochem Pharmacol <strong>2012</strong> accepted.<br />
5. Schneider P, Tanrikulu Y, Schneider G., Curr Med Chem 2009, 16: 258–66.<br />
6. Knox C, Law V, Jewison T, Liu P, Ly S, Frolkis A, et. Al., Nucleic Acids Res 2011, 39: D1035–1041.<br />
56 Sessionvorträge Targets
Inhibitors of class I histone deacetylases induce 5-lipoxygenase expression<br />
Steinhilber D<br />
Goethe-University Frankfurt, Institute of Pharmaceutical Chemistry/ZAFES, Max-von-Laue Str. 9, D-60438 Frankfurt, Germany<br />
5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes. We have previously shown that the histone<br />
deacetylase (HDAC) inhibitor trichostatin A (TSA) activates 5-LO transcription via recruitment of Sp1, Sp3 and RNA<br />
polymerase II to the proximal promoter [1, 2].<br />
In order to identify the HDACs involved in the regulation of 5-LO promoter activity isoform-specific HDAC inhibitors<br />
were applied. 5-LO promoter activity and mRNA expression was upregulated by the class I HDAC inhibitors apicidin<br />
and MS-275 but not by class II inhibitors.<br />
In order to analyze the chromatin modifications at the 5-LO promoter associated with HDAC inhibition, the time<br />
course of 5-LO mRNA induction by trichostatin A was analyzed and the concomitant changes in histone<br />
modifications at the 5-LO promoter in HL-60, U937 and Mono Mac6 cells was determined. ChIP analysis revealed<br />
that trichostatin A increases acetylation of histones H3 and H4 at the 5-LO core promoter in HL-60 and U937 cells<br />
whereas no significant changes were observed in Mono Mac6 cells. The appearance of H3 and H4 acetylation<br />
preceded the 5-LO mRNA induction whereas in all three cell lines, induction of 5-LO mRNA expression correlated<br />
with histone H3 lysine 4 trimethylation (H3K4me3), a marker for transcriptional activity of gene promoters.<br />
Induction of 5-LO mRNA expression by class I HDAC inhibitors coincides with the upregulation of 5-LO promoter<br />
activity as determined by reporter gene assays. The data suggest that in contrast to calcitriol and TGFβ [3], induction<br />
of 5-LO mRNA expression by HDAC inhibition mainly occurs on the 5-LO promoter level.<br />
Acknowledgments: We gratefully acknowledge financial support from the Else Kröner-Fresenius-Stiftung and DFG (GRK757).<br />
References:<br />
1. Schnur, N., Seuter, S., Katryniok, C., Rådmark, O. and Steinhilber, D., Biochim Biophys Acta 2007, 1771: 1271-1282.<br />
2. Klan, N., Seuter, S., Schnur, N., Jung, M. and Steinhilber, D., Biol Chem 2003, 384: 777-785.<br />
3. Stoffers, K.L., Sorg, B.L., Seuter, S., Rau, O., Radmark, O. and Steinhilber, D., J Mol Biol 2010, 395: 884-896.<br />
Sessionvorträge Targets 57
Session Thrombozytenfunktion und Gerinnung<br />
Novel oral anticoagulants – State of the art and aspects of their application<br />
Alban S<br />
Institute of Pharmacy, Christian-Albrechts-University of Kiel, Gutenbergstr. 76, 24118 Kiel, Germany<br />
Since thromboembolic diseases, i.e. myocardial infarction, stroke and pulmonary embolism (PE), represent the major<br />
cause of death in the Western world, anticoagulants are a class of life-saving drugs. Whereas there have been some<br />
advances in short- and medium-term anticoagulation during the last two decades, the only option for long-term<br />
anticoagulation were the vitamin K antagonists (VKA) so far.<br />
This has changed in 2008 with the approval of two novel oral anticoagulants (NOA): the direct thrombin inhibitor<br />
(DTI) dabigatran etexilate (Pradaxa ® ) und the first direct Factor Xa-Inhibitor (DXI) rivaroxaban (Xarelto ® ) for<br />
prevention of venous thromboembolism (VTE) after hip and knee replacement operation. In 2011, apixaban<br />
(Eliquis ® ), another oral DXI, was approved for this proof of concept indication and dabigatran etexilate and<br />
rivaroxaban were licenced for stroke prevention in atrial fibrillation and rivaroxaban additionally for treatment of deep<br />
vein thrombosis (DVT) and prevention of recurrent DVT and PE following acute DVT.<br />
Therefore, it’s time for an update: How the anticoagulation in clinical practice changes with the NOA? What are the<br />
differences between the NOA and the VKA as well as between the three NOA? Which patients are candidates for<br />
switching from VKA to NOA? What is about practical aspects like monitoring anticoagulant efficacy, interruption for<br />
surgical or invasive procedures and management of bleeding? Finally, are Marcumar ® and heparin injections about<br />
to fold?<br />
58 Sessionvorträge Targets
Novel platelet inhibitors – State of the art and aspects of their application<br />
Greinacher A<br />
Sessionvorträge Targets 59
Naturally occurring PF4/polyanion complexes prime for heparin-induced thrombocytopenia as a<br />
misdirected host defense mechanism<br />
Krauel K1,2 ; Weber C3 ; Jaax M1 ; Hackbarth C 1 ; Brandt S2 ; Block S2 ; Hammerschmidt S3 ; Greinacher A1 1 Institut für Immunologie und Transfusionsmedizin<br />
2 Zentrum für Innovationskompetenz – Humorale Immunreaktionen bei kardiovaskulären Erkrankungen<br />
3 Abteilung Genetik der Mikroorganismen, Interfakultäres Institut für Genetik und Funktionelle Genomforschung, <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-<strong>Universität</strong><br />
<strong>Greifswald</strong>, Germany<br />
Heparin-induced thrombocytopenia (HIT) is caused by highly immunogenic complexes of negatively charged heparin<br />
and positively charged platelet factor 4 (PF4). Some of the resulting antibodies against PF4/heparin complexes<br />
activate platelets via the platelet FcγIIa receptor, leading to thrombin generation and a prothrombotic state. We have<br />
recently proposed that HIT might be the result of a misdirected bacterial defense mechanism, as PF4 bound to<br />
bacteria also exposes the epitopes to which PF4/heparin antibodies bind [1]. Consistently, we showed that<br />
PF4/heparin antibodies are highly significantly associated with periodontitis, one of the most prevalent human<br />
infections mostly induced by Gram-negative bacteria [2].<br />
We now identified potential PF4 binding structures on the surface of Gram-negative bacteria using different bacterial<br />
mutants with defined variations in their lipopolysaccharide (LPS) sequences [3]. Binding of biotinylated PF4 was<br />
quantified by flow cytometry, and exposure of the antigenic epitope was assessed by adsorption and elution of<br />
PF4/heparin antibodies. Bacterial mutants lacking the O-antigens and the core of LPS but still exposing lipid A<br />
showed strongest PF4 binding, mediated by the phosphate groups of lipid A. As both the lipid A on the surface of<br />
Gram-negative bacteria and the amino acids of PF4 contributing to polyanion binding are highly conserved, our<br />
results further support the hypothesis that neoepitope formation on PF4 after binding to bacteria is an ancient host<br />
defense mechanism. The distance of two phosphate groups on lipid A is rather similar to the distance of phosphate<br />
groups on DNA or RNA. We therefore assessed the interaction of PF4 and nucleic acids. By enzyme-immunoassay<br />
and functional studies, we found cross-reactivity of PF4/heparin antibodies with PF4/DNA complexes in vitro.<br />
Systematic assessment of different RNA- and DNA-constructs revealed length and number of double-stranded<br />
segments of nucleic acids as risk factors for complex formation with PF4. This has consequences for drug<br />
development, as we accordingly found immunogenicity as a class effect of aptamers (DNA/RNA based therapeutics).<br />
PF4/aptamer complexes bound PF4/heparin antibodies in vitro and induced PF4/aptamer antibodies in a mouse<br />
model.<br />
We next asked the question, which structural changes of PF4 may trigger B-cell activation using circular dichroism<br />
(CD) spectroscopy. Pronounced structural changes of PF4, leading to expression of anti-parallel beta sheets,<br />
occurred during the interaction with highly charged polyanions known to be immunogenic in vivo. Only minor or no<br />
changes were found for weakly charged or neutral polymers. This allows to predict immunogenicity of drugs exposing<br />
polyanions by CD spectroscopy.<br />
Through desulfation of heparin, the new compound 2-O, 3-O desulfated heparin (ODSH, ParinGenix) has low<br />
anticoagulant activity but the residual negative charge still allows interaction with PF4. We found that ODSH inhibits<br />
platelet activation by PF4/heparin antibodies by disruption of PF4/heparin complexes [4]. In addition, ODSH does not<br />
induce the conformational changes of PF4 analyzed by CD spectroscopy. Therefore a mixture of heparin and ODSH<br />
might be an interesting option to reduce the risk of HIT which has to be addressed in clinical studies.<br />
In summary, polyanions induce structural changes of PF4 leading to an adverse immune reaction, which is a<br />
misdirected humoral host defense mechanism. CD spectroscopy can be applied to screen new drugs for their<br />
potential to induce immunogenic structural changes of PF4 and partially desulfated heparin can be applied to<br />
antagonize the adverse biological effects of PF4/polyanion complex antibodies.<br />
References:<br />
1. Krauel, K. et al.: Blood 2011, 117(4): 1370-1378.<br />
2. Greinacher, A. et al.: Blood 2011, 118(5): 1395-1401.<br />
3. Krauel, K. et al.: Blood <strong>2012</strong>, epub.<br />
4. Krauel, K. et al.: Blood <strong>2012</strong>, 119(5): 1248-1255.<br />
60 Sessionvorträge Targets
Session Neue Targets und Testsysteme<br />
PfGSK-3: A target for new antimalarial drugs?<br />
Kunick C1 , Fugel W1 , Ratin M2 , Kruggel S3 , Oberholzer A E4 , Dzikowski R5 , Pressburger N5 , Ferandin Y2 , Lemcke T3 ,<br />
Meijer L2 1 Technische <strong>Universität</strong> Braunschweig, Institut für Medizinische und Pharmazeutische Chemie, Beethovenstraße 55, 38106 Braunschweig,<br />
Germany<br />
2 Protein Phosphorylation & Human Disease Group, CNRS, Station Biologique, Place Georges Teissier, B.P. 74, 29682 Roscoff, France<br />
3 <strong>Universität</strong> Hamburg, Institut für Pharmazie, Bundesstraße 45, 20146 Hamburg, Germany<br />
4 Structural Biology Community Laenggasse (sbcl), 3000 Bern, Switzerland<br />
5 Department of Microbiology & Molecular Genetics, The Kuvin Center for the Study of Infectious and Tropical Diseases, IMRIC, The Hebrew<br />
University-Hadassah Medical School, Jerusalem 91120, Israel<br />
Malaria is one of the most threatening infectious diseases in tropical areas, causing hundreds of thousands fatal<br />
cases each year. [1] Although efficacious medicines are available for treatment and prophylaxis, the emergence of<br />
resistance is urging for new drugs with alternative pharmacological mechanisms. The inhibition of plasmodial protein<br />
kinases has repeatedly been suggested as a strategy for the development of antimalarial drugs throughout the last<br />
decade. [2-4] In spite of several efforts in this direction, protein kinase inhibitors have not yet reached clinical trials as<br />
antimalarial drugs. PfGSK-3, the plasmodial homologue of the human glycogen synthase kinase-3 (HsGSK-3), was<br />
recently identified from Plasmodium falciparum, the causative agent of malaria tropica. With a view to evaluate<br />
PfGSK-3 as potential target for new antimalarial agents, several known GSK-3 inhibitors were tested on PfGSK-3. [5]<br />
Unfortunately, these compounds were either none-selective or showed selective inhibition of the human enzyme.<br />
Therefore, compound libraries were tested to identify new selective PfGSK-3 inhibitors. A hit compound from this<br />
campaign was employed as structural template for the design of a congener series. Analysis of structure-activity<br />
relationships and structure-guided design enabled the preparation of selective PfGSK-3 inhibitors with in vitro<br />
antiplasmodial activity in the single-digit micromolar concentration range.<br />
Acknowledgments: The work was funded in part by the Commission of the European Communities (Contracts LSHB-CT-2004-503467 and<br />
Health-F3-2008-223414, to L. M. and C. K.). This joint project was financially supported by the State of Lower-Saxony, Hannover, Germany (to<br />
R. D., N. P., and C. K). S. K. was supported by a PhD grant from the University of Hamburg, Germany.<br />
References:<br />
1. WHO: World Malaria Report 2010 (http://www.who.int/malaria/world_malaria_report_2010/worldmalariareport2010.pdf).<br />
2. Doerig, C., Meijer, L.: Expert Opin Ther Targets 2007, 11(3): 279–290.<br />
3. Zhang, V. M., Chavchich, M., Waters, N. C.: Curr Top Med Chem <strong>2012</strong> 12(5): 456–472.<br />
4. Jirage, D., Keenan, S. M., Waters, N. C.: Infect Disord Drug Targets 2010, 10(3): 134–146.<br />
5. Droucheau, E. at al.: Biochim Biophys Acta 2004, 1697: 181–196.<br />
Sessionvorträge Targets 61
Chemical Oncology – Converging Cancer Genetics, Structural Biology and Medicinal Chemistry<br />
Rauh D<br />
Technische <strong>Universität</strong> Dortmund, Fakultät Chemie, Chemische Biologie, Otto-Hahn-Str. 6, 44227 Dortmund<br />
The 512 protein kinases encoded by the human genome are a prime example of nature's ability to create diversity by<br />
introducing variations to a highly conserved theme. Layers of regulatory mechanisms involving different combinations<br />
of post-translational modifications, intramolecular contacts, and intermolecular interactions control the activity of each<br />
kinase domain. Ultimately, they all achieve their effect by favoring particular conformations that promote or prevent<br />
the kinase domain from catalyzing protein phosphorylation. The central role of kinases in various diseases has<br />
encouraged extensive investigations of their biological function and three-dimensional structures, yielding a more<br />
detailed understanding of the mechanisms that regulate protein kinase activity by conformational changes.[1] I will<br />
discuss these regulatory mechanisms and show how conformational changes can be exploited for the design of<br />
specific inhibitors that lock protein kinases in inactive conformations.[2, 3] In addition, I highlight our recent<br />
developments to monitor ligand-induced structural changes in protein kinases and for screening and identifying<br />
inhibitors that stabilize enzymatically incompetent kinase conformations.[4-8]<br />
References:<br />
1. Rabiller, M., et al., Arch Pharm 2010, 343(4): 193-206.<br />
2. Getlik, M., et al., J Medicinal Chem 2009, 52(13): 3915-26.<br />
3. Getlik, M., et al., Eur J Med Chem <strong>2012</strong>, 48: 1-15.<br />
4. Simard, J.R., et al., Nature Chem Biol 2009, 5(6): 394-6.<br />
5. Simard, J.R., et al., J Am Chem Soc 2009, 131(51): 18478-88.<br />
6. Simard, J.R., et al., J Am Chem Soc 2010, 132(12): 4152-60.<br />
7. Simard, J.R., et al., J Am Chem Soc 2009, 131(37): 13286-96.<br />
8. Schneider, R., et al., J Am Chem Soc <strong>2012</strong>, 134(22): 9138-41.<br />
62 Sessionvorträge Targets
Development of lysine mimic quinazoline-based selective inhibitors of a Jmj-C histone demethylase<br />
family<br />
Rotili D1 ; Cheng X2 ; Mai A1 1 Pasteur Institute–Cenci Bolognetti Foundation, Department of Chemistry and Technology of Drugs, “Sapienza” University of Rome, Aldo Moro<br />
Square 5, 00185 Rome, Italy<br />
2 Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA<br />
The histone proteins are subjected to various post-translational modifications, such as methylation, acetylation,<br />
phosphorylation, and ubiquitylation. These modifications, interfering with RNA polymerase activity and DNA<br />
transcription, represent one of the main mechanisms of the epigenetic modulation of gene expression. Aberrant<br />
expression of histone-modifying enzymes has been linked to various human diseases, such as neurological<br />
disorders and cancer. While studies on histone acetylation are known from years, studies on histone<br />
methylation/demethylation and relative modulators are at their infancy [1].<br />
N<br />
N<br />
HN<br />
HN<br />
MeO<br />
N<br />
MeO<br />
N<br />
MeO N N<br />
N<br />
H2N O N NH<br />
N<br />
BIX-01294 MC3076<br />
N<br />
H2N MeO<br />
O<br />
HN<br />
N<br />
N Cl<br />
H2N<br />
MeO<br />
O<br />
N<br />
N<br />
N NH<br />
N<br />
MC3073 MC3074<br />
BIX-01294, a diazepin-quinazoline-amine derivative able to inhibit G9a and G9a-like protein (GLP) lysine<br />
methyltransferases and to reduce methylation levels of H3K9 at several G9a target genes [2], was our starting point<br />
to design novel putative inhibitors of histone methyltransferases and/or demethylases acting on H3K9, (G9a/GLP and<br />
KIAA1718, respectively). Indeed, G9a and KIAA catalyze opposite reactions on H3K9 (the first adding and the latter<br />
removing the Me2 histone mark), and may recognize Lys in either un-methylated or methylated state, it being the<br />
substrate or reaction product, alternatively. Thus, we replaced the C7-methoxy group of BIX with a Lys-mimic side<br />
chain (5-aminopentyloxy chain), obtaining increased G9a/GLP inhibition (MC3076) respect to BIX [3]. Since MC3076<br />
as well as BIX were found by us able to inhibit also the demethylase KIAA1718, on the basis of crystallographic<br />
studies performed on MC3076 complexed with either GLP or KIAA, we prepared a new series of quinazoline analogs<br />
in order to improve their potency and selectivity against KIAA demethylase.<br />
We proceeded to the simplification of the MC3076 structure by insertion of small substituents at the C2 and/or C4<br />
quinazoline ring positions, so obtaining new quinazolines (MC3073, MC3074, etc.) highly potent against KIAA1718<br />
and devoid of any GLP inhibiting activity [4]. Some of these derivatives (MC3074) were also inactive against the<br />
JARID family of Jmj-C histone demethylases so demonstrating a promising capability to discriminate between<br />
different Jmj-C histone demethylase families [4].<br />
References:<br />
1. Varier, R.A.; Timmers, H.T.M. Biochim Biophys Acta 2011, 1815(1): 75-89.<br />
2. Kubicek, S. et al.: Mol Cell 2007, 25(3): 473-481.<br />
3. Chang, Y. et al.: J Mol Biol 2010, 400(1): 1-7.<br />
4. Upadhyay, A.K. et al.: J Mol Biol <strong>2012</strong>, 416(3): 319-327.<br />
Sessionvorträge Targets 63
Profiling of epigenetic targets using peptide microarrays<br />
Schutkowski M; Rauh D; Roessler C; Masch A<br />
Martin-Luther-University Halle-Wittenberg, Institute of Biochemistry & Biotechnology, Department Enzymology, Kurt-Mothes-Strasse 3, 06120<br />
Halle, Germany<br />
Acetylation of lysine residues in proteins is one of the most frequently occurring post-translational modifications<br />
playing a major role in protein-protein-interaction as well as in regulating DNA transcription and metabolic processes.<br />
Using acetyl coenzyme A as donor for the acetyl group, lysine acetyltransferases are transferring the acetyl group<br />
onto the ε-amino-group of lysine residues in proteins. The positive charge of the side chains is neutralized, thus<br />
remodeling biological activity of enzymes and protein-protein-interaction. Bromodomains as protein interaction<br />
modules are recognizing specifically the acetylated lysine side chains.<br />
To understand substrate specificity of lysine acetyltransferases and binding specificity of bromodomains, we created<br />
a high-density peptide microarray displaying 6800 peptides derived from all known acetylation sites in human<br />
proteins in both unmodified and acetylated form resulting in more than 13.600 different features per microarray pair<br />
(acetylome microarray). The acetylome microarrays were treated with lysine acetyltransferases in the presence of<br />
acetyl-CoA followed by an optimized mixture of anti-acetyl-lysine-antibodies.<br />
Additionally, treatment of the acetylome microarray with Sirtuins, NAD + -dependent protein lysine-deacetylases<br />
involved in regulation of central physiological functions, such as energy metabolism, cell cycle progression, and<br />
aging processes, resulted in decrease of signals for several of the acetylated peptides in comparism to the control<br />
experiment in the absence of NAD + . We were able to identify subsite specificities and substrate sequences for all of<br />
the seven human Sirtuin isoforms. Besides known substrates our results provide novel substrate candidates for the<br />
different Sirtuin isoforms which could be confirmed in subsequent solution phase experiments.<br />
Principally, each enzyme transfering residues to the side chains of lysines could be analysed with the acetylome<br />
microarray using either radioisotopic labeling (lysine-methyltransferases and labelled AdoMet, poly-ADPribosyltransferases<br />
and labelled NAD + ) or antibody-based readout (poly-ADP-ribosyltransferases and ethenoNAD +<br />
followed by anti-ethenoNAD + -antibody, ubiquitin/SUMO transfering enzymes followed by anti-ubiquitin/SUMOantibodies).<br />
64 Sessionvorträge Targets
Session Tumortherapie und Resistenzmechanismen<br />
Anti-infectives research to combat drug resistant tumors?<br />
Hartmann R W 1,2<br />
1 Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, 66123 Saarbrücken, Germany<br />
2 Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123 Saarbrücken, Germany<br />
In the last decades there have been great efforts to develop new tumor therapeutics and in the last 25 years<br />
approximately 100 new drugs (NCEs) have been admitted to the market. The clinical situation however has only<br />
improved slightly which is due to the fact that the tumors develop resistance against the new drugs as well. The big<br />
amounts of money that have been invested into basic research to elucidate resistance mechanisms have brought<br />
new interesting findings but (so far) have not resulted in more efficient drugs. Also in antibiotics therapy the<br />
emergence of resistant bacterial strains is a severe problem and research is focused on strategies to investigate how<br />
the development of resistant bacterial strains can be avoided. In this context recent results from our group will be<br />
presented aiming at a novel therapy for Pseudomonas aeruginosa infections by interference with its unique PQS<br />
quorum sensing system. Finally the question will be raised whether insights from antiinfectives research could help to<br />
discover novel strategies to combat drug resistant tumors.<br />
A new class of glutathione peroxidase inhibitors is able to reverse resistance to chemotherapeutics<br />
in human B-cell lymphoma cell lines<br />
Schulz R1 ; Emmrich T1 ; Lemmerhirt H1 ; Hirt C2 ; Kiefer T2 ; Link A1 ; Bednarski P J1 1 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong> University <strong>Greifswald</strong>, 17489 <strong>Greifswald</strong>,<br />
Germany<br />
2 Clinic for Haematology and Oncology, <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong> University <strong>Greifswald</strong>, 17475 <strong>Greifswald</strong>, Germany<br />
The development of chemoresistance in cancer cells poses a major risk for chemotherapy failure. A combination of<br />
anticancer drugs and agents that suppresses adaptation in cancer cells is a potent method to increase the efficiency<br />
of therapies. GUMBUS and DOGUM, two cell lines from patients with non-Hodgkin lymphomas, were used to<br />
investigate the process of drug resistance. Cells were treated with the anticancer drugs methotrexate, etopiside,<br />
cisplatin and bortezomib to induce resistance. Assays showed a significantly increased amount of glutathione<br />
peroxidase 1 (GPx1) in treated cells compared to untreated. The approach now is to inhibit GPx1 to target the antioxidative<br />
pathway in these cancer cells. A virtual screening of a self-composed compound library docked to the<br />
active site of the X-ray crystal structure of bovine GPx1 yielded promising structures. In an enzyme assay a class of<br />
acylhydrazone heterocyclic proved to be GPx inhibitors. Furthermore, lymphoma cells lost their resistance to anticancer<br />
drugs when treated with these hydrazones. This concept could be refined to a new strategy in<br />
chemotherapies.<br />
Sessionvorträge Targets 65
Chemoresistance of ovarian cancers against platinum complexes<br />
Kassack M U; Hamacher A<br />
Heinrich-Heine-<strong>Universität</strong> Düsseldorf, Institut für Pharmazeutische und Medizinische Chemie, Pharmazeutische Biochemie,<br />
<strong>Universität</strong>sstrasse 1, 40225 Düsseldorf, Germany. matthias.kassack@uni-duesseldorf.de<br />
Ovarian cancer is the most lethal gynaecological cancer. Surgery and a combination chemotherapy including<br />
carboplatin (cisplatin) are the primary treatment options. Initially, most ovarian cancers are chemosensitive. However<br />
rather soon, resistance mechanisms develop and lead to relapse and therapeutic failure. Many resistance<br />
mechanisms against platinum compounds are known but this has not yet been translated into improved therapy. One<br />
reason is a lack of systematic studies to overcome chemoresistance using resistance mechanisms as additional<br />
therapeutic targets and second, relevant mechanisms driving the development of chemoresistance have only partly<br />
been studied. Comprehensive analysis of cancer cells on a genomic, transcriptional and translational level show that<br />
the development of chemoresistance is a complex biological process involving both, selection and adaptation<br />
processes (1, 2).<br />
cisplatin resistance factor<br />
7<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
1 2 3 4 5 6 7 8 9 10 12 16 20 24<br />
No of treatment cycles [weeks]<br />
Resistance development over 6 months in A2780 ovarian cancer cells<br />
To study the development of chemoresistance, we<br />
have undertaken a novel approach of generating<br />
resistant cell lines. Ovarian cancer cell lines were<br />
treated intermittently according to a clinically<br />
relevant dosing scheme over a period of 6<br />
months. By that procedure, we have obtained<br />
pairs of sensitive and resistant cell lines that were<br />
analysed using MTT assay, whole genome<br />
expression microarrays, and (phospho) proteome<br />
profiling of kinases. Bioinformatic analysis<br />
revealed a number of potential novel targets to<br />
overcome chemoresistance. Subsequent inhibitor<br />
studies in combination with carbo/cisplatin could<br />
demonstrate a reversal of chemoresistance<br />
against platinum compounds.<br />
In conclusion, bioinformatic analysis allows establishing drug resistance signatures with potential prognostic value.<br />
Furthermore, novel therapeutic targets have emerged establishing a new approach to overcome chemoresistance<br />
against carbo/cisplatin in ovarian cancer.<br />
References:<br />
1. Eckstein N, et al. Cancer Res. 2009; 69(7): 2996-3003.<br />
2. Gosepath EM, et al. Int J Cancer. 2008; 123(9): 2013-9<br />
66 Sessionvorträge Targets
Medicinal Chemistry with Organometallics – From Catalysts to Anticancer Drugs?<br />
Ott I<br />
Institute of Medicinal and Pharmaceutical Chemistry, Technische <strong>Universität</strong> Braunschweig, Beethovenstr. 55, 38106 Braunschweig, Germany<br />
Despite the tremendous success of platinum based cancer chemotherapeutics, transition metal based drugs are rare<br />
on the current drug market as well as in medicinal chemistry and drug design. However, metal complexes offer<br />
interesting opportunities for drug development based on their structural and kinetic properties, which differ in many<br />
ways from traditional “organic” compounds.[1]<br />
Organometallics are metal complexes with metal-carbon bonds, which have been attracting increasing attention in<br />
general chemistry over the last decades and enabled an enormous progress in the field of catalysis. Not surprisingly<br />
this has led to a broader knowledge of the properties of available organometallic functional groups and thereby also<br />
new approaches for inorganic medicinal chemistry have emerged.[2,3]<br />
We have been working over the last years on several types of organometallics including metal complexes with Nheterocyclic<br />
carbene (NHC) ligands or gold alkynyl complexes as new antitumor drugs (see the figure for some<br />
examples).[4-6] For many of these compounds we could confirm distinct biological properties such as antiproliferative<br />
effects in cultured tumor cells, selective inhibition of the enzyme thioredoxin reductase, triggering of reactive oxygen<br />
species (ROS) formation or anti-angiogenic effects. Binding to albumin, cellular uptake and intracellular distribution<br />
have been studied for selected examples by high resolution atomic absorption spectroscopy (HRCS-AAS) in order to<br />
provide a more detailed picture of their behaviour on the cellular level.<br />
In the presentation a short overview of the topic will be given and selected examples of our current projects will be<br />
presented.<br />
N<br />
N<br />
Au<br />
Cl<br />
O<br />
Au P<br />
Figure: examples for bioactive organometallics<br />
Sessionvorträge Targets 67<br />
N<br />
N<br />
Ru<br />
Cl Cl<br />
Acknowledgments: Financial support by the DFG (project FOR 630, “Biological Function of Organometallic Compounds”) is gratefully<br />
acknowledged.<br />
References:<br />
1. Alessio, E: Bioinorganic Medicinal Chemistry (Wiley) 2011.<br />
2. Gasser G, Ott I, Metzler-Nolte N: J Med Chem 2011, 54(1): 3–25.<br />
3. Noffke, AL et al.: Chem Comm <strong>2012</strong>, 48: 5219–5246.<br />
4. Rubbiani, R et al.: J Med Chem 2011, 54(24): 8646–8657.<br />
5. Oehninger, L et al.: ChemMedChem 2011, 6: 2142–2145.<br />
6. Meyer, A et al.: Angew Chem <strong>2012</strong>, accepted.
NHC-Silver(I) and NHC-gold(I) complexes as bioorganometallic anticancer and antibacterial drugs<br />
Tacke M; Hackenberg F; Patil S; Lally G; Streciwilk W<br />
UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland<br />
The synthesis of N-heterocyclic carbene (NHC) silver(I) acetate complexes with varying lipophilic benzyl-substituents<br />
at the 1 and 3 positions of the (benz)imidazole ring was achieved by reaction of silver(I) acetate with the<br />
corresponding (benz)imidazolium bromide or iodide salts [1-7]. These NHC-silver(I) acetate derivatives exhibit<br />
interesting structural motifs in the solid state and proof to be soluble and stable in biological media. The preliminary<br />
antibacterial activity of all the compounds was studied against Gram-negative bacteria Escherichia coli, and Grampositive<br />
bacteria Staphylococcus aureus using the Kirby-Bauer disk-diffusion method. Almost all the NHC-silver(I)<br />
acetate complexes have shown high antibacterial activity compared to the NHC-precursors. The antibiotic lead<br />
compound SBC3, which is synthesised from 4,5-diphenyl-1,3-dibenzyl-imidazole, reaches an antibacterial activity<br />
comparable to marketed modern antibiotics. In addition, the NHC-silver complexes had their cytotoxicity investigated<br />
through MTT based preliminary in vitro testing on the human renal cancer cell line Caki-1 in order to determine their<br />
IC50 values. Here, NHC-silver(I) acetate complexes were found to have IC50 values as low as 1.2 μM for (1-methyl-<br />
3-(p-cyanobenzyl) benzimidazole-2-ylidene) silver(I) acetate, which has become the anticancer lead structure SBC1.<br />
The best corresponding NHC-Au(I)Cl complex SBC2 reaches an IC50 value of 10 μM against the cancer cell line<br />
CAKI-1 [8,9]. In addition, results of SBC1 against drug-resistant cancer cell lines, transport and target assays as well<br />
as in vivo testing in zebrafish and mouse will be communicated.<br />
The authors acknowledge IRCSET and UCD for funding.<br />
References:<br />
1. F. Hackenberg, A. Deally, G. Lally, S. Malenke, H. Müller-Bunz, F. Paradisi, S. Patil, D. Quaglia, M. Tacke, Int J Inorg Chem <strong>2012</strong>,<br />
DOI: 10.1155/<strong>2012</strong>/121540<br />
2. S. Patil, A. Deally, B. Gleeson, F. Hackenberg, H. Müller-Bunz, F. Paradisi, M. Tacke., Z Allg Anorg Chem 2011, 637: 386–396.<br />
3. S. Patil, A. Deally, B. Gleeson, H. Müller-Bunz, F. Paradisi, M. Tacke. Novel Benzyl-Substituted N-Heterocyclic Carbene–Silver Acetate<br />
Complexes: Synthesis, Cytotoxicity and Antibacterial Studies, Metallomics, 2011, 3: 74–88.<br />
4. S. Patil, K. Dietrich, A. Deally, B. Gleeson, H. Müller-Bunz, F. Paradisi, M. Tacke, Helv Chim Acta 2010, 93: 2347–2364.<br />
5. S. Patil, A. Deally, B. Gleeson, H. Müller-Bunz, F. Paradisi, M. Tacke, Appl Organomet Chem 2010, 24: 781–793.<br />
6. S. Patil, J. Claffey, A. Deally, B. Gleeson, M. Hogan, L. M. Menéndez Méndez, H. Müller-Bunz, F. Paradisi, M. Tacke, Eur J Inorg Chem<br />
2010, 1020–1031.<br />
7. S. Patil, M. Tacke., „NHC-Silver(I) Acetates as Bioorganometallic Anticancer and Antibacterial Drugs. Insights into Coordination,<br />
Bioinorganic and Applied Inorganic Chemistry.“ Edited by M. Melník, P. Segľa, M. Tatarko., Press of Slovak University of Technology,<br />
Bratislava, 2011: 555–566.<br />
8. L. Kaps, B. Biersack, H. Müller-Bunz, K. Mahal, J. Münzner, M. Tacke, T. Mueller, R. Schobert., J Inorg Biochem <strong>2012</strong>, 106: 52–58.<br />
9. S. Patil, A. Deally, F. Hackenberg, L. Kaps, H. Müller-Bunz, R. Schobert, M. Tacke., Helv Chim Acta 2011, 94: 1551–1562.<br />
68 Sessionvorträge Targets
Sessionvorträge Tabletten<br />
Session Nanomedizin<br />
Nanomedicines for targeted drug delivery to the inflamed intestinal mucosa<br />
Collnot E M1 ; Leonard F2 ; Hare E1,3 ; Mell N1 ; Ali H2 ; Lehr C M1,2 1 Department of Drug Delivery (DDEL), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infectious<br />
Research (HZI), Saarland University, Campus A4.1, 66123 Saarbruecken, Germany<br />
2 Biopharmacy and Pharmaceutical Technology, Saarland University, 66123 Saarbruecken, Germany<br />
3 Hebrew University of Jerusalem, Faculty of Medicine, School of Pharmacy, Institute for Drug Research, Jerusalem 91120, Israel<br />
Inflammatory bowel diseases (IBD) such as Crohn’s disease or Ulcerative colitis are autoimmune, chronic, episodic,<br />
inflammatory conditions of the gastrointestinal tract. Classical therapy of IBD with steroids or immunomodulators<br />
delivered via pellets, capsules, or tablets is often inefficient mainly due to limited drug release time and enhanced<br />
elimination of drug carriers as the result of diarrhoea. The emergence of biologicals has significantly increased the<br />
treatment options for IBD in recent years. Still, these new therapeutics have to be applied systemically resulting in<br />
sometimes severe adverse effects. Nanomedicine may enhance the efficacy of conventional IBD therapeutics and<br />
open up new routes of application for next generation drugs: Passively targeting the inflamed intestinal areas,<br />
nanoparticles of ~100 nm size have been shown to accumulate in the inflamed tissue, while bigger particles of 1 or<br />
10 µm showed no specificity for diseased intestinal areas [1]. Moreover, polylactide-co-glycolide (PLGA)<br />
nanoparticles loaded with rolipram showed higher and prolonged anti-inflammatory activity and reduced central<br />
nervous adverse effects in a TNBS rat model [1].<br />
Further investigating this EPR (enhanced permeability and retention) – like effect we developed a co-culture model of<br />
the inflamed intestine based on Caco-2 enterocytes and blood derived macrophages and dendritic cells [2]. Via<br />
addition of the cytokine interleukin-1β a reversible inflammation can be induced. In response to the stimulus a<br />
decrease in barrier function, a re-organization of tight junctions, a release of inflammation markers and increased<br />
mucus production can be observed mimicking pathophysiological phenomena observed in IBD in vivo.<br />
Different nanoformulations of the glucocorticoid budesonide were applied in the in vitro system and evaluated for<br />
their mechanism of accumulation and anti-inflammatory activity [3]. Both budesonide loaded PLGA nanoparticles and<br />
the free drug solution were able to recover epithelial barrier function (quantified via TEER) and to reduce release of<br />
inflammation marker IL-8. However, treatment with free budesonide solution was only effective for a short time as<br />
after 48 h a rebound of IL-8 release was observed. In contrast PLGA budesonide treated cells retained low IL-8<br />
levels. The prolonged activity of the nanoparticulate formulation can be ascribed to the formation of local drug<br />
depots, as particles accumulate between the cells in the area of the tight junctions. A liposomal budesonide<br />
formulation seemed to be preferentially taken up and processed by the immune cells in the co-culture model. The<br />
resulting dose dump negatively affected the barrier function and resulted in increased IL-8 levels.<br />
Aiming to further enhance the efficacy of nanocarrier accumulation, active targeting ligands for the inflamed intestinal<br />
mucosa were investigated. In the inflamed colonic mucosa of colitis patients an increased expression of transferrin<br />
receptor (TfR) has been reported both in the basolateral aspects of crypts and on the apical surface [4]. Indeed anti-<br />
TfR immunoliposomes applied the luminal side showed greatly enhanced adherence in vivo compared to nonspecific<br />
immunoliposomes.<br />
As a first topical application of a biological in IBD therapy a nanoformulation of the potent, anti-inflammatory cytokine<br />
IL-10 was developed. IL-10 is a promising IBD therapeutic, but failed in clinical trials upon subcutaneous injection,<br />
due to its short serum half-life and severe adverse effects at higher doses. Drug loaded albumin nanoparticles were<br />
prepared by spray drying and embedded in Eudragit S-100 microparticles to ensure stability during gastrointestinal<br />
passage. The nanoparticle in microparticle oral system succeeded in protecting IL-10 integrity during the preparation<br />
process and releasing bioactive cytokine at the desired pH of 7.4.<br />
References:<br />
1. Collnot, E.M., H. Ali, and C.M. Lehr, J Control Release <strong>2012</strong>.<br />
2. Leonard, F., E.M. Collnot, and C.M. Lehr, Mol Pharm 2010. 7(6): 2103–2119.<br />
3. Leonard, F., et al., ALTEX <strong>2012</strong>, 29(3): 275–285.<br />
4. Harel, E., et al., PLoS ONE 2011, 6(9): e24202.<br />
Sessionvorträge Tabletten 69
Selective gene vehicles for enhanced DNA and siRNA delivery<br />
Schümmelfeder J2 , Dayyoub E1 , Marxer E1 , Brüßler J1 , Schäfer J1 , Bakowsky U1 1Marburg University, Department of Pharmaceutical Technology and Biopharmaceutics, Ketzerbach 63, D-35037 Marburg, Germany<br />
2Department of Cardiology, St. Georg Hospital Eisenach, Mühlhäuser Strasse 94/95, D-99817 Eisenach, Germany<br />
Gene therapy is believed by many to be the therapy of the 21st century. Successful gene therapy or gene<br />
knockdown depends on the delivery of nucleic acid molecules into cell. While viral vectors have shown high gene<br />
transfer efficacies, their drawbacks include immunogenic/inflammatory responses, limited loading capacity and<br />
issues with regards to large scale manufacturing and quality control. Consequently, numerous non-viral gene delivery<br />
vectors including liposomes (lipoplexes), polycationic polymers (polyplexes) and organic or inorganic nanoparticles<br />
(nanoplexes) have been developed and reported in recent years [1,2,4]. General issues in non-viral transfection<br />
systems are the delivery of DNA for gene transfection into the nucleus or small interfering RNAs (siRNAs) for the<br />
induction of RNA interference (RNAi). RNAi is a powerful method that has been applied successfully for the downregulation<br />
of genes in both functional genetic analysis and gene therapy. It relies small interfering RNAs (siRNAs),<br />
and since all other components of the RNAi machinery are provided by the target cell /target tissue, the efficient<br />
siRNA delivery is of critical importance and has proven to be the bottleneck for successful RNAi applications.<br />
Atherosclerosis is characterized by the buildup of lipid-rich plaques within the blood vessel walls of large arteries, and<br />
underlies the clinical conditions of myocardial infarction, chronic stable angina, stroke and peripheral vascular<br />
disease. The process of atherogenesis is induced by a low-grade inflammatory process in the vascular wall, leading<br />
through various steps to the eventual formation of atheromatous plaque. These plaques may also become<br />
progressively unstable due to local reaction to pro-inflammatory cytokines and proteinases (e.g. IFN-gamma,IL-<br />
18,CD40–CD154, or IL 1 etc.), leading to plaque ulceration or rupture. Additionally activated endothelial cells express<br />
adhesion molecules such as P-selectin and E-selectin, VCAM or ICAM leading to a pro-inflammatory environment<br />
and a mediated activation of clotting cascade [3]. The gene overexpression of such target molecules make them<br />
promising candidates for the treatment by siRNA formulations.<br />
The aim of this research was to develop a gene-transfection system based on site specific lipoplexes and<br />
lipopolyplexes which would possess a nano-scaled size below 200 nm. For the preliminary experiments a luciferase<br />
reporter gene bearing plasmid DNA (pcmv beta) was used instead of siRNA for the therapeutic application (down<br />
regulation of the targets TNF-alpha, VEGF). The pDNA was pre-condensed with cationic polymers [1] and/or<br />
complexed [2,4] with different lipid mixtures (DPPC, DOPC, DSPC, Saint, Tetraetherlipids or Cholesterol etc.) to<br />
improve the stability/efficiency and lowering the toxicity. The gene vehicles were targeted to E-selectin via surface<br />
modification with E-Sel antibodies. The transfection efficacy and cell viability was investigated on the HUVEC and<br />
CHO-E cells under shear flow conditions.<br />
Acknowledgments: This study was supported by “Deutscher Akademischer Austauschdienst“ (DAAD) and by “Deutsche Forschungsgemeinschaft”<br />
(Forschergruppe FG 495 and FG 629, UB). We are grateful to Prof. Dr. J. Aigner for the development of the polyplex vehicles<br />
and the technical support.<br />
References:<br />
1. Schäfer J, Höbel S, Bakowsky U, Aigner A: Biomaterials 2010, 31(26): 6892–6900.<br />
2. Kneuer C et al.: J Nanosci Nanotechnol 2006, 6(9-10): 2776–2782.<br />
3. Kneuer C et al.: Drug Discov Today 2006, 11 (21-22): 1034–1040.<br />
4. Tabatt K et al.: J Controlled Release 2004; 97: 321–332.<br />
70 Sessionvorträge Tabletten
Modulating immune responses of human cells by adjuvant loaded particulate carriers<br />
Wischke C1,2 , Mathew S1,2 , Roch T1 , Frentsch M2 , Lendlein A1,2 1Center for Biomaterial Development, Helmholtz-Zentrum Geesthacht, Kantstr. 55, 14513 Teltow, Germany<br />
2Berlin-Brandenburg Centre for Regenerative Therapies, Berlin and Teltow, Germany<br />
Building an efficient immunological memory to avoid infections remains a challenge<br />
for various types of pathogens, particularly when purified, poorly immunogenic antigenic<br />
structures should be used for safety reasons. Therefore, antigens are often<br />
combined with cocktails of immunostimulating compounds, which in a rather uncontrolled<br />
process induce a proinflammatory environment and frequently have led to<br />
discussions about adjuvant induced severe vaccine reactions. For enabling tailored<br />
immune responses including efficacy against intracellular pathogenes, carrier systems<br />
are of high interest, as they may co-deliver the antigen(s) and a specific adjuvant<br />
[1]. Particles from poly[(rac-lactide)-co-glycolide] (PLGA) have shown their<br />
suitability as protein carriers as they are very efficiently taken up into cells by<br />
Fig. 1: Intracellular localization of<br />
MP, reproduced from [3] with<br />
permission, Copyright 2006.<br />
phagocytosis [2] (Fig. 1) and, moreover, facilitated phagolysosomal escape to the cytosol as relevant to induce<br />
immunity against intracellular pathogens. The carrier size should be in the upper nanosize range or, preferentially,<br />
microparticles (MP) < 10 µm, in order to efficiently enable phagocytosis as uptake mechanism and deliver sufficient<br />
levels of payload.<br />
Importantly, the MP carriers themselves were shown to be inert and did not induce activation of antigen presenting<br />
cells (APC) such as human primary dendritic cells (DC) [3], which is required to tailoring the immune response by the<br />
selectively added adjuvant. When poly[(riboinosinic acid)-co-(ribocytidylic acid)] as a Toll-like receptor 3 (TLR3)<br />
agonist was bound to surface-functionalized MP [3] in a layer-by-layer approached, only ligand loaded but not blank<br />
particles induced a full antigen-independent maturation of DC [4].<br />
As the safety profile of TLR ligands in general is occasionally subject to<br />
concerns and addressed presently in clinical studies, alternative defined<br />
adjuvants may be required. Therefore, in this study, N-acetylmuramyl–<br />
L-alanyl–D-isoglutamine (MDP) and γ-D-glutamyl-meso-diaminopimelic<br />
acid (iE-DAP) as ligands of cytosolic receptors sensing nucleotide and<br />
oligomerization domains (NOD) were evaluated for their capability to<br />
induce DC maturation and cytokine release after encapsulation into<br />
PLGA MP [5]. This concept was rationalized by the carriers’ capability<br />
to support phagolysosomal escape, thus making available their payload<br />
to the compartment where NOD receptors are expressed. Microencapsulated<br />
NOD agonists but not blank MP or soluble NOD agonists<br />
induced a dose-dependent maturation of DC (Fig. 2), illustrating the<br />
efficiency of the particulate carrier in combination with phagocytosis as<br />
transport mechanism. A proinflammatory DC phenotype with<br />
Fig. 2: Dose dependent maturation of DC. remarkable high levels of released cytokines particularly for iE-DAP<br />
were detected. Importantly, for selectively detecting effects of the studied adjuvant, a strategy is required to exclude<br />
immunogenic impurities and prevent erroneous interpretations of cell activation in the case of impurity-burdened MP.<br />
By different reporter cell lines not sensing MDP and iE-DAP but selectively expressing other pathogen associated<br />
molecular pattern, the purity of the employed carriers and the specificity of results were proven. Based on this, further<br />
exploration of these carriers is planned in vivo.<br />
Acknowledgments: D. Lorenzen, Institute for Tumor Therapy, Duderstadt, Germany, for support in previous cell studies [Fig. 1; Ref. 2-4].<br />
References:<br />
1. Rappuoli, R., Aderem, A.: Nature 2011, 473: 463–469.<br />
2. Wischke, C. et al.: Eur J Pharm Biopharm 2006, 62: 247–253.<br />
3. Wischke, C. et al.: J Contr Rel 2006, 114: 359–368.<br />
4. Wischke, C. et al.: Int J Pharm 2009, 365: 61–68.<br />
5. Wischke, C. et al.: <strong>2012</strong> manuscript in revision.<br />
6. Mathew, S. et al.: <strong>2012</strong> manuscript submitted.<br />
Sessionvorträge Tabletten 71
Acid-Degradable Dextran Particles for the Delivery of Biotheropeutics<br />
Wich P R1 ; Fréchet J M J2 1 Institut für Pharmazie und Biochemie, Johannes Gutenberg-<strong>Universität</strong> Mainz, Staudingerweg 5, 55128 Mainz, Germany<br />
2 College of Chemistry, University of California Berkeley, CA 94720-1460, USA<br />
Polymer-based drug, vaccine and gene delivery systems can be easily modified and are particularly attractive<br />
because of their ability to perform several critical functions simultaneously. These include delivering therapeutic or<br />
other bioactive agents to a specific site via a targeting mechanism, increasing the circulation times of drugs in the<br />
body, protecting drugs from degradation, or increasing the bioavailability of poorly soluble drugs.<br />
Recently, a new class of modular and acid-degradable, polymeric particles using acetal-modified dextran (Ac-DEX)<br />
was developed.[1,2] This biocompatible material can be formulated into particles using a variety of common<br />
emulsion-based techniques enabling control over both size and morphology. Due to their pH sensitivity, Ac-DEX<br />
particles can selectively and rapidly release their encapsulated payload under mildly acidic conditions including those<br />
found in sites of inflammation, tumor tissue, or endocytic vesicles. Upon hydrolytic degradation, Ac-DEX reverts back<br />
to FDA-approved dextran without the generation of acidic byproducts that could damage the encapsulated payload or<br />
lead to inflammation, as has been observed with some other commonly used polymer systems. In addition, Ac-DEX<br />
particle degradation rate can be easily tuned within the time scale of relevant cellular processes. This release<br />
tunability combined with their low-toxicity and payload versatility make Ac-DEX particles an ideal platform for a wide<br />
range of biotherapeutic delivery applications including immunotherapy [2] and gene delivery.[3]<br />
References:<br />
1. Bachelder E.M. et al.: J Am Chem Soc 2008, 32: 10494–10495.<br />
2. Broaders, K.E. et al.: Proc Natl Acad Sci USA, 2009, 106: 5497–5502.<br />
3. Cohen, J. L. et al.: Bioconj Chem 2011, 13: 1902–1905.<br />
72 Sessionvorträge Tabletten
Characterization of Bacterial Nanocellulose as Drug Delivery System for Low and High Molecular<br />
Weight Drugs<br />
Müller A1 ; <strong>Moritz</strong> S1 ; Wiegand C 2 ; Wesar F 3 ; Munteanu M 4 ; Hessler N 4 ; Kralisch D 4 ; Müller F A 3 ; Hipler U-C 2 ;<br />
Fischer D1 1 Department of Pharmaceutical Technology, Friedrich-Schiller-University Jena, Otto-Schott-Str. 41, 07745 Jena, Germany<br />
2 Department of Dermatology, Friedrich-Schiller-University Jena, Erfurter-Str. 35, 07743 Jena, Germany<br />
3 Institute of Materials Science and Technology, Friedrich-Schiller-University Jena, Loebdergraben 32, 07743 Jena, Germany<br />
4 Institute of Technical Chemistry and Environmental Chemistry, Friedrich-Schiller-University Jena, Lessingstr. 12, 07743 Jena, Germany<br />
Synthesized by bacteria of the genus Gluconacetobacter the biopolymer bacterial nanocellulose (BNC) has already<br />
been proven to be advantageous for various biomedical applications as tissue engineering, implant materials, skin<br />
substitutes and wound care [1, 2]. The use of this biomaterial as drug delivery system offers the opportunity to<br />
combine its beneficial material properties (e.g. high purity, mechanical stability and biocompatibility) with the positive<br />
effects of incorporated drugs and opens up new promising fields of application.<br />
Comparative loading and release studies with high molecular weight protein drugs (bovine serum albumin: BSA,<br />
luciferase) as well as different low molecular weight antiseptics (polihexanide: PHMB, PVP-iodine, octenidine) were<br />
performed using planar BNC fleeces produced in a static cultivation process. Therefore BNC samples were loaded<br />
by immersion in aqueous drug solutions and released after different incubation times. Influence of drug<br />
concentration, water content of the BNC samples, incubation time as well as temperature were investigated.<br />
Quantification of the different drugs was performed by UV spectrophotometry (BSA, 278 nm; PHMB, 235 nm; PVPiodine,<br />
352; octenidine, 281 nm), BCA assay and luminescence measurements (luciferase assay system). Drug<br />
distribution in the samples was analyzed by staining of BSA loaded samples with BCA assay reagent and the use of<br />
red brown coloured PVP-iodine. Sample morphology was studied by SEM analysis. Furthermore, drug release data<br />
were examined according to Ritger-Peppas equation. Polyacrylamide gel electrophoresis of released BSA as well as<br />
measurement of biological activity of released luciferase was conducted to study protein stability. Antibacterial<br />
efficacy of antiseptic BNC extracts prepared according to DIN EN ISO 10993-12 against Staphylococcus aureus was<br />
tested by microplate laser nephelometry. Furthermore, a direct challenge test (JIS L 1902:2002) was performed for<br />
measuring the antibacterial efficacy of PVP-iodine loaded samples. Cell proliferation of human HaCaT keratinocytes<br />
incubated with extracts of BNC loaded with antiseptics was quantified using a luminometric ATP-luciferase assay for<br />
cytotoxicity studies. The proteins as well as the low molecular antiseptic drugs showed the same release pattern,<br />
characterized by a burst in the beginning followed by a slower release rate until 48 hours. An overlay of diffusion- and<br />
swelling-controlled drug transport mechanisms was found by mathematical analysis (Ritger-Peppas equation) for<br />
both types of drugs. Optimized loading techniques allowed a homogenous drug distribution in the loaded samples.<br />
Protein stability could be maintained during loading and release as shown by gel electrophoresis as well as biological<br />
enzyme activity. Extracts of BNC hydrogels loaded with PHMB and octenidine showed a strong inhibition of<br />
Staphylococcus aureus growth. Antiseptic efficacy of PVP-iodine loaded BNC could be confirmed with the direct<br />
challenge test. Cell proliferation of HaCaT keratinocytes in cytotoxicity assays was found to be dependent on<br />
antiseptic drug type and extraction ratio. Whereas PVP-iodine extracts had no influence on cell proliferation, PHMB<br />
and octenidine extracts exhibited a decrease of cell proliferation at different extraction ratios (PHMB: 0.1 g:50 mL,<br />
octenidine: 0.001 g:50 mL), which has to be considered for determination of active loading concentration in wound<br />
dressing development.<br />
In conclusion, obtained results characterize drug loading to and release from the BNC biomaterial and confirm the<br />
suitability of BNC for drug delivery applications for high molecular as well as low molecular weight drugs.<br />
Acknowledgments: This study was funded by the Thuringian Ministry of Education, Science and Culture (B714-10032) as well as the European<br />
Fund for Regional Development. Additionally, the authors would like to thank Ramona Brabetz as well as Elena Pfaff for the excellent technical<br />
assistance.<br />
References:<br />
1. Kralisch, D. et al.: Biotechnol Bioeng 2010, 105(4): 740–747.<br />
2. Klemm, D. et al.: Angew Chem Int Ed 2011, 50(24): 5438–5466.<br />
Sessionvorträge Tabletten 73
Biomedical applications of magnetite in the respiratory system – Toxicological considerations and<br />
mechanistic studies in A549 lung cells<br />
Könczöl M1,2 , Gminski R1 , Gieré R3 , Merfort I2 , Mersch-Sundermann V1 1 Institut für Umweltmedizin und Krankenhaushygiene, <strong>Universität</strong>sklinikum Freiburg<br />
2 Institut für Pharmazeutische Biologie und Biotechnologie, <strong>Universität</strong> Freiburg<br />
3 Institut für Geowissenschaften, <strong>Universität</strong> Freiburg<br />
Magnetite, a ferromagnetic iron oxide, represents a promising material within the broad field of nanomaterials for<br />
biomedical applications [1]. Beside its use in magnetic resonance imaging or hyperthermia, research has been<br />
focused on its potential to serve as an inhalative carrier system for drugs or vaccines. The lack of consistent data on<br />
pulmonary toxicity requires more systematic investigation of magnetite nano- and micrometer particles. In a former<br />
study, magnetite was thoroughly investigated with regard to its potential to induce toxic effects and to influence<br />
signaling pathways. It was clearly demonstrated that reactive oxygen species (ROS)-formation led to mitochondrial<br />
damage and genotoxic effects in A549 cells [2]. Based on these findings, we here report studies which elucidate the<br />
origin of magnetite-mediated ROS-formation and their influence on cell cycle of A549 human lung epithelial cells.<br />
Additionally, effects on p53 and p21 activation were evaluated by western blots.<br />
After exposure of A549 to magnetite particles, an increased superoxide production, measured by electron<br />
paramagnetic resonance (EPR), was detected, while the glutathion (GSH) level decreased significantly. Moreover,<br />
the particles were able to induce ROS-formation in an acellular environment. Mitochondria or soluble iron did not<br />
contribute considerably to the ROS production. We could show that incubation of A549 cells with inhibitors of<br />
NADPH-oxidase (NOX) prior to particle treatment led to decreased ROS formation measured by EPR and DCFH-DA<br />
assay. Analysis of cell cycle distribution yielded a pronounced sub-G1-peak, which cannot be linked to increased cell<br />
death. Western blot analysis showed no activation of p53 but upregulation of p21 in A549. Surprisingly, exposure to<br />
magnetite leads to p21-mediated G1-like arrest, which has only reported before for low concentrations of microtubule<br />
stabilisation drugs, but so far never after particle exposure. Importantly, the arrested sub-G1 cells were viable and<br />
showed no caspase activation. In summary, magnetite induced a variety of cellular responses in A549 cells in vitro.<br />
Since it cannot be excluded that these toxic effects may be also of relevance in vivo, a fundamental safety profile has<br />
to be compiled before considering nanomaterials for biomedical use in human.<br />
References:<br />
1. Kim, J. et al., Arch Toxicol <strong>2012</strong>, 86(5): 685–700.<br />
2. Könczöl, M. et al., Chem Res Toxicol 2011, 24(9): 1460–1475.<br />
74 Sessionvorträge Tabletten
Session Pharmaverfahrenstechnik<br />
Formulation screening with colloidal drug carrier systems<br />
Bunjes H<br />
Institute of Pharmaceutical Technology, TU Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany<br />
As a large fraction of newly identified drug candidates is poorly water soluble the use of enabling formulation<br />
technologies becomes increasingly important. In principle, this applies to all potentially relevant routes of<br />
administration but dosage forms for systemic administration, in particular via the parenteral (especially intravenous)<br />
and oral route are usually of special interest. Usually, the development of viable dosage forms for poorly soluble<br />
substances is a comparatively time-consuming process. This creates a kind of dilemma in regard to the rapid<br />
availability of a rather large number of potential drug candidates by modern drug discovery methods. The situation is<br />
further complicated by the usually very limited amount of drug substance available for formulation studies in early<br />
development. In order to rapidly provide suitable formulations for the required tests with poorly water soluble drug<br />
candidates the availability of formulation screening methods for the fast development of formulations with a minimum<br />
amount of drug candidate substance would thus be highly desirable. Ideally, the formulations identified this way<br />
would also bear the potential to be developed into marketable products.<br />
Colloidal carrier systems like drug nanosuspensions or different types of lipid dispersions (such as liposomes, mixed<br />
micellar systems or several types of lipid nanoparticles) are a prominent formulation option for poorly soluble drug<br />
candidates. Properly designed colloidal formulations are, in principle, applicable via all potential routes of<br />
administration and may also be developed into drug products. Compared with more conventional dosage forms (e.g.,<br />
drug solutions) most of them are, however, also comparatively demanding, in particular with regard to their way of<br />
preparation and their physicochemical stability. Only a few of them can be prepared by simple mixing (e.g. mixed<br />
micellar solutions) that would allow for easy screening. For others (like liposomes), small scale dispersion methods<br />
are well established but the whole process of formulation is still quite time-consuming.<br />
The preparation of colloidal carrier systems with micro- and nanostructured devices like by high-pressure<br />
microchannel or premix membrane emulsification [1, 2] or in miniaturized milling devices [3] offer novel approaches<br />
for small scale formulation preparation. Beyond formulation screening with a drug candidate of interest, they are also<br />
very useful for the optimization of formulations and in basic formulation research. The work with such preparation<br />
methods may, however, still require several experiments to determine the loading capacity of a lipid carrier system<br />
for a specific drug. In order to solve this task in a more efficient way, loading of pre-formed colloidal carrier systems is<br />
an interesting option [4]. This presentation will introduce and discuss different approaches for formulation screening<br />
with a special focus on the development of colloidal lipid carrier systems for poorly water soluble substances.<br />
References:<br />
1. Finke, J. et al.: Chem Eng J <strong>2012</strong>, accepted for publication.<br />
2. Joseph, S., Bunjes, H.: J Pharm Sci <strong>2012</strong>, 101(7): 2479–2489.<br />
3. Juhnke, M., Berghausen, J., Timpe, C.: Chem Eng Technol 2010, 33(9): 1412–1418.<br />
4. Kupetz, E. et al.: 8 th PBP World Meeting <strong>2012</strong>, Istanbul, Turkey.<br />
Sessionvorträge Tabletten 75
Pharmaceutical Extrusion Technology – Recent developments<br />
Thommes M<br />
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitaetsstrasse 1, 40225 Duesseldorf, Germany<br />
Pharmaceutical extrusion technology has become an important processing technology within the last few decades.<br />
For this reason, academia, as well as industry, is focusing research efforts on this area. It is likely that extrusion will<br />
become one of the most important technologies for many pharmaceutical applications.<br />
In the last few years the co-rotating twin screw extruder has become the common extruder type due to its versatility.<br />
Its modular setup allows for broad manufacturing process customization to meet different product requirements.<br />
Such flexibility allows multiple unit operations to be performed in a single machine, thereby increasing efficiency.<br />
Based on the plastification method, extrusion processes can be differentiated as wet, hot melt, and cold extrusion.<br />
These have been investigated and used during the past 30 years. Each of these methods has its own advantages<br />
leading to specific applications, such as pelletization, taste masking, and solubility enhancement.<br />
Recent investigations deal with the process understanding (QbD) and downstream processing such as film extrusion<br />
and injection moulding. A lot of research efforts also deal with down-scaling of extrusion equipment to make it<br />
suitable for small-scale production and early development.<br />
Pharmaceutical extrusion technology is an innovative technology that continues to become more relevant, due to its<br />
many assets as compared with other pharmaceutical technologies.<br />
76 Sessionvorträge Tabletten
Hot melt extruded dosage forms<br />
Rein H<br />
Pharmazeutische Technologie, <strong>Universität</strong> Bonn, Gerhard-Domagk-Straße 3, D-53121 Bonn-Endenich, Germany<br />
1. Introduction<br />
Hot melt extrusion is gaining more and more interest in pharmaceutical industry. One of the main advantages, apart<br />
from improving the bioavailability of an API, is the possibility to move away from batch processing towards<br />
continuous production. Mostly the extrusion process itself is already well adjusted. One of the major problems right<br />
now is giving shape to the final products.The aim of our work was to develop continuous processes for the<br />
production of pellets and extruded tablets (extrudets).<br />
2. Pellets [1]<br />
Spherical starch pellets were directly and continuously produced using hot-melt extrusion and die-face pelletisation.<br />
In contrast to conventional pelletisation procedures, a discontinuous spheronisation step is not necessary. Pellets<br />
were produced based on different starches and several other polysaccarids, four different active ingredients<br />
(ibuprofen, paracetamol, phenazon and tramadol-HCl) and various additives. The resulting pellets exhibit a large<br />
mechanical stability, low porosity, small surface area and a a very narrow particle size distribution even in the micron<br />
scale. The drug is either dispersed or dissolved in the polysaccharid melt. Drug loadings of up to 80% are<br />
achievable. The dissolution rate depends on pellet size, pellet porosity, the velocity with which the penetrant diffuses<br />
into the pellet-core, the water solubility of the API, and the molecular structure, composition and the swelling behavior<br />
of the used polysaccarid. The fractional drug release from non eroding starch pellets can be described with the<br />
Noyes-Whitney equation. [2]<br />
3. Tablets/extrudets<br />
Biplan tablets were produced by using a co-rotating twin-screw extruder (ZSE 27 HP-PH, Leistritz AG, D-Nürnberg).<br />
For the final shaping of the tablets, the still elastic strands were cut with a rotary fly-knife cutting machine (Dynamat,<br />
Metzner, D-Neu Ulm), which is generally used in plastics industry for cutting of wires, cables or seals. [3] Another<br />
important aspect of our work concerns data-acquisition during the cutting process. Monitoring the energy of each cut<br />
gives an interesting insight into the process. This might be applied as a useful in-process tool.<br />
Tablets were produced based on several starches and various different polysaccharides, which were well known in<br />
the food industry, e.g. gelatine, gummi arabicum, pectine or locust bean gum and several additives. Depending on<br />
the used polysaccharide the production of melting tablets, lozenges or dosage forms with fast or prolonged API<br />
release is possible.<br />
Acknowledgments:<br />
1, Roquette, F-Lestrem, donation of starches.<br />
2. Leistritz Extrusionstechnik, D-Nürnberg, allocation of a twin-screw extruders (ZSE 18 HP-PH and ZSE 27 HP-PH).<br />
3. Metzner Maschinenbau, D-Neu-Ulm, allocation of a rotary fly-knife cutting machine (Dynamat 20/40).<br />
References:<br />
1. Bialleck S, Rein H,: Eur J Pharm Biopharm 2011, 79: 440–448.<br />
2. Bialleck S, Rein H,: Stärke <strong>2012</strong>, 64: 408–419.<br />
3. Kudernatsch, H., 2011, Pharm Ind 2011, 73: 2050–2060.<br />
Sessionvorträge Tabletten 77
Nanogrinding of naproxen: influence of process parameters on product quality<br />
Bitterlich A1 , Komoß C2 , Dengler M, Bunjes H 2 , Kwade A1 1Institute for Particle Technology, TU Braunschweig, Volkmaroder Str. 5, 38104 Braunschweig, Germany<br />
2Institute of Pharmaceutical Technology, TU Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany<br />
Nanosized active pharmaceutical ingredients (APIs) increasingly attract interest in pharmaceutical industries. Up to<br />
now, five drug-containing nanoparticulate formulations are on the market [1]. In micronized form, all corresponding<br />
APIs exhibit a poor water solubility and, consequently, a low bioavailability after oral administration. By reducing the<br />
particle size, the dissolution rate can be strongly increased which leads to an improved biopharmaceutical profile.<br />
Wet media milling is often used to produce API nanoparticle suspensions. In this study the influence of the process<br />
parameters on product quality during the nanogrindnig process was investigated. A planetary ball mill (PM400,<br />
Retsch GmbH) was modified according to [2] to enable parallel small scale screening with 12 samples of 25 mg API<br />
each. Inside the grinding chambers, the anti-inflammatory API naproxen (kindly provided by Novartis Pharma AG) in<br />
aqueous suspension experienced high shear and compression forces between grinding media of different size,<br />
material and shape which were brought into relative motion by centrifugal forces.<br />
In a first step screening experiments were conducted, using various polymers and surfactants as stabilizers to<br />
identify an optimum formulation against agglomeration phenomena. With this formulation (10 % naproxen, 2.5 %<br />
Kollidon ® 30) the influence of process parameters on product quality was investigated. Within a certain range, the<br />
product quality (regarding particle size and distribution width) increased by using small grinding beads, spherical<br />
grinding media, high centrifugal forces and a high density of the grinding media.<br />
In addition, some interesting stabilizer and process parameter dependent phenomena were observed during the<br />
grinding process: Different product shapes were obtained by varying the stabilizer. When using PVP (Kollidon ® 30) or<br />
PVA (Mowiol ® 3-85) grades, the product particles exhibited a round morphology after the grinding process. By using<br />
Tween ® 80 or HPMC (Pharmacoat ® 603) as stabilizer, however, the product particle shape was more needle-like<br />
(figure 1). Furthermore, the product particle shape could be influenced by adjusting the process parameters: By<br />
applying grinding media made from yttrium stabilized zirconia with a diameter of 100 µm at high centrifugal forces of<br />
the mill, even the morphology of the product particles stabilized with Kollidon ® 30 changed from “round” over “needlelike”<br />
to finally “disc-like” over a grinding time of 960 min in the modified planetary ball mill. Apparently, the high<br />
(mechanic) energy transferred to the product led to a growth of the API crystals under these process conditions<br />
rather than to a real comminution process.<br />
Figure 1: TEM micrograph of a Mowiol ® 3-85 (left) and Tween ® 80 (right) stabilized<br />
naproxen suspension after 4 h grinding time<br />
References:<br />
1. Merisko-Liversidge, E., Liversidge, G.G.: Adv Drug Deliv Rev 2011, 63: 427–440.<br />
2. Juhnke, M., Berghausen, J., Timpe, C.: Chem Eng Technol 2010, 33: 1412–1418.<br />
78 Sessionvorträge Tabletten
Session Bioanalytik: Testsysteme und Datenqualität<br />
Image Processing in medicinal microscopy<br />
Matz M1 , Schumacher K2 , Rustenbeck I2 , Baumann K1 1Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany.<br />
2Institute of Pharmacology, Toxicology and Clinical Pharmacy, TU Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany.<br />
In the last decade, total internal reflection fluorescence microscopy (TIRF-M) was established as a standard method<br />
for observing molecular phenomena close to the cell membrane. It is used here to study the behavior of insulin<br />
granules. The obtained TIRF images and video sequences are usually evaluated with commercial software<br />
packages, which allow counting of the labeled cell components and tracking of single labeled particles (i.e. insulin<br />
granules).<br />
Here we present a new and sophisticated program for the exploration of TIRF video sequences, enabling the user to<br />
simultaneously count and track all visible labeled objects. Moreover, the tracked objects can be characterized with<br />
respect to velocity, size, kinetic behavior, and exocytosis. The program starts with background subtraction. Next, a<br />
3D convolution is carried out for noise reduction. Afterwards all fluorescing objects are tracked down and are being<br />
followed. A specifically tailored clustering routine in combination with an edge filter, which detects jumps, is used<br />
exocytosis detection.<br />
While methods for object identification and single particle tracking are well established in the literature, this talk<br />
focuses on new methods for the characterization of granule tracks. The goal is to estimate whether a particular object<br />
moves due to diffusion or some kind of directed movement.<br />
A detailed analysis of the detection of exocytitic events will also be presented. Simply using an edge filter on the<br />
tracks yields too many false positive and false negative events. The clustering routine employed here, is able to<br />
identify even vague increases in fluorescence intensity, which is highly related to membrane fusion and the release<br />
of vesicle content.<br />
Sessionvorträge Tabletten 79
The use of microscale thermophoresis (MST) for protein characterization and formulation<br />
Besheer A1 , Abstiens K1 , Winter G1 , Baaske P2 , Duhr S2 1 Pharmaceutical Technology and Biopharmaceutics, Department of Pharmacy, Ludwig-Maximilians-<strong>Universität</strong>, Butenandtstrasse 5-13, D-<br />
81377 Munich, Germany.<br />
2 NanoTemper Technologies GmbH, Flößergasse 4, D-81369 Munich, Germany.<br />
Microscale thermophoresis (MST) is the drift of dissolved molecules along a micron-sized temperature gradient [1]<br />
(Figure 1). The strength and direction of this molecular thermal diffusion depend mainly on the molecular surface<br />
area, the strength of ionic shielding and on the molecule’s hydration shell [2]. Changes in these properties lead to<br />
alterations in the thermophoretic motion, which make MST a very sensitive tool to analyse structural changes in<br />
proteins. Accordingly, MST was successfully applied for the measurement of macromolecular interactions and<br />
binding affinities [3]. By Applying MST to investigate protein unfolding, we found out that the changes in protein<br />
hydration accompanying unfolding lead to significant changes in thermophoresis, allowing the determination of the<br />
protein’s melting point (Tm). For instance, it was possible to determine Tm for recombinant human granulocyte colony<br />
stimulating factor (rh-GCSF) at different pHs and sucrose concentrations, and validate the results by fluorescence<br />
and microcalorimetric measurements. In this respect, MST offers a number of advantages as it is a label-free<br />
technique, requiring very small sample volumes (< 3 µl) and is able to measure protein thermophoresis over a wide<br />
concentration range (nM to mM). Furthermore, it is an entirely optical method, which is contact-free and therefore<br />
minimizes contamination of the sample. In comparison to other methods, it is not dependant on the surroundings of<br />
tryptophan and does not require the use of external fluorescent dyes like in fluorescence-based techniques, nor is it<br />
limited by low concentration ranges like microcalorimetry and circular dichroism, or the need for relatively<br />
concentrated solutions as in ATR-FTIR. The potential of MST to deliver information about protein unfolding can thus<br />
be exploited over a wide range of applications in protein science.<br />
Figure 1. A) Experimental setup<br />
for label-free MST involves the<br />
use of UV-LED fluorescence for<br />
concentration measurement<br />
through the intrinsic tryptophan<br />
fluorescence. An IR-Laser<br />
produces a temperature<br />
gradient on the order of<br />
0.1 K/µm. Capillaries containing<br />
< 3 µl samples are heated to<br />
the desired temperature.<br />
Samples can show B) a<br />
thermophobic behaviour, where<br />
molecules diffuse away from the<br />
IR-Laser or C) a thermophilic<br />
behaviour moving towards it.<br />
References<br />
1. Iacopini, S., Rusconi, R., & Piazza, R., Eur Phys J E Soft Matter 2006 19: 59–67.<br />
2. Duhr, S. & Braun, D., Proc Natl Acad Sci USA 2006 103: 19678–19682.<br />
3. Wienken, C .J., Baaske, P., Rothbauer, U., Braun, D., & Duhr, S., Nat Commun 2010 (1): 100.<br />
80 Sessionvorträge Tabletten
High-Throughput Protein Melting Temperature Analysis by Differential Scanning Fluorimetry<br />
Menzen T, Frieß W<br />
Ludwig-Maximilians-<strong>Universität</strong> München, Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics; Butenandtstraße 5, D-<br />
81377 Munich, Germany<br />
Unfolding and aggregation of therapeutic proteins are severe problems in drug formulation, because they are linked<br />
to immunogenic reactions and reduced efficiency. Thus, the investigation of stabilizing conditions is essential during<br />
development. The protein melting temperature (Tm) resembling the temperature at which half of the protein is in a<br />
denatured state, is commonly used to assess overall thermal stability. Stabilizing conditions will shift Tm to higher<br />
values whereas destabilizing effects reduce Tm. Due to the wealth of formulation parameters, such as buffers, pH,<br />
excipients, and combinations thereof a high-throughput Tm assay would be beneficial. Differential Scanning<br />
Calorimetry (DSC) as the gold standard for Tm analysis even in an automatized capillary system is limited to a few<br />
samples per day of several hundred microliters of volume. Differential Scanning Fluorimetry (DSF) was introduced in<br />
2001 by Pantoliano and coworkers and allows the determination of Tm by tracking the thermal unfolding of the<br />
protein by means of an environmental sensitive fluorescent dye [1]. The application of DSF for high-throughput Tm<br />
analysis of therapeutic proteins with low consumption of both time and material has been described [2-4]. But this<br />
method to determine Tm is limited in presence of surfactants which are frequently used as stabilizers in protein<br />
formulation. SYPRO® Orange is commonly utilised because it shows bright fluorescence after binding to the<br />
exposed hydrophobic structures of the unfolded protein. However, the unfolding transition of the protein is covered by<br />
a high background fluorescence of the dye trapped inside the hydrophobic core of the micelles formed by the<br />
surfactants present. In this study we demonstrate how to overcome this limitation to determine Tm of a therapeutic<br />
monoclonal antibody (MAb) with DSF in presence of surfactants typically used as stabilizing agent polysorbate 20,<br />
polysorbate 80, and poloxamer 188 in concentrations above the CMC. The molecular rotor probe DCVJ (4-<br />
(dicyanovinyl)julolidine) is sensitive to changes in protein structure and shows less interactions with micelles [5]. By<br />
careful correction of the background fluorescence of corresponding placebo samples, Tm values of surfactant<br />
containing protein formulations can be determined, whereas it is not possible solely based on the original sample<br />
traces. The resulting Tm values correlate very well with Tm values obtained by DSC and in surfactant free<br />
formulations. Tm values could be determined in a concentration range between 4 and 40 mg/ml MAb with DCVJ and<br />
SYPRO® Orange in presence of surfactants. Despite the limited sensitivity of DCVJ compared to SYPRO® Orange,<br />
DCVJ allowed Tm determination in various cases in which SYPRO® Orange failed. Additionally, we observed that<br />
the appearance of turbidity marking the formation of aggregates of this MAb is linked to the second unfolding<br />
transition (Tm2). This indicates that unfolding of specific domains is decisive for aggregation. We conclude that DSF<br />
is a very promising method for high-throughput Tm analysis during therapeutic protein formulation development and<br />
can also be used in presence of surfactants, a previously critical limitation.<br />
References:<br />
1. Pantoliano, M. W. et al.: J Biomol Screen 2001, 6(6): 429–440.<br />
2. He, F. et al.: J Pharm Sci 2010, 99(4): 1707–1720.<br />
3. Goldberg, D. S. et al.: J Pharm Sci 2011, 100(4): 1306–1315.<br />
4. Li, Y. et al.: J Pharm Sci 2011, 100(6): 2120–2135.<br />
5. Hawe, A. et al.: Pharm Res 2010, 27(2): 314–326.<br />
Sessionvorträge Tabletten 81
NMR spectroscopy of high molecular weight protein complexes<br />
Nagaraj M1 ; Schubeis T1 ; Ahmed M1 ; Zimmer A1,2 ; Ritter C1 1 Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany<br />
2 current address: Karolinska Institutet, SE-171 77 Stockholm, Sweden<br />
High-resolution structural information of protein targets is of crucial importance for rational drug design approaches.<br />
X-ray crystallography and solution Nuclear Magnetic Resonance (NMR) are standard techniques to determine the<br />
structures of soluble proteins. However, large protein complexes are still extremely challenging to study, as they are<br />
difficult to crystallize or inherently non-crystalline, as is the case e.g. for fibrillar proteins. Recent advances in NMR<br />
spectroscopy, isotope labelling schemes and protein sample preparation strategies have enabled new approaches to<br />
obtain atomic-resolution structural details, and even high-resolution structures, of ordered, high molecular weight<br />
protein complexes. Examples include fibrillar aggregates, microcrystalline samples, oligomeric assemblies and<br />
membrane proteins.<br />
Here, we present solution- and solid state NMR based approaches [1-3] and specific isotope labelling strategies to<br />
study the structural details of the amyloid-like bacterial virulence factor curli. Amyloid fibrils are ordered aggregates<br />
that have a high content of specific eta-sheet secondary structure. Long associated with fatal human diseases such<br />
as Alzheimer’s, Parkinson’s and prion diseases, the amyloid fold is now known to be formed natively by a diverse set<br />
of proteins without toxic side-effects. Amyloid fibrils appear to be particularly abundant as adhesive surface<br />
structures on bacteria forming multi-cellular communities called biofilms. Understanding the structural properties of<br />
these amyloid fibrils, as well as the details of their biogenesis, is therefore of great interest for infection research. In<br />
addition, they allow the straight-forward correlation of structure and function within the native organism. They are<br />
thus promising targets to study general aspects of amlyoid formation.<br />
T. Schubeis greatly acknowledges a scholarship from the HZI Graduate School. M. Ahmed greatly acknowledges the funding from the German<br />
Research Foundation.<br />
References:<br />
1. Ritter, C. et al.: Nature 2005, 435: 844–848.<br />
2. Siemer, A.B. et al.: Angew Chem Int Ed 2005, 44: 2441–2444.<br />
3. Wasmer, C. et al.: J Mol Biol 2010, 402(2): 311–325.<br />
82 Sessionvorträge Tabletten
Session Spezielle Darreichungsformen<br />
Orodispersible dosage forms – novel concepts, innovative products, new therapeutic options<br />
Breitkreutz J<br />
Heinrich Heine University Düsseldorf, Institute of Pharmaceutics and Biopharmaceutics, 40225 Düsseldorf, Germany<br />
Orodispersible dosage forms are pharmaceutical drug formulations which rapidly disintegrate into multiple particles in<br />
the oral cavity. Whereas oral lyophilisates 1 and orodispersible tablets (ODTs) 2 have been used for years in various<br />
indications throughout the world, orodispersible films (ODFs) 3 and orodispersible minitablets (ODMTs) 4 have been<br />
licensed most recently for the European and the national German market. Orodispersible films have been recently<br />
included in the Ph.Eur. for the first time. Some further production methodologies such as electrospinning are<br />
currently under investigation, but have not reached industrial production scale yet.<br />
The gained popularity of orodispersible drug formulations is due to the fact that these rapidly disintegrating dosage<br />
forms combine beneficial properties of both liquid and solid dosage forms. They enable improved drug stability over<br />
the shelf-life and usually do not contain any potential harmful ingredients like preservatives or antioxidants, but offer<br />
precise dosing, ease of administration and swallowing. Hence, they are ideal dosage forms for patients with diseaserelated<br />
swallowing issues 5 , in particular for the paediatric and geriatric population 6 .<br />
Some recent advances in material science and improved engineering processes seem to overcome the previously<br />
used energy and time consuming freeze-drying technology for preparing orodispersibles. Ready-to-use tabletting<br />
excipients such as Ludiflash ® , Pharmaburst ® , Pearlitol Flash ® or Parteck ® ODT promise easy and safe production by<br />
simple mixing and direct compression. However, the resulting disintegration times are usually much higher as for the<br />
oral lyophilisates. More recently the ODMT concept was introduced by our group using orodispersible minitablets of<br />
only 2 mm in diameter or even less made from ready-to-use excipients or self-prepared mixtures which exhibit short<br />
disintegration times below 5 s which is very similar to the marketed oral lyophilisates. As the total minitablet mass is<br />
restricted to about 7 mg, the maximum drug load is also limited to approximately 2.5 mg. Complying with the Ph.Eur.<br />
specifications regarding uniformity of dosage units is a major issue for low-dosed drug substances. A subsequent<br />
granulation step may improve the content uniformity. Orodispersible films can be produced by various methods, such<br />
as solvent-casting, melt extrusion or drug printing. Disintegration times are much higher than for lyophilisates and<br />
ODMTs, but often the film is not sensed at the application site and therefore, the disintegration times might be of<br />
minor importance for patients’ acceptance and therapy adherence. The drug dose is defined by the film area and<br />
thickness, usually containing up to 15-20 mg drug substance. Inclusion of taste masking agents such as ionexchange<br />
resins or cyclodextrins may further lower the maximum drug load.<br />
Although the recent developments and products are quite promising and offer new therapeutic options in various<br />
conditions, the specifications in the pharmacopoeias and the requirements of the regulatory bodies are still not<br />
harmonized and mostly even useless. Especially mechanical properties of the orodispersible dosage forms,<br />
disintegration and dissolution testing need new validated equipment and analytical methods to ensure the quality of<br />
the advanced drug dosage forms.<br />
References:<br />
Seager H., J Pharm Pharmacol 1998, 50: 375–382.<br />
Brown D., Drug Deliv Technol 2003, 3: 58–61.<br />
Hoffmann E.M., Breitenbach A., Breitkreutz J., Exp Opin Drug Deliv 2011, 8: 299–316.<br />
Stoltenberg I., Breitkreutz J., Eur J Pharm Biopharm 2011, 78: 462–469.<br />
Stegemann S., Gogol M., Breitkreutz J., Int J Pharm <strong>2012</strong>, 430: 197–206.<br />
Breitkreutz J., Boos J., Exp Opin Drug Deliv 2007, 4: 37–45.<br />
Sessionvorträge Tabletten 83
Challenges in Developing Gastroretentive Dosage Forms<br />
Neumann M; Schneider F, Weitschies W<br />
Institute of Biopharmaceutics and Pharmaceutical Technology, Department of Pharmacy, University of <strong>Greifswald</strong>, Center of Drug Absorption<br />
and Transport, F.-Hausdorff-Str. 3, 17487 <strong>Greifswald</strong>, Germany<br />
Similarly to food, solid oral dosage forms show highly variable gastric emptying, depending e.g. on their size, volume<br />
and viscosity of gastric contents, intragastric flows, pH-profiles in the gastrointestinal (GI) tract or caloric content of<br />
coadministered food [1]. Furthermore, due to the interdigestive migrating motor complex (IMMC), especially the<br />
housekeeping waves, both particles and monolithic dosage forms are subject to antral forces in the stomach [2].<br />
Once they are emptied through the pylorus, absorption of drugs from the human intestine is a complex and also<br />
highly variable process. Hence, consideration of human physiology is the major prerequisite in the design of dosage<br />
forms for drugs that may benefit from the targeted delivery in the stomach.<br />
Since years, it is an ongoing challenge to overcome physiological processes of gastric emptying, and thereby<br />
achieving prolonged gastric residence times of oral dosage forms in a controlled and reproducible manner.<br />
In general, so called gastroretentive dosage forms are supposed to remain in the stomach over a long period of time,<br />
and thus enable controlled drug delivery by delayed gastric emptying. Furthermore, gastroretention is suitable for<br />
drugs that exhibit e.g. regio specific absorption in the upper small intestine (absorption windows), low solubility or<br />
stability at high pH-values or short plasma half-lives [3]. By means of prolonging gastric residence time the<br />
bioavailability of drugs can be increased.<br />
During the last decades different gastroretentive dosage forms have been designed, which can be classified into four<br />
major categories: (i.) floating systems that are able to swim on top of gastric contents; (ii.) mucoadhesive systems<br />
that attach to the stomach walls; (iii.) high density systems that deposit in the antrum; and (iv.) unfolding or<br />
expanding systems that enlarge in size, and thus cannot pass the pylorus [4].<br />
Our aim is to develop and evaluate new gastroretentive dosage forms, which remain in the stomach in a controlled<br />
manner. On one side, we focus on the design of fast expandable hydrogels of different shapes and the formulation of<br />
a swelling gastroretentive dosage form for pH independent release of acyclovir. This antiviral drug is widely used in<br />
therapy and prophylaxis of herpes infections and exhibits low bioavailability of only 10-30 % primarily due to its<br />
narrow absorption window in the upper small intestine [5].<br />
On the other side, another challenge is the development of in vitro and in vivo test methods to evaluate potential<br />
gastroretentive dosage forms. Critical physiological conditions cannot be reflected by the application of rather simple<br />
in vitro tests as they were used in the past. However, data from tracking of dosage forms in the human GI tract via<br />
commonly applied techniques like gamma scintigraphy, X ray, MRI and magnetic marker monitoring (MMM) raised<br />
the need for more biorelevant in vitro test methods [6]. Therefore, our dosage forms are tested by use of a novel,<br />
mechanical stomach model developed in our laboratory, which simulates antral pressures and housekeeping waves.<br />
Financial support from the German Federal Ministry of Education and Research is gratefully acknowledge (FKZ 13N11370).<br />
References:<br />
1. Streubel, A., Siepmann, J., Bodmeier, R.: Curr Opin Pharmacol 2006, 6: 501–508.<br />
2. Wilson, C.G., Crowley, P.J. (eds): Controlled Release in Oral Drug Delivery (Controlled Release Society) 2011.<br />
3. Klausner, E.A. et al.: J Control Release 2003, 90: 143–162.<br />
4. Bardonnet, P.L. et al.: J Control Release 2006, 111: 1–18.<br />
5. Arnal, J. et al.: J Pharm Sci 2008, 97(12): 5061–5073.<br />
6. Kagan, L., Hoffman, A.: Expert Opin Drug Deliv 2008, 5(6): 681–692.<br />
84 Sessionvorträge Tabletten
Innovative nanoparticular dosage forms – advantages in pharmacokinetics and administration<br />
Rischer M<br />
Losan Pharma GmbH, Department of Drug Delivery & Innovation Projects, Otto-Hahn-Strasse 13, 79395 Neuenburg am Rhein, Germany<br />
Oral and parenteral dosage forms containing nanoparticles in the pharmaceutical development have been proposed<br />
and used over more than 10 years. However, very often results have been published for the application of<br />
nanosuspensions only not considering the difficulties (e.g. agglomeration, loading, scale-up, manufacturing costs)<br />
which may appear if the downprocessing to the final dosage form is performed. In addition sometimes surfactants<br />
or polymers as stabilizers have been used which could not be regarded as safe and toxicological assessments or<br />
studies need to be performed. Basically, these reasons should be mainly responsible that only a few dosage forms<br />
containing nanoparticles have entered the final commercial market yet. Losan Pharma has now completed several<br />
studies with up-to-date Active Pharmaceutical Ingredients (BCS II compounds) and also projects with New<br />
Chemical Entities from Research collaborations and has implemented a process of a cost-effective production of<br />
nanoparticles in oral dosage forms (e.g. tablets). Key figures of this innovative process will be presented as well as<br />
case studies proving the advantages of nanoparticulate dosage forms. Also a comparison of in-vitro with in-vivo<br />
data will be made for certain cases. Finally, new convenient water-free “on the go” applications for such<br />
formulations will be presented. Although, many research companies nowadays try to avoid insoluble new<br />
compounds, there is still a majority of API´s present which do need new and innovative formulations and<br />
applications. Nanoparticulate formulations do also help to reduce environmental contamination by reducing the<br />
dosage for application an aspect which is be more and more considered (e.g. by AMNOG) for future developments.<br />
Sessionvorträge Tabletten 85
Development and pharmaceutical technological applications of Diels-Alder hydrogels<br />
Brandl F1,2 ; Kirchhof S2 ; Hammer N2 ; Messmann V; Goepferich A2 1 Massachusetts Institute of Technology, David H. Koch Institute for Integrative Cancer Research, Cambridge, MA 02139<br />
2 Universitaet Regensburg, Department of Pharmaceutical Technology, 93040 Regensburg<br />
Since the pioneering work of Otto Wichterle in the 1950s, polymeric hydrogels received much interest due to their<br />
chemical versatility and favorable properties. Hydrogels essentially consist of water-insoluble polymers with infinite<br />
molecular weight that bind large amounts of water. Their rheological behavior is between those of liquids and solids,<br />
which makes them ideal candidates for injectable soft tissue implants. In fact, hydrogels have been proposed as<br />
wound dressings, as carrier systems for the controlled release of drugs, as three-dimensional scaffolds for cell<br />
transplantation, and for many other biomedical or technical applications. However, despite many years of research,<br />
finding suitable cross-linking reactions is still an issue. On the one hand, the chosen reaction has to be highly<br />
efficient in order to obtain high cross-linking densities. But on the other hand, the cross-linking reaction must occur<br />
under mild conditions to be compatible with peptides, proteins, or living cells.<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O<br />
N<br />
O<br />
O<br />
O<br />
N<br />
During our research on hydrogels, the Diels-Alder reaction came into our focus. The Diels-Alder reaction is a [4+2]<br />
cycloaddition reaction between a conjugated diene and a substituted alkene which occurs in water without any<br />
catalysts or initiators. The high efficiency and selectivity makes this reaction an ideal, biocompatible cross-linking<br />
reaction. In our group, star-shaped poly(ethylene glycol) is functionalized with furyl and maleimide groups yielding<br />
two complementary macromonomers. Upon mixing the two macromonomers, highly elastic hydrogels with low<br />
swelling ratio are formed within minutes. Currently, we are investigating the suitability of these hydrogels as carrier<br />
systems for the controlled release of pharmacologically active proteins. Special emphasis is put on the delivery of<br />
antibodies, e.g. to treat common eye diseases such as age-related macular degeneration. Other potential<br />
applications are the treatment of postoperative wounds or in the field of tissue engineering.<br />
Acknowledgements: Financial support from Deutsche Forschungsgemeinschaft (grant number GO 565/16-1) is gratefully acknowledged.<br />
O<br />
O<br />
86 Sessionvorträge Tabletten<br />
O<br />
O<br />
N<br />
H<br />
O<br />
N<br />
H<br />
O<br />
O<br />
O<br />
O
Session Optimierte Patientenversorgung<br />
Medication safety research as challenge for pharmaceutical scientists<br />
Jaehde U<br />
Institute of Pharmacy, Clinical Pharmacy, University of Bonn<br />
Various studies have shown that drug therapy is a high-risk process demanding particular attention by all health care<br />
professionals. Many adverse drug events are caused by medication errors, such as overdosing in patients with organ<br />
dysfunctions or inadequate consideration of drug-drug interactions. Whereas there are strict regulatory requirements<br />
to assure the safety of drugs before and after approval (product safety), there are only few systematic approaches to<br />
enhance the safety of the entire medication (patient safety). The Federal Ministry of Health (BMG) has therefore<br />
published an action plan for "Improving medication safety in Germany” which defines specific measures to be taken<br />
by professional institutions and organizations dealing with drug therapy.<br />
As part of this action plan the BMG supports various individual projects and encourages scientists to initiate research<br />
aiming at developing new concepts and models for the improvement of medication safety. At the University of Bonn<br />
various research projects have been conducted in collaboration with pharmacy practitioners as well as physicians<br />
from various medical disciplines. Based on two examples, the challenges and opportunities of medication safety<br />
research for pharmaceutical scientists will be discussed.<br />
Clinical pharmacists to optimise patient safety in drug therapy<br />
Bertsche T<br />
Department of Clinical Pharmacy, University of Leipzig, Germany<br />
Objectives<br />
Drug-related problems (DRP) are frequent in hospitals. Particularly, they occur in prescription and administration and<br />
lead to death, prolonged hospital stay, and severe adverse drug reactions (ADRs). Different reasons, such as<br />
knowledge deficits, account for DRPs. In a setting of limited resources, however, intervention strategies to improve<br />
patient safety have to be prioritised to the most frequent and severe DRPs and have to be tailored to the causes.<br />
Participants and methods<br />
At a university hospital, we identified DRPs in routine care by instructed monitors (clinical pharmacists) and assessed<br />
knowledge deficits by a questionnaire survey. DRPs were classified according to their prevalence, potential risk, and<br />
reasons for their occurrence using a decision-matrix model. Tailored interventions such as teaching sessions, clinical<br />
pharmacist interventions, and newly developed clinical decision support systems were implemented in hospital<br />
settings including e.g. intensive care units, paediatric wards, or cancer patients.<br />
Results<br />
In drug administration, we identified physico-chemical incompatibilities and drug administration by gastric tube as<br />
most prevalent DRPs (n = 1376 processes). DRP prevalence decreased by 59% (p = 0.003, incompatibilities, adult<br />
ICU patients, n = 1108 drug pairs) and by 94% (p < 0.001, gastric tube administration in children, n = 1164<br />
processes). In a follow-up analysis these effects were sustainable. In drug prescription, guideline adherence in pain<br />
treatment for cancer patients increased by 81% (p < 0.001, n = 100 patients), ADRs caused by drug interactions<br />
decreased by 43% (p = 0.001, n = 265 patients) and excessive dosing in patients with renal insufficiency by 51%<br />
(p < 0.001, n = 68).<br />
Conclusions<br />
Tailored interventions in routine care by clinical pharmacists based on teaching sessions and newly developed<br />
electronic systems decreased rates of DRPs and DRP-related ADRs.<br />
Sessionvorträge Tabletten 87
Safety of pharmacotherapy in nursing homes<br />
Thürmann P A<br />
Clinical Pharmacology, Faculty of Health (Department of Human Medicine), HELIOS Clinical Centre, 42283 Wuppertal, Germany<br />
Nursing home residents are seen as a vulnerable population, due to their old age, multimorbidity, high frequency of<br />
dementia and subsequent polypharmacy. Moreover, nursing home residents receive the highest number of<br />
psychotropics. Many of these psychotropic drugs are so-called potentially inappropriate medication for the elderly<br />
(PIM) as defined by Beers (1) and adopted to the German market by Holt et al. (2). Moreover, nursing home<br />
residents are prone to experience drug-related problems (DRPs) and adverse drug-related events (AEs) (3). So far,<br />
no data were available on the frequency and severity of AEs and DRPs in German nursing homes. We thus aimed to<br />
investigate DRPs and AEs in German nursing homes and to develop a concept for an inter-disciplinary intervention in<br />
order to improve safety of drug therapy in nursing homes.<br />
Methods: Drug- related problems and drug-related adverse events were detected by trained clinical pharmacists in<br />
nursing homes willing to participate over a period of four weeks. The study protocol was approved by the responsible<br />
ethics committee, written informed consent was obtained from the residents or their responsible caregivers. Medical<br />
documentation and nurses notes were screened by trained pharmacists, medication was documented and potential<br />
DRPs and AEs were documented in an ACCESS database. Potential AEs were discussed in a multi-disciplinary<br />
team, were causality assessment was performed. Frequency of ongoing and new AEs during the 30-days period was<br />
calculated as the 30-day-prevalence of AEs per 100 nursing home residents months (NHRMs), whereas the 30-daysincidence<br />
included only those AEs which started during the 30-days period and were also related to 100 NHRMs.<br />
Results of the cross-sectional survey were presented in a multi-disciplinary expert workshop and served to develop a<br />
concept to improve safe prescribing and monitoring of pharmacotherapy in nursing homes. This intervention was<br />
implemented in 4 nursing homes, where the prescribing general practitioners, nurses and pharmacists received<br />
special training in geriatric pharmacotherapy. In nursing homes Drug Safety Teams (DSTs) were built consisting of<br />
trained nurses and pharmacists, who followed a pre-specified protocol of medication checks, residents monitoring<br />
and communication with physicians.<br />
Results: 778 nursing home participants in 11 nursing homes participated in the first survey. In total 102 AEs were<br />
detected in 80 seniors (10.3 %). 37 of these AEs (59.7 %) were judged as preventable by the experts and 4 AEs (6.5<br />
%) were ameliorable. The 30-days-prevalence of AEs was estimated with 12.94 AEs/100 NHRMs, the incidence<br />
came to 7.87 AEs/100 NHRMs. 1.493 DRPs were detected, where almost 50 % concerned the documentation, 35 %<br />
the storage and 15 % dispension of drugs. Three months after the implementation intervention the 30-daysprevalence<br />
came to 15.93 AEs per 100 NHRMs and the incidence to 7.62 AEs/100 NHRMs. In contrast to the first<br />
cross-sectional survey a much higher number of adverse events concerning the central nervous system was<br />
observed, probably due to the increased awareness of the nurses. The intervention proved to be feasible, most<br />
nurses would recommend it for other nursing homes.<br />
Discussion: Data on frequency of AEs in nursing homes obtained in this study are in accordance with data published<br />
in the literature (3). A prospective, cluster-randomized trial will be performed in two different regions of Germany to<br />
demonstrate the effectiveness of the intervention with reduction of AEs as primary outcome variable.<br />
The study was funded by the German Ministry of Health, grant no.: 1501/54401.<br />
References:<br />
1. Beers MH, Ouslander JG, Rollingher I, Reuben DB, Brooks J, Beck JC., Arch Intern Med 1991, 151: 1825–32.<br />
2. Holt S, Schmiedl S, Thürmann PA., Dtsch Arztebl In. 2010, 107: 543–51.<br />
3. Gurwitz JH, Field TS, Judge J et al., Am J Med 2005, 118: 251–258.<br />
88 Sessionvorträge Tabletten
Identification of predictors for drug related problems in the context of home medication review<br />
Fiß T1 ; Hoffmann W2 1 German Center for neurodegenerative diseases (DZNE), site Rostock/<strong>Greifswald</strong>; Ellernholzstreet 1/2; 17487 <strong>Greifswald</strong>, Germany<br />
2 University <strong>Greifswald</strong>, Institute for Community Medicine; Ellernholzstreet 1/2 , 17487 <strong>Greifswald</strong><br />
Introduction: Drug related problems (DRP) are affecting success of pharmacotherapy, cause drug-induced<br />
hospitalisations high costs, and limit health related quality of life. Identification of DRP and their predictors is needed<br />
for focussed intervention and implementation of innovative health supply concepts. Several data sources are<br />
available for identification of predictors for DRP. Especially routine data from the statutory health insurances have a<br />
very strong role in this field. However, these data are limited due to incomplete data. Routine data only reflect health<br />
care services which are paid by the statutory health insurance. Clinical tests as plasma level of several vital<br />
parameter or cognitive tests are not available. Hence there is a need for drug utilisation studies in the context of<br />
health services research.<br />
Methods: We used datasets from two studies (AGnES: Arzt-entlastende, Gemeinde-nahe, E-Health-gestützte,<br />
systemische Intervention; IDemUck: inegriertes Demenznetzwerk Uckermark) to evaluate determinants for drug<br />
related problems and estimated needs for a structured medication management. Patients received a structured<br />
medication review1 in their households and subsequent intervention. The pharmaceutical care was delegated in the<br />
AGnES-studies to the local pharmacy whereas the patient’s GP conducted intervention in IdemUck.<br />
Results: We found a high proportion of drug related problems. Especially the intake of potentially inappropriate<br />
medication as well as intake of anticholinergig drugs was found as problematic. Out of the AGnES-patients aged > 65<br />
years a total of 18% (n= 134) of the patients received 163 inappropriate drugs according to the Beers’ criteria. The<br />
intake of PIM was slightly associated with self-reported falls (Phi: 0.1074; P= 0.0244). Multivariate logistic regression<br />
showed significant results for the number of taken substances (OR = 1.176; 95% CI 1.121-1.234, P< 0.001).<br />
Additionally we found the intake of anticholinergics (according to the ACBS list) as determinant for impaired cognition<br />
(OR 10; 95-CI 1.2-85.3). As a 2nd problem we identified the occurrence of potentially clinically relevant drug-drug<br />
interaction. In n=454 patients (58.3%) we found at least one drug-drug interaction with serious or moderate<br />
relevance. Adjusted for age and gender, multiple binary logistic regression showed significant results for the number<br />
of taken active substances (OR 1.48; 95%-CI 1.38-1.58), metabolic diseases (OR 1.52; 95%-CI 1.04-2.2), or a<br />
diagnose of the muscular-skeletal disease (OR 1.74; 95%-CI 1.2-2.5).<br />
Due to the pharmacist’s intervention in the AGnES-studies we found significantly improved DRP (adherence, drugdrug<br />
interaction). In contrast to these results we found an increased prevalence of potentially clinically relevant drugdrug<br />
interaction from 42.1% to 47.4% in the IDemUck-study.<br />
Discussion: In the context of home medication reviews we identified a large array of DRP. Due to several additional<br />
tests we could find some associations to clinical outcomes as falls and impaired cognition. Overall the number of<br />
taken drugs was the main risk factor for the occurrence of DRP. Subsequent intervention by a pharmacist is<br />
necessary to improve DRP.<br />
However data are limited due to small number of patients and short follow-up period. We could only find first clues for<br />
determinants which must be evidenced in further analysis. Hence linkage with routine data could help for long term<br />
follow-up. Clinical outcomes as hospitalisation and mortality could be measured with these data.<br />
As one model of evaluate efficacy of tailored intervention including long term effects we have included medication<br />
management into the study DelpHi-MV (dementia: life and person centred help in Mecklenburg Western Pomerania,<br />
clinicaltrials.gov identifier NCT01401582). Results of the analysis of determinants for the occurrence of DRP were<br />
integrated into the study algorithms.<br />
References:<br />
1. Fiss T, Ritter CA, van den Berg N et al. Pa Wo Sci 2010, 32(5):566-74.<br />
Sessionvorträge Tabletten 89
Session Arzneimittelüberwachung/Regulatory Affairs<br />
Analyzing the use of (Q)SAR models for the evaluation of potential genotoxic impurities: A<br />
cookbook in support of ICH M7<br />
Sutter A<br />
Bayer AG<br />
• Assessing the (Q)SAR systems used<br />
• Defining what exactly is expert knowledge<br />
• Evaluating current practice in pharma companies: PhRMA and EFPIA surveys<br />
• Discussing (Q)SAR reporting<br />
Control strategy for an active coating process based on systematic process development (DoE) and<br />
in-line Raman spectroscopy (PAT)<br />
Funke A<br />
Bayer Pharma AG, Global Pharmaceutical Development, Müllerstraße 178, 13353 Berlin, Germany<br />
Introducing active ingredients into pharmaceutical dosage forms via film coating onto tablets (active coating) is<br />
unusual and challenging due to high quality requirements with regard to both intra-tablet coating uniformity and<br />
accuracy of average coating amount per tablet. The variability has been reduced by systematic process development<br />
using design of Experiments (DoE). A process control strategy has been elaborated using in-line Raman<br />
spectroscopy as an IPC for the endpoint determination of the coating process (PAT). As a result, the active coating<br />
process reproducibly leads to a drug product fulfilling the predefined quality requirements.<br />
90 Sessionvorträge Tabletten
Human Tissues and Cells – Advanced Therapies Medicinal Products<br />
Astner I1, 2<br />
1 GAA, Petzvalstr. 18, 38104 Braunschweig, Germany.<br />
2 ZLG – EFG 04, Sebastianstr. 189, 53115 Bonn, Germany.<br />
Tissues and Cells for transplantation offer a wide and expanding field of medicinal products for treating a variety of<br />
diseases in humans. Alike the better known organ transplants tissues and cells are exchanged worldwide to<br />
overcome transplant shortages or to ensure better donor–recipient matching.<br />
Furthermore human or xenogenous tissues and cells are used as starting material for manufacturing (personalized)<br />
advanced therapies medicinal products (ATMPs) or Medical Devices. Gene therapy, somatic cell therapy and tissue<br />
engineering medicinal products offer new treatment opportunities for human diseases or dysfunctions like<br />
regeneration, repairing or replacement of human tissues.<br />
For tissues, cells and ATMPs it is essential to establish high standards of quality and safety for donors and patients,<br />
on the one hand and to provide exchange of these products within the (sometimes short) expiry date of the cell<br />
products, on the other hand.<br />
As human and xenogenous materials are heterogeneous similar to biologicals the quality management and the<br />
validated manufacturing process is an essential part of the product.<br />
References:<br />
1. DIRECTIVE 2004/23/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 31 March 2004 on setting standards of quality<br />
and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells: OJ L 102,<br />
2004: 48<br />
2. COMMISSION DIRECTIVE 2006/17/EC of 8 February 2006 implementing Directive 2004/23/EC of the European Parliament and of the<br />
Council as regards certain technical requirements for the donation, procurement and testing of human tissues and cells, OJ L 38, 2006: 38<br />
3. REGULATION (EC) No 1394/2007 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 13 November 2007 on advanced<br />
therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004, OJ L324, 2007: 121<br />
Sessionvorträge Tabletten 91
Analysis of illegal medicinal products using NMR<br />
Schramek N<br />
Bavarian Health and Food Safety Authority, Veterinärstr. 2, Oberschleißheim, 85764, Germany<br />
The number of adulterated food supplements and illegal medicinal products to be analyzed at the Official Medicinal<br />
Control Laboratories raises steadily. More and more often unknown substances can be found as illicit additives in<br />
food supplements [1]. Fast and feasible analytical methods are required to deal with this challenge. Despite its lack of<br />
sensitivity Nuclear Magnetic Resonance (NMR) spectroscopy can be used as a fast screening method. Moreover<br />
structural information can be obtained from NMR experiments required for the identification of unknown substances<br />
[2, 3].<br />
This presentation gives a short overview on how 1- and 2-dimensional NMR spectroscopy can be useful to official<br />
medicinal control laboratories. A novel sildenafil-analogue, found as adulterant in a food supplement is presented [4].<br />
References:<br />
1. Venhuis B. J., de Kaste D.: J Pharm Biomed Anal <strong>2012</strong>, DOI:10.1016/j.jpba.<strong>2012</strong>.02.014.<br />
2. Trefi S., Gilard V., Balayssac S., Malet-Martino M., Martino R.: Magn Reson Chem 2009, 47: 163–173.<br />
3. Holzgrabe U., Malet-Martino M.: J Pharm Biomed Anal 2011, 55: 679–687.<br />
4. Wollein U., Eisenreich W., Schramek N.: J Pharm Biochem Anal 2011, 56: 705–712.<br />
92 Sessionvorträge Tabletten
Poster
001<br />
An in vitro test system to examine release in the vitreous humor<br />
Loch C1; Nagel S1; Guthoff R²; Seidlitz A1; Weitschies W1 1 University of <strong>Greifswald</strong>; Center of Drug Absorption and Transport (C_DAT), Institute of Pharmacy, Felix-<br />
Hausdorff-Strasse 3, <strong>Greifswald</strong>, 17487; Germany<br />
2 University of Rostock; Department of Ophthalmology, Doberaner Strasse 140, Rostock, 18055; Germany<br />
Introduction<br />
The vitreous body has become an interesting location for the administration of injections or<br />
implants in modern ophthalmology [1]. Such intravitreal dosage forms are loaded with<br />
active agents, which are released over a prolonged period of time. A test method simulating<br />
the situation in vivo is needed to observe the release behaviour of these new dosage forms.<br />
To simulate this situation the Vitreous Model (VM) was developed.<br />
Methods<br />
The corpus of the VM consists of glass. Size and shape are assimilated to the in vivo<br />
situation. Gel insertion and sampling can be performed via a vent at the top. To simulate<br />
the movement of the eye the VM was placed on an altered orbital shaker. The modified<br />
polyacrylamide gel (PAA-gel), which is used as vitreous substitute in this study, consists of<br />
a 30% solution of acryl amide and standard Ringer buffer solution. The viscose solution<br />
was filled in the corpus and the acryl amide monomers were cross-linked. The water<br />
content, pH, relative density, refractive index and viscosity of the PAA-gel were examined<br />
and compared experimentally to porcine and to human vitreous humor by literature<br />
analysis. Furthermore, during a period of 3 hours the distribution behaviour of fluorescein<br />
sodium solution (1.25 mM) injected into the PAA-gel and in the porcine vitreous humor was<br />
investigated by using the VM.<br />
Results<br />
The water content (99%) and pH (7.4) of the PAA-gel were equal to the values obtained for<br />
porcine and human vitreous humor. Also, the results for the relative density (1.0013) and<br />
refractive index (1.3385) showed good accordances (deviation < 1%). Slight deviations<br />
were found for the viscosity. The shape and volume of the VM correspond to the natural<br />
vitreous body.<br />
In the distribution study further agreements were achieved. For analysis the porcine<br />
vitreous body and the substitute were divided in two halves. 82% of the detected amount of<br />
fluorescein sodium was found at the side at which the injection occurred for the PAA-gel<br />
and 75% for the natural vitreous humor.<br />
Conclusion<br />
The results of the distribution study and the tested physical and chemical properties of the<br />
PAA-gel were in good accordance with the properties of porcine and human vitreous<br />
humor. Along with the related shape of the VM, the new model seems suitable to perform in<br />
vitro experiments, which are capable of simulating in vivo conditions. Through the<br />
possibility of insertion of intravitreal injections and implants in the VM, biorelevant<br />
distribution and release studies may become feasible.<br />
Acknowledgments: The authors would like to thank to the staff members of the glazier’s workshop of the<br />
Faculty of Mathematics and Natural Sciences for their excellent technical assistance. This project was<br />
funded by the Federal Ministry of Education and Research (BMBF) within REMEDIS “Höhere Lebensqualität<br />
durch neuartige Mikroimplantate” (FKZ: 03IS2081).<br />
References:<br />
1. Baino, F. et al.: Acta Biomaterialia 2011, 7(3): 921-935.<br />
002<br />
Diversified influences of arginine and guanidine on proteins<br />
Redweik S; Wätzig H<br />
Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstr. 55, 381106<br />
Braunschweig, Germany<br />
Although arginine and guanidine exhibit the same partial structure their influence on<br />
proteins is very diversified [1]. In this study these influences on the proteins BSA and ßlactoglobulin<br />
were analyzed by Affinity Capillary Electrophoresis (ACE). These influences<br />
on the proteins were also tested in the presence of urea in the Tris-buffer system because<br />
there is a concentration dependent inhibition of arginine to the urea induced protein<br />
unfolding of BSA [1].<br />
ACE is a powerful tool to detect interactions between proteins and various ligands due to<br />
mobility shifts of the protein [2]. Therefore the difference of the protein electrophoretic<br />
mobility is detected without any ligand in the buffer and with the addition of a defined ligand<br />
concentration. In order to compensate for changes on the migration time, which are not<br />
due to ligand binding, the mobility ratio of an EOF-marker and the protein is used [3]. Six<br />
repeats were done with a very good precision due to the use of a special rinsing procedure<br />
[4]. The differences of the mobility ratios Rf (without addition of ligand) and Ri (in presence<br />
of a defined ligand concentration) were normalized and the confidence intervals were<br />
estimated to demonstrate the significance of these interactions. The achieved small<br />
confidence intervals showed clearly different influences of the tested ions on BSA and ßlactoglobulin<br />
with and without urea in the buffer.<br />
References:<br />
1. Gosh, R. et al.: Biochemistry 2009, 48(5): 1135-1143.<br />
2. Busch, M. H. A. et al.: J Chromatogr A 1997, 777(2): 311-328.<br />
3. Bose, S. et al.: J Chromatogr B 1997, 697(1-2): 77-88.<br />
4. El-Hady, D. et al.: J Pharm Biomed Anal 2010, 52(2): 232–241.<br />
003<br />
The new cardioactive compounds from Leonurus cardiaca L. (Ph.Eur.)<br />
Savtschenko A1 2, Dhein S1, Rauwald H W2 1 Clinic for Cardiac Surgery, University of Leipzig, Strümpellstraße 39, D-04289 Leipzig, Germany<br />
2 Department of Pharmaceutical Biology, University of Leipzig, Johannisallee 21–23, D-04103 Leipzig,<br />
Germany.<br />
Just recently, aqueous preparations of Leonurus cardiaca – such as a newly developed<br />
refined extract – were reported to exert a series of effects on cardiac electrophysiology,<br />
such as ICa.L blockade, a reduction of the repolarising current IK.r, as well as the prolongation<br />
of both AP-duration and the activation time constant of the If current, while INa was strikingly<br />
unaffected [1]. This synergistic action on multiple electrophysiological targets may limit<br />
potential adverse effects, especially the risk of proarrhythmia, and is thus in accordance<br />
with the traditional use of this medicinal plant as an antianginal in European herbalism [2].<br />
Therefore, several extract samples were examined for substances potentially contributing<br />
to these pharmacological activities. In this screening procedure, ferulic acid (4-hydroxy-3methoxy-trans-cinnamic-acid)<br />
and stachydrine were detected as cardioactive compounds of<br />
the refined extract. Although a variety of pharmacological actions on the cardiovascular<br />
system have been reported for this caffeic acid derivative [3], specific molecular<br />
pharmacological actions on cardiac ion channels – as observed for the L. cardiaca refined<br />
extract [1] – have not been reported for the isolated compound up to now. In subsequent<br />
voltage clamp experiments carried out according to a protocol previously developed in our<br />
group [4], ferulic acid at concentration of 3 µM influences neonatal rat ventricular<br />
cardiomyocytes by ICa.L blockade and AP-duration shortening while INa was not affected.<br />
Epicardial mapping experiments on isolated rabbit hearts perfused according to the<br />
Langendorff technique showed an increase of basic cycle length and relative coronary flow,<br />
as well as a reduction of left ventricular pressure. Thus mimicking parts of the effect of the<br />
previously described L. cardiaca refined extract. In voltage clamp experiments stachydrine<br />
at concentration of 1 mM and depending on cell type increases the ICa.L current and<br />
prolongs the AP-duration of neonatal rat ventricular cardiomyocytes, while INa was<br />
unaffected. Thus, ferulic acid and stachydrine contribute to the cardiac effects of L.<br />
cardiaca; however, other compounds from L. cardiaca may also contribute according to a<br />
‘multi-component / multi-target’ active principle [1].<br />
References:<br />
1. Ritter, M. et al.: Planta Med. 2010, 76: 572-82.<br />
2. Fuchs, L.: New Kreutterbuch (Basel edit on) 1543.<br />
3. Wang, BH., Ou-Yang JP.: Cardiovascular Drug Reviews 2005, 23(2): 161-172.<br />
4. Scheffler, A. et al.: J. Ethnopharmacol. 2008, 120: 233-240.<br />
004<br />
Quantification of tumor-promoting and skin irritating diterpene esters in<br />
homeopathic mother tinctures<br />
Kummer, J., Melzig, M. F.<br />
Institute of Pharmacy - Pharmaceutical Biology, Freie <strong>Universität</strong> Berlin, Königin-Luise-Str. 2+4,<br />
14195 Berlin, Germany<br />
Some plants from the family Euphorbiaceae were used in the past as traditional remedies.<br />
With ingenol melbutate there is now a highly active therapeutic for the topical field therapy<br />
of actinic keratosis [1]. The active ingredients are diterpene esters, which are known to be<br />
tumor-promoting and skin irritating since Hecker et al. discovered TPA (12-Otetradecanoylphorbol-13-acetate)<br />
in the oil of Croton tiglium L. seeds [2].<br />
There are two basic chemical structures such as the alcohols phorbol and ingenol. These<br />
two alcohols form a great variety of different esters with different activities. Available<br />
preparations of these esters are homeopathic mother tinctures. For standardization and the<br />
monographic description in pharmacopoeias it is necessary to establish an easy method to<br />
quantify these esters from herbal preparations, e.g. homeopathic mother tinctures.<br />
To avoid the difficult analysis of the different esters and to quantify all the toxic esters it´s<br />
necessary to hydrolyse them and to determine the resulting alcohols by HPLC. The alkaline<br />
hydrolysis is not suitable for all structures because of a bad recovery [3].<br />
We will discuss the different approaches for gentle methods to hydrolyze the esters. The<br />
enzymatic hydrolysis with different enzymes such as pectinase from Aspergillus niger and<br />
esterase from pig liver and Bacillus subtilis didn t result in complete hydrolysis. Other<br />
esterases like enzymes from rabbit or rat liver seem to be more selective for diterpene<br />
esters [4].<br />
Ingenol Phorbol<br />
References:<br />
1. Lebwohl, M. et al., N Engl J Med <strong>2012</strong>, 366: 1010-1019.<br />
2. Hecker, E., Schmidt, R., Prog Chem Org Nat Prod 1974, 31: 377-467.<br />
3. Gläser, S., Untersuchungen zu einem möglichen Gesundheits- und Krebsrisiko durch pflanzliche<br />
Arzneimittel sowie industriell genutze Rohstoffe aus Euphorbiaceen – Quantitative Bestimmung von<br />
irritierenden und tumorpromovierenden Diterpenestern und Evaluierung durch biochemische und<br />
biologische Tests (Thesis, <strong>Universität</strong> Heidelberg) 1991.<br />
4. Saito, M., Egawa, K., J Biol Chem 1984, 259: 5821-5826.<br />
005<br />
Various Modes of High Performance Liquid Chromatography for Analysis of<br />
Monoclonal Antibodies<br />
Grotefend S1; Wätzig H1 1 Technische <strong>Universität</strong> Braunschweig, Institute of Medicinal and Pharmaceutical Chemistry, Beethovenstr.<br />
55, 38106 Braunschweig, Germany<br />
Protein quantitation as well as the detection of protein aggregates becomes more and more<br />
important since protein based pharmaceuticals (biologicals), especially monoclonal<br />
antibodies (mAb), were established as potent drugs in anticancer therapy or for treatment<br />
of autoimmune based diseases. Accurate and precise methods have to be found for this<br />
purpose, due to the high efficiency of these drugs and its behaviour to form aggregates,<br />
which may lead to adverse effects like anaphylaxis or injection side reactions after drug<br />
application even in small amounts [1]. Quality control purposes could not been reached by<br />
classic protein quantification methods such as the Bradford assay or SDS-PAGE, yet. In<br />
contrast different HPLC separation modes possess great selectivity as well as high<br />
precision in monoclonal antibody analysis. An IgG1-type antibody could be precisely<br />
analyzed (RSD % Monomer = 2.54 % day-to-day precision, n=60) by using High<br />
Performance Size Exclusion Chromatography (HP-SEC), which also enables the complete<br />
separation from monomer, aggregates and a low molecular weight fragment [2]. Weak<br />
Cation Exchange Chromatography (WCX), which is also already successfully applied in<br />
protein, especially antibody analysis, will be analyzed for its suitability in precise and<br />
selective monoclonal antibodies analysis. The high selectivity of this mode allows<br />
separating antibodies with different C-terminal lysine residues (proved by carboxypeptidase<br />
B addition) [3]. The separation modes will be compared in terms of precision, selectivity,<br />
analysis time as well as effort during sample and mobile phase preparation.<br />
94 Poster
References:<br />
1. Carpenter, J.F. et al, J. Pharm. Sci. 2010, 99 (5): 2200-2208.<br />
2. Grotefend, S. et al, Oral presentation on HPLC 2011 Budapest, Hungary.<br />
3. Weitzhandler, M. et al, Proteomics 2001, 1: 179-185.<br />
006<br />
Synthesis of novel Janus Kinase Inhibitors via Copper Catalyzed Azide-Alkyne<br />
Cycloaddition<br />
Gehringer, M. and Laufer, S.<br />
University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany<br />
Janus Kinases (JAKs) are non-receptor protein tyrosine kinases mediating signalling<br />
through the JAK-STAT (Signal Transducer and Activator of Transcription) pathway. The<br />
Janus Kinase family has four members: JAK1,2,3 and TYK2. Being crucial signal<br />
transducers for a variety of cytokines, growth factors, and interferons, JAKs are involved in<br />
numerous pathologies including malignancies, myeloproliferative disorders and<br />
autoimmune diseases.<br />
In contrast to the ubiquitous expression of the other JAK family members, JAK3 is<br />
predominantly expressed in hematopoietic cells. In mammals, the lack of functional JAK3<br />
causes immunodeficiencies while not disrupting the function of non-immune cells.<br />
Therefore, targeting JAK3 is a promising strategy to generate a novel class of<br />
immunosupressant drugs with limited side effects. [1]<br />
Recently, Ruxolitinib a small-molecule JAK1/2 inhibitor was approved by the US Food and<br />
Drug Administration for the treatment of patients with intermediate or high-risk<br />
myelofibrosis. [2]<br />
In search for novel JAK3 inhibitors we replaced the Ruxolitinib pyrazole ring by a 1,4disubstituted<br />
1,2,3-triazole accessible thought copper catalyzed azide-alkyne<br />
cycloaddition. [3] Compared to the laborious synthesis of the corresponding pyrazoles, click<br />
chemistry offers a rapid and efficient access to triazols with various substitution patterns<br />
facilitating their optimization towards JAK3 inhibition.<br />
References:<br />
[1] Cornejo , M. G. et al., Int. J. Biochem. Cell. Biol. 2009, 41, 2376-2379.<br />
[2] Mesa, R. A. et al., Nature Rev. Drug Discov. <strong>2012</strong>, 11, 103-104.<br />
[3] Sharpless, K.B. et al., Angew. Chem. Int. Ed., 2002, 41, 2596–2259.<br />
007<br />
Synthesis of water soluble aziridine-2,3-dicarboxylate-based cysteine protease<br />
inhibitors with leishmanicidal activity<br />
Fey, P. 1; Schad, C. 2; Schurigt, U. 3; Moll, H. 3; Schirmeister, T. 1<br />
1Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, 55099 Mainz,<br />
Germany<br />
2Institute of Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, 97074<br />
Wuerzburg, Germany<br />
3Institute for Molecular Infection Biology, Infection Immunology Unit, University of Wuerzburg,<br />
Josef-Schneider-Str. 2, 97080 Wuerzburg, Germany<br />
Cysteine proteases of Leishmania are proven targets for disease control of leishmaniasis,<br />
because they play a key role in parasite growth, differentiation, pathogenicity and<br />
virulence.[1]<br />
Starting from two selective protease inhibitors RV122C (Boc-(S)-Leu-(R)-Pro-(S,S)-<br />
Azi(OBn)2) and RV212C (Boc-(R)-Leu-(S)-Pro-(S,S)-Azi(OBn)2) of cathepsin L and CL-like<br />
proteases, a series of structural isomers, stereoisomers and derivatives was synthesized<br />
and tested for antileishmanial activity against L. major. [2] Out of this series CS128 (Boc-<br />
(S)-Leu-(R)-Pro-(R,R)-Azi(OBn)2) is the most promising candidate. The improvement of the<br />
water solubility of this compound is the main goal of the present work.<br />
According to docking results with RV122C in the active site of human cathepsin L[3] the<br />
Boc protecting group as well as one of the benzyl esters are not necessary for productive<br />
interactions with the enzyme. CS128, only differing in the configuration at the aziridine ring<br />
carbons, is proposed to have a comparable binding mode and therefore will be modified at<br />
the identified positions.<br />
We first focussed on the synthesis of aziridin-2,3-dicarboxylates with different ester<br />
moieties. One of these is used to yield a free acid-group after hydrogenolysis, which will<br />
increase water solubility. The other is designed to be stable against hydrogenolysis, but<br />
enabling the introduction of different liphophilic esters increasing affinity by deeply<br />
extending into the hydrophobic S2-pocket of the enzyme.<br />
Additionally, substitution of the protecting group with hydrophilic groups like glycols was<br />
chosen as alternative possibility to increase water solubility.<br />
O<br />
O<br />
HN<br />
(S)<br />
N<br />
O<br />
(R)<br />
N<br />
HO<br />
O<br />
R<br />
O O<br />
(S S) + (R R)<br />
References<br />
1. Ponte-Sucre, A. et al., Antimicrob. Agents Chemother. 2006, 50, 2439-2447.<br />
2. Schurigt U. et al., Antimicrob. Agents Chemother. 2010, 54, 5028-5041.<br />
3. Vicik R. et al., ChemMedChem 2006, 1, 1126-1141.<br />
008<br />
Synthesis, characterization and screening of Dengue Virus Type 2 NS2B-NS3<br />
protease inhibitors<br />
Holloway, S. 1; Wu, H. 1; Bodem, J. 2; Snitko, M. 2; Welker, A. 3; Sotriffer, C. 3; Klein, C. 4;<br />
Schirmeister, T. 1<br />
1 Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55099 Mainz,<br />
Germany<br />
2 Institute of Virology and Immunology, University of Würzburg, Versbacher Strasse 7, D-97078<br />
Würzburg, Germany<br />
3 Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074<br />
Würzburg, Germany<br />
4 Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer<br />
Feld 364, D-69120 Heidelberg, Germany<br />
Dengue, a mosquito-borne viral infection of humans transmitted by Aedes aegypti and<br />
Aedes albopticus mosquitoes, has become a continuing global threat, since 2.5 billion<br />
people living in tropical and subtropical regions of the world (predominantly in Asia, Africa<br />
and the Americas) are at risk of infection. [1,2] It is caused by a flavivirus with a single<br />
positive-stranded RNA genome [3,4] and four distinct serotypes. [5] The dengue virus<br />
infection can result in classic dengue fever, dengue hemorrhagic fever (DHF) or dengue<br />
shock syndrome (DSS). [6,7] The dengue virus genome encodes a serine protease with a<br />
classical catalytic triad (His51, Asp75 and Ser135) [8,9] which is responsible for the posttranslational<br />
proteolytic processing of the polyprotein precursor and essential for the viral<br />
replication [10,11], making it an important and attractive therapeutic target. [12]<br />
Our projects include syntheses, characterization and testing of DENV-2 NS2B-NS3pro inhibitors based on the structure of a) cinnamic acid amides, with the aim to analyze the<br />
influence on enzyme inhibition due to a variation of the amine residue on the one hand and<br />
the aromatic cinnamic acid residue on the other hand, and b) diaryl thioethers. The possible<br />
binding modes are analyzed by docking studies. The synthesized compounds are screened<br />
in fluorometric enzyme assays and HPLC-based assays using a fluorogenic AMC-derived<br />
substrate. Since a good specifity for the DENV-2 NS2B-NS3pro should be achieved, other<br />
serine proteases, such as trypsin, chymotrypsin and thrombin are included in our screening<br />
procedures.<br />
References<br />
1. Guzman, M. G. et al.: Nat. Rev. Microbiol. 2010, 8: S7–S16.<br />
2. Halstead, S. B.: The Lancet 2007, 370: 1644–1652.<br />
3. Li, H. et al.: J. Virol. 1998, 73(4): 3108–3116.<br />
4. Chambers, T. J. et al.: Annu. Rev. Microbiol. 1990, 44: 649–688.<br />
5. Morita, K., Tanaka, M., Igarashi, A.: J. Clin. Microbiol. 1991, 29: 2107–2110.<br />
6. Gubler, D. J.: Clin. Microbiol. Rev. 1998, 11(3): 480–496.<br />
7. Rigau-Pérez, J. G. et al.: The Lancet 1998, 352: 971–977.<br />
8. Bazan, J. F., Fletterick, R. J.: Virology 1989, 171: 637–639.<br />
9. Melino, S., Paci, M.: FEBS Journal 2007, 274: 2986–3002.<br />
10. Falgout, B. et al.: J. Virol. 1991, 65: 2467–2475.<br />
11. Zhang, L., Mohan, P. M., Padmanabhan, R.: J. Virol. 1992, 66: 7549–7554.<br />
12. Zheng, Y. et al: Bioorg. Med. Chem. Lett. 2006, 16: 36–39.<br />
009<br />
Substituted vinyl sulfones as covalent and reversible cysteine protease<br />
inhibitors<br />
Kesselring, J. 1; Schneider, T. 1; Engels, B. 2; Sotriffer, C.; 3 Schirmeister, T. 1<br />
1 Institute of Pharmacy and Biochemistry, University of Mainz, GERMANY<br />
2 Institute of Physical and Theoretical Chemistry, Universiy of Würzburg, GERMANY<br />
3 Institute of Pharmacy and Food Chemistry, University of Würzburg, GERMANY<br />
Cysteine proteases play vital roles for the life cycles, nutrition and pathogenesis of a variety<br />
of parasites causing infectious tropical diseases [1,2]. Therefore a promising strategy for<br />
the treatment of these diseases is the inhibition of these proteases.<br />
At present a large number of potent and irreversible cysteine protease inhibitors of different<br />
classes, including peptidyl vinyl sulfones, have been reported [3]. The inhibition mechanism<br />
of such vinyl sulfones results from the addition of the enzyme’s active site cysteine residue<br />
in a Michael type reaction [4].<br />
QM and QM/MM calculations have proposed substituted vinylsulfones which should be able<br />
to form a covalent, but reversible bond with the cysteine sulfur of the protease’s active site.<br />
Such a reversible reaction should be possible by choosing suitable substituents and leaving<br />
groups attached to the α- and β-position of the vinyl sulfone moiety, e.g. nitrile groups, and<br />
thiolates, respectively. This leads to a thermoneutral or slightly endergonic vinylic<br />
substitution or addition reaction. To confirm these calculations a series of highly<br />
functionalized vinyl sulfones with varying peptidic residues were synthesized and reacted<br />
with phenylethanethiol as an analogue of the active site’s cysteine residue. The progression<br />
of the reaction was monitored by NMR- and quantitative IR spectroscopy, which allows the<br />
determination of rate constants, equilibrium constants, and reaction energies. Furthermore<br />
the inhibitory potencies of the synthesized compounds were evaluated by enzyme assays<br />
with various cysteine proteases.<br />
References:<br />
[1] R.P. Hanzlik, J. Med. Chem. 1984, 27(6), 711.<br />
[2] P.J. Rosenthal and coworkers, Ant microb. Ag. Chemother. 2003, 47(1), 154.<br />
[3] J.C. Powers, J.L. Asgian, O.D. Ekici, K.E. James Chem. Rev. 2002, 12, 4639.<br />
[4] J.T. Palmer, D. Rasnick, J.L. Klaus, D. Bromme J. Med. Chem. 1995, 38 (17), 3193.<br />
010<br />
Genome-inspired drug discovery in the predatory bacterium Cupriavidus<br />
necator<br />
Nett, M. 1; Kreutzer, M. F. 1; Kage, H. 1<br />
1 Junior Research Group “Secondary Metabolism of Predatory Bacteria”, Leibniz Institute for<br />
Natural Product Research and Infection Biology / Hans-Knöll-Institute, Beutenbergstr. 11a, 07745<br />
Jena, Germany<br />
Whole genome sequencing has illuminated the secondary metabolic proficiency of<br />
Poster 95
microbes – underappreciated for years based on conventional isolation programs – and has<br />
helped set the foundation for a new natural product discovery paradigm based on genome<br />
mining [1]. Furthermore, it is becoming increasingly evident that the potential for the<br />
biosynthesis of bioactive compounds is more abundant than previously anticipated. In<br />
recent years, many neglected microorganisms have emerged as alternative resources of<br />
chemical diversity [2].<br />
Bacteria that prey on other organisms as a food source are particularly appealing in this<br />
context. We assume that a large fraction among these micropredators evolved or acquired<br />
antibiotic biosynthesis genes to promote their feeding strategy. As a proof-of-concept, we<br />
analyzed the genome of the predatory bacterium Cupriavidus necator for the presence of<br />
such genes. C. necator had previously been reported to secrete a peptidic molecule, which<br />
sequesters copper from the environment and is important for the killing of competing<br />
predacious species [3], but the structure of the involved metabolite was never resolved. We<br />
now identified a promising gene cluster that could account for the biosynthesis of this<br />
copper chelating natural product. The candidate locus includes genes for the nonribosomal<br />
assembly of a peptide as well as for the incorporation of bidentate ligand groups. To<br />
retrieve the associated natural product, we disrupted one of the biosynthesis genes by<br />
insertional mutagenesis and subsequently compared the metabolite profiles of mutant and<br />
wild-type strain. This approach led to the discovery of a family of linear lipopeptides,<br />
exemplified by the main metabolite cupriachelin [4]. When tested in an agar diffusion assay,<br />
cupriachelin and its congeners exhibited strong effects against numerous bacteria,<br />
including all identified prey organisms of C. necator.<br />
Acknowledgments: We gratefully acknowledge the Bundesministerium für Bildung und Forschung for<br />
supporting our research involving genomics and genome-inspired drug discovery (BMBF GenoMik-Transfer<br />
programme; grant no. 0315591A).<br />
References:<br />
1. Nett, M.; Ikeda, H.; Moore, B. S.: Nat. Prod. Rep. 2009, 26(11): 1362-1384.<br />
2. Winter, J. M.; Behnken, S.; Hertweck, C.: Curr. Opin. Chem. Biol. 2011, 15(1): 22-31.<br />
3. Casida, L. E.: Appl. Environ. Microbiol. 1987, 53(7): 1515-1518.<br />
4. Kreutzer, M. F.; Kage, H.; Nett, M.: J. Am. Chem. Soc. <strong>2012</strong>, 134(11): 5415-5422.<br />
011<br />
Aziridine-based inhibitors of HIV-1 protease<br />
Grün, J. 1; Rieger, M. 2; Welker, A. 3; Bodem, J. 4; Schneider, T. 1; Engels, B. 2 ; Sotriffer, C. 3;<br />
Kiefer, W. 1; Schirmeister, T. 1;<br />
1 Institute of Pharmacy and Biochemistry, University of Mainz, GERMANY<br />
2 Institute of Physical and Theoritical Chemistry, Universiy of Würzburg, GERMANY<br />
3 Institute of Pharmacy and Food Chemistry, University of Würzburg, GERMANY<br />
4 Institute of Virology and Immunology, University of Würzburg, GERMANY<br />
The HIV-1 protease represents an aspartate protease essential for proper virion assembly<br />
and maturation. [1-3] Many competitive inhibitors of this protease are available with FDA<br />
approval. [4,5] But there is a rapid rise of strains that encode mutant proteases resistant to<br />
these reversible protease inhibitors. [6,7] We explored the ability of QM/MM models to<br />
accurately describe the inhibition reaction of the HIV-1 protease by epoxide- and aziridinebased<br />
inhibitors. In contrast to their epoxide counterparts, the mechanisms and binding<br />
modes of aziridine-based inhibitors have much more rarely been the subject of<br />
investigations, e.g. no X-ray data for complexes with HIV-1 PR or SIV PR exist.<br />
Computations [8-11] predict their inhibition mechanism to be similar to that of epoxides, but<br />
differences result from the stronger basicity of the aziridine. Accordingly, aziridine-based<br />
inhibitors should be ideally suitable for aspartate proteases which act in more acidic<br />
environments. [12] This was indeed shown by recent work. Employing docking approaches<br />
the HIV-1 PR structure is used to predict possible substitution patterns of such new<br />
inhibitors with improved binding affinities. We present synthetic approaches for these new<br />
optimized aziridine-based inhibitors.<br />
References:<br />
1. Frankel, A. D.; Young, J. A. T. Annu Rev B ochem 1998, 67, 1.<br />
2. Seelmeier, S.; Schmidt, H.; Turk, V.; Vonderhelm, K. P Natl Acad Sci USA 1988, 85, 6612.<br />
3. Brik, A.; Wong, C. H. Org Biomol Chem 2003, 1, 5.<br />
4. Rose, J. R.; Craik, C. S. Am J Resp Crit Care 1994, 150, 176.<br />
5. Wlodawer, A.; Erickson, J. W. Annu Rev Biochem 1993, 62, 543.<br />
6. Metzner, K. J.; Rauch, P.; von Wyl, V.; Leemann, C.; Grube, C.; Kuster, H.; Boni, J.; Weber, R.;<br />
Gunthard, H. F. J Infect Dis 2010, 201, 1063.<br />
7. Bihani, S. C.; Das, A.; Prashar, V.; Ferrer, J. L.; Hosur, M. V. Biochem Bioph Res Co 2009, 389, 295.<br />
8. Helten, H.; Schirmeister, T.; Engels, B. J Phys Chem A 2004, 108, 7691.<br />
9. Helten, H.; Schirmeister, T.; Engels, B. J Org Chem 2005, 70, 233.<br />
10. Vicik, R.; Helten, H.; Schirmeister, T.; Engels, B. Chemmedchem 2006, 1, 1021.<br />
11. Vicik, R.; Busemann, M.; Baumann, K.; Schirmeister, T. Curr Top Med Chem 2006, 6, 331.<br />
12. Dunn, B. M. Chem Rev 2002, 102, 4431.<br />
012<br />
Bicyclic acetals – potential inhibitors of Golgi α-mannosidase II<br />
Juchum, M. 1; Kiefer, W. 1; Schneider, T. 1; Seibel, J. 2; Sotriffer, C. 2; Engels, B. 3; Schirmeister,<br />
T. 1;<br />
1 Institute of Pharmacy and Biochemistry, University of Mainz, GERMANY<br />
2 Institute of Pharmacy and Food Chemistry, University of Würzburg, GERMANY<br />
3 Institute of Physical and Theoretical Chemistry, University of Würzburg, GERMANY<br />
Cancer – predominantly trachea, bronchus and lung cancer – causes 5.9 % (3rd place in the<br />
top ten after ischemic heart disease and stroke) of deaths in high-income countries and 2.4<br />
% (7 h place) of deaths in the world [1]. Unregulated spreading of cells, invasion of healthy<br />
tissue and especially metastasis characterize the pathology of these diseases [2].<br />
Selectins, carbohydrate recognizing proteins, play a crucial role in binding metastasizing<br />
cancer cells [3]. Ligands for selectins are often modified glycosylation patterns on the<br />
surface of the cancer cells [4]. Studies have shown an overexpression of different sugar-<br />
hydrolyzing enzymes in these cells, making such enzymes promising targets for new anticancer<br />
drugs [5]. Especially inhibition of the Golgi alpha-mannosidase II (GM II) has shown<br />
tumor repression [6].<br />
GM II, a glycosyl hydrolase, is a 125 kDa type II transmembrane protein [7] that plays an<br />
essential role in the N-glycosylation pathway of asparagine side chains. The high specific<br />
cleavage of two mannose units (α-(1,3) and α-(1,3)) of the intermediate<br />
GlcNAcMan5(GlcNAc)2 takes place in the active site of the enzyme, two aspartate residues<br />
and a zinc cation involved. GM II is a retaining glycosidase and cleaves the sugars in a twostep-SN2-mechanism<br />
that preserve the configuration of the anomeric C-atom [8].<br />
Already available inhibitors, mostly derivatives of swainsonine, have side effects, due to low<br />
selectivity [9]. The goal of the presented project is the synthesis of selective, covalentreversible<br />
inhibitors with a long resting time in the catalytic site of the enzyme. QM<br />
calculations and docking simulations have shown that bicyclic acetals are promising<br />
candidates, in terms of both, high affinity to the target enzyme and reaction kinetics. Based<br />
on L-gulose, we synthesized 1-2 and 1-6 bridged species. New strategies like cycloaddition<br />
and especially olefin ring-closing metathesis (RCM) were also evaluated as new<br />
synthetic pathways to yield the desired acetals.<br />
References:<br />
1. World Health Organization. Media Centre: The top ten causes of death (Updated June 2011).<br />
http://www.who.int/mediacentre/factsheets/fs310/en/index.html (accessed March 19, <strong>2012</strong>).<br />
2. Kohn, E. C., Liotta, L. A.: Cancer Res 1995, 55: 1856-1862.<br />
3. Kim, Y. J. et al.: PNAS 1998, 95: 9325-9330.<br />
4. Kim, Y. J., Varki, A.: Glycoconjugate J. 1997, 5: 569-576.<br />
5. Rambaruth, N. D., Dwek, M. V.: Acta Histochem. 2011, 113: 591-600.<br />
6. Goss, P. E. et al.: Clin. Cancer Res, 1997, 3: 1077-1086.<br />
7. Moremen, K. W., Touster, O.: J. Biol. Chem. 1986, 261: 10945-10951.<br />
8. Petersen, L. et al.: J. Am. Chem. Soc. 2010, 132: 8291-8300.<br />
9. Kuntz, D. A. et al.: ChemBioChem 2010, 11: 673-680.<br />
013<br />
Tacrine Derivatives and its dimers as Potential Antiprotozoic Agents<br />
Schmidt I1; Schurigt U2; Pradel G2; Holzgrabe U1 1Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074<br />
Würzburg, Germany<br />
2Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Str. 2, 97080<br />
Würzburg, Germany<br />
Three of the most important tropical infectious diseases are Malaria with an estimated 655<br />
000 deaths in 20101, Leishmaniasis with an estimated incidence of 2 million new cases per<br />
year2 and Human African Trypanosomiasis with 7139 new cases as reported in 20103. The<br />
development of new and cheap agents for the treatment is urgently needed. In our previous<br />
investigations antiprotozoal activity of tacrine (e.g. IC50 = 12.5 µM for Plasmodium<br />
falciparum) has been shown. Its homodimeric hexyl-linked derivative N1,N6-bis(1,2,3,4 tetrahydroacridin-9-yl)hexane-1,6-diamine, R1 = H, (1) was found to have increased activity<br />
(e. g. IC50 = 0.095 µM for Plasmodium falciparum). This dimer and derivatives with<br />
carbonyl- and hydroxyfunctions at the saturated ringsysteme (R1) exhibit antiprotozoal<br />
activity partially in nanomolar range of concentration, but associated with moderate<br />
cytotoxicity. Hence our first task was to identify the optimal length of the linking alkyl chain.<br />
Therefore we synthesized the above mentioned compounds with various chain length from<br />
C6 to C12. The antiprotozoal activities of this dimers against Plasmodium falciparum,<br />
Leishmania major, Trypanosoma brucei and cytotoxicity on macrophages J774.1 were<br />
determined in vitro and showed that the length of 7 to 9 methylene units is most suitable.<br />
Furthermore our results demonstrated that the introduction of substituents like nitro or<br />
chlorine in the aromatic moiety leads to a similar antiprotozoal effect and reduced<br />
cytotoxicity. Therefore our future goal will be the design and synthesis of active compounds<br />
with decreased cytotoxicity.<br />
1<br />
Acknowledgments: We acknowledge the Deutsche Forschungsgemeinschaft (SFB 630) for support.<br />
References:<br />
1. WHO; www.who.int; World Malar a report 2011.<br />
2. WHO; www.who.int; Technical Report Series 949.<br />
3. WHO; www.who.int; Fact Sheet N° 259.<br />
014<br />
Development of MIP-Inhibitors<br />
Seufert F1; Juli C1; Norville I H2; Jenner D2; Sarkar-Tyson M2; Weiwad M3; Schweimer K4; Rösch P4; Holzgrabe U1 1 Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074<br />
Würzburg, Germany<br />
2 Defence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, UK<br />
3 Research Center for Enzymology of Protein Folding, Max-Planck Institute Halle, Weinbergweg 22,<br />
06120 Halle, Germany<br />
4 Department of Biopolymers, University of Bayreuth, <strong>Universität</strong>sstrasse 30, 95447 Bayreuth,<br />
Germany<br />
Major virulence factors known as macrophage infectivity potentiators (MIPs) facilitate the<br />
infection of cells with different pathogenic bacteria like Legionella pneumophila or<br />
Burkholderia pseudomallei. Both pathogens manifest in different diseases, L. pneumophila<br />
is the causative agent of Legionnaires‘ disease, whereas B. pseudomallei induces<br />
melioidosis which emerges in Southeast Asia and Northern Australia. An infection with<br />
B. pseudomallei may occur by inoculation through skin lesions or occasionally by<br />
inhalation. Human infection with Legionellosis transpires as well by inhalation of<br />
contaminated aerosols, typically being air-conditioning systems, whirlpools or even<br />
showers, i.e. systems with stagnant, warm water. The MIP of L. pneumophilia assists the<br />
Legionella to transmigrate epithelial cells and extracellular matrix (ECM) of lung tissue and<br />
to intrude alveolar macrophages. Both MIPs of L. pneumophila and B. pseudomallei show<br />
peptidyl propyl cis/trans isomerase (PPIase) activity [1,2]. These surface proteins belonging<br />
to the family of the FKBP binding protein form a stable complex with immunosuppressive<br />
96 Poster
drugs FK506 or rapamycin which inhibit the enzymatic function of the mentioned MIPs [3].<br />
Due to the immunosuppressive effects Legionnaires‘ disease and melioidosis can not be<br />
treated with these drugs, thus novel inhibitors had to be applied. With the aid of<br />
computational analysis, subsequent syntheses and biological screening molecules with a<br />
pipecolin acid moiety (A, B) were established. These compounds showed an activity<br />
against L. pneumophila with an IC50 of 9.0 µM for A and 32 µM for B [1]. Also, a satisfying<br />
activity against B. pseudomallei was detected for both substances. In HSQC-NMR binding<br />
studies an interaction of synthesized compounds A, B and the MIP of L. pneumophila could<br />
be verified. Furthermore, an interaction was found with substance B and the MIP of<br />
B. pseudomallei. To continue studies NMR binding experiments with compound A and the<br />
MIP of B. pseudomallei will be carried out.<br />
Acknowledgments: Financial support DFG (SFB 630)<br />
References:<br />
1. Juli, C. et al. J. Med Chem. 2011, 54(1): 277–283.<br />
2. Norville, I.H. et al. Infection and Immunity 2011, 79(11): 4299–4307.<br />
3. Fischer, G. et al. Mol. Microbiol. 1992, 6(10): 1375–1383.<br />
015<br />
Discovery of novel protease inhibitors from the Caribbean sponge Plakortis<br />
halichondrioides.<br />
Oli S, 1 Tabares P, 2 Hentschel-Humeida U, 2 Schirmeister T3 1 Institut für Pharmazie und LMC, <strong>Universität</strong> Würzburg, Am Hubland, 97074 Würzburg, Germany<br />
2 Julius-von-Sachs Institut, <strong>Universität</strong> Würzburg, Julius-von Sachs Platz 3, 97082 Würzburg,<br />
Germany<br />
3 Institut für Pharmazie und Biochemie, Johannes Gutenberg-<strong>Universität</strong> Mainz, Staudinger Weg 5,<br />
55128 Mainz, Germany<br />
Marine sponges of the family Plakinadae are known to be rich sources of structurally<br />
unique and biologically active metabolites.[1] In this study, bioactive compounds with<br />
protease inhibiting properties were discovered from the sponge Plakortis halichondrioides<br />
following bioactivity-guided fractionation. The structures of the endoperoxides plakortide E<br />
and plakortin were identified by means of MS and NMR spectroscopy. Plakortide E showed<br />
inhibitory activities against papain-like proteases such as cathepsins B and L, as well as<br />
against SARS-CoV PLpro and rhodesain. The ester plakortin was identified due to its activity<br />
against the parasite protease rhodesain from Trypanosoma brucei rhodesiense.<br />
Procedures of isolation, spectroscopic data, enzyme inhibition data as well as in vitro<br />
results are presented.<br />
Plakortide E<br />
Plakortin<br />
References:<br />
[1] Noriko Sata. et al.: J. Nat. Prod. 2005, 68, 1400-1403.<br />
016<br />
New therapy against HIV: Syntheses of inhibitors of the vif - elongin-C<br />
interaction<br />
Menrath, C. 1; Matz, F. 2; Welker A. 2; Nowotny, B. 3; Bodem, J. 3; Rethwilm, A. 3, Sotriffer, C. 2,<br />
Schirmeister, T. 1<br />
1 Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55099 Mainz,<br />
Germany<br />
2 Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074<br />
Würzburg, Germany<br />
3 Institute of Virology and Immunology, University of Würzburg, Versbacher Strasse 7, D-97078<br />
Würzburg, Germany<br />
AIDS (acquired immunodeficiency syndrome) is a disease caused by HIV-1 and HIV-2<br />
(human immunodeficiency virus). In the end of 2010, about 33 millions of people were<br />
infected with HIV. 1<br />
The today’s most successful therapy is HAART (highly active anti-retroviral therapy) using<br />
combinations of drugs inhibiting essential viral enzymes thus blocking virus replication.<br />
In the human immune system, the proteins of the APOBEC3 family play essential roles in<br />
the defence against retroviral infections, in case of HIV especially APOBEC3G and 3F are<br />
involved. These proteins possess cytidine deaminase acivity thus inducing various<br />
mutations in the viral nucleic acid. Such hypermutation ultimately destroys the coding and<br />
replicative capacity of the virus, resulting in nonviable virions. However, the viral protein vif<br />
(viral infectivity factor) interacts with APOBEC3G and triggers the ubiquitination and<br />
degradation of APOBEC3G via the proteasomal pathway, thus overcoming its native<br />
antiviral activity. APOBEC3G and vif complex formation also involves binding of the human<br />
protein elongin C.<br />
On the basis of the crystal structure of elongin C, we identified new inhibitors of the vif-eloC<br />
interaction by means of virtual screening and docking studies. We present syntheses and<br />
test results of these compounds exhibiting a new mechanism of action against retroviral<br />
infections. 2 3 4 5 6<br />
References:<br />
1. Epidemie Report Update (UNAIDS 2010).<br />
2. K. N. Bishop,R. K. Holmes, A. M. Sheehy, M. H. Malim Science 2004, 305 (5684), 645.<br />
3. S. L. Sawyer, M. Emerman, H. S. Malik, PLoS. Biol. 2004, Sep; 2(9), E275.<br />
4. A. M. Sheehy, N. C. Gaddis, J. D. Choi, M. H. Malim, Nature 2002, 418 (6898), 646 – 50.<br />
5. R. S. Harris, M. T. Liddament, Nat. Rev. Immunol. 2004, 4, 868 – 877.<br />
6. M. J. Wichroski, K. Ichiyama, T. M. Rana, J. Biol. Chem. 2005, 280 (9), 8387 – 8396.<br />
017<br />
Synthesis of 1-benzoxepines as NR2B selective NMDA receptor antagonists<br />
Wendling, F. 1; Wünsch, B. 1<br />
1 Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms <strong>Universität</strong><br />
Münster, Hittorfstraße 58-62, 48149 Münster, Germany<br />
While the NMDA receptor is responsible for a number of physiological effects, including<br />
neuronal development, overactivation of NMDA receptors has been associated with<br />
pathological conditions such as Alzheimer’s Disease, Parkinson’s Disease and depression.<br />
[1]<br />
Functional NMDA receptors are composed of four subunits containing at least one NR1(ah)<br />
subunit assembled with NR2 (A-D) and/ or NR3 (A, B). [2] The NR2 subunits vary in their<br />
distribution throughout the CNS. As the NR2B subunit is less distributed in the cerebellum<br />
[3], addressing it selectively has been reported to result in decreased cognitive and motoric<br />
side effects. [4]<br />
Therefore design, synthesis and structure-affinity relationships of NR2B selective<br />
antagonists are of great interest as these compounds represent possible therapeutic agents<br />
for various neurological conditions.<br />
Using Ro-25,6981 1 as a lead compound, which binds with high affinity (Ki = 6 nM) at the<br />
NR2B subunit [5], we develop structurally related molecules with various substituents and<br />
stereochemistry.<br />
On this poster we present a Dieckmann cyclisation as key step for the synthesis of<br />
benzoxepines 2 (X=O), which we developed as conformationally restricted Ro-25,6981<br />
analogues.<br />
1 2<br />
References:<br />
1. Dingledine, R., et al.: Pharmacol. Rev. 1999, 51(1): 7-62.<br />
2. Chatterton, J.E., et al.: Nature 2002, 415(6873): 793-798.<br />
3. Gogas, K.: Curr. Opin. Pharmacol. 2006, 6 (1):68-74.<br />
4.Nikam, S.S., Meltzer, N.T.: Curr. Pharm. Design 2002, 8(10): 845-855.<br />
5. Pinard, E., et al.: Bioorgan. Med. Chem. Lett. 2001, 11(16):2173-2176.<br />
018<br />
Identification of the bioactive principles underlying the vascular barrierprotecting<br />
action of the Crataegus extract WS ® 1442<br />
Fuchs, S. 1; Willer, E. A. 1; Hartung, E. 1; Vollmar, A. M. 1; Potterat, O. 2; Hamburger M. 2; Fürst,<br />
R. 1<br />
1 Department of Pharmacy, Center for Drug Research, Pharmaceutical Biology, University of<br />
Munich, Butenandtstr. 5-13, 81377 Munich, Germany<br />
2 Department of Pharmaceutical Sciences, Pharmaceutical Biology, University of Basel,<br />
Klingelbergstr. 50, 4056 Basel, Switzerland<br />
We have recently discovered that the hawthorn (Crataegus spp.) special extract WS ® 1442,<br />
widely used for the treatment of mild forms of heart failure, effectively prevents<br />
inflammation-induced endothelial hyperpermeability in vitro and in vivo [1]. WS ® 1442<br />
affects key systems of endothelial barrier regulation, such as the F-actin cytoskeleton,<br />
adherens junctions (VE-cadherin), the contractile machinery (myosin light chain), and the<br />
intracellular calcium concentration [1]. The extract targets two major endothelial signaling<br />
cascades: it blocks the barrier-destabilizing Ca2+/PKC/RhoA pathway and activates the<br />
barrier-protecting cAMP/Rap1/Rac1 signaling [1]. In the search for the constituents<br />
responsible for these effects, 4 fractions (A-D) of WS ® 1442 were prepared (Sephadex LH-<br />
20 column chromatography, kindly provided by Dr. Willmar Schwabe GmbH & Co. KG,<br />
Karlsruhe, Germany). We could show that the two signaling cascades affected by the<br />
extract are separately targeted by two different fractions: only fraction B, containing small<br />
phenolic compounds, flavonoids, and lipophilic constituents, blocked the calcium signaling<br />
and only fraction C, consisting of oligomeric proanthocyanidins (OPCs), activated the cAMP<br />
pathway in endothelial cells.<br />
Intention of the present study was to identify the bioactive compounds by bioguided<br />
fractionation. Regarding fraction B, 9 subfractions (preparative HPLC) were analyzed for<br />
their influence on a thrombin-evoked rise of the intracellular calcium concentration in<br />
primary human endothelial cells (ratiometric, microscopic analysis using Fura-2). Only one<br />
subfraction blocked the thrombin effect and was thus judged as active. A preliminary HPLC-<br />
DAD-MS study revealed that this fraction contains lipophilic, UV inactive substances,<br />
suggesting that phenolic compounds and flavonoids are not responsible for the observed<br />
action on calcium levels. A detailed phytochemical analysis of this subfraction (isolation,<br />
structure elucidation) is in progress.<br />
Fraction C was divided into 6 subfractions (preparative HPLC). The microscopical analysis<br />
of endothelial cortactin phosphorylation, a downstream event of the cAMP-triggered<br />
signaling evoked by WS ® 1442, served as read-out parameter for bioactivity. 2 fractions<br />
were highly active, 3 exhibited a slight activity, and one was completely inactive. Further<br />
separation (Sephadex LH-20 column chromatography) of the two highly active fractions<br />
resulted in 10 subfractions, of which 8 were active. Due to this very broad activity<br />
distribution and the well-known difficulties in precisely separating OPCs, we decided to test<br />
isolated OPCs of WS ® 1442. Procyanidin B2 (an OPC dimer) and procyanidin C1 (an OPC<br />
trimer) were confirmed to trigger endothelial cAMP signaling (cAMP ELISA, phosphocortactin<br />
Western blots and microscopical analysis).<br />
In summary, we could provide first insights into the bioactive principles that underlie the<br />
polypharmacological action of WS ® 1442. The inhibition of calcium signaling seems to be<br />
evoked by lipophilic, non-phenolic compounds, which we currently aim to identify.<br />
Moreover, we could show the OPCs procyanidin B2 and C1 to be responsible for the<br />
activation of endothelial cAMP signaling by WS ® 1442.<br />
Poster 97
Acknowledgments: This work is supported by the German Research Foundation (DFG, FU691/7-1). WS ®<br />
1442 was kindly provided by Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany.<br />
References:<br />
1. Bubik, M. F. et al.: J. Mol. Cell. Cardiol. 2011, 52(1): 196-205.<br />
019<br />
HPTLC of purified hyaluronan oligosaccharides: reagent-free derivatization<br />
and densitometric quantification on amino-modified silica, TLC–ESI–Q-TOF-MS<br />
coupling on normal phase<br />
Rothenhöfer, M. 1; Scherübl, R. 2; Bernhardt, G. 1; Heilmann, J. 2; Buschauer, A. 1<br />
1 Lehrstuhl für Pharmazeutische/Medizinische Chemie II, <strong>Universität</strong> Regensburg, 93040<br />
Regensburg, Germany<br />
2 Lehrstuhl für Pharmazeutische Biologie, <strong>Universität</strong> Regensburg, 93040 Regensburg, Germany<br />
By the development of powerful high performance thin layer chromatography (HPTLC)<br />
techniques, planar chromatography has become a cost-effective and convenient alternative<br />
to high performance liquid chromatography (HPLC). It allows parallel analysis of multiple<br />
samples and, owing to a multitude of derivatization possibilities, attractive options for the<br />
detection of unfavorable analytes. For instance, oligosaccharides derived from enzymatic<br />
cleavage of hyaluronan by hyaluronidases pose a particular analytical challenge.<br />
Nevertheless, there is a strong need for purified hyaluronan fragments as pharmacological<br />
tools to shed light on the physiological and pathophysiological role of hyaluronidases,<br />
hyaluronan, and its degradation products.<br />
Therefore, we developed and validated an efficient HPTLC method for qualitative and<br />
quantitative analysis of small saturated hyaluronan oligosaccharides consisting of 2 to 4<br />
hyalobiuronic acid moieties. Uniform and reagent-free in situ derivatization is enabled by<br />
the use of amino-modified silica as stationary phase. Subsequent quantification by<br />
densitometric scans in reflectance mode revealed very low limits of detection of 7 to 19<br />
pmol per band, depending on the oligosaccharide.<br />
In view of the applicability of HPTLC for the quality control of hyaluronan oligosaccharides,<br />
proof of principle was provided for unambiguous identification of the substances by direct<br />
ESI–Q-TOF-MS coupling. For this purpose standard silica TLC plates were used and high<br />
resolution mass spectra were obtained for all three analytes.<br />
020<br />
Effects of STW 5 and STW 6 on rat ileal and colonic preparations: A<br />
comparative study<br />
Nieber, K1, Voß, U. 1, Michael, S. 2, Kelber, O. 3, H. Abdel-Abziz3 Weiser, D. 3<br />
1Institut für Pharmazie, <strong>Universität</strong> Leipzig, 04013 Leipzig,<br />
2Löwen-Apotheke-Waldheim, 04736 Waldheim,<br />
3Wissenschaftliche Abteilung, Steigerwald Arzneimittelwerk GmbH, 64295 Darmstadt<br />
STW 5 (Iberogast ) is a fixed combination of nine plant extracts with Iberis amara (STW 6)<br />
as one of its components. It is successfully used for treatment of functional dyspepsia and<br />
irritable bowel syndrome (IBS). Because clinical data suggest inflammatory involvement in<br />
the etiology of IBS the influence of STW 5 and STW 6 on tone and acetylcholine (ACh)induced<br />
contractions of intact and inflamed intestinal preparations was examined. We used<br />
1 - 1.5 cm long ileum and colon preparations of male Wistar rats to analyze region specific<br />
differences. The inflammation was induced by intraluminal instillation of 2,4,6trinitrobenzene<br />
sulfonic acid (TNBS, 10 mM, 30 min). Incubation with STW 5 (64 – 512<br />
µg/ml, 30 min) concentration dependently reduced the tone and decreased ACh-induced<br />
contractions of untreated ileal and colonic preparations. STW 6 in equivalent concentrations<br />
(3 – 24.1 µg/ml) neither affected the tone nor the contractility. TNBS-induced inflammation<br />
was accompanied by a significant reduction of ACh-induced contractions and<br />
morphological disturbances. Co-incubation of TNBS with STW 5 (512 µg/ml) or STW 6<br />
(24.1 µg/ml) partially normalized the TNBS-induced attenuation of ACh-induced<br />
contractions and the morphological damage in ileum preparations, whereas in inflamed<br />
colon segments only the co-incubation of TNBS with STW 6 in a high concentration (24.1<br />
µg/ml) revealed similar effects.<br />
In conclusion, STW 5 influenced ACh-induced contractions and tone in untreated ileal and<br />
colonic preparations, whereas STW 6 did not contribute to these effects. In TNBS-inflamed<br />
ileum preparations STW 5 as well as STW 6 normalized morphological and contractile<br />
disturbances, while in colon preparations STW 6 but not STW 5 was effective. Our study<br />
confirms region specific effects of STW 5 and its fresh plant component STW 6.<br />
021<br />
The PI3/Akt signaling pathway is involved in endothelium-dependent relaxation<br />
by Salix spp. in porcine coronary arteries<br />
Kaufeld, A. M.; Pertz, H. H.; Kolodziej, H.<br />
Institut für Pharmazie, Freie <strong>Universität</strong> Berlin, Königin-Luise-Str. 2+4, 14195 Berlin<br />
Numerous lines of evidence have shown cardioprotective and cardiotonic effects of<br />
polyphenols [1]. Preliminary studies demonstrated that 2,3-cis procyanidins of Nelia meyeri<br />
Schwant. induced an endothelium-dependent relaxation of isolated porcine coronary<br />
arteries [2]. The aim of the present study was to gain insight into the molecular mechanism<br />
by which 2,3-trans procyanidins of willow bark elicit blood vessel relaxation.<br />
The willow bark procyanidin sample comprising a mixture of dimeric to hexameric 2,3-trans<br />
flavan-3-ols [3] induced a concentration-dependent relaxation in pre-contracted<br />
endothelium-intact coronary arterial rings. The effect was inhibited by NG-nitro-L-arginine methyl ester (L-NAME; inhibitor of eNOS) and rotenone (inhibitor of mitochondrial electron<br />
transport chain). Furthermore, the extract-induced relaxation was abolished by mechanical<br />
removal of the endothelium, addition of wortmannin (inhibitor of phosphoinositide 3-kinase;<br />
PI3K), as well as by manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; cell<br />
permeable mimetic of superoxide dismutase). Similarly, the response was completely<br />
inhibited by the combination of charybdotoxin, apamin (Ca2+-activated K + channel blockers)<br />
and L-NAME. This showed that the endothelium-derived hyperpolarizing factor (EDHF) and<br />
NO (EDRF) were involved in the relaxant response to Salix spp.. In contrast, the addition of<br />
ICI 182,780 (estrogen receptor antagonist) or pertussis toxin (inhibitor of Gi proteins) did not<br />
affect the relaxation. These findings indicated that willow bark procyanidins triggered the<br />
activation of the PI3/Akt pathway. Western Blot analysis using human umbilical vein<br />
endothelial cells (HUVEC) demonstrated that Salix procyanidins concentration-dependently<br />
induced Akt and eNOS phosphorylation. The addition of wortmannin, MnTMPyP or<br />
rotenone significantly inhibited key phosphorylation sites.<br />
In conclusion, the present data confirm procyanidin-induced production of potent<br />
vasoprotective factors including NO and EDHF. The results support the view that<br />
polyphenols have beneficial effect on endothelial cell functions of blood vessels.<br />
References:<br />
1. Rasmussen, S.E. et al.: Mol. Nutr. Food Res. 2005, 49: 159–174.<br />
2. Kaufeld, A.M., Pertz, H.H., Kolodziej, H.: Planta Med. 2011, 77: 1403.<br />
3. Kolodziej, H.: Phytochemistry 1989, 29: 955–960.<br />
022<br />
Development of NR2B-selective NMDA receptor antagonists<br />
Lütnant I. 1; Wünsch B. 1<br />
1 Institut für Pharmazeutische und Medizinsche Chemie der Westfälischen Wilhelms-<strong>Universität</strong><br />
Münster, Hittorfstraße 58-62, D-48149 Münster, Germany<br />
The N-Methyl-D-Aspartate (NMDA) receptor is an excitatoric amino acid receptor belonging<br />
to the class of ionotropic glutamate receptors. The receptor is a ligand-gated ion channel<br />
highly permeable for Ca2+-ions consisting of four subunits. Seven subunits named NR1,<br />
NR2 A-D and NR3 A, B have been identified. A functional NMDA receptor contains at least<br />
one NR1-subunit and one NR2-subunit. The receptor is distributed all over the brain, but its<br />
composition differs depending on the region in the brain in which it is expressed. [1, 2]<br />
The NMDA receptor shows unique properties such as a co-agonism of (S)-glutamate and<br />
glycine, permeability for Na +-, K +-, Ca2+-ions and a voltage-dependent Mg2+-block. When the<br />
channel is opened Ca2+-ions enter the cell and cause activation of several pathways<br />
leading to different effects amongst others neuronal development. Overstimulation of the<br />
NMDA receptor causes cell death and therefore “neurodegeneration”. [3, 4]<br />
Ifenprodil (1) is a non-competitive NMDA receptor antagonist with a high affinity towards the<br />
ifenprodil binding site on the NR2B subunit. Due to the fact that ifenprodil also binds to 5-<br />
HT1A, 5-HT2 and σ- receptor, it causes many adverse effects.<br />
To reduce the adverse side effects on the one hand and to improve the neuroprotective<br />
effects on the other hand, compounds with increased selectivity have been developed<br />
derived from the lead structure ifenprodil (1). [4]<br />
NR2B-selective NMDA receptor antagonists were found to be of value because the NR2B<br />
subunit is not expressed in the cerebellum. [5] Therefore NR2B-selective antagonists lead<br />
to reduced motoric side effects. They seem to be useful compounds considering the<br />
therapy of Parkinson´s and Alzheimer´s disease because they are capable to inhibit the<br />
effects of an overstimulation of the NMDA receptor.<br />
We develop a new group of potential NR2B-selective NMDA receptor antagonists with a<br />
benzimidazolone bioisoster (2) of the phenol of ifenprodil (1).<br />
References:<br />
1. Dingledine, R. et al.: Pharmacol. Reviews 1999, 51(1).<br />
2. Goebel, D.J., Poosch, M.S.: Mol. Brain Res. 1999 69: 164-170.<br />
3. Stark, H, Graßmann, S., Reichert, U.: Pharm. In unsrere Zeit 2000, 29(3): 159-166.<br />
4. Stark, H, Graßmann, S., Reichert, U.: Pharm. In unsrere Zeit 2000, 29(4): 228-236.<br />
5. Monyer, H. et al.: Neuron. 1994, 12: 529-540.<br />
023<br />
Chain modified oxa-analogs of primaquine and some of its derivatives<br />
Leven, M. 1; Held, J. 2; Tschan, S. 2; Delves, M. 3; Plouffe, D. 4; Mordmüller, B. 2; Kurz, T. 1<br />
1 Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine <strong>Universität</strong> Düsseldorf,<br />
<strong>Universität</strong>sstr. 1, 40225 Düsseldorf, Germany<br />
2 Institut für Tropenmedizin, Eberhard Karls <strong>Universität</strong> Tübingen, Wilhelmstr. 27, 72074 Tübingen,<br />
Germany<br />
3 Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7<br />
2AZ, United Kingdom<br />
4 Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San<br />
Diego, CA 92121, United States of America<br />
The 8-aminoquinoline antimalarial primaquine 1 affects multiple life cycle stages of<br />
Plasmodium species. It exhibits activity against developing and dormant liver stages of P.<br />
vivax and P. ovale. Thus it is used for the treatment of symptomatic patients with P. vivax or<br />
P. ovale malaria and for the presumptive anti-relapse therapy in persons heavily exposed to<br />
these plasmodia. As 1 also influences developing liver stages of P. falciparum, it is used as<br />
a primary prophylactic agent. Due to its activity against gametocytes of all species of<br />
human malaria, 1 is recommended as a transmission-blocking agent in endemic regions. Its<br />
weak inhibitory activity against asexual blood stages of all Plasmodium species calls for a<br />
combination therapy with an effective blood schizontocidal antimalarial agent. Serious<br />
adverse effects including hemolysis in persons with glucose-6-phosphate dehydrogenase<br />
(G6PD) deficiency and methemoglobinemia limit the therapeutic potential of 1. Its<br />
application requires a previous test for G6PD activity. 1 is contraindicated in pregnancy and<br />
in children under the age of four years [1,2].<br />
In contrast to 1, clinical phase III drug candidate tafenoquin 2 demonstrates potent<br />
inhibitory activity against blood stages of P. falciparum and P. vivax. Preliminary data<br />
suggest that 2 is less toxic than 1, but hemolysis in persons with G6PD deficiency has also<br />
been reported.<br />
The (-)-enantiomer NPC1161B 3, currently in preclinical trials, affects effectively blood<br />
stages of P. berghei in mice and is less hematotoxic in beagle dogs than 1. In addition, 3<br />
inhibits effectively sporogony in vitro [3,4].<br />
98 Poster
In order to improve activities and to reduce side effects numerous derivatives of 1 have<br />
been prepared and biologically evaluated. An established concept for the modification of<br />
lead structures is the bioisosteric replacement of methylene groups in alkyl chains by<br />
bivalent atoms like oxygen and sulfur. In an attempt to optimize structural features of 1 we<br />
now report on the synthesis and biological evaluation of side chain modified oxa-analogs of<br />
1 and some of its derivatives.<br />
References:<br />
1. Hill, D. R. et al.: Am. J. Trop. Med. Hyg. 2006, 75(3): 402–415.<br />
2. Vale, N., Moreira, R., Gomes, P.: Eur. J. Med. Chem. 2009, 44(12): 937-953.<br />
3. Nannayakara, N. P. D. et al.: Antimicrob. Agents Chemother. 2008, 52(6): 2130-2137.<br />
4. Delves, M. et al.: PLoS Med. <strong>2012</strong>, 9(2): e1001169. doi:10.1371/journal.pmed.1001169.<br />
024<br />
Enantioselective Synthesis of Spirocyclic σ1 Ligands<br />
Holl, K. 1; Wünsch, B. 1<br />
1 Institut für Pharmazeutische und Medizinische Chemie, Westf. Wilhelms-<strong>Universität</strong> Münster,<br />
Hittorfstr. 58-62, D-48149 Münster, Germany<br />
σ receptors represent a unique class of receptors, consisting of two subtypes, σ1 and σ2<br />
receptors. They are widely distributed in the human body, including the central nervous<br />
system, as well as in many tissues of the periphery, e.g. heart, lung, liver, kidney [1]. The σ1<br />
receptor has been associated with various neuronal diseases, such as schizophrenia,<br />
depression and Alzheimer’s disease. In addition to that, an overexpression of σ1 receptors<br />
in certain tumor cells was reported [2]. Therefore, high-affinity σ1 ligands have potential for<br />
the therapy and diagnosis of CNS diseases and cancer. Fluorinated ligands can be used as<br />
PET tracers [3].<br />
A variety of spirocyclic ligands with high affinity for the σ1 receptor has been synthesized,<br />
but so far, only racemic mixtures have been characterized. We report on the first<br />
enantioselective synthesis of this kind of ligands, in order to detect relationships between<br />
the absolute configuration and the σ1 affinity.<br />
Starting from 2-bromostyrene (1), a series of spirocyclic σ1 ligands with a benzopyrane<br />
structure was synthesized, using Sharpless asymmetric dihydroxylation as key step.<br />
Furthermore, several variations in position 4 were carried out, including fluoro derivatives<br />
Br<br />
OH<br />
N<br />
R<br />
OH<br />
OH<br />
1 2 3<br />
Acknowledgments: We thank Deutsche Forschungsgesellschaft for financial support.<br />
References:<br />
1. Matsumoto, R. R. et al.: Eur. J. Pharmacol. 2003, 469(1-3): 1-12.<br />
2. Maurice, T. Su, T.: Pharmacol. Ther. 2009, 124(2): 195-206.<br />
3. van Waarde, A. et al.: Curr. Pharm. Des. 2010, 16(31): 3519-3537.<br />
025<br />
Comparative effect of proanthocyanidin enriched extracts on the in vitro<br />
inhibition of bacterial and viral neuraminidase<br />
Quosdorf, S. J.; Kolodziej, H.<br />
Institut für Pharmazie, Freie <strong>Universität</strong> Berlin, Königin-Luise-Str. 2+4, 14195 Berlin<br />
Influenza infections are still a major threat to human health and emergence of resistance to<br />
anti-influenza drugs is a cause for concern. Expanding the range of antiviral drugs that<br />
effectively inhibit influenza viruses is therefore a matter of urgency. Neuraminidase (NA), a<br />
key enzyme in the influenza life cycle and pathogenesis, has been established as a primary<br />
drug target. Accordingly, NA inhibition assays represent a promising strategy for finding<br />
new antiviral compounds. However, the substrates used in such functional assays are<br />
recognized by both bacterial and viral NAs [1]. Due to limited comparative data, IC50 values<br />
obtained from either setup must be regarded with caution. The current study was carried<br />
out to examine the validity and significance of the origin of NAs in a functional NA inhibition<br />
test system.<br />
Using an established fluorescence based in vitro assay, the inhibitory potential of a series<br />
of polyphenolic samples (Pelargonium sidoides DC, Salix spp, Nelia meyeri Schwant. and<br />
Diospyros kaki L.) was measured for bacterial (Vibrio cholerae) and viral NA (Baculovirus).<br />
Zanamivir (Relenza ®) and oseltamivir carboxylic acid served as positive controls. Selection<br />
of the test substances is based on preliminary studies regarding prominent in vitro inhibition<br />
of bacterial NA and differences in their proanthocyanidin patterns [2]. In this assay the<br />
substrate 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (MUNANA) is cleaved by NA<br />
to produce the fluorescent compound 4-methylumbelliferone (MU) [3]. The quantity of MU is<br />
inversely proportional to the inhibitory activity of the tested sample.<br />
The results demonstrate that the inhibitory potential of the proanthocyanidin enriched<br />
extracts differ significantly and depend strongly on the origin of NA under the same<br />
experimental conditions. All extracts were more effective against Vibrio cholerae NA (IC50<br />
ca. 1 µg/mL – ca. 19 µg/mL) compared to Baculovirus NA (IC50 ca. 19 µg/mL – ca. 58<br />
µg/mL). Interestingly and in contrast, zanamivir and oseltamivir carboxylic acid exhibited<br />
much stronger inhibitory activities against the viral NA (IC50 ca. 4 µg/mL and ca. 1 µg/mL,<br />
respectively) than against the bacterial NA (IC50 ca. 16 µg/mL and ca. 40 µg/mL,<br />
respectively). Therefore it appears reasonable to select preferably viral NAs when testing<br />
samples for anti-influenza potentials or searching for promising antiviral compounds. On the<br />
OR<br />
N<br />
Bn<br />
O<br />
other hand, the test system should be adapted to a given pathogenic condition that may<br />
well include bacterial NAs. The observed difference in susceptibility of the NAs is an<br />
important parameter affecting generated results in the applied test system.<br />
References:<br />
1. Grienke, U., et al.: Nat. Prod. Rep. <strong>2012</strong>, 29(1): 11–36.<br />
2. Janecki, A., Kiderlen, A.F., Kolodziej, H.: Planta Med. 2009, 75(9): 989.<br />
3. Potier, M., et al.: Analytical Biochemistry. 1979, 94(2): 287–296.<br />
026<br />
Spirocyclic isochromane-piperidines as new lead structures against<br />
atherosclerosis<br />
Strunz, A. K. 1; Wünsch, B. 1<br />
1Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-<strong>Universität</strong><br />
Münster, Hittorfstraße 58-62, D-48149 Münster, Germany<br />
The chemokine receptor 2 (CCR2) is a G-protein coupled receptor (GPCR) and part of the<br />
chemokine system, a complex network consisting of soluble proteins and their receptors.<br />
Chemokines and their receptors mediate effects on the cellular signalling pathway. The<br />
CCR2 receptor is expressed on various immune cells and regulates cellular movement and<br />
activation.<br />
The interaction of the endogenous ligand MCP-1(monocyte chemoattractant protein 1) with<br />
the CCR2 receptor plays a crucial role in several inflammatory processes [1]. In the chronic<br />
inflammatory disease of atherosclerosis, this interaction leads to the mobilization of<br />
activated monocytes into the subendothelial tissue [2].<br />
On one hand CCR2 antagonists can stop the growth and progression of atherosclerotic<br />
plaques by inhibition of early key steps, on the other hand [ 18F]-labeled CCR2 antagonists<br />
can be applied in PET (positron emission tomography) studies to analyze atherosclerotic<br />
plaques.<br />
The synthesis of new CCR2 antagonists with a spirocyclic substructure will be presented on<br />
this poster. The main building block is a spirocyclic system which consists of an<br />
isochromane (A) with a piperidine ring (B), prepared by Oxa-Pictet-Spengler reaction [3].<br />
The cyclization is performed with various phenylethanol derivatives. Different benzylamines<br />
(D) lead to different test compounds. The introduction of an aromatic substituent R5 might<br />
lead to compounds with increased CCR2 affinity [4]. The four variable building blocks (A-D)<br />
can be combined in different ways.<br />
References:<br />
1. Gerard, C., Rollins, B. J.: Nat. Immunol. 2001, 2 (2): 108-115.<br />
2. Galkina, E., Ley, K.: Annu. Rev. Immunol. 2009, 27: 165-197.<br />
3. Larghi, E. L., Kaufman, T. S.: European Journal of Organic Chemistry 2011, 2011 (27): 5195-5231.<br />
4. Butora, G. et al.: Bioorg. Med. Chem. Lett. 2006, 16 (18): 4715-4722.<br />
027<br />
Reverse α-aryl substituted β-thia-analogs of fosmidomycin as 1-deoxy-Dxylulose<br />
5-phosphate reductoisomerase (Dxr) inhibitors<br />
Lienau, C. 1, Kunfermann, A. 2, Hähn, S. 1, Konzuch, S. 1, Behrendt, C. T. 1, Illarionov, B. 3,<br />
Held, J. 4, Mordmüller, B. 4, Fischer, M. 3, Groll, M. 2, Kurz, T. 1<br />
1 Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine <strong>Universität</strong> Düsseldorf,<br />
<strong>Universität</strong>sstr. 1, 40225 Düsseldorf, Germany<br />
2 Center for Integrated Protein Science Munich, Lehrstuhl für Biochemie, Technische <strong>Universität</strong><br />
München, Lichtenbergstr. 4, 85747 Garching, Germany<br />
3 Hamburg School of Food Science, <strong>Universität</strong> Hamburg, Grindelallee 117, 20146 Hamburg,<br />
Germany<br />
4 Institut für Tropenmedizin, Eberhard Karls <strong>Universität</strong> Tübingen, Wilhelmstr. 27, 72074 Tübingen,<br />
Germany<br />
Inhibition of enzymes of the non-mevalonate pathway (MEP pathway) is a promising<br />
concept for the development of novel anti-infective drugs [1,2]. The non-mevalonate<br />
pathway of isoprenoid biosynthesis is the only source of the isoprenoid precursors<br />
isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAP) in several pathogenic<br />
bacteria and apicomplexan protozoa including the causative agents of malaria, Plasmodium<br />
spp.. The MEP pathway is absent in mammalians but essential for various pathogenic<br />
bacteria and Plasmodium spp.. A validated target for the development of novel<br />
antimalarials within the MEP pathway is the 1-deoxy-D-xylulose 5-phosphate<br />
reductoisomerase (Dxr) [3,4]. Reverse α-aryl substituted carba- and oxa-analogs (1,2) of<br />
the natural product fosmidomycin are highly active Dxr inhibitors and show potent<br />
antiplasmodial activity [3,4]. Further structural modifications of analogs (1,2) led to<br />
previously unreported reverse α-aryl substituted β-thia-isosters of fosmidomycin.<br />
References:<br />
1. Jomaa, H. et al.: Science 1999, 285: 1573-1576.<br />
2. Rohmer, M. et al.: Curr. Opin. Investig. Drugs 2004, 5: 154-162.<br />
3. Behrendt, C. T. et al.: J. Med. Chem. 2011, 54: 6796-6802.<br />
Poster 99
4. Behrendt, C. T. et al.: ChemMedChem 2010, 5(10):1673-1676.<br />
028<br />
Influence of the indirubin derivative 6BIO on proliferation and metastasis of<br />
breast cancer cells<br />
Braig S. 1, Kressirer C. 1, Bischoff F. 1, Meijer L. 2, Zahler S. 1, Vollmar A.M. 1<br />
1Department of Pharmacy, Center for Drug Research, Butenandtstr.5-13, 81377 Munich, Germany<br />
2CNRS, Station Biologique, Roscoff, France<br />
Initially, indirubins were discovered as the active component of a well known traditional<br />
Chinese medicine. Since then, it was shown that natural indirubins and also its synthetic,<br />
cell permeable derivative 6BIO (6-bromo-indirubin-3-oxime) display a remarkable potential<br />
to inhibit glycogen synthase kinase 3 (GSK-3) and cyclin-dependent kinases (CDKs) by<br />
interacting with the ATP-binding pocket of these kinases.<br />
By an inverse virtual screening approach phosphoinositide-dependent kinase 1 (PDK1) was<br />
proposed by us as a yet unknown target of 6BIO. Western blot analysis and confocal<br />
microscopy revealed that treatment of breast cancer cells with 6BIO resulted in a reduced<br />
phosphorylation of AKT, an important downstream target of PDK1. Since about 70% of all<br />
breast cancer carcinomas express moderate to high levels of activated PDK1, we<br />
investigated the impact of 6BIO treatment on different breast cancer cell lines by analyzing<br />
the anti-proliferative and cytotoxic effects as well as the influence on reduction of metastatic<br />
capabilities of these cells.<br />
As determined by crystal violet staining and xCELLigence experiments, incubation of<br />
SkBr3, MDA-MB-231, MCF7 and AKT overexpressing SkBr3 breast cancer cell lines with<br />
increasing concentrations of 6BIO led to a dose-dependent inhibition of proliferation. In<br />
addition, 6BIO treated breast cancer cells showed a strongly diminished long-term<br />
clonogenic survival rate.<br />
Furthermore, Nicoletti assays and Hoechst staining indicated that stimulation of breast<br />
cancer cell lines with high doses of 6BIO led to an induction of apoptosis.<br />
Cells treated with sub-toxic doses of 6BIO displayed a strongly reduced attachment<br />
capacity, as shown by adhesion assays on fibronectin and xCELLigence experiments.<br />
Scratch assays as well as chemotaxis assays revealed a significantly diminished migratory<br />
potential of 6BIO treated breast cancer cells compared to control cells. Furthermore, 6BIO<br />
significantly disrupted the invasion capacity of the cells. The impact of 6BIO treatment on<br />
the migration and invasion inhibition might be due to altered localization of the well-known<br />
migration associated molecules FAK and Rac1 within the cells, as shown by confocal<br />
microscopy.<br />
In summary, we were able to show that 6BIO inhibits PDK1 signal transduction in different<br />
breast cancer cell lines and reduces proliferation, clonogenic survival, attachment,<br />
migration and invasion of the cells. Thus, the indirubin derivative 6BIO might serve as a<br />
highly promising potential therapeutic agent for metastatic breast cancer cells.<br />
029<br />
Discovery of the first antagonists of PqsR to interrupt Pseudomonas<br />
aeruginosa cell-to-cell communication<br />
Steinbach, A. 1; Lu C1; Kirsch, B. 1; Zimmer, C. 1; de Jong, J.C. 1; Henn, C. 1; Maurer, C.K. 1;<br />
Müsken, M. 2; Häussler, S. 2 3; Hartmann, R.W. 1<br />
1 Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123 Saarbrücken,<br />
Germany<br />
2 Twincore, Department of Pathophysiology of Bacterial Biofilms, Feodor-Lynen-Str. 7, 30625<br />
Hannover, Germany<br />
3 Helmholtz Centre for Infection Research, Chronic Pseudomonas Infection Research Group,<br />
Inhoffenstr. 7, 38124 Braunschweig, Germany<br />
P. aeruginosa coordinates group behaviors such as virulence factor expression and biofilm<br />
formation via a cell-to-cell communication system known as quorum sensing (QS). The<br />
transcription of target genes are regulated via a pqs QS system that functions via the signal<br />
molecules PQS and its precursor HHQ that interact with their receptor PqsR. Targeting<br />
PqsR to develop quorum sensing inhibitors is considered as novel strategy to attenuate P.<br />
aeruginosa pathogenicity [1]. In order to discover PqsR antagonists, a ligand-based drug<br />
design approach was followed to synthesize a set of HHQ and PQS derived compounds.<br />
HHQ (R = H) R 1 = ED or EW groups<br />
PQS (R = OH) R 2 = alkyl or alkylether<br />
Agonistic or antagonistic properties were determined in a heterologous β-galactosidase<br />
reporter gene assay in E. coli to examine effects of side chain modifications and<br />
substitutions at the benzene moiety. An n-heptyl chain in 2-position was found to be<br />
optimal. Most importantly, the introduction of strong electron withdrawing functions like CN,<br />
NO2 or CF3 in 6-position of HHQ led to the discovery of the first antagonists (IC50 values of<br />
259 nM, 51 nM and 54 nM), while HHQ analogues with the same substituents in 7- or 8position<br />
or other substituents and all PQS derivatives were moderate to weak agonists.<br />
Direct evidence for the binding of a selected antagonist (6-CN HHQ) to PqsR was provided<br />
by surface plasmon resonance (SPR) biosensor experiments (KD=57 nM). Moreover, 6-CF3<br />
HHQ reduced pyocyanin production by 74 % (3 µM) in the target organism P. aeruginosa<br />
[2]. The aqueous solubility of the antagonists could be improved by the introduction of O<br />
into the alkyl side chain or CONH2 into 3-position.<br />
The first antagonists of PqsR are highly valuable scientific tools for in-depth study of<br />
protein-ligand interactions and provide a promising starting point for the development of a<br />
new anti-infective strategy.<br />
References:<br />
Deziel E et al., Mol. Microbiol. 2005, 55: 998–1014.<br />
Lu, C. et al.; Chem. Biol. <strong>2012</strong>, 19: 381–390.<br />
030<br />
Diarylpyrrolizines as Inhibitors of Microsomal Prostaglandin E2 Synthase-1<br />
(mPGES-1) or as Dual Inhibitors of mPGES-1 and 5-Lipoxygenase (5-LO)<br />
Keck, P. R. W. E. F. 1; Liedtke, A. J. 2; Koeberle, A. 3; Werz, O. 3; Schollmeyer, D. 4; Laufer, S.<br />
A. 1<br />
1 Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, University of<br />
Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany<br />
2 Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4,<br />
53121 Bonn, Germany<br />
3 Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, University of<br />
Jena, Philosophenweg 14, 07743 Jena, Germany<br />
4 Institute Organic Chemistry, University Mainz, Duesbergweg 10-14, 55099 Mainz, Germany<br />
Prostaglandin E2 (PGE2) is one of the most abundant and powerful prostanoids with diverse<br />
biological activities. As a key cyclic lipid mediator derived by the cyclooxygenase (COX)<br />
from arachidonic acid (AA), it is involved in the development and perpetuation of<br />
inflammation seen in diseases such as rheumatoid arthritis (RA). PGE2 has been implicated<br />
in the development of peripheral and central sensitization during nociceptive processing<br />
(e.g., hyperalgesia, allodynia) and in tumorigenesis[1]. Nonsteroidal anti-inflammatory<br />
drugs (NSAIDs), including selective COX-2 inhibitors, have analgesic, antipyretic and antiinflammatory<br />
properties. The main mechanism of action of NSAIDs is the inhibition of the<br />
COX isoenzymes. However, inhibition of the COX pathway by NSAIDs also reduces gastro<br />
protective PG synthesis, resulting in the well-known gastrointestinal side effects. In<br />
addition, the resulting increase in leukotriene (LT) biosynthesis due to shunting of AA to the<br />
5-lipoxygenase (5LO) pathway activated by the 5-lipoxygenase activating protein (FLAP)<br />
causes additional gastrointestinal injury[2,3].<br />
A dual COX/FLAP inhibition by Licofelone (ML3000) is an approach to minimize the side<br />
effects[2]. Licofelone also shows an inhibitory activity on the mPGES-1[4]. Going<br />
downstream, we synthesized and evaluated inhibitors for the mPGES-1, based on the<br />
diarylpyrrolizine scaffold of Licofelone while conserving the activity on the FLAP respective<br />
5-LO[1].<br />
Additionally, two crystal structure have been obtained to determine the angle of the aryl<br />
substituents[5,6].<br />
References:<br />
1. Liedtke, A. J. et al. J. Med. Chem. 2009, 52(15): 4968-4972.<br />
2. Laufer, S. A. et al. J. Med. Chem. 1994, 37(12): 1894-1897.<br />
3. Laufer, S. A.; Gay, S.; Brune, K.: Inflammation and Rheumatic Diseases (Thieme) 2003.<br />
4. Koeberle, A. et al. J. Pharmacol. Exp. Ther. 2008. 326(3): 975-982.<br />
5. Keck, P. R. W. E. F.; Schollmeyer, D.; Laufer, S. Acta. Cryst. E 2011, E67(9): 2292.<br />
6. Keck, P. R. W. E. F.; Schollmeyer, D.; Laufer, S. Acta. Cryst. E 2011, E67(9): 2417.<br />
031<br />
The actin cytoskeleton as potential antitumor target: Chondramide and its<br />
effects on breast cancer cells<br />
Förster F1; von Schwarzenberg K; Müller R2; Vollmar A<br />
1 Department of Pharmacy - Center for Drug Research, Pharmaceutical Biology, LMU Munich,<br />
Butenandtstaße 5-13, 81377, München<br />
2 Institut für Pharmazeutische Biotechnologie, <strong>Universität</strong> des Saarlandes, Postfach 15 11 50,<br />
66041, Saarbrücken”<br />
The actin cytoskeleton is a crucial component to maintain cellular homeostasis. It is<br />
important in processes like muscle contraction, trafficking of cellular compartments, mitosis,<br />
invasion and migration. Thus, actin-binding compounds like Chondramide A (Chon A), a<br />
cyclic depsipeptide of myxobacterial origin could be developed as potential anticancer<br />
therapeutics. Aim of this study was to characterize antitumoral effects of Chon A in<br />
mammary cancer cells in vitro.<br />
In fact we could show that Chon A inhibits proliferation of two breast tumor cell lines MCF-7<br />
and MDA-MB-231 in a low nanomolar range. The long term survival of these cell lines was<br />
impaired by Chon A as the clonogenic survival was also decreased by about 60 % (MDA-<br />
MB-231) and 80 % (MCF-7), respectively. To proof if the observed effects are due to actin<br />
overpolymerization, the Triton-X 100 soluble fraction was analyzed for its actin content and<br />
showed a time (24h – 72h) and dose dependent decrease, which suggested that Chon A<br />
overpolymerized actin to its filamentous form that is not soluble in the Triton-X-100 fraction.<br />
Clarifying the mechanisms underlying the antiproliferative effects of Chon A, we examined<br />
whether Chon A induces apoptosis and/or premature cellular senescence. Annexin V/PI<br />
costaining revealed a concentration dependent apoptotic cell death induced by 500 nM<br />
Chon A in both cell lines. Cleavage of PARP, a known substrate of caspase-3, detected by<br />
western blotting, supported the induction of apoptosis by Chon A, too. The induction of<br />
cellular senescence was cell line specific, as MCF-7 showed an increased β-galactosidase<br />
activity, compared to MDA-MB-231 which did not display this hallmark of senescence. The<br />
cdk-inhibitor p21, which is important for senescence induction, was also increased with<br />
Chon A 500 nM treatment in MCF-7 cells. These results suggest, that the inhibition of<br />
growth in MDA-MB-231 is only due to apoptosis induction, whereas in MCF-7 cells both<br />
processes apoptosis and senescence are involved. Furthermore, phosphorylation of<br />
histone γH2A.X, an established sign for DNA damage, treated with Chon A was observed<br />
in both cell lines. To this end, Chon A induces pronounced cell cycle arrest in G2-phase (40<br />
% versus 20 % in untreated cells).<br />
100 Poster
Our investigations reveal that Chon A is a potent inhibitor of breast cancer cell proliferation<br />
via induction of apoptosis and premature cellular senescence. Induction of senescence has<br />
not yet been reported for actin – binding drugs. Thus, further experiments will focus on the<br />
mechanism underlying senescence induction. One major tool will be PCR and micro-array<br />
technologies to possibly find new and valuable players and targets in senescence and<br />
growth arrest signaling.<br />
Acknowledgments: Supported by the DFG: FOR 1406 Vo 376/15-1<br />
032<br />
Phenylamino-substituted 5,11-dihydrodibenzo[a,d]cyclohepten-10-ones and<br />
11H-dibenzo[b,f]-oxepin-10-ones as novel p38 MAP kinase inhibitors<br />
Dorn, A. 1; Martz, K. E. 1; Laufer, S. A. 1<br />
1 Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-<strong>Universität</strong>, Auf der<br />
Morgenstelle 8, 72076 Tübingen<br />
The p38 MAP kinase is a key player in signalling pathways regulating the biosynthesis of<br />
pro-inflammatory cytokines like TNF-α and IL-1β. Small molecule p38 inhibitors suppress<br />
the production of these cytokines making p38 a promising drug target for novel antiinflammatory<br />
drugs.<br />
We recently reported a novel series of dibenzepinone inhibitors belonging to the class of so<br />
called linear binders [1]. Upon binding of the dibenzepinone inhibitor, the p38 MAP kinase<br />
undergoes a Gly110 flip. The glycine flip is a small conformational rearrangement in the<br />
hinge region of the p38 MAP kinase induced by the inhibitor and it provides selectivity for<br />
p38 over other kinases with less flexible, non-glycine residues at this position. Hence the<br />
carbonyl functionality of the dibenzepinone inhibitor is essential for the inhibitory activity<br />
and selectivity as it forms two hydrogen bonds towards Met109 and Gly110 in the hinge<br />
region.<br />
In this study, we report the design, synthesis, and SAR of novel N-substituted 11Hdibenzo[b,f]oxepin-10-ones<br />
and 5,11-dihydro-dibenzo[a,d]cyclohepten-10-ones as p38α<br />
inhibitors. The main aim was to conserve the key interaction: the bidentate hydrogen bond<br />
of the carbonyl oxygen of the inhibitor to the backbone NH of Met109 and the backbone NH<br />
of Gly110. Docking studies predicted alternative positions for the carbonyl oxygen within<br />
the scaffold of the inhibitor. Within these inhibitor structures, the carbonyl functionality was<br />
moved on the bridge side and the linker atoms X and Y were varied to obtain distinctive<br />
molecular geometries and additional interaction opportunities.<br />
Initial investigations of the inhibitory activities and structure-activity-relationships of<br />
phenylamino-substituted dibenzo[b,f]oxepin-10(11H)-one, 5,11-dihydro-(10H)-dibenzo[a,d]cyclohepten-10-one<br />
and dibenzo[b,f][1,4] oxazepin-11(10H)-one inhibitors for p38 MAP<br />
kinase were accomplished. The promising structural variations suggested by our docking<br />
experiments did not result in any novel compounds as active as the lead compound. While<br />
our initial hypothesis that substitution of a tricyclic scaffold would result in a favorable<br />
position for the carbonyl functionality was not supported by our data, we did identify some<br />
structural determinants that may be useful for the development of future p38 MAPK<br />
inhibitors.<br />
References:<br />
1. Laufer, S. A. et al.: J. Med. Chem. 2006, 49: 7912-7915.<br />
033<br />
Long-Range Intramolecular S → N Acyl Migration: A Study of the Formation of<br />
Native Peptide Analogues via 8-, 11-, 13-, 14-, 15-, and 16-Membered Cyclic<br />
Transition States<br />
Hansen, F. K. 1 2; Ha, K. 2; Chahar, M. 2; Monbaliu, J.-C. M. 2 3; Oliferenko, A. A. 2; Katritzky, A.<br />
R. 2<br />
1 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University of Düsseldorf,<br />
<strong>Universität</strong>sstr. 1, 40225 Düsseldorf, Germany. Email: finn.hansen@uni-duesseldorf.de<br />
2 Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville,<br />
FL 32611-7200, USA.<br />
3 Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience<br />
Engineering, Ghent University, B-9000 Ghent, Belgium.<br />
Native chemical ligation (NCL) is the most widely used chemoselective ligation technique<br />
based on a rearrangement concept nowadays. NCL involves the covalent condensation of<br />
a peptide-thioester with N-terminal cystein peptide, followed by S to N acyl shift to form a<br />
native amide linkage.[1] In recent years, various chemical ligation methodologies such as<br />
sugar-assisted ligation, nucleotide-based ligation and chemical ligation using click<br />
chemistry have been developed. The classical NCL method is limited to peptides<br />
possessing an N-terminal cysteine residue. Unfortunately, cysteine is a relatively rare<br />
amino acid (1.3% average content) and is not always available in a terminal position.<br />
Moreover, some amino acid-cysteine bonds such as Pro-Cys, Asp-Cys, Glu-Cys and Lys-<br />
Cys can be difficult to access by chemical ligation.[2]<br />
To overcome this requirement of a specifically placed cysteine residue one approach is the<br />
use of thiol ligation auxiliaries, but removable cysteine mimics can sterically hinder ligation<br />
and difficulties can arise at the stage of auxiliary removal.[3]<br />
A long-range S to N acyl migration methodology would avoid the requirement of a Nterminal<br />
cysteine residue. We have now studied the feasibility of chemical ligation of Sacylated<br />
cysteine peptides via 8-, 11-, 13-, 14-, 15-, and 16-membered cyclic transition<br />
states.[4,5] Our microwave-assisted methodology allows the synthesis of native peptides<br />
from non-terminal cysteine residues without the need of ligation auxiliaries.[4,5]<br />
Computational studies provide mechanistic evidence for the ligation process.[5] The impact<br />
of pH on the long-range S to N acyl migration is exemplified using a S-acyl tetrapeptide.[5]<br />
Acknowledgments: The authors gratefully acknowledge the Kenan foundation for financial support.<br />
References:<br />
1. Dawson, P. E. et al.: Science 1994, 266(5186): 776-779.<br />
2. Haase, C. and Seitz, O.: Eur. J. Org. Chem., 2009, (13): 2096-2101.<br />
3. Marinzi, C. et al.: Bioorg. Med. Chem. 2001, 9(9): 2323-2328.<br />
4. Hansen, F. K. et al.: Org. Biomol. Chem. 2011, 9(20): 7162-7167.<br />
5. Ha, K. et al.: J. Org. Chem., <strong>2012</strong>, 77(6): 2637-2648.<br />
034<br />
Synthesis and pharmacological evaluation of thiazole-featured pirinixic acid<br />
derivatives acting as dual 5-lipoxygenase and microsomal prostaglandin E2<br />
synthase-1 inhibitors<br />
Hanke, T. 1; Popella, S.-D. 2; Liening, S. 2; Dehm, F. 2; Lämmerhofer, M. 3; Werz, O. 2;<br />
Schubert-Zsilavecz, M. 1<br />
1Goethe University of Frankfurt am Main, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany<br />
2Friedrich Schiller University of Jena, Philosophenweg 14, 07743 Jena, Germany<br />
3Eberhard Karls <strong>Universität</strong> Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany<br />
Imbalances in the lipid signaling network contribute to the pathogenesis of a large number<br />
of human diseases. The metabolic fate of arachidonic acid (AA) plays a crucial role within<br />
this network and is associated with pathophysiological conditions such as inflammation,<br />
analgesia, asthma and cancer.<br />
The metabolic pathway of AA can be divided into two different ways: The formation of<br />
prostaglandins (PGs) catalyzed by cyclooxygenases (COXs) and the biosynthesis of<br />
leukotrienes (LTs) initialized by 5-lipoxygenase (5-LO). Non steroidal anti-inflammatory<br />
drugs (NSAIDs) and COX-2 selective inhibitors (coxibs) are the most wide-spread drugs in<br />
the therapy of inflammation. However, especially long-term use of these drugs is closely<br />
related to severe side effects such as gastrointestinal and renal complications (NSAIDs) or<br />
an increased cardiovascular risk (coxibs) due to the suppression of physiological relevant<br />
prostaglandins [1]. Consequently, new pharmacological strategies for anti-inflammatory<br />
therapy are urgently needed. One promising approach is the selective inhibition of<br />
downstream-acting enzymes such as the microsomal prostaglandin E2 synthase-1<br />
(mPGES-1), which catalyzes the formation of PGE2 from PGH2. PGE2 is the most<br />
prominent mediator in inflammatory pain. On the other hand, LTs produced by 5-LO are<br />
important inflammatory mediators which act as bronchoconstrictors and increase vascular<br />
permeability. The dual inhibition of 5-LO and mPGES-1 is considered as a novel strategy to<br />
avoid COX-related side effects such as the analgesic asthma syndrome and to maintain the<br />
physiological prostaglandin levels.<br />
The structural basis of the presented compounds is pirinixic acid, which is inactive on both,<br />
mPGES-1 and 5-LO. Especially the introduction of n-alkyl chains in α-position led to potent<br />
dual 5-LO / mPGES-1 inhibitors. Furthermore, a broad modification of the lipophilic<br />
backbone is tolerated with an equal or increased activity [2]. Herein we present a novel class<br />
of pirinixic acid derivatives featuring a thiazole-scaffold at the lipophilic backbone. The<br />
resulting thiazole-substituted derivatives show balanced dual inhibition of mPGES-1 and 5-<br />
LO with IC50 values mainly in the submicromolar range.<br />
References:<br />
1. Koeberle, A. et al.: Curr. Med. Chem. 2009, 16: 4274–4296.<br />
2. Koeberle, A. et al.: J. Med. Chem. 2008, 51: 8068–8076.<br />
035<br />
Optimization of transport conditions for the shipping of 3D human cornea<br />
constructs used for in vitro drug absorption studies<br />
Beißner N1; Reichl S1 1 Institut für Pharmazeutische Technologie, Technische <strong>Universität</strong> Carolo-Wilhelmina zu<br />
Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig<br />
In vitro data of transcorneal absorption behaviour of ophthalmic drugs may be obtained by<br />
permeation studies using either excised cornea taken from experimental animals or corneal<br />
cell culture models. In recent years we developed serum-free cultivated 3D cornea<br />
constructs (HC) based on immortalized human corneal cells exhibiting similar barrier<br />
properties as excised porcine and rabbit cornea [1]. In a prevalidation study we generated<br />
SOPs for HC cultivation and permeation procedures and transferred SOPs in two other cell<br />
Poster 101
culture labs. Furthermore, in this study using seven ophthalmic drugs we demonstrated that<br />
our cornea model exhibits lower intra- and inter-laboratory variances than excised animal<br />
cornea [2]. The wide use of validated cell culture models normally requires valid cultivation<br />
in few labs and standardized shipment without loss of their functionality. In the present<br />
study we evaluated optimal transport conditions for the shipping of our cornea construct.<br />
Human cornea constructs were cultivated under serum-free conditions on permeable<br />
polycarbonate filters (Transwells ®) using SV40 immortalized human keratocytes (HCK-Ca)<br />
and human corneal epithelial cells (HCE-T) as described before [1]. After finishing of<br />
cultivation HC were kept in medium under submerged conditions and stored in different<br />
transport containers for various time periods changing shipment parameters as temperature<br />
and CO2 content. Cell viability after simulated transport was evaluated via MTT testing.<br />
Barrier properties of HC expressed as epithelial integrity was estimated by transepithelial<br />
electrical resistance (TEER) measurements.<br />
Both storage temperature and CO2 content had great impact on viability and TEER after 24<br />
and 48 h of transport simulation. Storage at 4 °C and 20 °C without additional CO2 support<br />
resulted in decreased TEER values. In addition, the viability was diminished at 4 °C. In<br />
contrast, keeping cornea constructs at lowered temperature in 5% CO2 atmosphere yielded<br />
viable HC exhibiting constant TEER values. However, lowering CO2 content resulted in<br />
strongly decreased TEER values, which couldn’t be recovered after cultivation for additional<br />
24 h in incubator (37 °C, 5% CO2, humidified atmosphere). Sealing of storage container led<br />
to TEER values similar to those obtained for HC stored under incubator conditions at same<br />
temperature. The combination of sealed transport containers and lowered temperature (22<br />
– 24 °C) caused only marginal reduction of TEER values after 24 h storage. Various<br />
preparatory treatments of cell culture medium as pre-warming to 37 °C or 24 h equilibration<br />
in incubator atmosphere (5% CO2) before use seemed to have no significant influence on<br />
the resulting corneal barrier properties after shipment simulation. Additionally, the container<br />
material (polystyrene, polyamide) didn’t show significant impact.<br />
Our results suggest CO2 atmosphere and medium pH, respectively, as critical parameters<br />
during transport of HC. Thus, an optimized transport system should ensure constant CO2<br />
atmosphere during shipment. In the next step appropriate transport container will be<br />
developed to ensure these requirements. Furthermore, real time transport experiments<br />
have to prove the usefulness of our developed shipment SOP.<br />
Acknowledgments: We are grateful to the German Federal Institute for Risk Assessment (BfR), which<br />
funded this work under grant no. 3-1328-488.1.<br />
References:<br />
1. Hahne, M., Reichl, S., International Journal of Pharmaceutics, 2011, 416, 268-279<br />
2. Hahne, M. et al., Journal of Pharmaceutical Science (accepted and in press,) <strong>2012</strong>, Prevalidation of a<br />
human construct as an alternative to animal corneas for vitro drug absorption studies<br />
036<br />
Type-I-allergic diseases and a possible way to treat them: new clozapinederived<br />
Histamine H1-/H4-receptor-ligands and their pharmacological<br />
characterization<br />
Gobleder S. 1, Wittmann H.-J. 2, Elz S. 1 Straßer A. 3<br />
1 Department of Pharm./Med. Chemistry I, <strong>Universität</strong>sstr. 31, 93040 Regensburg<br />
2 Faculty of Chemistry and Pharmacy, <strong>Universität</strong>sstr. 31, 93040 Regensburg<br />
3 Department of Pharm./Med. Chemistry II, <strong>Universität</strong>sstr. 31, 93040 Regensburg<br />
The number of patients that suffer from allergic rhinitis and conjunctivitis (the so called<br />
“hay-fever”) increases every year, a trend that indicates the need of good pharmaceutical<br />
treatment. A very common and well-tried therapy is the combined p.o. and local application<br />
of “antihistamines”, drugs with antagonistic effects at H1R, such as cetirizine and azelastine<br />
to name only a few. Recent research results pointed out, that not only the H1R but also the<br />
H4R is involved in type-I-allergic diseases. [1] Good therapeutic response could be<br />
achieved in the acute murine asthma model with a combined application of mepyramine<br />
(H1-antagonist) and JNJ7777120 (H4-antagonist). [2] These results suggest that addressing<br />
both, H1R and H4R, could be a new therapy-option of hay-fever and other type-I-allergic<br />
diseases like histamine induced itch or inflammatory asthma.<br />
Screening compounds with good affinity to H1R and H4R, we choose the antipsychotic drug<br />
clozapine (1) which has high antagonistic affinity to H1R and moderate agonistic affinity to<br />
H4R. [3] Soft structure modifications, e.g. exchanging the 5-nitrogen by a 5-oxygen and<br />
shifting the 8-chlorine to position 7 lead to an optimized clozapine-derivative (2) with<br />
increased agonistic affinity at H4R and constant high antagonistic affinity to H1R. [4] The<br />
aim of the project was to get a closer insight into structure-activity relationships at H1R and<br />
H4R and, according to our results, to convert the agonism at H4R into an antagonism or<br />
inverse agonism by modifying the clozapine-molecule in different ways.<br />
In a first part of the project we reduced the 5H-dibenzo[b,e][1,4]diazepine-structure of<br />
clozapine to an “open” (E)-N-((phenyl)methylene)aniline structure and varied the kind and<br />
position of substituents on both aromatic rings. Examples of the synthesized compounds<br />
are shown in (3), (4) and (5). Since our compounds showed, compared to clozapine, a<br />
decreased affinity to hH1R and hH4R, we continued synthesizing compounds with<br />
dibenzo[b,f][1,4]oxazepine-structure and added new non-halogenide substituents to this<br />
optimized clozapine derivatives (6), (7) and (8). All compounds were tested at the guinea<br />
pig ileum in presence of histamine and in a competition binding assay at hH1R and hH4R.<br />
For selected compounds ((6), (7) and (8)) we performed a GTPγS assay for hH2R, hH3R<br />
and hH4R to get information about receptor-selectivity and potency. First data revealed<br />
selectivity towards hH1R and/or hH4R for all tested compounds. To explain structure-activity<br />
relationships on a molecular level, molecular modelling studies at appropriate models of<br />
hH1R and hH4R were performed.<br />
R 1<br />
R 2<br />
N N<br />
X<br />
N<br />
R1 Cl,R2 H,X NH 1<br />
R 1 H,R 2 Cl,X O 2<br />
R 1<br />
R 2<br />
N N<br />
N<br />
R1 R2 H,R3<br />
R3<br />
Cl 3<br />
R1 Cl,R2 R3 H 4<br />
R1 H,R2 Cl,R3 NH2 5<br />
R 1<br />
R 2<br />
pA2 pKi pKi<br />
gpH1R hH1R hH4R<br />
1 n.d. 8,6 5,9<br />
2 7,6 7,8 7,0<br />
N N<br />
O<br />
N<br />
NH2<br />
R1 Cl,R2 H 6<br />
R 1 H,R 2 Cl 7<br />
R1 R2 H 8<br />
3 6,6 6,1 4,7<br />
4 6,9 6,0 4,9<br />
5 5,0 4,1 6,6<br />
6 8,9 8,2 6,1<br />
7 7,9 7,7 6,5<br />
8 n.d. 8,0 5,1<br />
References:<br />
1. Thurmond, R.L. et al.: Nature Reviews Drug Discovery 2008, 7 : 41-53.<br />
2. Deml, K.-F. et al.: Mol. Pharmacol. 2009, 76 : 1019-1030.<br />
3. Appl, H. et al.: Nauyn-Schmiedeberg’s Arch. Pharmacol. <strong>2012</strong>, 385(2) : 145-170.<br />
4. Smits, R. A. et al.: J. Med. Chem. 2006, 49 : 4512-4516.<br />
037<br />
Optimization of capillary electrophoresis-based analysis of 5-lipoxygenase<br />
metabolites.<br />
Abromeit H; Schaible A M; Werz O, Scriba G K E<br />
Department of Pharmaceutical/Medical Chemistry, Friedrich-Schiller-<strong>Universität</strong>, Philosophenweg<br />
14, 07743 Jena, Germany<br />
A new sensitive method using α-cyclodextrin-modified micellar electrokinetic<br />
chromatography has been developed to separate and quantify arachidonic acid metabolites<br />
of the lipoxygenase pathways in human polymorphonuclear leukocytes, i.e. leukotriene B4,<br />
6-trans-leukotriene B4, 6-trans-12-epi-leukotriene B4, 5(S)-hydroxy-6-trans-8,11,14-ciseicosatetraenoic<br />
acid, 12(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, and 15(S)hydroxy-6-trans-8,11,14-cis-eicosatetraenoic<br />
acid. The electrophoresis system was<br />
optimized with regard to the pH, boric acid, SDS and α-cyclodextrin concentration as well<br />
as separation voltage and temperature using a three level resolution IV fractional factorial<br />
design and a five level circumscribed central composite design. The optimized conditions<br />
included 80 mM sodium borate buffer, pH 10.07, containing 16.6 mM sodium dodecyl<br />
sulfate and 15 mM α-cyclodextrin, using a separation voltage of 12.5 kV at 23 °C.<br />
Sensitivity was enhanced employing head-column field amplified sample stacking which<br />
resulted in limits of quantification between 30 and 50 ng/mL and limits of detection between<br />
10 and 17 ng/mL after solid phase extraction of the lipoxygenase products. The method<br />
was validated according to the recommendations of the International Conference on<br />
Harmonization and applied to the determination of the lipoxygenase metabolites in<br />
polymorphonuclear leukocytes upon stimulation with Ca2+-ionophore A23187 and<br />
arachidonic acid. Robustness was confirmed using a three level resolution IV fractional<br />
factorial design. In conclusion, the novel method is suitable for the analysis of various<br />
arachidonic acid metabolites produced by living cells and may be used for evaluation of<br />
lipoxygenase inhibitors.<br />
038<br />
Carnosol and Carnosic acids from Salvia officinalis inhibit microsomal<br />
Prostaglandin E2 Synthase-1<br />
Bauer, J1., Kuehnl, S. 2, Rollinger, J. M. 2, Scherer, O. 4, Northoff, H. 3, Stuppner, H. 2, Werz,<br />
O. 4, Koeberle, A. 4<br />
1 Department for Pharmaceutical Analytics, Pharmaceutical Institute, University of Tuebingen,<br />
Tuebingen, Germany);<br />
2 Institute of Pharmacy / Pharmacognosy and Center for Molecular Biosciences Innsbruck,<br />
University of Innsbruck, Innsbruck, Austria<br />
3 Institute for Clinical and Experimental Transfusion Medicine, University Medical Center,<br />
Tuebingen, Germany<br />
4 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University of Jena, Jena,<br />
Germany<br />
Prostaglandin E2 (PGE2), the most relevant eicosanoid promoting inflammation and<br />
tumorigenesis, is formed by cyclooxygenases (COXs) and PGE2 synthases from free<br />
arachidonic acid. Preparations of the leaves of Salvia officinalis are commonly used in folk<br />
medicine as an effective antiseptic and anti-inflammatory remedy and possess anticancer<br />
activity. Here, we demonstrate that a standard ethyl acetate extract of S. officinalis<br />
efficiently suppresses the formation of PGE2 in a cell-free assay by direct interference with<br />
microsomal PGE2 synthase (mPGES)-1. Bioactivityguided fractionation of the extract<br />
yielded closely related fractions that potently suppressed mPGES-1 with IC50 values<br />
between 1.9 and 3.5 µg/ml. Component analysis of these fractions revealed the diterpenes<br />
carnosol and carnosic acid as potential bioactive principles inhibiting mPGES-1 activity with<br />
IC50 values of 5.0 µM. Using a human whole-blood assay as a robust cell-based model,<br />
carnosic acid, but not carnosol, blocked PGE2 generation upon stimulation with<br />
lipopolysaccharide (IC50 9.3 µM). Carnosic acid neither inhibited the concomitant<br />
biosynthesis of other prostanoids [6-keto PGF1a, 12(S)-hydroxy-5-cis-8,10-transheptadecatrienoic<br />
acid, and thromboxane B2] in human whole blood nor affected the<br />
activities of COX-1/2 in a cell-free assay. Together, S. officinalis extracts and its ingredients<br />
carnosol and carnosic acid inhibit PGE2 formation by selectively targeting mPGES-1. We<br />
conclude that the inhibitory effect of carnosic acid on PGE2 formation, observed in the<br />
physiologically relevant whole-blood model, may critically contribute to the antiinflammatory<br />
and anticarcinogenic properties of S. officinalis.<br />
039<br />
Investigation of the degradation of the model tetrapeptide Gly-Phe-Asp-GlyOH<br />
at alkaline pH by capillary electrophoresis including identification of the<br />
degradation products by CE-ESI-MS<br />
Brückner, C. 1; Scriba, G. K. E. 1<br />
1 Friedrich Schiller University, Department of Pharmaceutical and Medicinal Chemistry, School of<br />
Pharmacy, Philosophenweg 14, 07743 Jena, Germany<br />
Asparagine and aspartic acid are prone to chemical degradation such as deamidation,<br />
isomerization, and enantiomerization. [1-3] Enantiomerization and isomerization proceed<br />
via the formation of an aminosuccinimidyl intermediate (Asu). Hydrolysis of the Asu peptide<br />
yields native Asp and β-Asp (iso-Asp) peptides favouring the β-Asp peptide over the Asp<br />
peptide by a ratio of 3-5:1. [1,2] Differentiation of the Asp isomers by mass spectrometry<br />
can be achieved by the ratio of the complementary b- and y-ions generated during peptide<br />
fragmentation. [4] In contrast, diastereomeric Asp peptides, i.e. peptides containing an L-<br />
Asp or D-Asp moiety could not be distinguished based on their MS spectra.<br />
102 Poster
The degradation of the model tetrapeptide Gly-L-Phe-α-L-Asp-GlyOH was investigated at<br />
80°C and 25°C in sodium borate buffer pH 10. Separation and quantification of the<br />
degradation products was achieved using a validated capillary electrophoresis assay. Peak<br />
identification was obtained by CE-ESI-MS experiments and by co-injection of synthetic<br />
reference compounds. Due to the differences in the MS spectra the degradation products<br />
could be clearly assigned to the α-Asp or β-Asp linkage. While for α-Asp peptides b2-ion<br />
formation competes with y2-ion formation, in case of β-Asp peptides the intensity of the y2ion<br />
considerably exceeded that of the b2-ion. At 80°C a rapid decline of the starting peptide<br />
was observed yielding a mixture of α-Asp and β-Asp as well as L-Asp and D-Asp containing<br />
peptides. The β-aspartyl peptides exceeded the α-Asp linkage after 288 h by a ratio of<br />
4.0:1. Furthermore, the peptides containing the D-Asp moiety were favored over the L-Asp<br />
residues. At 25°C the tetrapeptide Gly-L-Phe-α-L-Asp-GlyOH proved to be relatively stable<br />
compared to 80°C. After an incubation interval of 2350 h 88 % of the starting peptide was<br />
still present in the incubation solution. Nevertheless, formation of the enantiomerization and<br />
isomerization products could be demonstrated yielding β-Asp residues in higher amounts<br />
as the respective α-D-Asp peptide.<br />
References:<br />
1. Aswad DW, CRC Press. 1995, 279.<br />
2. Geiger T, Clarke S, J. Biol. Chem. 1987, 262(2):785-794.<br />
3. Manning MC, Patel K, Borchardt RT, Pharm. Res. 1989, 6(11):903-918.<br />
4. Lehmann, WD. et al., Protein Sci. 2000, 9(11):2260-2268.<br />
040<br />
Evaluation of the cytoprotective effects of individual and combined<br />
constituents of the combination drug STW 5<br />
Hoser S1; Winkelmann V1; Kelber O2, Abdel-Aziz H2, Weiser D2, Nieber K1 1 University of Leipzig, Institute of Pharmacy, Talstr. 33, 04103 Leipzig, Germany<br />
2 Steigerwald Arzneimittelwerk GmbH, Scientific Department, Havelstr. 5, 64295 Darmstadt,<br />
Germany<br />
STW 5 (Iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of<br />
functional dyspepsia and irritable bowel syndrome (IBS). We investigated the cytoprotective<br />
action of individual herbal extracts of STW 5 in CaCo-2 cells. The CaCo-2 cells were<br />
incubated with the herbal extracts and stimulated with LPS (10 ng/ml) for 2 hours. Possible<br />
synergistic effects were determined by isobologram analysis of LPS-induced LDH release<br />
as a measure for cell toxicity using a commercially available kit.<br />
STW 5 (500.5 µg/ml) significantly inhibited LPS-induced LDH release by 74 %.<br />
Comparable effects were found with lemon balm, whereas in equivalent concentrations to<br />
STW 5 the effects of Iberis amara, peppermint, chamomile, angelica and milk thistle were<br />
less potent. Caraway and celandine had stimulatory effects. Isobolgram analysis indicated<br />
that a 2 h exposure to peppermint and Iberis amara (ratio 0.5/0.5) resulted in a synergistic<br />
effect of 28 %. Less pronounced synergy was observed using the component ratio<br />
0.75/0.25. An additive effect was present in the ratio 0.25/0.75. Application of peppermint<br />
together with milk thistle (ratio 0.5/0.5) showed ratio-independent synergistic effects of 13-<br />
16 % whereas the combined application of chamomile and Iberis amara had ratioindependent<br />
additive effects. The combination of chamomile and angelica (ratio 0.5/0.5)<br />
exerted an additive effect, the ratio 0.75/0.25 showed an antagonistic effect which<br />
increased using the ratio 0.25/0.75.<br />
Our observations confirm that the components of STW 5 contribute to different extents to its<br />
cytoprotective effect in CaCo-2 cells and clearly indicate that the individual herbal extracts<br />
can contribute synergistically, additive and antagonistically to the overall effects of STW 5.<br />
The type of effect was determined in some cases by the ratio of the constituents.<br />
041<br />
Novel synthetic analogues of toxiferine I and their pharmacological<br />
characterization at α7 nAChRs, muscle-type nAChRs, and the allosteric<br />
binding site of muscarinic M2 receptors<br />
Zlotos, D.P1; Gündisch, D2;Tränkle, C3; Holzgrabe, U4; Jensen, A.A5 1 The German University in Cairo, Dept. of Pharmaceutical Chemistry, New Cairo City, 11835<br />
Cairo, Egypt<br />
2 Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawai'i at Hilo, Hilo,<br />
HI 96720, USA<br />
3 Pharmacology and Toxicology Section, Institute of Pharmacy, Rheinische Friedrich-Wilhelms-<br />
University, D-53121 Bonn, Germany<br />
4Institut für Pharmazie and Lebensmittelchemie, <strong>Universität</strong> Würzburg, Am Hubland, 97074<br />
Würzburg, Germany<br />
5 Department of Medicinal Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100<br />
Copenhagen, Denmark<br />
Toxiferine I is a major bisquaternary alkaloid of calabash-curare and Strychnos species. [1]<br />
Its semi-synthetic diallyl analogue alcuronium (Alloferin ®) is in clinical use as muscle<br />
relaxant. Both compounds display high binding affinities for the muscle-type nAChRs from<br />
the electric organ of Torpedo californica. [2] In addition to their neuromuscular-blocking<br />
activity, toxiferine I and alcuronium have been reported to be moderately potent antagonists<br />
at α7-nAChRs [3], and to possess moderate to high affinity for the allosteric binding site of<br />
M2-mAChRs, respectively. [4] Interestingly, the structurally related caracurine V analogues<br />
show a different pharmacological profile displaying considerably lower affinity for the<br />
muscle-type nAChRs, [2] higher antagonistic activity at α7-nAChRs, [3] and similar<br />
allosteric potency at M2-mAChRs, [4] when compared to the correspondingly substituted<br />
bisnortoxiferine analogues. Here, we describe the synthesis and pharmacological<br />
evaluation of novel bisquaternary toxiferine I analogues with structurally modified side<br />
chains. In particular, a series of non-symmetrical derivatives lacking only one alcohol group<br />
(1a-c), and of symmetrical compounds lacking both alcohol functions (2a-c) have been<br />
examined. The findings help delineate the structural requirements for the action at different<br />
AChRs and design ligands selective for one of the subtypes.<br />
Poster 103<br />
OH<br />
H<br />
N<br />
R<br />
N H<br />
H<br />
H<br />
H N<br />
R<br />
N<br />
H<br />
toxiferine I, R Me<br />
alcuronium, R allyl<br />
OH<br />
2Cl -<br />
H<br />
N H H<br />
H<br />
O<br />
O<br />
H<br />
H<br />
H N<br />
H<br />
N<br />
R +<br />
+ R<br />
N<br />
caracurinium V analogues<br />
R Me, allyl, pNO 2 -benzyl<br />
2X -<br />
1<br />
R<br />
H<br />
2 N<br />
R<br />
N H<br />
H<br />
H<br />
H N<br />
2<br />
R<br />
N<br />
H<br />
CH 3<br />
2X -<br />
1a c R 1 -CH 2 OH, R 2 Me, allyl, pNO 2 -benzyl<br />
2a c R 1 -CH 3 , R 2 Me, allyl, pNO 2 -benzyl<br />
Acknowledgments: Bonn University, Mechthild Kepe, Würzburg University, Elfriede Ruckdeschel, Matthias<br />
Grüne<br />
References:<br />
1. Battersby, A.R.; Hodson, H.F. Q. Rev. Chem. Soc. 1960, 14, 77-103.<br />
2. Zlotos, D.P. et al.: Bioorg. Med. Chem., 2004, 12, 6277-6285.<br />
3. Jensen, A.A.; Zlotos, D.P.; Liljefors, T. J. Med. Chem. 2007, 50, 4616-4629.<br />
4. Zlotos, D. P. et al.: J. Med. Chem. 2004, 47, 3561-3571.<br />
042<br />
Ligand based approaches to reveal the active conformation of highly flexible<br />
azecine derivative<br />
Enzensperger C1, Robaa D2, Schulze M3, Lehmann J1 1 Institute for Pharmacy, Department of Medicinal Chemistry, Philosophenweg 14, D-07743 Jena,<br />
Germany<br />
2 Institute for Pharmacy, Department of Medicinal Chemistry, Wolfgang-Langenbeck Str. 4 D-06120<br />
Halle-Wittenberg, Germany<br />
3 Temporarily at ‘Institute de Genomique Fonctionnelle-CNRS’, Montpellier cedex 05, France<br />
Annulated azecine derivatives are highly flexible compounds and some of them exert very<br />
high affinity for dopamine and serotonin receptors. In the crystal structure there are<br />
obviously two enantiomers present, which give rise to the question about the eutomer,<br />
dystomer and the active conformation (in figure 1 LE300 (1) is shown). We succeeded to<br />
synthesize, screen and X-ray an enantiopure chiral benzindolo azecine (2).[1]<br />
Figure 1: Both enantiomers of LE300 (1) present 1:1 in the crystal (left). The absolute<br />
configuration and conformation of the chiral eutomer (2) (right)<br />
This crystal structure is the only conformation that allows a rigid alignment with<br />
conformationally constricted and stereochemically defined templates like asenapine (3) and<br />
ecopipam (4). In that case, the indole moiety can only be superimposed with the aromatic<br />
ring not containing a chloro subtitutent.<br />
O<br />
Cl<br />
1<br />
H<br />
N<br />
H<br />
N CH3<br />
3<br />
H<br />
N CH3<br />
S<br />
CH3<br />
Cl<br />
HO<br />
N<br />
H<br />
N CH3<br />
Figure 2: Rigid alignment of the X-ray structure of the chiral eutomer (2) with asenapine (3)<br />
and ecopipam (4)<br />
This hypothesis is also supported by observed SARs in the class of benzazepine- and<br />
benzindolo- azecine derivatives. The most important structural features of these<br />
compounds seem to be the shape and the site of protonation of the basic nitrogen. We are<br />
now also able to explain, why azecine derivatives with a different annulation pattern are<br />
inactive.[2]<br />
References:<br />
1. Robaa, D.et al., J Med Chem 2011, 54: 7422-6.<br />
2. Schulze, M. et al., Bioorg Med Chem 2009, 17: 6898-907.<br />
2<br />
1<br />
N CH3<br />
CH3<br />
S<br />
043<br />
Design, synthesis, biological testing and SAR of 5-Pyridinyl-2-thioimidazole<br />
derivatives as selective JNK3-inhibitors over p38α<br />
S. Klos1, F. M. Muth1, M. Goettert / S. Bauer / K. Bauer1, S. Laufer1 1 Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-<br />
University, Auf der Morgenstelle 8, 72076 Tuebingen, Germany<br />
Many inflammatory conditions are driven by both p38α and JNK3 MAPK.[1] To differentiate<br />
the contribution of each kinase, selective inhibitors are necessary. For p38α this might be<br />
the case[2], but as of today only few selective JNK3 inhibitors with good ADME (Absorption,<br />
Distribution, Metabolism, Excretion) properties are available.<br />
JNK-kinases are c-jun NH2-terminal serine/threonine mitogen activated kinases which are<br />
Cl<br />
HO<br />
4<br />
N CH3<br />
Cl<br />
H<br />
O<br />
2<br />
H<br />
N CH3
mainly activated by cytokines and environmental influences.[3][4] JNK3-kinases are<br />
believed to play a central role in the pathology of neurologic diseases such as<br />
cerebrovascular accidents, Parkinson's and Alzheimer's disease.[5] Therefore it has<br />
become an attractive and valid drug target.<br />
5-Pyridinyl-2-thioimidazole derivatives are known as p38α-inhibitores.[6] Due to the<br />
sequential and steric similarity of p38α- and JNK3-kinase, we assumed to gain active and<br />
selective JNK3-inhibitores by introducing different substitution patterns for R1, R2, and R3.<br />
At the edge of the hydrophobic region II in JNK3-kinase, there are Asn152 and Gln155,<br />
whereas Asp112 and Asn152 are shown in p38α-kinase.[3] In order to create repulsion<br />
between the Asp112 and p38α, we introduced anionic substituents at the aminopyridine<br />
scaffold.<br />
Furthermore, we tried to hit the Asp112[3] by introducing carboxylic moieties at the<br />
imidazole nitrogen. In order to target the conserved but steric diverse Arg107 and<br />
Asn194[3], we synthesised carboxylic substituents linked by a sulfide at R1 resulting in 50<br />
nM inhibition of JNK3 with about 10 fold selectivity against p38α.<br />
References:<br />
1. Kamenecka T.,: J. Biol. Chem. 2009, 284(19): 12852-61.<br />
2. Koebler S. C., et al., Nat. Chem. Biol. <strong>2012</strong>, 8(2): 141-3.<br />
3. Scapin G. L., et al., Chem. Biol. 2003, 10: 705-712.<br />
4. Johnson G. L., et al., Science 2002, 298: 1911-1912.<br />
5. Bogoyevitch M. A., Trends Mol. Med. 2005, 11: 232-239.<br />
6. Laufer S. A., et al., J. Med. Chem. 2008, 51(14), 4122-4149.<br />
044<br />
8-Substitued Indolo[3,2-f][3]Benzazecines Receptor Antagonists: Synthesis<br />
and Activity of Racemic and Enantiopure Derivatives<br />
Robaa, D. 1; Enzensperger, C. 2; AbulAzm, S.; 3 Lehmann, J. 2<br />
1Institute for Pharmacy, Department of Medicinal Chemistry, Wolfgang-Langenbeck Str. 4, 06120<br />
Halle<br />
2Institute for Pharmacy, Department of Medicinal Chemistry, Philosophenweg 14, 07743 Jena,<br />
Germany<br />
3Department of Medicinal Chemistry, Faculty of Pharmacy, El Khartoum Square 1, 21521<br />
Alexandria, Egypt<br />
Partially hydrogenated indolo[3,2-f][3]benzazecines represent a structurally novel class of<br />
dopamine receptor antagonists, distinguished by nanomolar affinities as well as their<br />
preference for the receptors of the D1 family [1,2].<br />
Derivatives of the lead indolo[3,2-f][3]benzazecine derivative LE 300 (1), substituted at<br />
position 8 with three different residues, namely methyl, hydroxymethyl and carboxylic acid,<br />
were prepared. The 8-methyl and 8-hydroxymethyl derivatives were obtained as the<br />
separated R- and S- enantiomer as well as the racemic mixture, while the amino acid<br />
derivative could only be obtained in a racemized form. The separate enantiomers showed<br />
significantly different affinities; the (8S)-methyl and (8R)-hyroxymethyl derivatives where the<br />
substituents point below the reference plane of the indolo[3,2-f][3]benzazecine scaffold<br />
were markedly more active than their enantiomeric counterparts. The racemic 8-carboxy<br />
derivative was highly interesting since it showed a pronounced selectivity for the D5receptor,<br />
even against D1.<br />
11<br />
10<br />
12<br />
13 N<br />
H<br />
1<br />
1<br />
(LE300)<br />
9<br />
8<br />
CH3 N 6<br />
7<br />
15 5<br />
1<br />
2<br />
3<br />
N<br />
H<br />
R<br />
CH3 N<br />
Derivatives with substituents<br />
pointing below the projection<br />
place<br />
References:<br />
1. Hoefgen, B. et al.: J. Med. Chem. 2006, 49, 760–769.<br />
2. Witt, T.; Hock, F. J.; Lehmann, J. J. Med. Chem. 2000, 43, 2079–2081.<br />
N<br />
H<br />
R<br />
CH3 N<br />
Derivatives with substituents<br />
pointing above the projection<br />
place<br />
045<br />
Influence of API and Surfactant Selection on Nanosuspensions Properties<br />
Comparing Two Different Top Down Methods<br />
Lestari, Maria L.A.D1; Müller, R.H1; Möschwitzer, J.P 1 2 *<br />
1 Freie <strong>Universität</strong> Berlin, Institute of Pharmacy, Biopharmaceutics & NutriCosmetics, Kelchstrasse<br />
31, 12169 Berlin, Germany<br />
2 Pharmaceutical Development, Abbott GmbH &Co.KG, Ludwigshafen, Germany<br />
* Corresponding author: Telephone: 030-838-50703, email: info@janmoeschwitzer.de<br />
High Pressure Homogenization (HPH) and wet ball milling (WBM) are two well known top<br />
down methods for producing nanosuspension. Both methods are based on different<br />
diminution principles. These different principles may affect the stabilization of the<br />
nanosuspension as well as process efficiency. Besides these two factors also the active<br />
pharmaceutical ingredients (API) and surfactants are playing an important role for the<br />
particle size reduction process. Amphotericine B and resveratrol were used as model drugs<br />
in this study. Various surfactants (anionic, cationic, nonionic, polymeric and combination)<br />
were tested at specified concentration level. Nanosuspensions were produced with low<br />
energy wet ball milling using yttrium stabilized milling beads (diameter 0.5 mm) with electric<br />
stirring system at 900 rpm for 72h. HPH was done by two pre-milling cycles at 150 and 500<br />
bar and 20 cycles at high pressure of 1500 bar. The nanosuspensions were characterized<br />
using photon correlation spectroscopy (PCS), laser diffractometry (LD), light polarized<br />
microscopy and laser doppler anemometry (zeta potential). Results showed that LE-WBM<br />
of resveratrol produced nanosuspensions with mean particle sizes below 500 nm and a<br />
narrow size distribution. In case of WBM only TPGS led to a mean particle size larger than<br />
1000nm. In contrast, HPH of resveratrol resulted in general in larger particle sizes above<br />
1000 nm. Only nanosuspensions stabilized with sodium cholate had particle sizes below<br />
1000 nm. For amphotericine B, some nanosuspensions had similar particle sizes<br />
irrespectively of the employed technique. Sodium cholate and a combination of sodium<br />
cholate-poloxamer were identified as most suitable stabilizers for both techniques. In<br />
conclusion, both models behaved differently regarding the optimal stabilizer applied. When<br />
LE-WBM technique was used, stabilizer selection was less important since most of the<br />
nanosuspension produced had small mean particle sizes. For the HPH method, the<br />
selection of the right stabilizer system in combination with the API was more important.<br />
046<br />
inSARa: SAR interpretation by network navigation<br />
Wollenhaupt, S. 1; Baumann, K. 1<br />
1 Institut für Medizinische und Pharmazeutische Chemie, Technische <strong>Universität</strong> Braunschweig,<br />
Beethovenstr. 55, D-38106 Braunschweig, Germany<br />
Structure-Activity-Relationship (SAR) analysis of small molecules is a fundamental and<br />
challenging task in drug discovery. The knowledge of this relationship between structural<br />
determinants and bioactivity is of high value for the medicinal chemist e.g. in the leadoptimization<br />
process or de-novo-design. For the elucidation of SARs the recognition of<br />
molecular similarities is a crucial step due to the assumption that molecules with similar<br />
properties or features exhibit similar biological activities [1].<br />
The question arising from this is how to define molecular similarity ideally. Fingerprintbased<br />
similarity is an established and widely used concept [2]. Fingerprints represent<br />
molecules in the form of binary vectors which show the presence or absence of<br />
substructures or other structural features. Although fingerprints are fast to compute and well<br />
suited for processing large data sets, the interpretation of the results is difficult and the<br />
concept appears less intuitive to a medicinal chemist. A promising and more intuitive<br />
alternative is the maximum common substructure (MCS), the largest common substructure<br />
of a pair of molecules. One drawback is the high computational complexity which prevents<br />
large-scale applications such as the analysis of high-throughput-screening data. Moreover,<br />
exact atom matching is a limiting factor which decreases the scaffold hopping potential and<br />
hinders the identification of structurally less similar bioisosteric substructures.<br />
To tackle these limitations our in-house approach inSARa (intuitive networks for Structure-<br />
Activity-Relationships analysis) combines MCS similarity with the reduced graph (RG)<br />
concept [3]. Reduced graphs promise a higher degree of abstraction as they provide a<br />
conceptual representation of physicochemical properties and pharmacophoric features of a<br />
molecule by reducing groups of atoms to single pseudoatoms while retaining the topology<br />
between these functional units.<br />
inSARa networks are derived by combining RG-MCS-calculations and iterative super- and<br />
substructure searches. By linking the molecules with bioactivity data the networks can be<br />
used for SAR interpretation. This opens up the possibility to display molecules sharing<br />
common features as a function of bioactivity. Furthermore, inSARa networks are of great<br />
value for the detection of so called activity cliffs [4], where minor structural changes lead to<br />
a major change in bioactivity. Even though no bioactivity information is used to generate the<br />
inSARa networks, they can also be used for compound classification and bioactivity<br />
prediction.<br />
References:<br />
1. Johnson, M. A., Maggiora, G. M.: Concepts and applications of molecular similarity (Wiley) 1990.<br />
2. Wawer, M. et al.: J. Med. Chem. 2008, 51(19): 6075-6084.<br />
3. Gardiner, E. J. et al.: J. Chem. Inf. Model. 2007, 47(2): 354-366.<br />
4. Stumpfe, D., Bajorath, J.: J. Med. Chem. <strong>2012</strong>, 55(7): 2932-2942.<br />
047<br />
Dibenzosuberones as highly potent p38α MAP kinase inhibitors: Efficient use<br />
of a hydrophobic pocket to improve activity<br />
Fischer S1, Wentsch H1 , Karcher S1, Dorn A1, Laufer S1 1 Institute of Pharmacy, Department of Pharmaceutical Chemistry, University of Tuebingen, Auf der<br />
Morgenstelle 8, 72076, Germany<br />
The dysregulation of inflammatory cellular signal transduction cascades is known to be<br />
involved in the development of many diseases including chronic inflammatory and<br />
autoimmune diseases like rheumatoid arthritis (RA) and inflammatory bowel disease (IBD).<br />
p38α MAP kinase is a key enzyme regarding the regulation of the pro-inflammatory<br />
cytokines TNF-α and IL-1β and is therefore a promising target for the treatment of many<br />
inflammatory diseases. 1 2<br />
By now, successful development of p38α MAP kinase inhibitors as drugs has been<br />
compromised by poor selectivity and / or high toxicity. 3 The ATP-binding pocket of kinases<br />
is highly conserved, offering almost no possibilities to achieve selectivity. Therefore our<br />
efforts concentrated on exploiting unique features of the enzyme. One of them is the ability<br />
to perform the so called “glycine-flip”, which is already enforced by the suberone template.<br />
Another one is the “selectivity-pocket”. This region is located adjacent to the ATP-bindingpocket.<br />
Figure 1: Proposed binding mode of dibenzosuberones and essential interaction sites<br />
(Hinge Region, Selectivity Pocket, Deep Pocket)<br />
In contrast to other kinases, the “selectivity-pocket” is accessible because of the small<br />
gatekeeper residue Thr106. As it is mostly surrounded by lipophilic residues, we had to<br />
104 Poster
design moieties fitting exactly in this location. By extending these moieties towards the<br />
deep pocket, we were able to achieve high potency in the p38α MAP kinase enzyme assay<br />
down to an IC50 of 1nM.<br />
References:<br />
1. Badger et al., J. Pharmacol. Exp. Ther. 1996, 279, 1453-1461.<br />
2. Boehm et al., J. Med. Chem. 1996, 39, 3929-3937.<br />
3. Dominguez et al., Curr. Opin. Drug Discov. Devel. 2005, 8, 421-430.<br />
048<br />
Different column classification systems applied to calixarene- and<br />
resorcinarene-bonded stationary phases<br />
Chamseddin, C.; Jira, T.<br />
Institute of Pharmacy, Pharmaceutical/Medicinal Chemistry, <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-University of<br />
<strong>Greifswald</strong>, Friedrich-Ludwig-Jahn-Strasse 17, <strong>Greifswald</strong> D-17487, Germany.<br />
The recently dramatic increase in the number of reversed phase columns could be an<br />
advantage of this liquid chromatographic technique. However, due to the insufficiency of<br />
information that is available from manufacturers in terms of the exact functionality of their<br />
phases, it is problematic to determine which could be the best column for a given situation.<br />
There is not a “super column” that will work best for all applications. As a result, there have<br />
been several attempts to develop testing strategies to characterize column chemistries.<br />
These column classification systems are typically based on one or more test mixtures to<br />
identify the so called “column parameters”. Three different systems, originally developed for<br />
reversed phases, were applied to evaluate the chromatographic properties of the<br />
calixarene- and resorcinarene-bonded stationary phases. These are: Tanaka, USP, and<br />
Snyder/Dolan approaches [1,2,3]. Although all column classification systems aim to<br />
evaluate the “more or less” same characteristics, each system uses different test mixtures<br />
in different chromatographic conditions. All of that make the resulted “column parameters”<br />
using these systems deviate from one to another. Calixarene- and resorcinarene-bonded<br />
stationary phases are reversed phases; however they show, depending on the analytes,<br />
some additional interactions, since there steric, polar and ionic properties are different<br />
compared to those of conventional alkyl-bonded phases.<br />
The following table summarizes the parameters used by each system.<br />
System<br />
Tanaka<br />
USP Snyder/Dolan<br />
Parameter<br />
Hydrophobicity<br />
Hydrophobicity<br />
Hydrophobicity<br />
KPB<br />
KEB<br />
H<br />
Hydrophobic<br />
interactions<br />
Hydrophobic selectivity<br />
αCH2 = KPB/ KBB<br />
Hydrophobic selectivity<br />
αCH2 = KEB/ KT<br />
Steric<br />
Shape selectivity<br />
___ Steric resistance<br />
interactions αT/O = KT/ KO<br />
S*<br />
Hydrogen bonding Activity toward bases Hydrogen-bond<br />
Hydrogen<br />
capacity<br />
KAm and TAm<br />
acidity<br />
bonding<br />
αC/P = KC/ KP<br />
A<br />
and<br />
ionic interactions<br />
Acidic ion-exchange<br />
αB/P = KB/ KP (pH 2.7)<br />
Hydrogen-bond<br />
basicity<br />
Chelation<br />
B<br />
Total ion-exchange<br />
TQ<br />
Cation-exchange<br />
αB/P = KB/ KP (pH 7.6)<br />
activity<br />
C<br />
Am: Amitriptyline; B: Benzylamine; BB: n-butylbenzene; C: Caffeine; EB: Ethylbenzene; O: oterphenyl;<br />
P: Phenol; PB: n-pentylbenzene; Q: Quinizarin (1,4-dihydroxyanthraquinone); T:<br />
Triphenylene.<br />
Acknowledgments: Chamseddin, C. has a PhD scholarship from the DAAD and the Syrian ministry of high<br />
education.<br />
References:<br />
1. Kimata, K. et al.: J. Chromatogr. Sci.1989, 27: 721-728.<br />
2. Bidlingmeyer, B. et al.: Pharmacopeial Forum. 2005, 31(2): 637-645.<br />
3. Snyder, L.R. et al.: J. Chromatogr. A. 2004, 1057: 49-57.<br />
049<br />
Structure-based discovery of novel allosteric modulators addressing the<br />
chemokine receptor CXCR3<br />
Tschammer, N. 1; Bernat, V. 1; Schmidt, D. 2; Kolb, P. 2<br />
1 Friedrich Alexander-University Erlangen/Nürnberg, Department of Chemistry and Pharmacy,<br />
Chair of Medicinal Chemistry, Schuhstr. 19, 91052 Erlangen, Germany<br />
2 Philipps University Marburg, Pharmaceutical Chemistry, Wilhelm-Roser-Str. 2, 35032 Marburg,<br />
Germany<br />
Small soluble proteins chemokines and their G protein-coupled receptors are the key<br />
mediators of immune system function in health and disease. Irregularities in the regulation<br />
and response of the chemokine receptor CXCR3 are associated with numerous pathologies<br />
including autoimmune diseases (e.g. multiple sclerosis, rheumatoid arthritis, and psoriasis),<br />
cancer, vascular disease and transplant rejection. Allosteric modulators that inhibit this<br />
attractive pharmacological target could be thus used to treat or prevent a range of<br />
pathologies.<br />
We began the search for novel allosteric modulators addressing the chemokine receptor<br />
CXCR3 with a structure-based virtual screen [1]. Recently resolved X-ray structure of<br />
CXCR4 [2] served us as a template on which a homology model of CXCR3 was build. The<br />
homology model of CXCR3 was further refined using a large library of known CXCR3<br />
ligands in the combination with a set of non-binders. The homology model of CXCR3<br />
characterized by the best docking score for recent clinical candidate AMG487 was chosen<br />
as a basis for virtual screening. Nearly one million of “lead-like” compounds from ZINK, a<br />
free database of commercially available compound for virtual screening, were docked.<br />
From the 500 best ranked after visual inspection, a set of eight compounds was subjected<br />
to biological testing measuring [ 35S]GTPγS incorporation. To facilitate the identification of<br />
novel CXCR3 ligands we synthesized also a tritium labelled allosteric modulator of CXCR3<br />
RAMX3, which was based on an 8-azaquinazolinone core of AMG487 [3]. The study of<br />
allosteric modulators namely needs to differentiate between the ligand affinity toward the<br />
receptor from the cooperativity exhibited towards the endogenous agonist like chemokine,<br />
as the two properties are not correlated [4]. We discovered six novel chemical scaffolds<br />
modulating the function of CXCR3. Interestingly, although we refined the homology model<br />
with negative allosteric modulators of CXCR3, we identified also positive (PAM) and silent<br />
(SAM) besides negative (NAM) allosteric modulators, indicating the delicate balance<br />
between a molecules' function as SAM, NAM or PAM.<br />
Our investigations demonstrate that a carefully refined homology model can provide a<br />
valuable template for the discovery of novel pharmacophores for allosteric modulators even<br />
for such challenging targets as chemokine receptors.<br />
Acknowledgments: N.T. was supported by the German Research Foundation (DFG, TS 287/2-1).<br />
References:<br />
1. Kolb, P. et al.: P. Natl. Acad. Sci. USA 2009, 106(16): 6843-6848.<br />
2. Wu, B. Et al.: Science 2010, 330(6007): 1066-1071.<br />
3. Bernat, V. et al.: ChemMedChem <strong>2012</strong>, Early View, doi: 10.1002/cmdc.<strong>2012</strong>00184.<br />
4. Keov, P. et al.: Neuropharmacology 2011, 60 (1) : 24-35.<br />
050<br />
Structural variations on the thiazolone derivative C06 as potent 5-lipoxygenase<br />
inhibitor<br />
Lill A1, Barzen S1, Rödl C B1, Steinhilber D1, Hofmann B1, Stark H1 1Goethe University, Institute of Pharmaceutical Chemistry, Frankfurt am Main, Germany<br />
(h.stark@zafes.de)<br />
Arachidonic acid released from cytoplasmic membrane upon external stimuli is the source<br />
of one important group of inflammatory mediators: the leukotrienes (LTs). The key enzyme<br />
of their biosynthesis is the iron-containing, heme-free 5-lipoxygenase (5-LO). LTs play a<br />
pivotal role in inflammation, allergic disorders, asthma, cardiovascular diseases and cancer.<br />
Up to now, only two LT-interfering drugs are marketed with limited success: the LT-receptor<br />
antagonist Montelukast (Singulair®) and the iron chelating 5-LO inhibitor Zileuton (Zyflo®).<br />
The need to discover new active ligands for anti-leukotriene therapy is still urgent [1,2].<br />
Based on early virtual screening [3] and medium-throughput screening we previously<br />
identified 5-(4-methoxybenzylidene)-2-p-tolylthiazol-4(5H)-one, C06, as a promising new<br />
direct inhibitor of 5-LO [4,5].<br />
S<br />
N<br />
O<br />
C06 IC = 300nM<br />
50<br />
Poster 105<br />
H3C O<br />
CH 3<br />
R<br />
S<br />
N<br />
O<br />
Our previous studies pointed out that 5-benzylidene-2-phenyl-thiazolinones are a new class<br />
of 5-LO inhibitors. By variation of the substitution pattern we designed and synthesized<br />
novel derivatives of C06 leading to a promising new inhibitors.<br />
The compounds were either prepared by one-pot domino reaction using 2-sulfanylacetic<br />
acid and the corresponding benzaldehyd and benzonitrile or in a two-step synthetic<br />
procedure. All compounds were tested for their 5-LO inhibitory efficiency using a cell-free<br />
and whole-cell (PMNL cells) based assay systems [4,5].<br />
Kindly supported by LOEWE Schwerpunkte LIFF, OSF, NeFF, Anwendungsorientierte<br />
Arzneimittelforschung (Fraunhofer IME) and EU COST Actions CM1103, BM1107, BM0806.<br />
References:<br />
1 M. Peters-Golden and W. Henderson, N. Engl. J. Med. 2007, 357: 1841-1854<br />
2 O. Werz, O and D. Steinhilber, Pharmacol. Ther. 2006, 112: 701-718<br />
3 G. Schneider et al., ChemMedChem 2008, 3: 1535-1538<br />
4 B. Hofmann et al., J. Med. Chem. 2011, 54: 1943-1947<br />
5 S. Barzen et al., Bioorg. Med. Chem. <strong>2012</strong>, 20: 3575-3583<br />
051<br />
Myrrhinil-Intest � chamomile component affects contraction and morphology of<br />
rat ileum/jejunum ex-vivo preparations in a concentration dependent manner<br />
Vissiennon, C. 1; Raszek, M. 1; Goos, K.-H. 2; Nieber, K. 1<br />
1 University of Leipzig, Institute of Pharmacy, Talstraße 33, 04103 Leipzig, Germany<br />
2 REPHA GmbH Biologische Arzneimittel, Alt-Godshorn 87, 30855 Langenhagen, Germany<br />
Myrrhinil-Intest � is a prescribed phytotherapy for the treatment of inflammatory intestinal<br />
diseases. However, how its functional effects are exerted has not yet been fully elucidated.<br />
One of its active ingredients, lyophilised extract of chamomile flowers, was tested on the<br />
acute inflammation of rat ileum/jejunum preparations, experimentally induced by 2,4,6trinitrobenzene<br />
sulfonic acid (TNBS, 10 mM, 30 min). At low concentrations tested (8.75-70<br />
µg/mL) chamomile exhibited a basal tone enhancement of both the untreated controls and<br />
TNBS-treated preparations. At higher concentrations this trend subsided at first with the<br />
inflamed segments only (140 µg/mL), with subsequent inhibition of basal tone for both<br />
untreated and TNBS-treated preparations (210-280 µg/mL).<br />
Histological analysis revealed that the concentration-dependent effect correlated with a<br />
protective function of chamomile from TNBS-induced morphological damage (100 mM, 30<br />
min). Thus whilst pre-treatment with 70 µg/mL exhibited protective function, higher levels of<br />
chamomile (280 µg/mL) did not. 10 mM TNBS also appeared to have 1.6-fold reduction<br />
effect on acetylcholine (ACh)-induced contraction (EC50 0.9 µM vs. 0.58 µM for untreated<br />
controls). This could be reversed with chamomile pre-treatment. However, this observation<br />
was not statistically significant.<br />
The importance of a chamomile treatment concentration-dependence is further<br />
underscored by the fact that TNBS-inflamed segments also exhibit increased sensitivity to<br />
chamomile inhibition of ACh-induced contractions (IC50 106 µg/mL vs. 175 µg/mL for<br />
untreated controls), particularly in the higher concentration range.<br />
052<br />
Four novel antifungal compounds from the Baltic Sea cyanobacterium<br />
Anabaena sp.<br />
Bui, T. H. 1; Wray, V. 2; Nimtz, M. 2; Fossen, T. 3; Schröder, G. 4; Wende, K. 1; Mundt, S. 1<br />
1Institute of Pharmacy, Department of Pharmaceutical Biology, <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-University,<br />
Friedrich-Ludwig-Jahnstr. 17, D-17487 <strong>Greifswald</strong><br />
2Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38214 Braunschweig, Germany<br />
3Department of Chemistry and Centre for Pharmacy, University of Bergen, N-5007 Bergen, Norway<br />
R
4 Friedrich Loeffler Institute of Medical Microbiology, University Medicine <strong>Greifswald</strong>, Martin-Luther-<br />
Str. 6, D-17475 <strong>Greifswald</strong><br />
18 Vietnamese and 4 German cyanobacterial strains were screened with the agar diffusion<br />
assay for their antifungal activity to human pathogenous fungi. It was found that Anabaena<br />
sp. strain Bio 33 collected from The Baltic Sea (Rügen Island, Germany) exhibited an<br />
excellent and specific antifungal activity with inhibition zones from 21 to 32 mm against<br />
Candida albicans, Candida krusei, Candida maltosa, Aspergillus fumigatus, Microsporum<br />
gypseum, Trichophyton rubrum and Mucor sp. No cytotoxic activity against the human<br />
bladder carcinoma cell line 5637 (IC50 > 500 μg/mL) and no antibacterial activity against<br />
Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa<br />
were observed. Bioassay-guided fractionation of the 50% methanolic water extract by silica<br />
gel column chromatography followed by repeated reversed phase HPLC (column<br />
LiChrospher RP-18, gradient of MeOH in H2O with 0.05% TFA) resulted in four white<br />
amorphous solids. The structures were elucidated by 1D ( 1H) and 2D spectra (COSY,<br />
TOCSY, NOESY, HSQC, HSQC edited TOCSY and gHMBC), HR-ESI-MS and confirmed<br />
by amino acid analysis and sugar analysis. Spectroscopic data analysis afforded an<br />
unambiguous sequence of R.CHO(S1).CHOH.CONH-Thr(1)-Thr(2)-Thr(3)-HOTyr(4)-<br />
DeThr(5)-Gln(6)-Gly(7)-ThrNMe(8)(S2)-GlnCOOH(9) in which S1 is arabinose-(3-1)galacturonic<br />
acid, S2 is a mannose and R is a aliphatic residue. The four compounds<br />
distinguish from their cyclic/linear core and the presence of chlorine in the aliphatic part.<br />
Culture optimization to enhance the yield of these antifungal peptides is currently<br />
developed.<br />
053<br />
Synthesis of inhibitors of glutathione peroxidases for reversal of cancer drug<br />
resistance<br />
Wilde F1; Lemmerhirt H1; Emmrich T1; Schulz R1; Bednarski P J1, Link A1 1 <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong> University of <strong>Greifswald</strong>, Friedrich-Ludwig-Jahn-Str. 17, 17489 <strong>Greifswald</strong>,<br />
Germany<br />
Glutathione peroxidases (GPx) contribute substantially to cell protection by reducing<br />
reactive oxygen species (ROS) such as H2O2 and organic hydroperoxides and hence<br />
regulating the redox status of cells to prevent cell death [1]. While H2O2 plays a vital role in<br />
the induction of apoptosis [2], over-expression of GPx has been observed in cancer cells<br />
that have acquired resistance to anticancer agents.<br />
Following a combinatorial chemistry approach, a small library of test candidates was built<br />
starting from a lead compound identified by a molecular modeling project [3].<br />
deletion<br />
of<br />
substituent<br />
replacement<br />
of<br />
basic<br />
heterocycle<br />
To determine the activity of the potential inhibitors, a recently established microtiter method<br />
based on the inhibition of bovine erythrocyte GPx was applied, which allowed for a rapid<br />
screening of compounds.<br />
Here we present the general synthesis of the lead compound and the library members.<br />
Characterization of this class of compounds is hampered by the formation of isomers<br />
leading to complex NMR spectra. A rational for this finding is discussed.<br />
References:<br />
1. Takebe, G. et al.: J. Biol. Chem. 2002, 277(43): 41254-8.<br />
2. Lopez-Lazaro, M.: Cancer Lett. 2007, 252(1): 1-8.<br />
3. Schulz, R.: Diploma thesis, 2008.<br />
adjustment<br />
of<br />
spacer length<br />
modification<br />
of<br />
substitution<br />
pattern<br />
variation<br />
of<br />
aromatic system<br />
054<br />
Structural Insights into Retinal Guanylylcyclase / GCAP-2 Interaction<br />
Determined by Cross-Linking and Mass Spectrometry<br />
Jens Pettelkau1; Thomas Schröder2; Christian Ihling1; Björn E. S. Olausson2; Knut Kölbel1; Christian Lange2; Andrea Sinz1 1 Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther<br />
University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle-Saale, Germany<br />
2 Department of Technical Biochemistry, Institute of Biochemistry and Biotechnology, Martin-Luther<br />
University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle-Saale, Germany<br />
Two isoforms of retinal guanylylcylase (ROS-GC 1 and 2), localized in photoreceptor cells,<br />
are responsible for the synthesis of cGMP, the second messenger in phototransduction [1].<br />
ROS-GC 1 and 2 are regulated in a Ca2+-dependent manner from the intracellular site by<br />
guanylylcyclase-activating proteins (GCAPs) [2]. GCAPs are hydrophobic N-terminally<br />
myristoylated proteins containing four EF-hand motifs, which are responsible for Ca2+ sensitivity [3]. Genetic disorders in GCAP genes or parts of the regulatory regions in ROS-<br />
GC genes lead to different retinopathies [4]. Therefore, a detailed knowledge of the<br />
complex formation between GCAPs and ROS-GC is crucial for the development of novel<br />
drugs.<br />
Interactions between a ROS-GC 1 peptide (amino acids 965-981 from the catalytic domain<br />
[5]) and GCAP-2 were investigated applying two complementary types of cross-linking<br />
chemistry. For mapping larger distances, the homobifunctional amine-reactive cross-linker<br />
bis(sulfosuccinimidyl)glutatrate (BS2G) was used. To determine spatially close interactions,<br />
the unnatural photoreactive amino acid photo-leucine that is activated by UV-A radiation,<br />
was incorporated into ROS-GC peptides. After the cross-linking reaction the cross-linked<br />
GCAP-2/GC peptide complexes were enzymatically digested and analysed by highresolution<br />
mass spectrometry.<br />
In the presence of Ca2+, several intramolecular cross-links were identified within N-<br />
terminally myristoylated GCAP-2, all of which were in agreement with the published NMRstructure<br />
(pdb 1JBA) of non-myristoylated GCAP-2. The C-terminal part of GCAP-2, which<br />
is not resolved in the NMR-structure, seems to be highly flexible as indicated by the<br />
numerous intramolecular cross-links within GCAP-2. Analysis of cross-linking samples<br />
without Ca 2+ yielded a limited number of intramolecular cross-linking products of GCAP-2<br />
itself. Several cross-links between the N-terminus of GC peptide and different lysines of<br />
GCAP-2 were identified both in the presence and absence of Ca 2+. After photo-crosslinking,<br />
a limited number of cross-links were obtained pointing to a structurally well-defined<br />
interaction between GCAP-2 and the GC peptide in the presence of Ca 2+. In the absence of<br />
Ca 2+ the structure of GCAP-2 appeared to be much more flexible as indicated by a large<br />
number of diverse photo cross-linking products between GCAP-2 and GC peptide. Based<br />
on the distance contraints imposed by the cross-links it was possible to create a model of<br />
the complex between myristoylated GCAP-2 and the GC peptide that is formed in the<br />
presence of Ca 2+ (Figure 1).<br />
Figure 1: Structural model of the GCAP-2/GC peptide complex. The backbone of the most<br />
representative GC peptide is colored red along with GCAP-2, which is shown as a light blue<br />
cartoon. Cross-linking sites in GCAP-2 obtained from photoaffinity labeling, which were<br />
used for modeling, are shown as light green spheres. Cross-linking sites obtained with<br />
BS2G, which were used for modeling, are shown as light orange spheres. The reactive<br />
sites in the GC peptide are shown as dark green (photo-Leu at positions 5 and 13) and<br />
brown (Tyr-1) spheres.<br />
References:<br />
1. Yang R.B. et al.: Proceedings of the National Academy of Science of the United States of America 1995,<br />
92(2): 602-606.<br />
2. Sharma R.K.: Mol.Cell. Biochem. 2010, 334(1-2): 3–36.<br />
3. Olshevskaya E.V. et al.: J. Biol. Chem. 1997, 272(22): 14327-14333.<br />
4. Sato M. et al.: Graefes Arch. Clin. Exp. Ophthalm. 2005, 243(3): 235-242.<br />
5. Duda T. et al.: Biochemistry 2005, 44(19): 7336-7345.<br />
055<br />
Triterpenes from the fungus Piptoporus betulinus<br />
Alresly, Z. 1; Lindequist, U. 1; Lalk, M. 1; Porzel, A. 2; Arnold, N. 2 ; Wessjohann, L. A. 2<br />
1Institute of Pharmacy, <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-University, Friedrich-Ludwig-Jahn-Straße 17, D-17489<br />
<strong>Greifswald</strong>, Germany<br />
2Leibniz – Institute of Plant Biochemistry, Department of Bioorganic Chemistry (NWC), Weinberg 3,<br />
06120 Halle (Saale , Germany<br />
The mushroom Piptoporus betulinus (Bull. : Fr.) P. Karst. (Polyporaceae), birch polypore,<br />
growth as trunk parasit and saprophyt on Betula pendula Roth. and B. pubescens Ehrh.<br />
(Betulaceae). Fruit bodies of this fungus are used in the European ethnomedicine for the<br />
treatment of cancer and stomach diseases. The mushroom is also known as fungus of the<br />
“iceman” from the Copper Age found in 1991, who carried P. betulinus fruiting bodies<br />
attached to his clothing on his journey in the Alps [1].<br />
The phytochemical knowledge about this mushroom and the justification of its<br />
ethnomedicinal use are limited. Aim of our studies was the phytochemical examination of<br />
extracts prepared from the fruiting bodies of collected mushrooms and the investigation of<br />
isolated compounds for antimicrobial activity.<br />
Fractionation of the ethyl acetate extract led to the isolation of a new bioactive triterpene<br />
with a lanostane basic structure. By the help of spectroscopic methods it could be identified<br />
as 3β-acetoxy-16 hydroxy-24- oxo-5a- lanosta- 8(9) diene -30- oic acid (Fig. 1). In addition,<br />
seven known triterpenes, polyporenic acid C, betulinic acid, betulin, ergosterol peroxide,<br />
9,11dehydroxyergosterol peroxide, 24-methylene-3-oxo-lanosta-8-en-21-oic acid, and<br />
fomefficinic acid, could be isolated and identified by comparison with data from the<br />
literature. All isolated compounds were tested for antimicrobial activity against some Grampositive<br />
and Gram-negative bacteria as well as against some microscopic fungi. The new<br />
triterpene, polyporenic acid C, and fomefficinic acid showed antimicrobial activity against<br />
Staphylococcus aureus.<br />
106 Poster<br />
CH 3<br />
CH3 OH<br />
CH3 CH3 CH3 O O<br />
H3C CH3 Fig. 1: 3β-acetoxy-16α hydroxyl-24-oxo-5α-lanosta-8(9) diene-30-oic acid<br />
References:<br />
1. Lindequist, U.; Niedermeyer, T.H.J.; Jülich, W.D.: eCAM 2005, 2(3): 285–299.<br />
056<br />
A1-barrigenol and rare 17,22-seco oleanolic acid glycosides from the leaves of<br />
Pittosporum angustifolium.<br />
Bäcker, C. 1; Jenett-Siems, K. 2; Siems, K. 3; Wurster, M. 1; Bodtke, A. 4; Chamseddin, C. 4;<br />
Crüsemann, M. 1a; Lindequist, U. 1<br />
1 Department of Pharmaceutical Biology, Institute of Pharmacy, <strong>Ernst</strong> <strong>Moritz</strong> <strong>Arndt</strong> University<br />
<strong>Greifswald</strong>, Friedrich-Ludwig-Jahn-Str. 17, 17489 <strong>Greifswald</strong>, Germany<br />
2 Department of Pharmaceutical Biology, Institute of Pharmacy, Free University of Berlin, Königin-<br />
O<br />
OH<br />
O<br />
CH 3
Luise-Str. 2+4, 14195 Berlin, Germany<br />
3 AnalytiCon Discovery GmbH, Hermannswerder Haus 17, 14473 Potsdam, Germany<br />
4 Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, <strong>Ernst</strong> <strong>Moritz</strong> <strong>Arndt</strong><br />
University <strong>Greifswald</strong>, Friedrich-Ludwig-Jahn-Str. 17, 17489 <strong>Greifswald</strong>, Germany<br />
1a Present address: Kekulé Institute of Organic Chemistry and Biochemistry, Rheinische Friedrich<br />
Wilhelms University Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany<br />
Pittosporum angustifolium LODD. (Pittosporaceae), a small tree known colloquially as<br />
"gumby gumby", occurs in inland areas of most states of Australia. Especially the leaves<br />
are used by Aboriginals for various medical applications [1]. Recently beneficial effects of<br />
“gumby gumby” as a supportive agent for cancer treatment have been observed [2].<br />
Phylogenetic studies described P. angustifolium as a distinct species, which previously was<br />
considered as a variety of P. phillyreoides (syn. P. phillyraeoides; var. microcarpa) [1].<br />
Phytochemical investigation of the leaves collected in Queensland, Australia, resulted in the<br />
isolation of triterpene saponins known as eryngioside I [3] and new compounds named<br />
pittangretosides A-I. Their structures were elucidated and characterized by NMR,<br />
HRESIMS, hydrolysis and CD measurement. Eight saponins were identified as<br />
A1-barrigenol derivatives whereas two compounds exhibit an unusual 17,22-seco backbone<br />
of oleanolic acid.<br />
Acknowledgments: We sincerely thank Dr. Rudolf Kunze, Berlin, Germany, for the stimulation of this work<br />
and his continuous support as well as Dr. Cornelia Krasser, Yepoon, Australia and Mr. Klaus von<br />
Gliszczynski for the collection and provision of the plant material. We also thank all of the colleagues at the<br />
Institute who were involved in supporting this work.<br />
References:<br />
[1] Cayzer, L.W., Crisp, M.D., Telford, R.H., Aust. Syst. Bot. 2000, 13: 845-902.<br />
[2] Patent, 2009. WO 2009/037225 A2; DE 10 2007 044 094 A1.<br />
[3] Zhang, Z. et al., Phytochemistry 2008, 69: 2070-2080.<br />
057<br />
Homology Modeling of the Kinase Domain of DLK<br />
Hasenpusch, D. 1; Oetjen, E. 2; Lemcke, T. 1<br />
1 Institut für Pharmazie, <strong>Universität</strong> Hamburg, Bundesstr. 45, 201146 Hamburg, Germany<br />
2 Institut für Klinische Pharmakologie und Toxikologie, <strong>Universität</strong>sklinikum Eppendorf, Martinistr.<br />
52, 20246 Hamburg, Germany<br />
An interesting subgroup of protein kinases are the mixed-lineage kinases (MLKs). They<br />
belong to the tyrosine kinase-like kinases (TKL) and show phylogenetic similarity to both<br />
typical serine/threonine kinases and tyrosine kinases. Functional they are a member of the<br />
serine/threonine kinases.<br />
Two interesting members of this group are the DLK (dual leucine zipper kinase) and LZK<br />
(leucine zipper kinase), which form homodimers mediated via hydrophobic interactions of<br />
their leucine zippers. Acting as a MAP3K DLK was shown to result in the activation of the<br />
downstream kinases p38 and JNK (jun-N-terminal kinase). In the insulin producing β-cells<br />
within the pancreatic islets DLK was shown to mediate apoptosis induced by<br />
immunosuppressants and by the cytokine TNFα and to inhibit insulin gen transcription [1–<br />
4]. Given the importance of maintaining β-cell function and mass to prevent the<br />
pathogenesis of diabetes mellitus, the inhibition of DLK represents an interesting and novel<br />
target for developing potential drugs for the therapy of this chronic disease.<br />
So far no crystal structure data of the DLK is available. As a starting point for a structure<br />
based project to identify potential selective DLK inhibitors, we constructed threedimensional<br />
homology models of the kinase domain of DLK. Suitable templates, which<br />
represented different conformations of the kinases, namely the open and closed<br />
conformation of the activation loop, were found in the phylogenetic neighborhood of DLK.<br />
The actual model building was accomplished with MODELLER [5] using standard<br />
procedures and subsequent loop refinement by the loop model routine. Models were<br />
afterwards evaluated using a workflow that combines individual scorings of different<br />
evaluation algorithms (e.g. PROCHECK, ProSA, ERRAT, MODELLER) in a rank-by-rank<br />
ConsensusScoring [6]. Top ranking models were submitted to molecular dynamic<br />
simulations of at least 2 ns, to test for general stability and to allow assuming their final<br />
three-dimensional shape. These models will be used in a future virtual screening<br />
experiment for the search of DLK inhibitors.<br />
References:<br />
[1] Plaumann, S. et al., Mol. Pharmacol. 2008, 73, 652–659.<br />
[2] Klimpel, C., Boerchers, S., Oetjen, E., Arch Pharmacol. 2009, 379, 37-37.<br />
[3] Oehmen, M.-J. et al., Arch. Pharmacol. 2010, 381, 36-36.<br />
[4] Phu do, T. et al. Cell Signal. 2011, 23, 344-353.<br />
[5] Sali, A., Blundel, T. L., J. Mol. Biol. 1993, 234, 779–815.<br />
[6] Kruggel, S., Lemcke, T., Arch. Pharm. (Weinheim) 2009, 342, 327–332.<br />
058<br />
ChemicalToolBoX in action!<br />
Grüning, B. A. 1; Lucas, X. 1; Chbeib, M. 1, Patel, H. 1, Günther, S. 1<br />
1 Institute of Pharmaceutical Sciences, Hermann-Herder-Strasse 9, 79104, Germany<br />
ChemicalToolBox is a set of tools integrated into a workflow-management system to enable<br />
researchers easy-to-use, reproducible, and transparent access to cheminformatic libraries<br />
and drug discovery tools. It includes standard applications for similarity and substructure<br />
searches, clustering of compounds, prediction of properties and descriptors, filtering, and<br />
many other tools that range from drug-likeliness classification to fragmentation and<br />
fragment-merging.<br />
ChemicalToolBox is based on open-source software, web-accessible, freely available, and<br />
easily expandable. It can be downloaded and easily deployed locally or on a cluster.<br />
Here we present some use-cases for the ChemicalToolBox that we have developed in our<br />
lab to demonstrate its capabilities.<br />
As a first application we have created ChemicalBoX, a comprehensive library of about 40<br />
million unique small compounds collected from freely-accessible repositories. The<br />
compounds included in ChemicalBoX have been filtered by physico-chemical properties<br />
and clustered by similarity to allow fast navigation throughout millions of compounds<br />
belonging to many different chemical classes.<br />
In a similar project, called PurchasableBoX, a comprehensive library consisting of several<br />
millions of purchasable small compounds was assembled. Both libraries are unique and, to<br />
the best of our knowledge, are the largest freely-available libraries.<br />
With the fragmentation tool included in ChemicalToolBox, we can build specialized<br />
fragment libraries using databases of compounds as input. Those collections provide the<br />
foundation for calculating focused Mass Spectrometry libraries and for enabling fast and<br />
efficient screening of large amounts of MS data. Furthermore, these fragments can be the<br />
input of a workflow we have created for fragment-based drug design: it merges the<br />
fragments according to predefined synthetic route rules to form new promising compounds<br />
that enlarge the known chemical space.<br />
The use cases presented here provide an insight into the capabilities of ChemicalToolBoX.<br />
By combinations of the various tools many more powerful applications can be designed.<br />
059<br />
The influence of saponins on the cell membrane-cholesterol<br />
Böttger, S. 1, Melzig, M. F. 1<br />
1 Institute of Pharmacy – Pharmaceutical Biology, Freie <strong>Universität</strong> Berlin, Königin-Luise-Str. 2+4,<br />
14195 Berlin, GERMANY<br />
As secondary plant compounds with amphiphilic properties saponins possess surface<br />
activity. They can interact with physical surfaces such as those of aqueous solutions and<br />
lower the surface tension [1]. They also interfere with biologic surfaces such as cell<br />
membranes. The latter case may result in haemolysis and cytotoxicity as a consequence of<br />
perturbation of the membrane integrity at higher saponin concentrations.<br />
The mechanism behind this saponin characteristic is not yet completely understood, but<br />
there are different indications that the membrane-cholesterol is decisively involved in this<br />
process. Saponins have been described to bind/to complex cholesterol [1, 2, 3]. As<br />
cholesterol is known to carry out a cell membrane stabilizing function [4], a loss of the<br />
cholesterol itself or the loss of the ability to carry out this function will lead to cell membrane<br />
instabilities and/or membranolysis.<br />
We developed a cell culture model using ECV-304 urinary bladder carcinoma cells<br />
involving 3H-marked cholesterol and investigated the influence of different types of<br />
saponins on the cell membrane-cholesterol.<br />
References:<br />
1. Böttger, S., Hofmann, K., Melzig, M. F.: Bioorg. Med. Chem. <strong>2012</strong>, 20(9): 2822-2828.<br />
2. Glauert, A. M., Dingle, J. T., Lucy, J. A.: Nature 1962, 196(4858): 953-955.<br />
3. Schlösser, E.: Can. J. Physiol. Pharmacol. 1969, 47(5), 487-490.<br />
4. Meyer, T. et al.: Biospektrum, <strong>2012</strong>, 18(2), 142-145.<br />
060<br />
Synthesis and testing of novel 2,4-thiazolidinedione derivatives as inhibitors of<br />
the enoyl-ACP-reductase InhA of Mycobacterium tuberculosis<br />
Vogel, S. 1; Wagner, S., Waltenberger, C., Sotriffer, C.A., Schirmeister, T. 2<br />
1 Institute of Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, 97074<br />
Würzburg , Germany<br />
2 Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55128 Mainz,<br />
Germany<br />
InhA is part of the fatty acid synthase complex type II (FAS II) and catalyzes the reduction<br />
of enoyl-acyl-[acyl-carrier-protein] derivatives of the chain length up to 56 carbons using the<br />
essential cofactor NADH.[1] When inhibited, unsaturated fatty acids accumulate, and the<br />
formation of the cell wall is disturbed.<br />
Computational methods have been applied in a step-wise fashion in order to search for new<br />
InhA inhibitors. Using ligand- and target-based approaches three pharmacophore models<br />
were defined. For screening purposes the ‘drug-like’ subset of the ZINC database and the<br />
vendor database distributed by CCG were used. Hits of the pharmacophore screening were<br />
subdued to hierarchical filtering and visually inspected. The most suitable candidates were<br />
selected for docking procedures with the programs MOE Dock and Autodock 3.0. For<br />
validation purposes, known active compounds were included in the docking simulations.<br />
The most promising hits were chosen for further studies and experimental testing of their<br />
inhibitory activity.<br />
For activity testing two enzyme assays based on the decrease of the absorption or<br />
fluorescence intensity upon reduction of the cofactor NADH were established and validated<br />
using triclosan as positive control. The enzyme InhA was overexpressed in E. coli and<br />
purified.[2] The substrate 2-trans-octenoyl-coenzyme A was synthesized via the mixed<br />
anhydride method.[3]<br />
Based on the results of the screening of a large library of compounds and subsequent<br />
docking experiments the most promising hits with 2,4-thiazolidinedione structure were<br />
chosen for further optimization.<br />
References:<br />
1. X.Hong et al.: Biomacromolecules 2004, 5: 1052-1065.<br />
2. Parikh et al.: Biochemistry 1999, 38: 13623-13634.<br />
3. Goldman/Vagelos: J.Biol.Chem. 1961, 10: 2620-2623.<br />
061<br />
3-Hydroxypyrroles or Pyrrolin-3-ones as novel p38 MAP kinase inhibitors<br />
Freitag, A. 1; Laufer, S. A. 1<br />
1 Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-<br />
<strong>Universität</strong>, Auf der Morgenstelle 8, 72076 Tübingen, Germany<br />
p38 MAP kinase controls a plethora of cellular responses to extracellular stimuli such as<br />
cellular stress. The kinase is part of a signalling pathway resulting in the biosynthesis of<br />
pro-inflammatory cytokines like IL-1β and TNF-α. Since their levels are elevated in<br />
chronical inflammatory diseases inhibition of p38 is a promising therapeutic target.<br />
The small molecule inhibitor SB-203580 is one of the first known selective inhibitors of p38.<br />
Poster 107
Due to X-ray crystal strucure analysis, the interactions of the pyridinylimidazole to the<br />
binding site of the kinase are established [1].<br />
Figure 1: left side: binding mode of SB-203580; right side: postulated binding mode of<br />
pyrrolin-3-ones<br />
Being iron-ligands, imidazoles lead to CYP-450 inhibition and probably hepatotoxicity. Our<br />
strategy is to replace the core-heterocycle in order to reduce hepatic toxicity and to improve<br />
the interaction with Lys53. These considerations led us to pyrrolin-3-ones.<br />
Since established synthetic approaches to build pyrroles don’t suit this substitution pattern<br />
a new route has been developed based on reference [2].<br />
One major challenge is tautomerism of the target compound. Depending on substituents<br />
and solvent 3-hydroxypyrrole and pyrrolin-3-one exist at equilibrium.<br />
In position 2 unsubstituted pyrrolinones are extremely tending to polymerize, but the 3hydroxypyrrole<br />
with an ethyl carboxylate substituent in position 2 could successfully be<br />
crystallized and tested in a p38α MAP kinase assay (IC50 0,139 µM).<br />
Acknowledgments: Katharina Bauer and Marcia Goettert for biological testing, Verena Schattel for molecular<br />
modelling.<br />
References:<br />
1. Gallagher, T. F. et al.: Bioorg. & Med. Chem. 1997, 5(1): 49–64.<br />
2. Bauer, H.: Justus Liebigs Annalen der Chemie 1970, 736:1-15.<br />
062<br />
On the Importance of Water Molecules on Ligand Binding in the ATPase of<br />
GyrB<br />
Münsterberg, M.; Lemcke, T.<br />
Institut für Pharmazie, <strong>Universität</strong> Hamburg, Bundesstr. 45, 20146 Hamburg, Germany<br />
Gyrase is a well-known ATP-dependent topoisomerase II, essential for prokaryotic cells. It<br />
is the only known topoisomerase, which catalyses the negative supercoiling of DNA and<br />
consists of two subunits GyrA and GyrB. While GyrA mainly processes the strand passage,<br />
the ATP-hydrolysis is done by the N-terminus of GyrB [1]. Because of its significance in<br />
prokaryotes, gyrase has become a widely used target in antibiotic therapy. Nevertheless,<br />
only fluoroquinolones, inhibitors of the DNA-GyrA-complex, are available for therapy.<br />
Although there are known inhibitors of the ATPase in GyrB, e. g. the aminocoumarines like<br />
novobiocin, there are no drugs on the market which use this target [2]. Meanwhile, the<br />
resistance of bacteria against all major antibiotics forces the development of new antibiotics<br />
with superior effects [3].<br />
Several x-ray crystal structures of GyrB complexes from the protein data bank [4], with<br />
chemically different ligands, contain conserved water molecules. It is well known that water<br />
molecules play an important role for the stability of protein-ligand complexes, because the<br />
presence of water in the ligand binding site can increase or weaken the binding affinity of a<br />
ligand [5,6]. The release of buried water molecules from hydrophobic binding site<br />
environments very often has a beneficial effect on the free energy of binding of an inhibitor.<br />
For a structure based virtual screening it is necessary to know, which water molecule is<br />
important for the complex stability and ligand binding, and which water molecule might be<br />
replaced during docking experiments.<br />
Through the investigation of the GyrB ATPase protein-ligand complexes with different<br />
methods and tools (MD simulations, SuperStar [7] and Rank and HINT [8]) we have<br />
assessed the water molecules in the ATP-binding site. Even though there are huge<br />
conformational differences within the structures, particular water molecules are necessary<br />
for ligand binding in all complexes and thus for successful docking. Other, also conserved,<br />
water molecules in hydrophobic environment might be replaced by a suitable ligand to<br />
perform a successful docking.<br />
Our results will be the foundation for the setup of a virtual high throughput screening in a<br />
structure based design campaign to identify new and selective GyrB ATPase inhibitors.<br />
References:<br />
1. Champoux J. J.: Annu. Rev. Biochem. 2001, 70(1): 369–413.<br />
2. Collin F., Karkare S., Maxwell A.: Appl. Microbiol. Biotechnol. 2011, 92(3): 479–497.<br />
3. Boucher H. W. et al.: Clin. Infect. Dis. 2009, 48(1): 1–12.<br />
4. Berman H. M. et al.: Nucleic Acids Res. 2000, 28(1): 235–242.<br />
5. Lam P. Y. et al.: Science 1994, 263(5145): 380–384.<br />
6. Mikol V., Papageorgiou C., Borer X.: J. Med. Chem. 1995, 38(17): 3361–3367.<br />
7. Verdonk M. L., Cole J. C., Taylor R.: J. Mol. Biol. 1999, 289(4): 1093–1108.<br />
8. Amadasi A. et al.: J. Med. Chem. 2008, 51(4): 1063–1067.<br />
063<br />
Natural surfactant of polyglycerol fatty acid esters type as stabilizers for lipid<br />
nanoparticles<br />
Keck CM1 2; Kovacevic A3; Müller RH1 1Institute of Pharmacy, Department of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Freie<br />
<strong>Universität</strong> Berlin, Kelchstrasse 31, 12169 Berlin, Germany<br />
2Applied Pharmacy Division, University of Applied Sciences Kaiserslautern, Campus Pirmasens,<br />
Carl-Schurz-Strasse 10-16, 66953 Pirmasens, Germany<br />
3Faculty of Pharmacy, Department of Pharmaceutical Technology and Cosmetology, University of<br />
Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia<br />
Polyglycerol fatty acid esters (PGE) are non-ionic surfactants with various applications in<br />
pharmaceutical and cosmetic industries. They have been used for many years as food<br />
additives and therefore designed as „orally safe” surfactants. The aim of the study<br />
presented here was to evaluate the performance of PGE as potential stabilizer for SLN and<br />
NLC. PGE may be formed by a polymerization of glycerol followed by an esterification with<br />
fatty acids. The obtained mixture might vary in polymerization degree, kind and position of<br />
esterified fatty acids. The surfactants investigated were differed in the kind and position of<br />
esterified fatty acid (Fig. 1).<br />
SLN composed of 10% Cutina ® CP as solid lipid and NLC containing 6% Cutina ® CP and<br />
4% Miglyol ® 812 as a liquid lipid were produced by hot high pressure homogenization [1].<br />
All samples were stabilized with 1% PGE. Characterization of the particles was performed<br />
by photon correlation spectroscopy (PCS) and laser diffraction (LD). To investigate the<br />
surface properties of the SLN and NLC zeta potential (ZP) was measured in distilled water<br />
adjusted to conductivity of 50 μScm −1. Crystallinity of the SLN and NLC and respective solid<br />
lipid i.e. solid lipid/liquid lipid blend used as a particle matrix was investigated by differential<br />
scanning calorimetry (DSC).<br />
The obtained results indicated that PGE showed a good efficiency in the stabilization of the<br />
SLN and NLC. All samples have revealed size (z-ave) below 200 nm, with narrow size<br />
distribution (polydispersity index < 0.15). The respective z-ave were in good agreement<br />
with LD diameters indicating narrow and unimodal size distribution. PGE based SLN and<br />
NLC possessed the same particle composition, but different crystallinity indices. Particles<br />
stabilized with PI and PDI possessed lower crystallinity than PS based samples. The<br />
differences in DSC parameters are obviously created by the different types of the<br />
surfactants i.e. different interaction with the particle matrix and different surface tension<br />
generated near to the surface of the particles. The differences in the internal structure of<br />
the particles were also reflected in ZP values. With higher melting enthalpy and higher<br />
crystallinity index higher ZP values were obtained.<br />
Conclusions: PGE are suitable stabilizers for SLN and NLC. However, the tendency of the<br />
lipid (used as a particle matrix) to create more crystalline matrix of the lipid nanoparticles<br />
was favored by polyglycerol-6 ester type of the surfactant (PD) whereas the influence of the<br />
polyglycerol-6 isoesters (PI, PDI) was not significant.<br />
A B<br />
C<br />
Chemical structures of the PGE-surfactants used: A- polyglycerol 6-distearate (PD), B-<br />
polyglycerol 6-diisostearate (PDI) and C- polyglycerol 6-monoisostearate (PI)<br />
References:<br />
1. Müller RH, Lucks JS. Arzneistoffträger aus festen Lipidteilchen - Feste Lipid Nanosphären (SLN). 1991,<br />
German patent application P 41 5 62.6.<br />
064<br />
Lectine Mimetics Based on Multivalent Boronic Acids<br />
Claes D1; Maison W1 1 Pharmaceutical and Medicinal Chemistry, Bundesstraße 45, 20146 Hamburg, Germany<br />
Carbohydrates are playing an essential role in biological systems. They are used for energy<br />
storage, as structural substances and for cellular recognition [1]. The so called glycocalix<br />
covers cells and its molecular structure is dependent on the cell type, its level of<br />
differentiation and the activity of carbohydrate modifying enzymes. The recognition of these<br />
differences is a key component of pathogen detection by the native immune system and<br />
makes carbohydrates promising targets for pharmaceuticals [2].<br />
Natural carbohydrate binders are lectins, the first line of defense in our immune system [3].<br />
Lectins show weak affinities to monosaccharides and are therefore binding carbohydrates<br />
multivalently to strengthen these interactions. The geometry, composition and density of<br />
binding epitopes has a profound influence on these binding processes [4]. Notably, many<br />
lectins recognize their carbohydrate ligands in C3-symmetric complexes.<br />
108 Poster<br />
HO<br />
B<br />
O<br />
peptide<br />
O<br />
NH<br />
B O<br />
HO<br />
scaffold<br />
HN O<br />
peptide<br />
O<br />
N<br />
H<br />
boronolectin s<br />
peptide<br />
O<br />
B OH<br />
We propose to mimic lectins with trivalent assemblies of boronic acids. For this purpose we<br />
have synthesized suitable trivalent scaffolds and benzboroxoles, which are known to bind<br />
to non reducing sugars with significant affinities [5].
References:<br />
1. Roseman S. J. Biol. Chem. 2001, 276(45): 41527–42542.<br />
2. Dube D. H., Bertozzi C. R. Nat. Rev. Drug. Dis. 2005, 4(6): 477–488.<br />
3. Lis H., Sharon N. Chem. Rev. 1998, 98(2): 637–674.<br />
4. Kiessling L. L., Gestwicki J. E., Strong L. E. Curr. Opin. Chem. Biol. 2000, 4(6): 696–703.<br />
5. Berube M., Dowlut M., Hall D. G. J. Org. Chem. 2008, 73(17): 6471–6479.<br />
065<br />
Heterologous expression of (putative) polyketide synthases and nonribosomal<br />
peptide synthetases in Bacillus subtilis<br />
Kumpfmüller, J. 1; Kabisch, J. 1; Wenzel, S. 2; Müller, R. 2; Pfeifer, B. 3; Nagel, S. 1; Hiddemann,<br />
L. 1; Lalk, M. 1 Schweder, T. 1<br />
1Institut für Pharmazie, <strong>Universität</strong> <strong>Greifswald</strong>, F.-L.-Jahnstr.17, 17489 <strong>Greifswald</strong>, Germany<br />
2Department of Chemical and Biological Engineering, Science and Technology Center, Tufts<br />
University, 4 Colby Street, Medford, MA 02155, USA<br />
3Institut für Pharmazeutische Biotechnologie, <strong>Universität</strong> des Saarlandes, <strong>Universität</strong>scampus,<br />
Gebäude C2 3, 66123 Saarbrücken, Germany<br />
Polyketides and non-ribosomal peptides constitute an important class of complex natural<br />
products with wide-ranging therapeutic value. They are produced by multifunctional megaenzymes,<br />
the polyketide synthases (PKSs) and the non-ribosomal peptide synthetase<br />
(NRPS), respectively. Looking for new drugs, especially from unculturable microorganisms,<br />
as many marine bacteria are, heterologous expression of putative PKS and NRPS clusters<br />
becomes more and more important. Hence, there is a need for new hosts that can serve as<br />
alternatives to existing expression systems.<br />
In this study, we investigated Bacillus subtilis’s ability to express heterologous<br />
PKSs/NRPSs. First three putative PKSs/NRPSs (RB386, RB6500 and RB11975) from the<br />
marine planctomycete Rhodopirellula baltica were successfully integrated into the<br />
chromosome of B. subtilis. Although all of the recombinant strains showed positive mRNA<br />
results, overexpression of the heterologous protein could be detected and verified via MS<br />
only for RB11975. HPLC-MS-analyses of cell and medium extracts are being used to<br />
identify specifically produced metabolites.<br />
Beside this, we chose the PKS deoxyerythronolide B synthase (DEBS) as a model gene<br />
cluster since it was already successfully expressed by the heterologous hosts Escherichia<br />
coli and Streptomyces coelicolor. The product, 6-deoxyerythronolide B (6dEB), is the<br />
polyketide core of the antibiotic erythromycin. Due to the complexity of the 30kb large<br />
DEBS-cluster it was necessary to develop a new method for its stepwise chromosomal<br />
integration.<br />
To optimize our expression host for the production of polyketides and non-ribosomal<br />
peptides we performed a knockout of two large clusters: the NRPS-operon srfA (26kb,<br />
coding for surfactin synthetase) and the PKS-cluster pksX (78kb, coding for bacillaene<br />
synthase). Hence, we hope to provide more substrates for the heterologous PKS/NRPS<br />
and to reduce the background for an enhanced detection of produced metabolites.<br />
Metabolite analyses of the mutant strains are being used to elucidate the effect of these<br />
mutants on the physiology of B. subtilis.<br />
066<br />
Fed Stomach Model – a novel test device for biorelevant simulation of<br />
intragastric drug dissolution after food intake<br />
Koziolek, M. 1; Görke, K. 1; Garbacz, G. 1, Weitschies, W. 1<br />
1 <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-<strong>Universität</strong> <strong>Greifswald</strong>, Institut für Pharmazie, Biopharmazie &<br />
Pharmazeutische Technologie C_DAT Center of Drug Absorption and Transport, Felix-Hausdorff-<br />
Straße 3, D-17487 <strong>Greifswald</strong><br />
Food ingestion is known to alter physicochemical as well as mechanical conditions in the<br />
upper gastrointestinal (GI) tract. Consequently, drug release from solid oral dosage forms<br />
can be affected, which may have an impact on drug plasma levels by changing one or more<br />
pharmacokinetic parameters. To predict food effects on drug release in the human<br />
stomach, we designed an in vitro dissolution device for biorelevant simulation of fed gastric<br />
conditions, the Fed Stomach Model (FSM). This study aimed at the examination of the<br />
technical functionality of the FSM and the evaluation of the individual effects of the factors<br />
applied.<br />
The construction of the FSM is based on USP paddle apparatus. It is equipped with six<br />
specially designed dissolution cells. Media circulation between dissolution cells and the<br />
vessels of USP paddle apparatus is provoked by a peristaltic pump. By the use of stepping<br />
motor and valve system, both controlled via custom-made computer software, the FSM<br />
enables the simultaneous simulation of predefined movement profiles, flow and pressures.<br />
These parameters are exerted by a central axis driven by the stepping motor and<br />
connected to a pressure regulation module. In the present study, the drug release from<br />
diclofenac extended release (ER) tablets in USP phosphate buffer pH 6.8 was investigated<br />
to examine the individual relevance of the factors applied. In this study, the FSM was<br />
operated in closed-loop mode and the amount of drug dissolved was measured on-line by<br />
fiber optics.<br />
It could be demonstrated with the aid of magnetically labeled tablets that the FSM was able<br />
to simulate intragastric transport velocities equivalent to in vivo data generated by our<br />
group [1]. Furthermore, a valve system connected to a pressure regulation module allowed<br />
the application of biorelevant pressures by air inflation or deflation of a balloon. In<br />
preliminary dissolution experiments, we demonstrated that drug release from diclofenac ER<br />
tablets depended on dosage form movement as well as on pressure. It is thus feasible to<br />
apply test algorithms specific for different gastric regions after food intake.<br />
The Fed Stomach Model can be a valuable tool for biorelevant dissolution testing due to its<br />
potential for precise and reproducible simulation of mechanical parameters present under<br />
fed gastric conditions. Media flow including secretion and emptying, movement patterns<br />
and pressures were integrated into test algorithms representing specific conditions in<br />
fundus and antrum. Thus, the intragastric location of solid dosage forms can be considered<br />
in the FSM. This enables a more realistic in vitro investigation of intragastric drug release.<br />
Further experiments are needed to provide detailed information on mixing behavior and<br />
hydrodynamics. In addition, the dynamics of gastric emptying will be considered in certain<br />
test programs.<br />
Acknowledgments: Federal Ministry of Education and Research (GASTROMAX, FKZ 13 N11368-370)<br />
References:<br />
1. Weitschies, W. et al.: J. Contr. Rel. 2005 108(2-3): 375-385.<br />
067<br />
Molecular Dynamics Simulations and MM/GBSA-Calculations of Docking<br />
Poses to predict the native Binding Mode of thieno[2,3-b]pyridines in PfGSK-3<br />
Poll, B. 1; Kruggel, S. 1; Kunick, C. 2; Brandt, W. 2; Meijer, L. 3, Lemcke, T. 1<br />
1 <strong>Universität</strong> Hamburg, Institut für Pharmazie, Bundesstraße 45, 20146 Hamburg, Germany<br />
2 Technische <strong>Universität</strong> Braunschweig, Institut für Medizinische und Pharmazeutische Chemie,<br />
Beethovenstraße 55, 38106 Braunschweig, Germany<br />
3 Protein Phosphorylation & Human Disease Group, CNRS, Station Biologique, Place Georges<br />
Teissier, B.P. 74, 29682 Roscoff, France<br />
In addition to HIV and Tuberculosis, Malaria is one of the most prevalent infectious<br />
diseases in developing countries, causing over 200 mio. infections every year [1]. Malaria<br />
tropica, the most serious form of malaria, is caused by Plasmodium falciparum. There are<br />
potent malaria therapeutics on the market, however rapid emergence of resistance and<br />
high costs necessitate the development of new drugs [2].<br />
In cooperation with the University of Braunschweig and the CNRS in Roscoff (France) a<br />
thieno[2,3-b]pyridine derivative has been identified as a potential lead structure to inhibit the<br />
plasmodial Glycogen Synthase Kinase 3 (PfGSK-3) [3,4].<br />
One of the crucial aspects of lead structure optimization is the identification of the correct<br />
binding geometry of putative inhibitors. Homology models of PfGSK-3 have been<br />
developed [5] and were used for docking- and 3D-QSAR-studies [6] as well as guide for<br />
structural variations [4]. Different binding modes have been identified and investigated [6].<br />
We used molecular dynamics simulations and MM/GBSA-calculations of the respective<br />
protein-ligand-complexes considering pre-optimized calculation parameters [7] to further<br />
evaluate the binding poses of a series of thieno[2,3-b]pyridine derivatives 1.<br />
Poster 109<br />
R1<br />
1<br />
The GBSA energies were correlated to the biologically measured IC50 values of the<br />
individual compounds for complexes with the proposed binding mode and give further<br />
evidence of the native binding mode [6]. The results of these investigations will be<br />
presented.<br />
References:<br />
1. WHO, World Malaria Report 2010.<br />
2. Noedl, H. et al.: N. Engl. J. Med. 2008, 359: 2619-2620.<br />
3. Droucheau, E. et al.: Biochim. Biophys. Acta. 2004, 1697: 181-196.<br />
4. Brandt, W.: Dissertation, Braunschweig 2009.<br />
5. Kruggel, S., Lemcke, T.: Arch. Pharm. Chem. Life. Sci. 2009, 342: 327-332.<br />
6. Kruggel, S.: Dissertation, Hamburg 2011.<br />
7. Poll, B. et al.: MM-PBSA and MM-GBSA Calculation for Estimating the Free Energy of Binding of<br />
Glycogen Synthase Kinase-3 Inhibitors 2011. Joint Meeting of the German and Austrian pharmaceutical<br />
societies. Innsbruck. Austria.<br />
068<br />
Pharmacometrics – methodologies, establishment and opportunities of a<br />
promising new discipline in Germany<br />
Minichmayr I1 2; Kloft C1 1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin,<br />
Kelchstr. 31, 12169 Berlin, Germany<br />
2 and Graduate Research Training program PharMetrX, Germany<br />
Background: The emerging science of pharmacometrics represents a multidisciplinary<br />
research field bridging clinical pharmacy, pharmacology, medicine, statistics, engineering<br />
and computational methods [mod. 1]. During the past years, pharmacometrics has been<br />
gaining pivotal importance in drug development, regulatory approval, study design and<br />
therapeutic decision making [2]. The present work presents an overview over different<br />
concepts and methodologies within pharmacometrics, outlines possible applications and<br />
gives examples of past pharmacometric accomplishments. Additionally, the current<br />
establishment of pharmacometrics and educational resources in Germany are presented.<br />
Methods: Seminal papers on pharmacometrics were reviewed. Prevalence of<br />
pharmacometrics research at German Institutes of Pharmacy was assessed by systematic<br />
literature search (PubMed, university homepages).<br />
Results: Pharmacometrics applies computational models of biology, anatomy, physiology,<br />
pharmacology, disease and systems biology to describe and quantify interactions between<br />
drugs and patients [mod. 1]. Ultimately, the qualitative, quantitative and temporal<br />
relationships between drug exposure (pharmacokinetics, PK), drug effects<br />
(pharmacodynamics, PD) and favourable or unfavourable responses in connection with<br />
disease and recovery progression are explored and predicted on a subcellular, cellular,<br />
tissue, organ and organism level.<br />
Various approaches exist to characterise drug-patient interactions, ranging from classical<br />
empirical to more mechanistic pharmacometric models. The most prominent concepts –<br />
often separately used in research – are:<br />
1) Classical drug data analysis comprises single individual analysis and population basedmethodologies,<br />
which allow for e.g. the assessment of both inter- and intra-individual and<br />
unexplained variability. Within the latter, the standard two stage approach requires data rich<br />
situations, whereas non-linear mixed effect (NLME) modelling is also apt for modelling<br />
sparse data, typically obtained in patient care, due to simultaneous analysis of several<br />
individuals.<br />
2) Physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) models do not<br />
start with study data but anatomical and physicochemical drug data, enabling a more<br />
mechanistic simulation of drug profiles in individual tissues and blood. By means of lumping<br />
techniques, a direct link to classical compartment models can be established [3].<br />
3) Systems biology models consider even more detailed knowledge of underlying complex<br />
biologic processes, e.g. cell signal transduction pathways and their subsequent gene<br />
regulatory network.<br />
Despite the widespread potential of pharmacometrics, educational resources (e.g. scientific<br />
societies, textbooks, journal articles) in this field are scarce. At German Institutes of<br />
R2
Pharmacy, pharmacometric modelling skills and competences are basically gained by<br />
postgraduate training within the scope of a PhD (Universities of Berlin, Bonn, Duesseldorf,<br />
Muenster, planned: Saarbruecken) or a structured graduate research training program<br />
(PharMetrx - Pharmacometrics and Computational Disease Modelling at the Freie<br />
Universitaet Berlin and the University of Potsdam, jointly with five industry partners).<br />
Conclusion: Ideally, all three concepts of pharmacometrics should be linked and<br />
combinedly integrated in future research. Given the tremendous progress and value of the<br />
various applications of pharmacometrics in drug development (“model-based drug<br />
development”), regulatory and therapeutic decisions (“model-based patient care”), the<br />
demand for pharmacometricians is substantially growing. To meet the need in Germany,<br />
several universities already provide postgraduate training. Nonetheless, further expansion<br />
of pharmacometric expertise, particularly in undergraduate pharmacy curricula, is desirable.<br />
References:<br />
1. Barrett JS. et al.: J. Clin. Pharmacol. 2008, 48(5): 632-49.<br />
2. Gobburu JV.: J. Clin. Pharmacol. 2010, 50(9 Suppl): 151-157.<br />
3. Pilari S., Huisinga, W.: J. Pharmacokinet. Pharmacodyn. 2010, 37(4): 365-405.<br />
069<br />
Comparison of the ATP-Binding Sites of IR and IGF-1R Kinase Crystal<br />
Structures and their corresponding TrK Homology Model Counterparts<br />
Löhr, J. H. 1; Lemcke, T. 1<br />
1 <strong>Universität</strong> Hamburg, Institut für Pharmazie, Bundesstraße 45, 20146 Hamburg, Germany<br />
Receptor tyrosine kinases (RTKs) have been pursued as promising targets in cancer<br />
therapy. Transforming extracellular signals into cellular responses, they take part in the<br />
regulation of cell growth and survival. Deregulation of certain RTKs has been linked to<br />
various human tumor conditions and already various small molecule inhibitors have been<br />
approved for therapeutic use.[1]<br />
The insulin-like growth-factor receptor 1 (IGF-1R) and the neurotrophic tyrosine kinase<br />
receptor (TrK) are examples of cancer associated RTKs, where inhibition may prove to be<br />
an effective therapeutic intervention.[2, 3]<br />
With plenty of crystal structures available, structure based design of small molecule<br />
inhibitors of the ATP-Binding site of the IGF-1R kinase is a feasible strategy. However, offtarget<br />
activity on the highly homologous insulin receptor kinase (IR) poses a challenge. In<br />
absence of published three-dimensional models of the TrK, construction and evaluation of<br />
homology models has to precede any structure-based efforts.<br />
Previously, homology models of both active and inactive states of the TrK-1 and TrK-2<br />
kinase domains were built with suitable IR and IGF-1R X-ray structures serving as<br />
templates. Using molecular interaction fields (MIFs) and the GRID/GOLPE approach, the<br />
MIFs were investigated for statistical differences among the different crystal structures of<br />
IR/IGF-1R and the corresponding TrK-1/2 homology models.[4-6] Based on these findings,<br />
geometric and volumetric measurements were evaluated in order to pinpoint structural<br />
differences that emerge from the set of over 30 crystal structures and 10 homology models.<br />
A recently published partly solved crystal structure showing the TrK-3 kinase domain for the<br />
very first time is also taken into account both to assess the homology models and to<br />
highlight structural differences.[7]<br />
References:<br />
1. Arora, A., Scholar, E.M.: J. Pharmacol. Exp. Ther. 2005, 315(3): 971–979.<br />
2. Li, R., Pourpak, A., Morris, S.W.: J. Med. Chem. 2009, 52(16): 4981–5004.<br />
3. Nakagawara, A.: Cancer Lett. 2001, 169(2): 107–114.<br />
4. Goodford, P.J.: J. Med. Chem. 1985, 28(7): 849–857.<br />
5. Kastenholz, M.A. et al.: J. Med. Chem. 2000, 43(16): 3033–3044.<br />
6. Baroni, M. et al.: Quant. Struct.-Act. Relat. 1993, 12(1): 9–20.<br />
7. Albaugh, P. et al.: ACS Med. Chem. Lett. <strong>2012</strong>, 3(2): 140–145.<br />
070<br />
Two-dimensional capillary gel electrophoresis (2-D-CGE) as powerful<br />
instrument for quantitation and characterisation of biomolecules<br />
Hahne, T.; Cianciulli, C.; Wätzig, H.<br />
Institute of Medicinal and Pharmaceutical Chemistry, Technische <strong>Universität</strong> Braunschweig,<br />
Beethovenstr. 55, 38106 Braunschweig, Germany<br />
Capillary gel electrophoresis (CGE) is a highly selective separation technique, used more<br />
and more in pharmaceutical industry for analysis of biomolecules. Thus, CGE is replacing<br />
conventional gel electrophoresis stepwise, because of some advantages such as ease of<br />
handling and time-saving. Even if CGE is a powerful and selective tool for bioanalysis the<br />
selectivity was increased by adding a second dimension. According to our approach the<br />
molecules are separated due to their isoelectric point in the first dimension and then due to<br />
their molecular weights in the following second dimension, as known from classical CGE. In<br />
this study a protein mixture consisting of bovine serum albumin (BSA), ovalbumin, ßlactoglobulin,<br />
myoglobin and a monoclonal antibody was used to investigate the<br />
performance of this innovative separation technique. Reproducibilities, resolutions, signalto-noise<br />
ratios and limits of quantitation with different concentrations of the biomolecules<br />
were investigated.<br />
071<br />
A comparison: Quantification of therapeutically used organic nitrates via LC-<br />
UV and LC-ESI-MS in biological samples after solvent and solid-phase<br />
extraction respectively<br />
Puerstinger, J. 1; Seeling, A. 1<br />
1 Institute of Pharmaceutical/Medical Chemistry, Friedrich-Schiller University, Philosophenweg 12,<br />
07747 Jena, Germany<br />
Pentaerithrityl tetranitrate (PETN), isosorbide dinitrate (ISDN) and glyceryl trinitrate (GTN)<br />
are organonitrate coronary vasodilators and, consequently, used in the treatment of<br />
coronary insufficiency and angina pectoris. Sustained therapeutical effects of PETN and<br />
ISDN are useful in the prophylaxis of attacks of angina pectoris but lead to a slower onset<br />
in comparison to GTN which plays an important role in the management of cardiac<br />
infarction. Furthermore, recent investigation points to a remarkable benefit of organic<br />
nitrates in the therapeutic fields of diabetes mellitus [1] and pulmonary hypertension [2].<br />
Bioactivation via several enzymatic reduction pathways [3] results in less nitrated polyols<br />
which contribute to the vasodilator potency [4].<br />
In order to quantify the therapeutically used organic nitrates and to monitor their<br />
denitrification processes, we developed miscellaneous chromatographic separation<br />
methods to determine PETN, ISDN and GTN as well as their less nitrated metabolites in<br />
biological liquids such as human plasma and whole blood samples using solvent or solidphase<br />
extraction for the segregation of the analytes and, subsequently, UV or ESI-MS for<br />
their detection.<br />
Acknowledgments: Measurements on LC-ESI-MS instruments could be realized in the research group of<br />
Prof Christian G. Huber, Department of Chemistry and Bioanalytics, University of Salzburg, Austria.<br />
References:<br />
1. Wenzel P. et al.: Arterioscler. Thromb. Vasv. Biol. 2007, 27(8): 1729-1735.<br />
2. Daiber A. et al.: Mol. Pharmacol. 2004, 66: 1372-1382.<br />
3. Munzel T., Daiber A., Mulsch A.: Circ. Res. 2005, 97(7): 618-628.<br />
4. Wenzel P. et al.: Br. J. Pharmacol. 2007, 150(4): 526-533.<br />
072<br />
Pulmonary deposition of three market dry powder inhalers using an idealized<br />
pediatric and adult mouth-throat model<br />
Below, A. 1; Bickmann, D. 2; Breitkreutz, J. 1<br />
1 Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, 40225 Düsseldorf,<br />
Germany<br />
2 Boehringer Ingelheim Pharma GmbH & Co. KG, 55216 Ingelheim am Rhein, Germany<br />
The efficiency of aerosol delivery to the patient´s lung depends on three main parameters:<br />
inhalation device, formulation and patient. Marketed inhalation products are in most cases<br />
developed according to the needs of adult patients and less for children. Changes in upper<br />
airway geometry and breathing pattern due to maturation from childhood to adolescent are<br />
usually not considered. Because of respiratory diseases like childhood asthma and cystic<br />
fibrosis even young children have to use inhaled medicines. The efficiency of inhaled<br />
products is especially in preschool children unknown. The complex interaction between<br />
inhaled product and patient is the scope of this study. In a first step the influence of the<br />
upper airway geometry in dependence of age on the performance of three market products<br />
[1-2] was investigated. Afterwards, also the impact of different flow rates was considered.<br />
For the in vitro assessment of particle deposition via next generation impactor (NGI), three<br />
commercially available multidose dry powder inhalers (Salbu Novolizer ® [1], SalbuHexal ®<br />
Easyhaler ® [2], Asmanex ® Twisthaler ®) were connected to two different inlets: idealized<br />
pediatric and adult upper airway model. The pediatric model represents 4-5-year-old<br />
children. In dependence of the device different steady flow rates through the model were<br />
achieved. The inhalation volume was adjusted to 4L (adult model) and 1L (adult and<br />
pediatric model). Deposited mass in the model (extrathoracic deposition) and at the model<br />
outlet (pulmonary deposition) was collected, determined by HPLC and related to label<br />
claim.<br />
An influence of inhalation volume on pulmonary deposition (PD) was observed for the<br />
Novolizer (15% vs. 22% at 75 L/min (4 kPa)). Easyhaler and Twisthaler PD were not<br />
influenced by inhalation volume accordingly. The comparison of adult and pediatric models<br />
has shown higher pulmonary doses using the adult mouth-throat model for all three<br />
products, e.g. Twisthaler 21% vs. 27% (46 L/min (4 kPa)). Different flow rates have an<br />
influence on product performance especially on Twisthaler performance. Using the pediatric<br />
mouth-throat model PD increased from 15% (30 L/min (2 kPa) to 21% (46 L/min (4 kPa)).<br />
Novolizer and Easyhaler performance is very robust; PD is almost the same for all<br />
investigated flow rates.<br />
Physical upper airway models as impactor inlets simulate the human upper airways more<br />
correctly than the USP/Ph.Eur. inlet. Using the adult mouth-throat model higher pulmonary<br />
doses were observed than for the pediatric model. Because there is still a lack of in vivo<br />
data of preschool children using dry powder inhalers in vitro in vivo correlation is not<br />
feasible. If in vivo deposition data will be available in future, the developed model may be<br />
validated. Based on different product performance we believe that for pediatric product<br />
developments child-appropriate models should be used.<br />
References:<br />
1. Below, A. et al.: 18th ISAM conference 2011<br />
2. Below, A.: 8th PBP World Meeting <strong>2012</strong>.<br />
073<br />
Biosynthesis of structurally diverse paracylophanes by Vietnamese<br />
cyanobacteria<br />
Preisitsch, M. 1, Neidhardt, I. 1, Pham, T. L. H. 1, Bodtke, A. 2, Niedermeyer, T. H. J. 3, Mundt,<br />
S. 1<br />
1 Institute of Pharmacy, Department of Pharmaceutical Biology, <strong>Ernst</strong> <strong>Moritz</strong> <strong>Arndt</strong> University<br />
<strong>Greifswald</strong>, Friedrich-Ludwig-Jahn-Str. 17, 17489 <strong>Greifswald</strong>, Germany<br />
2 Institute of Pharmacy, Department of Pharmaceutical/Medicinal Chemistry, <strong>Ernst</strong> <strong>Moritz</strong> <strong>Arndt</strong><br />
University <strong>Greifswald</strong>, Friedrich-Ludwig-Jahn-Str. 17, 17489 <strong>Greifswald</strong>, Germany<br />
3 Cyano Biotech GmbH, Magnusstraße 11, 12489 Berlin, Germany<br />
Cyanobacteria have been identified as a promising source of new, highly diverse, bioactive<br />
natural products in the research of novel pharmaceutical leads. Especially cyclic peptides<br />
and lipopeptides, alkaloids, lactones, amides, esters, derivatives of fatty and amino acids,<br />
biosynthesized by marine cyanobacteria, represent the largest part of recently isolated<br />
compounds with mainly antibiotic, antifungal, cytotoxic or enzyme-inhibiting activities [1]. In<br />
contrast to these metabolites, naturally occurring paracyclophanes have been isolated less<br />
frequently from cyanobacteria [2].<br />
Bioassay-guided separation of the methanol extract from the Vietnamese freshwater<br />
cyanobacterium Nostoc sp. CAVN 10 resulted in five [7.7]paracyclophanes,<br />
carbamidocyclophanes A-E, including carbamido moieties and a varying substitution<br />
pattern of the chlorinated butyl side chains. Carbamidocyclophanes A-C exhibited cytotoxic<br />
activity against MCF-7 (breast cancer cell line) and Fl cells (human amniotic epithelial cell<br />
line) and moderate antibacterial activity against Staphylococcus aureus (ATCC 6538)[3].<br />
Further screenings for carbamidocyclophanes in various cyanobacteria, in particular in<br />
Nostoc strains, revealed a second carbamidocyclophane producing strain, Nostoc sp.<br />
CAVN 2. HPLC-DAD/IT-TOF-HRMS analysis of separated CAVN 2 cyclophanes resulted in<br />
an identification of already published [2-4] and also previously unknown cyclophanes.<br />
Feeding experiments with Nostoc sp. CAVN 2, using different halogen salts, yielded in the<br />
first-time detection of brominated carbamidocyclophanes.<br />
An optimized, highly rapid one-step extraction and separation procedure for the detection of<br />
110 Poster
carbamidocyclophanes as well as closely related structural analogues and first insights into<br />
the phylogenetic relationship of axenic carbamidocyclophane biosynthesizing<br />
cyanobacteria are presented.<br />
Acknowledgments: The authors thank Dr. M. Meixner for 16S rDNA amplification and sequencing of<br />
investigated strains as well as Mr. S. Heiden for the assistance in the construction of the phylogenetic tree.<br />
References:<br />
1. Singh, R. K. et al.: J. Antibiot. 2011, 64(6):401-412.<br />
2. Chlipala, G. E. et al.: J. Nat. Prod. 2010, 73(9): 1529-1537.<br />
3. Bui, H. T. N. et al.: J. Nat. Prod. 2007, 70(4): 499-503.<br />
4. Kang, H.-S. et al.: Phytochem. <strong>2012</strong>, 79(0): 109-115.<br />
074<br />
Cannabinoids stimulate mesenchymal stem cell migration via a mitogenactivated<br />
protein kinase pathway<br />
Schmuhl, E. 1; Ramer, R. 1; Peters, K. 2; Hinz, B. 1<br />
1 Institute of Toxicology and Pharmacology, University of Rostock, Schillingallee 70, D-18057<br />
Rostock, Germany<br />
2 Department of Cell Biology, University of Rostock, Schillingallee 69, D-18057 Rostock, Germany<br />
Mesenchymal stem cells (MSC) are known to be involved in various regenerative<br />
processes such as cardiac, ocular, skin and bone tissue healing. However, little is known<br />
about the pharmacotherapeutical options aiming at tissue healing steps such as the<br />
mobilization and homing of MSC. Here, we show that cannabidiol (CBD), a nonpsychoactive<br />
cannabinoid, stimulates the migration of human adipose-derived MSC in both<br />
Boyden chamber and in vitro scratch wound assays. In Boyden chambers CBD (0.01 – 3<br />
µM) was shown to promote cell migration in a time- and concentration dependent manner.<br />
This promigratory action was inhibited by AM-630 (CB2 receptor antagonist) and by O-1602<br />
(G protein-coupled receptor [GPR] 55 agonist). Moreover, CBD activated the mitogenactivated<br />
protein kinase (MAPK) pathway as evidenced by increased phosphorylation of<br />
extracellular signal-regulated kinase (ERK) 1/2. Blockade of ERK activation by PD98059<br />
prevented CBD-stimulated MSC migration, whereas inhibition of p38 MAPK by SB203580<br />
was inactive in this respect. Furthermore, AM-630 and O-1602 were found to attenuate<br />
CBD-induced ERK activation. An ERK-dependent promigratory action was likewise<br />
demonstrated for the phytocannabinoid Δ9-tetrahydrocannabinol, the GPR55 antagonist O-<br />
1918 and the selective CB2 receptor agonist JWH-133. We conclude that cannabinoids<br />
promote MSC migration via receptor-dependent ERK activation, possibly contributing to<br />
tissue healing.<br />
075<br />
Accuracy temperature control as a meaningful tool for microwave assisted<br />
method development and optimization<br />
Härter A<br />
Anton Paar GmbH, Helmuth-Hirth-Str. 6, 73760 Ostfildern<br />
During the last decade microwave-assisted synthesis has become a powerful tool for<br />
pharmaceutical method development and optimization. Controlled microwave heating under<br />
sealed vessel conditions has - shown to dramatically reduce reaction times, increase<br />
product yields, and enhance product purities by reducing unwanted side reactions<br />
compared to conventional synthetic methods. Modern microwave reactors control and<br />
regulate important parameters such as temperature, pressure and power. Due to this<br />
plurality of control parameters, the essential of accurate temperature for reaction<br />
optimization is often unacknowledged.<br />
We will introduce the basics of optimal temperature measurement, including tips for heating<br />
- microwave transparent solvents, effects due to external cooling, the importance of reliable<br />
stirring and the influence of reactor stability. The concept of accurate temperature control is<br />
outlined, which enables performing optimized protocols in any applicable scale in any<br />
instrument of our modular equipment family without reoptimization. This increases the<br />
efficiency of microwave-mediated synthesis even further, speeding up the entire process of<br />
academic and industrial research.<br />
076<br />
Using sedimentation velocity centrifugation to study the aggregation process<br />
of proteins<br />
Wolff, M. 1 2; Nagel-Steger L. 1 2; Willbold, D. 1 2<br />
1 Heinrich-Heine-<strong>Universität</strong>, Institut für Physikalische Biologie, 40225 Düsseldorf, Germany<br />
2 Forschungszentrum Jülich, ICS-6, 52425 Jülich, Germany<br />
The pathogenic aggregation of proteins plays an important role in the development of a<br />
number of neurodegenerative diseases. For example in Alzheimer’s or Parkinson’s Disease<br />
the self-assembly of misfolded cellular proteins causes the formation of different oligomeric<br />
structures which finally leads to amyloid fibril formation. In order to identify different<br />
assemblies in the aggregation process we employ the method of sedimentation velocity<br />
(SV) analysis. Using SV analysis gives us the opportunity to study protein size and shape<br />
distributions in solution under defined conditions. The advantages of SV analysis are that 1)<br />
no interaction with liquid or solid phases influences the measurement as known for other<br />
separation techniques like e.g. size exclusion chromatography, and 2) that due to the<br />
fractionation power of the method large aggregates do not impair the results as in light<br />
scattering methods.<br />
One of the most important targets in Alzheimer’s therapy is the 39 to 43 amino acids long<br />
proteolytic fragment of the amyloid precursor protein, called amyloid β peptide for its<br />
property to form amyloid fibrils. Beginning with the characterization of the hydrodynamic<br />
properties of the monomeric Aβ peptide preparations, we will analyze early steps in<br />
aggregate formation in order to identify small Aβ assemblies of defined size, which are<br />
significantly populated in freshly prepared Aβ solutions. With this approach we want to gain<br />
insight into mechanistic details of the aggregation process. Moreover this approach is<br />
aimed at the identification of novel drug targets for Alzheimer’s disease therapy.<br />
077<br />
New Furanoid and seco-Labdanoid Diterpenes isolated from Leonurus<br />
cardiaca L.<br />
Brückner, A. 1; Hennig, L. 2; Rauwald, H. J. 1<br />
1University of Leipzig, Department of Pharmaceutical Biology, Johannisallee 21-23, D-04103<br />
Leipzig, Germany<br />
2University of Leipzig, Department of Chemistry, Institute for Organic Chemistry, Johannisallee 39,<br />
D-04103 Leipzig, Germany<br />
Continuing our search for possible cardioactive single constituents of primary and refined<br />
antiarrhythmic extracts of Leonurus cardiaca L. [1] we report herewith the isolation of three<br />
new diterpenes besides known substances, like leosibiricin, sitosterol and stigmasterol [2].<br />
The structures of isoleosibirin, leopersin F and 7-epi-leopersin F were determined by 1d/2d<br />
1H/ 13C NMR spectroscopical experiments. These labdane type diterpenes have been found<br />
only once in other plants of the genus leonurus, namely in the related Lamiaceae Leonurus<br />
sibiricus and Leonurus persicus [3, 4]. Thus these compounds may prove useful as<br />
analytical marker substances as well as possible pharmacologically active compounds, as<br />
for example shown for the vasorelaxant activity of similar diterpenes isolated of Marrubium<br />
vulgare [5].<br />
References:<br />
[1] Ritter, M et al.: Planta Med. 2010, 76: 572-582.<br />
[2] Knöss, W.: Plant Cell Reports 1995, 14: 790-793.<br />
[3] Tasdemir, D.; Wright, A.D.; Sticher, O.: J. Nat. Prod. 1996, 59: 131-134.<br />
[4] Savona, G.; Piozzi, F.; Rodriguez, B.: Phytochemistry 1982, 21 (11): 2699-2701.<br />
[5] El Bardai, S.; Morel, N.; Wibo, M.: Planta Med. 2003, 69: 75-77.<br />
078<br />
Characterization of the pharmacokinetic properties of visnagin and Ammi<br />
visnaga water extract following oral administration in rats<br />
Haug, K. G. 1; Weber, B. 1; Hochhaus, G. 1; Butterweck, V. 2<br />
1 Department of Pharmaceutics, College of Pharmacy, University of Florida, 1600 SW Archer<br />
Road, Gainesville, FL,32610, USA<br />
2 Institute for Pharma Technology, School of Life Sciences, University of Applied Sciences<br />
Northwestern Switzerland, Gruendenstrasse 40, 4132 Muttenz, Switzerland<br />
The furanochromone visnagin is one of the main compounds of Ammi visnaga L. (syn.<br />
Khella, Apiaceae) with potential effects on kidney stone prevention. After determination of<br />
the pharmacokinetic (PK) properties of visnagin after intravenous bolus administration in<br />
rats [1], the aim of the present study was the evaluation of the PK properties of visnagin<br />
and an aqueous Ammi visnaga extract (AVE) after oral administration in rats. In two<br />
separate experiments three doses of visnagin (2.5, 5, and 10 mg/kg) solubilized in 25%<br />
Captisol® and three doses of AVE (standardized on visnagin and containing equivalent<br />
amounts of visnagin) were administered by oral gavage to male Sprague-Dawley rats,<br />
respectively. Plasma samples were extracted and subsequently analyzed using a validated<br />
LC-MS/MS method [1]. Plasma concentration-time profiles (Figure 1) were explored by<br />
non-compartmental analysis. Visnagin plasma exposure (median AUClast and AUCinf) was<br />
significantly increased for all three doses (up to 10-fold for the low dose) when administered<br />
as extract compared to the pure agent (Figure 2). For both the AVE and the pure<br />
compound, AUClast and AUCinf increased disproportionately with an increase in dose (Figure<br />
1 & 2). Visnagin resided significantly longer in the body when given in form of AVE with up<br />
to three times longer median MRTlast and MRTinf for the low dose. Cmax values after AVE<br />
administration were elevated and occurred at later time points in comparison to equivalent<br />
doses of pure visnagin. The terminal half-life increased with dose for both AVE and pure<br />
visnagin, reaching a maximum value of 1.94 h for the 10 mg/kg pure compound group.<br />
In conclusion, exposure of visnagin is enhanced after extract administration and could<br />
result in a superior efficacy of AVE compared to an equivalent dose of visnagin.<br />
Figure 1: Visnagin plasma concentration (geometric mean � SEM) vs. time profiles after<br />
administration of the pure compound (a) and AVE (b), sorted by dosing group (Low: 2.5<br />
mg/kg, Medium: 5 mg/kg, High: 10 mg/kg).<br />
Figure 2: AUCinf (a) and AUClast (b) (median + SD) of visnagin after administration of pure<br />
compound vs. AVE, sorted by dosing group (Low: 2.5 mg/kg, Medium: 5 mg/kg, High: 10<br />
mg/kg), numbers represent Bonferroni adjusted p-values, AUCinf values that are based<br />
upon extrapolated AUC > 50% were excluded.<br />
Reference:<br />
Haug, K.G. et al.: Eur J Pharm Sci <strong>2012</strong>, 45 (1-2): 79-89<br />
079<br />
Synthesis and biological studies on novel water-soluble gold(I)alkynyl<br />
complexes as antitumor agents and thioredoxin reductase inhibitors.<br />
Andermark, V. 1; Meyer, A. 1; Ott, I. 1<br />
1 Institute of Medicinal and Pharmaceutical Chemistry, Technische <strong>Universität</strong> Braunschweig,<br />
Beethovenstraße 55, 38106 Braunschweig, Germany<br />
Poster 111
Besides the use of gold(I) complexes, i.e. auranofin, as disease modifying antirheumatic<br />
drugs (DMARDs)they are also known for their potential as antitumor drugs and highly<br />
efficient inhibition of thioredoxin reductase (TrxR)[1], an enzymewhich is overexpressed in<br />
tumor cells.TrxR contains a selenocystein in the active site, making it an excellent target for<br />
soft lewis acids like gold(I)-complexes[2]. Inhibition of TrxR leads to increased reactive<br />
oxygen species (ROS) levels, cell cycle arrest and can induce apoptosis [3].<br />
This work bases on recent results, which show that phosphinegold(I)alkynyl complexes can<br />
inhibit TrxR in the nanomolar concentration range and have anti-angiogenic properties in<br />
zebrafish embryos[4].To improve the water-solubility of the phosphinegold(I)alkynyl<br />
complexes the bulky, hydrophobic triphenylphosphine-ligand of the most active complexes<br />
will be replaced by more polar phosphine ligands, while the most promising alkynyl ligands<br />
will be kept.<br />
The tri-(2-furyl)phosphinegold(I)chloride (3)complex is prepared by the reaction of<br />
equimolar amounts of dimethylsulfidegold(I)chloride (1) and tri-(2-furyl)phosphine (2).<br />
Afterwards 3 is reacted with an equimolar amount ofan alkyne (4) in basic conditions,<br />
yielding aphosphinegold(I)alkynyl(5) complex (scheme 1). Synthesized compounds are<br />
confirmed by 1H, 13C, 31P NMRand their purity is evaluated by elemental analysis.<br />
The cytotoxicity of the synthesised complexes, as well asthe metal free alkynes and<br />
phosphinesis evaluated using HT-29 colon carcinoma cells and MCF-7 breast cancer cells<br />
in the colorimetric crystal violet assay. Additionallycurrent results onTrxR inhibition,<br />
glutathione reductase inhibition and cellular uptake into tumor-cells will be presented.<br />
Scheme 1.Synthesis of the compounds 3 and 5 as an example for the general synthetic<br />
route.<br />
Acknowledgments: Financial support bythe Deutsche Forschungsgemeinschaft (DFG, project FOR630)is<br />
gratefully acknowledged.<br />
References:<br />
1. Ott, I.: Coord. Chem Rev.2009, 253: 1670-1681.<br />
2. Gromer, S. et al.: J. Biol. Chem. 1998,273(32): 20096-20101<br />
3. Urig, S., Becker, K.: Semin.Cancer Biol. 2006,16: 452-465<br />
4. Meyer A. et al.:Angew. Chemie<strong>2012</strong>accepted<br />
080<br />
Influence of opioid receptor activation and inhibition on synaptic transmission<br />
under hypoxia<br />
Bloßfeld, M. 1; Merkenschlager, A. 2; Nieber, K. 1<br />
1 University Leipzig, Institute of Pharmacy, Pharmacology for Natural Sciences, Talstraße 33,<br />
04103 Leipzig, Germany<br />
2 <strong>Universität</strong>sklinikum Leipzig, Klinik und Poliklinik für Kinder und Jugendliche, Liebigstraße 20a,<br />
04103 Leipzig<br />
Activation of delta opioid receptors by [D-Ala2, D-Leu5] enkephalin (DADLE) has been<br />
reported to have neuroprotective effects on rat embryonic cortical neurons and PC12 cells<br />
under hypoxia. A neurotoxic impact has also been shown for micromolar concentrations<br />
[1,2]. There are limited information about effects of naloxon, a mu receptor antagonist,<br />
during hypoxia. The aim of the present study was to examine the influence of DADLE and<br />
naloxone on the amplitude of electrically evoked postsynaptic potentials (PSPs) under<br />
normoxic (95% O2, 5% CO2) and hypoxic (1% O2, 5% CO2, 94% N2) conditions. The<br />
experiments were done using intracellular recordings on cortical pyramidal cells in brain<br />
slices of adult rats. DADLE (1 µM) decreased the amplitude of PSPs. Naloxone (100 µM)<br />
inhibited synaptic transmission in neurons from 10 week old rats whereas in neurons of<br />
brain slices of rats older than 10 weeks no influence could be found. Naloxone (100 µM)<br />
inhibited completely the effect of DADLE (1µM). Both components had no influence on<br />
input resistance and membrane potential. Exposure of the slices to hypoxia (5 min) resulted<br />
in a decreased amplitude of PSPs. DADLE (1 µM), superfused 5 min before hypoxia, did<br />
not alter the hypoxia-decreased PSPs. Contrary, naloxone (100 µM) antagonised the PSP<br />
amplitude reduction. This effect was age-independent. We conclude that during hypoxia<br />
naloxon (100µM) but not DADLE (1µM) normalizes synaptic transmission as an indicativ of<br />
a neuroprotective effect.<br />
References:<br />
1. Su, D.S. et al.: Neurosci. Lett. 2007, 423, 113-117<br />
2. Sun K., Su D.S. and Wang X.: Brain Res. 2009, 1292, 100-106<br />
081<br />
Glutathione transferase isoenzymes in corneal tissues, human cornea<br />
construct and corneal cell lines<br />
Kölln C1; Reichl S1 1 Institut für Pharmazeutische Technologie, Technische <strong>Universität</strong> Carolo-Wilhelmina zu<br />
Braunschweig, Mendelssohnstr. 1, 38106 Braunschweig, Germany<br />
The human cornea represents the main barrier for active drug substances from ophthalmic<br />
formulations. Therefore, excised cornea and corneal cell culture models are used for in vitro<br />
drug absorption studies. However, little is known about drug metabolism during<br />
transcorneal passage. The purpose of this study was to determine expression and activity<br />
of glutathione transferases (GST) in human corneal cell lines and human cornea construct<br />
in comparison to excised animal corneas. Isoenzymes GSTO1 and GSTP1 are supposed<br />
to be considered in more detail.<br />
Presence of GST isoenzymes in corneal cell lines, cornea construct and excised corneal<br />
tissues was determined by western blot and immunohistochemical staining using specific<br />
antibodies against isoenzymes GSTO1 and GSTP1. In this study corneal cell lines HCE-T<br />
(epithelial), HCK-Ca (stromal) and HENC (endothelial) were investigated as well as tissue<br />
engineered human cornea construct [1, 2]. Porcine and rabbit corneas were studied in<br />
comparison. The activity was measured by spectrophotometrically methods. Total GST<br />
activity was profiled using 1-Chloro-2,4-dinitrobenzene (CDNB) [3], specific GSTO1 activity<br />
by using S-(4-Nitrophenacyl)-glutathione [4] and specific GSTP1 activity was investigated<br />
using ethacrynic acid [5].<br />
The presence of glutathione transferases in human cornea construct and all corneal cell<br />
lines was verified on protein level by western blot and immunohistochemical staining,<br />
respectively. In case of corneal cell lines, activity of investigated isoenzymes seems to be<br />
comparable in HCE-T and HCK-Ca, whereas total GST activity was found to be highest in<br />
HCE-T. In comparison, HENC showed lowest activity in all studies. Excised rabbit and<br />
porcine corneas showed higher specific GSTP1 and GSTO1 activity as well as total GST<br />
activity compared to human cornea construct.<br />
Known to be present in human cornea, glutathione transferase activity is detectable in all<br />
investigated human corneal cell lines, as well as in human cornea construct and excised<br />
animal corneas.<br />
Acknowledgments: We are grateful to the German Federal Institute for Risk Assessment (BfR), which<br />
funded this work under grant no. 3-1328-30652054369.<br />
References:<br />
1. Hahne M, Reichl S: Int J Pharm. 2011, 416(1): 268-279.<br />
2. Hahne M et al.: J Pharm Sci. <strong>2012</strong>, accepted<br />
3. Habig WH, Pabst MJ, Jakoby WB: J Biol Chem. 1974, 249(22): 7130-7139.<br />
4. Board PG et al.: Anal Biochem. 2008, 374(1): 25-30.<br />
5. Habig WH, Jakoby WB: Methods Enzymol. 1981, 77: 398-405.<br />
082<br />
Novel inhibitors of the arginine methyltransferase PRMT6<br />
Wagner, T. 1; Jung, M. 1<br />
1 Institute of Pharmaceutical Sciences, Albert-Ludwigs-<strong>Universität</strong> Freiburg, Albertstr. 25, 79110<br />
Freiburg, Germany<br />
Background<br />
Arginine protein methyltransferases (PRMTs) represent a SAM dependent class of protein<br />
modifying enzymes that are able to mono- and dimethylate the guanidino group of specific<br />
arginine residues within certain protein substrates e.g. histone proteins, p53, FOXO<br />
transcription factor and other protein modifying enzymes where PRMTs usually methylate<br />
glycine-alanine-arginine patches (GAR motifs). The human family of PRMTs consists of<br />
eleven members (PRMT1-11) which are devided into two classes. Type I<br />
methyltransferases are able to methylate arginine residues in an asymmetrical manner and<br />
type II methyltransferases catalyse the formation of symmetrically substituted arginine<br />
residues. PRMTs 1–4, 6 and 8 are type I enzymes and PRMTs 5, 7 and 9 are PRMTs of<br />
type II. [1] PRMTs are involved in the regulation of various physiological processes like<br />
apoptosis, cell differentiation, metabolism, DNA recombination and HIV tat transactivation.<br />
Thus, PRMT inhibitors are interesting potential compounds for drug discovery. [2]<br />
Inhibitor search is highly dependent on high throughput screening (HTS) which relies on<br />
suitable assay systems. Here we used a new homogeneous bead based in vitro assay<br />
system for screening new PRMT6 inhibitors. Therefore we surveyed some known PRMT1<br />
inhibitors that have been identified in our group previously.<br />
Material and Methods<br />
A method for the expression and purification of PRMT1 was set up in order to be able to<br />
screen larger amounts of compounds as potential inhibitors of this enzyme. Compounds<br />
have been screened and PRMT1 inhibitors have been identified by the use of an ELISA<br />
based, heterogeneous assay system (Delfia) in our group previously. In order to test the<br />
selectivity of the PRMT1 inhibitors towards other arginine methyltransferases we used an<br />
alphaLISA based method for exploring the enzyme PRMT6. Therefore a biotinylated<br />
peptide fragment of the histone protein H3 (amino acids 1-21) is used as substrate for the<br />
enzyme PRMT6. This peptide is able to bind to a streptavidine coated donor bead which<br />
contains phthalocyanine as a photosensitizer which converts ambient oxygen to singlet<br />
oxygen upon excitation with light at a wavelength of 680 nm. In the case of the peptide<br />
being methylated by PRMT6 at the arginine residue at position 2 of the peptide after<br />
addition of the cofactor S-adenosylmethionine (SAM) an antibody directed against this<br />
modification and coated to acceptor beads is able to bind to the modified substrate but not<br />
to the unmodified state. Thereby donor and acceptor beads are brought into close proximity<br />
so that the singlet oxygen emitted by the donorbeads is able to convert a thioxene<br />
derivative on the acceptor beads. The addition product degrades with chemoluminsecence<br />
which finally leads to the excitation of the lanthanide europium which is also present in the<br />
acceptor beads. The latter can be detected by time resolved fluorescence readout in an<br />
EnVision plate reader (commercial detection system by PerkinElmer) at a wave length of<br />
615nm.<br />
Results<br />
We were able to profile PRMT inhibitors against PRMT6 by using a new alphaLISA based<br />
assay system. Most compounds inhibit both PRMT1 and PRMT6 in low micromolar<br />
concentrations. But we could further show selectivity for PRMT1 towards PRMT6 for some<br />
compounds. We also identified one compound that seems to inhibit PRMT6 selectively<br />
compared to PRMT1. The presented assay protocols are also suitable for HTS of larger<br />
compound libraries.<br />
References:<br />
1. Bissinger, E.-M. et al.: Med. Chem. Commun. 2010, 1(2): 105–172.<br />
2. Bissinger, E.-M. et al.: Bioorg. Med. Chem. 2011, 19(12): 3717–3731.<br />
083<br />
The estrogen receptor as a target for anabolic effects of phytoecdysteroids in<br />
mammals<br />
Haupt, O1); Tchoukouegno Ngueu, S1); Diel, P1); Parr, MK1 2)<br />
1 Center for Preventive Doping Research, German Sport University, Cologne, Germany<br />
2 Institute of Pharmacy, Freie <strong>Universität</strong> Berlin, Germany<br />
Phytoecdysteroids, such as ecdysterone (chemical structure in figure 1), are structurally<br />
similar or identical to insect-moulting hormones and have been detected in various plant<br />
species. In mammals ecdysteroids are reported to produce a range of effects including the<br />
stimulation of protein synthesis and physical performance. Therefore, ecdysterone is<br />
promoted as active component in several over-the-counter supplements and may be<br />
misused by athletes to increase muscle mass.<br />
The anabolic-androgenic potency of ecdysterone was studied in-vivo. In a Hershberger<br />
assay analogue design male Wistar rats were treated for 21 days either with 5 mg/kg<br />
ecdysterone s.c. or placebo. After necropsy, the weights of the prostate, seminal vesicle<br />
and levator ani muscle were measured. The muscle fiber size of the soleus muscle and the<br />
gastrocnemius muscle was determined. The anabolic effect of ecdysterone resulted in an<br />
112 Poster
increase of the fiber size in both muscles. An androgenic effect could not be observed.<br />
In order to elucidate the molecular mechanism involved in the anabolic activity of<br />
ecdysterone, cell culture experiments were conducted using the mouse skeletal muscle cell<br />
line C2C12. Differentiation of C2C12 cells towards myotubes was induced. The resulting<br />
myotubes were incubated in the presence of ecdysterone, dihydrotestosterone (DHT) and<br />
antiandrogenic or antiestrogenic substances, such as flutamide (an antiandrogen) and ZK<br />
191703 (an antiestrogenic substance). Additionally co-incubation of with ecdysterone and<br />
the antagonists was performed. Cells were fixed and myotube diameters were determined<br />
by glutaraldehyde-induced autofluorescence and morphometry.<br />
The treatment of the cells with ecdysterone resulted in an increased myotube-diameter.<br />
This stimulation could be antagonized by an antiestrogen, which suggests an estrogen<br />
receptor mediated mechanism. Flutamide, an anti-androgen, could not antagonize this<br />
effect, which supports our hypothesis that the anabolic effect of ecdysterone is mediated by<br />
an estrogen receptor mediated pathway.<br />
In summary these observations are relevant for the development of new strategies for the<br />
treatment of skeletal muscle injuries such as sarcopenia and cachectic diseases but also to<br />
provide scientific data that allow for the classification of ecdysterone in preparations.<br />
Currently in Germany a categorization either as novel food or as pharmaceutical is<br />
discussed.<br />
Figure 1: Chemical structure of ecdysterone<br />
084<br />
Regulation of 5-lipoxygenase gene expression by AF4 and MLL as<br />
transcriptional regulators<br />
Ahmad, K. 1; Marschalek, R. 2; Steinhilber, D. 1<br />
1 Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main,<br />
Germany<br />
2 Institute of Pharmaceutical Biology, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany<br />
Besides its function in inflammation, recent findings support a role of 5-lipoxygenase (5-LO)<br />
as critical regulator of leukemia, creating a further link between inflammation and cancer<br />
[1]. The mechanism of this interaction still remains unclear.<br />
5-LO which is encoded by the ALOX5 gene is an enzyme that catalyses the first two steps<br />
in the biosynthesis of the pro-inflammatory leukotrienes derived from arachidonic acid [2]. It<br />
is known that ALOX5 gene expression is controlled by 1,25-dihydroxyvitamin D3 (calcitriol)<br />
and transforming growth factor β (TGFβ) [3]. The resulting upregulation of the 5-LO gene<br />
expression is mediated by the binding of TGFβ effector proteins SMADs and the vitamin D<br />
receptor (VDR) to response elements within the 5-LO gene and is due to transcript<br />
elongation [4,5].<br />
Concerning the transcript elongation it is described that the AF4 (ALL-1 fused gene on<br />
chromosome 4) protein is involved in the regulatory process of the transcript elongation and<br />
exhibits chromatin remodelling activities [6]. Furthermore the chromosomal translocation of<br />
the AF4 gene and the MLL (mixed lineage leukemia) gene leads to the production of der4<br />
(AF4-MLL) and der11 (MLL-AF4) fusion proteins. These fusion proteins are also associated<br />
with the pathomechanism of leukemia [7].<br />
To elucidate the involvement of AF4, MLL and their fusion proteins on the 5-LO gene<br />
expression, reporter gene assay experiments were performed. Thus different 5-LO reporter<br />
gene constructs were cotransfected with AF4, MLL, der4 or der11.<br />
The results revealed that both AF4 and MLL are involved in the control of 5-LO gene<br />
expression as transcriptional regulators. Furthermore the presented data suggest that der4<br />
and der11 are able to deregulate the 5-LO gene expression either in the step of transcript<br />
initiation or at the step of transcript elongation. Thus, MLL and its fusion proteins provide a<br />
potential link between 5-LO and leukemia development.<br />
Acknowledgments: Hans Kröner - Graduiertenkolleg<br />
References:<br />
1. Chen, Y. et al.: Nat Genet. 2009, 41(7): 783-92.<br />
2. Rådmark, O. et al.: Trends Biochem Sci. 2007, 32(7): 332-41<br />
3. Brungs, M. et al.: Proc. Natl. Acad. Sci. USA 1995, 92(1): 107-11<br />
4. Seuter, S., Sorg, BL., Steinhilber, D.: Biochem Biophys Res Commun. 2006, 348(4): 1403-10<br />
5. Stoffers, KL. et al.: J Mol Biol. 2010, 395(4): 884-96<br />
6. Bitoun, E., Oliver PL., Davies, KE.: Hum Mol Genet. 2007, 16(1): 92-106<br />
7. Gaussmann, A. et al.: Oncogene. 2007, 26(23): 3352-63<br />
085<br />
In vitro study of colonic microbial metabolization and absorption kinetics of<br />
olsalazine in the TIM-2 model simulating the conditions in the proximal large<br />
intestine<br />
Glöckl G1;Venema K2;Wilson CG3; Weitschies W1 1Institute of Pharmacy, <strong>Ernst</strong> <strong>Moritz</strong> <strong>Arndt</strong> University, <strong>Greifswald</strong>, Germany<br />
2TNO Healthy Living, Zeist, The Netherlands<br />
3Institute of Pharmacy and Biomedical Sciences, Strathclyde University, Glasgow, UK<br />
It is well-known that the colonic epithelium expresses a lot of transporters, either for uptake<br />
or efflux, as well as metabolizing enzymes. The epithelial metabolism is superimposed by<br />
the metabolic action of the microbiota colonizing the human colon. The complex interplay<br />
between these processes is the origin of the limited understanding of the colonic absorption<br />
process. The latter is of special importance when administering standard oral extended<br />
release formulations.The microbial metabolic activity is difficult to investigate in vivo<br />
(location) as well as in vitro (anaerobic environment). Therefore, TNO’s in vitro model of the<br />
large intestine (TIM-2) wasdesignedto mimic the conditions in the proximal part ofthe<br />
human colon [1].<br />
Some prodrugs, e.g. for the medication of chronic inflammatory bowel diseases, are<br />
specifically cleaved in the colon by means of microbial reducing enzymes. The aim of the<br />
presented study is to investigate in vitrohow the azo-prodrugolsalazineis cleaved by<br />
faecalmicrobiota and further absorbed using the TIM-2 model.<br />
The computer controlled TIM-2 system consists of glass tubes and flexible walls allowing<br />
the content to be mixed in a peristaltic movement. The device is continuously flushed with<br />
nitrogen generating an anaerobic environment and kept at body temperature. The pH is<br />
adjusted to 5.8 by adding NaOH. The system is further equipped with a set of hollow fiber<br />
membranes allowing the fermentation products (predominately short chain fatty acids) to be<br />
eliminated from the system. Therefore, a dialysis liquid containing electrolytes and vitamins<br />
in a phosphate buffer pH 5.8 is pumped through the system. The volume of the luminal<br />
content is kept at 120 mL using an integrated level sensor.For a continuous measurement<br />
of the luminal redox potential an electrode (InLab ® Redox, Mettler) was integrated in a<br />
specially designed glass compartment.<br />
The system is inoculated with standard pooled faecalmicrobiota obtained from healthy<br />
volunteers. For feeding, a standard ileal efflux medium mainly consisting of peptides and<br />
polysaccharides is continuously delivered to the system. Each day 25 mL of luminal<br />
contentare withdrawn and replaced by dialysis liquid to simulate passage of stool.<br />
Olsalazine was utilized as model drug to evaluate the metabolic function of the microbiota.<br />
The prodrugolsalazine is cleaved by microbial azoreductases to form two molecules of the<br />
active mesalazine. Both compounds can easily be quantified with UV/Vis spectrometry in<br />
the lumen as well as in the dialysate.<br />
After addition of 500 mg of olsalazineto the TIM-2 system via the sampling port we could<br />
measure the elimination of olsalazine and the appearance and subsequent elimination of its<br />
metabolite mesalazine. The elimination rate was inversely proportional to the molecular<br />
weight and thus faster for mesalazine.<br />
The integration of a redox electrode into the system allowed the continuous measurement<br />
of the intraluminal redox potential. After an initial drop of the potential in the growth phase<br />
of the microbiota(adaptation period of about 16 hours) a strongly reducing environment was<br />
generated (stable ecosystem maintained for at least 48 hours). During this period, the<br />
system is suitable to simulate the metabolization and absorption of the model drug<br />
olsalazine and probably of other actives. In future studies we will further check if these data<br />
can be correlated with in vivo data.<br />
Acknowledgments: G.G. thanks W. Borst, A. Maathuis, J. Lelieve d and M. Minekus (TNO) for their<br />
assistance and GalenusPrivatstiftung for a personal grant. The project was funded by the German federal<br />
ministry of education and research (InnoProfile, FKZ: 03IP612).<br />
References:<br />
1. Minekus, M. et al.:Appl. Microbiol. Biotechnol.1999, 53(1): 108-114.<br />
086<br />
Development of long-term stable hydrogel matrices for a vessel-simulating<br />
flow-through cell<br />
Semmling, B. 1; Seidlitz, A. 1; Nagel, S. 1; Schütt, R. 1; Brand, T. 1; Grabow, N. 2; Sternberg, K. 2;<br />
Weitschies, W. 1<br />
1 Institute of Pharmacy, EMA University of <strong>Greifswald</strong>, Felix-Hausdorff-Straße 3, 17487 <strong>Greifswald</strong>,<br />
Germany<br />
2 Institute for Biomedical Engineering, University of Rostock, Friedrich-Barnewitz-Straße 4, 18119<br />
Rostock, Germany<br />
Natural polymeric gels are often unstable and very sensitive to erosion. Therefore synthetic<br />
polymeric materials, gelling agents such as polyvinylalcohol (PVA) or polyacrylamide (PAA)<br />
are interesting due to various properties: stability, elasticity, diffusibility, hydrophilicity, nontoxicity.<br />
Modifications of the composition and the cell entrapment in the hydrogel are<br />
feasible. Drug dissolution as well distribution processes of coated drugs of drug-eluting<br />
stents (DES) were determined in the vessel-simulating flow-through cell (vessel-simulating<br />
FTC) [1]. The current study focuses on the approach of developing long-term stable<br />
hydrogel matrices for the vessel-simulating FTC by replacing the conventional polymer<br />
matrix (2 % (w / w) sodium alginate matrix, AM) by 15 % (w / w) PVA-hydrogel matrices<br />
(PVA-M), 10 % (w / w) PAA matrices (PAA-M) and 2 % (w / w) agarose hydrogel matrices<br />
(Ag-M). The distribution coefficient (DC) of the fluorescent model drug substance<br />
triamterene between the hydrogel and aqueous media, long-term stability of the novel<br />
hydrogel compartments and drug distribution between the three compartments: hydrogel,<br />
dissolution media and stent were investigated.<br />
The distribution coefficient of the model substance between the investigated long-term<br />
stable hydrogel matrices and dissolution media indicate differences in dependency on the<br />
respective hydrogel matrix (data Ag-M and PAA-M not shown): Compared to the reference<br />
matrix (DC of AM after 72 h was found to be about 1.4) the drug delivered into the hydrogel<br />
compartment accumulated up to 2.3 (after 72 h, PVA-M). Test results of the examination of<br />
the long-term stability of novel hydrogel matrices show no signs of gel erosion within 4<br />
weeks perfusion (data not shown) indicating mechanical long-term stability of the novel<br />
hydrogels under typical test conditions.<br />
Release profiles from triamterene coated DES (Eudragit ® RL / RS 30 / 70, dissolution<br />
media PBS 7.4, dissolution temperature 37 °C ± 0.5 °C, flow rate 35 mL / min [2]) obtained<br />
with the vessel-simulating FTC using the novel matrices (PAA-M, PVA-M and Ag-M)<br />
indicate a fast increase in the amount of dissolved drug in the dissolution media (Ag-M:<br />
82 % after 12 h) with a simultaneous fast decrease in residual drug amount in the DES<br />
coatings (Ag-M: 14 % after 12 h). After 12 hours 4 % of the total drug amount was detected<br />
in the agarose matrices. In comparison to release profile of fluorescent model drug using<br />
Ag-M, a slightly different dissolution profile of drug release using the reference hydrogel<br />
matrix (AM) could be observed. After 12 hours about 77 % of the total drug amount eluted<br />
from DES into dissolution media was detected. The residual drug amount in stent coating<br />
increases continuously and was found to be about 18 % after 12 hours. At the same<br />
sampling point the total drug amount in the sodium alginate hydrogel compartment<br />
accumulated up to 5 %. The difference in detected drug amount in the compartments<br />
dissolution media, hydrogel (AM, Ag-M, PAA-M and PVA-M) and DES may be attributed to<br />
different distribution coefficient of triamterene between the hydrogel compartments and<br />
PBS 7.4.<br />
In conclusion, the methods of hydrogel matrix preparation (PAA-M, PVA-M and Ag-M) were<br />
established successfully. The stability studies confirmed sufficient mechanical robustness<br />
of the matrices under long-term test conditions. Additionally, the developed matrices for the<br />
vessel-simulating FTC enable the determination of delivered drug not only in dissolution<br />
media representing the blood, but also in hydrogel matrices simulating the vessel wall as<br />
target organ structure. Consequently, the use of the gel compartment allows the adaption of<br />
the in vitro test closer to the physiological situation and therefore enables the estimation of<br />
the drug distribution of DES under more realistic test conditions.<br />
Poster 113
Acknowledgments: Financial support by the Federal Ministry of Education and Research (BMBF) within<br />
REMEDIS “Höhere Lebensqualität durch neuartige Mikroimplantate” (FKZ: 03IS2081) is gratefully<br />
acknowledged.<br />
References:<br />
1. Neubert, A. et al.: J. Control. Release 2008, 130(1): 2-8.<br />
2. Di Mario, C et al.: Am. J. Cardiol. 1993, 71(14): 54D-61D.<br />
087<br />
New titanium(IV) complexes as potential anticancer drugs<br />
Schur, J. 1; Manna, C.M. 2; Tshuva, E.Y. 2; Deally, A. 3; Tacke, M. 3; Köster, R. 4, Ott, I. 1<br />
1 Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstr. 55, 38106<br />
Braunschweig, Germany<br />
2 The Hebrew University of Jerusalem, Department of Inorganic Chemistry, 91904 Jerusalem,<br />
Israel<br />
3 University College Dublin, UCD School of Chemistry and Chemical Biology, Belfield, Dublin 4,<br />
Ireland<br />
4 Zoological Institute, Department of Cell Physiology, TU Braunschweig, Spielmannstr. 8, 38106<br />
Braunschweig, Germany<br />
Since the serendipitous discovery of the antitumor agent cisplatin in the 1960s, research on<br />
metal-based compounds has made remarkable progress. Nowadays, platinum-based<br />
metallodrugs (e.g. oxaliplatin) play a major role in medical anticancer treatment.<br />
Nevertheless, the activity is limited to a small spectrum of cancers because of increasing<br />
resistance phenomena of these drugs and several side effects caused by high toxicity.<br />
Therefore, in the last two decades, research has been extended to non-platinum<br />
metallodrugs.<br />
An innovative compound in the 1990s was the titanium-based metallodrug titanocene<br />
dichloride that reached clinical trials but failed because of decreased hydrolytic stability and<br />
formulation problems [1]. Based on these promising works and with the aim of improving<br />
cytotoxicity and hydrolytic stability better stabilized titanium-containing compounds e.g.<br />
titanocene Y and a new class of non-Cp diamino bis(phenolato) “salan” Ti(IV) complexes<br />
were recently developed and seem to hold a high potential for anticancer therapy [2,3].<br />
Figure 2: Metallodrugs: (a) Oxaliplatin, (b) Titanocene dichloride, (c) Titanocene Y, (d)<br />
Salan-Ti(IV) complex<br />
In our study the mentioned lead compounds were investigated biologically in comparison to<br />
titanocene dichloride. Very interesting properties for the novel class of salan-Ti(IV)<br />
complexes (see figure 1) and titanocene Y (as a substituted titanocene) could be obtained<br />
and may be a step forward in target identification of titanium-based anticancer drugs.<br />
Figure 3: Zebrafish embryos (94 hpf) exposed to 100 µM Salan-Ti(IV)<br />
The lead compound of new diamino salan-Ti(IV) complexes and titanocene Y have an<br />
improved cytotoxicity in the low micromolar range and a higher accumulation in cancer cells<br />
compared to titanocene dichloride. A relationship between the antiproliferative activity, the<br />
cellular uptake and the protein binding ability of the tested Ti-containing compounds was<br />
noted. In addition, the toxicity of the new titanium-based complexes in zebrafish embryos [4]<br />
was studied and no toxic effects could be observed up to a concentration of 100 µM (see<br />
figure 2). Further investigations were performed on the DNA-binding efficacy, the nuclear<br />
drug content and the drugs accumulation in mitochondria. Our results indicate that novel<br />
titanium(IV) complexes possibly have overcome the disadvantages of the well known<br />
titanocene dichloride, which underlines their high potential as anticancer drugs.<br />
References:<br />
1. Melendez, E., Crit. Rev. Oncol. Hematol. 2002, 42: 309–315.<br />
2. Sweeney, N.J. et al., J. Organomet. Chem. 2005, 690: 4537–4544.<br />
3. Peri, D. et al., Chem. Eur. J. 2009, 15: 2403–2415.<br />
4. Parng, C. et al., ASSAY Drug Dev. Technol. 2002, 1: 41–48.<br />
088<br />
Anti-infectives with novel mode of action: Interruption of P. aeruginosa cell-tocell<br />
communication by PqsD inhibitors<br />
Maurer, C. K. 1; Storz, M. P. 1; de Jong, J. C. 1; Weidel, E. 1; Zimmer, C. 1; Steinbach, A. 1;<br />
Hartmann, R. W. 1 2<br />
1 Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123 Saarbrücken,<br />
Germany<br />
2 Department of Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3,<br />
66123 Saarbrücken, Germany<br />
The opportunistic pathogen P. aeruginosa controls the coordinated production of virulence<br />
factors as well as biofilm formation via a cell density dependent cell-to-cell communication<br />
system known as quorum sensing (QS) [1]. The pqs QS system employs the signal<br />
molecules PQS and its precursor HHQ to activate PqsR [2], a transcriptional regulator that<br />
drives the expression of many genes involved in pathogenicity.<br />
PqsD is a key enzyme in the biosynthesis of HHQ and PQS [3]. Since reduction of HHQ<br />
production is accompanied by reduced mortality in infected mice [4], PqsD proves to be an<br />
interesting target for drug discovery.<br />
Two different drug design strategies were applied: (A) a ligand based approach including<br />
analogues of the natural substrate anthraniloyl-CoA (ACoA) and mimics of the<br />
corresponding transition state as well as (B) the development of compounds derived from<br />
known inhibitors of FabH, an enzyme, which is structurally and functionally related to PqsD<br />
[5,6]. Thereby, inhibitors with IC50 values in the nM to low µM range have been identified.<br />
SPR experiments revealed, that, while (2-nitrophenyl)methanol compounds derived from<br />
approach (A) resulted in time-dependent irreversible PqsD inhibition with binding within the<br />
anthraniloyl binding site, 2-benzamidobenzoic acid derivatives from (B) are reversible<br />
inhibitors that do not occupy the anthraniloyl binding site.<br />
The effect of representative compounds from both classes on the extracellular levels of the<br />
signalling molecules HHQ and PQS in P. aeruginosa PA14 was investigated. While<br />
2-benzamidobenzoic acids had no significant effect, (2-nitrophenyl)methanol derivatives<br />
strongly reduced extracellular HHQ and PQS levels without affecting cell viability.<br />
References:<br />
1. Dubern, J. F.; Diggle S. P.: Mol. BioSyst. 2008, 4: 882.<br />
2. Cao, H. et al.: Proc Natl Acad Sci USA 2001, 98(25): 14613-8.<br />
3. Pistorius, D. et al.: ChemBioChem. 2011, 12(6): 850-3.<br />
4. Lesic, B. et al.: PLoS Pathogens 2007, 3: 1229.<br />
5. Bera, A. K. et al.: Biochemistry 2009, 48: 8644-8655.<br />
6. Nie, Z. et al.: J. Med. Chem. 2005, 48: 1596-1609.<br />
089<br />
A Vascular function of Cyclin dependent kinase 5 (Cdk5)<br />
Liebl J. 1, Moser M. 2, Hager B. 1, Zhang S. 1, Fürst R. 1, Bibb J. A. 3, Adams R. H. 4, Vollmar A.<br />
M. 1, Zahler S. 1<br />
1Center of Drug Research, Ludwig-Maximilians University, Munich, Germany<br />
2Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany<br />
3Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, Texas<br />
4Department of Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, Münster,<br />
Germany<br />
Cyclin-dependent kinase 5 (Cdk5) is essential for CNS development and function. Whereas<br />
its role in neurons has been studied in detail, there still exists only limited knowledge about<br />
its functions in other systems. During recent years, more and more reports indicated<br />
functions of Cdk5 in non-neuronal tissues and we could show that Cdk5 regulates<br />
endothelial cell migration in vitro.<br />
This work elucidates an important function of Cdk5 in the vascular system in vivo. We<br />
specifically disrupted the Cdk5 gene in the mouse endothelium using the Cre-loxP system.<br />
Floxed Cdk5 mice were intercrossed with mice expressing Crerecombinase under control<br />
of the Tie2 promoter (Tie2Cre) or a tamoxifen-inducible VE-Cadherin promoter<br />
(Cdh5(PAC)-CreERT2), respectively. Breedings of Cdk5fl/wtTie2Cre males and Cdk5fl/fl<br />
females resulted in offspring in which only 15% pups harbored the Cdk5fl/flTie2Cre<br />
genotype instead of expected 25% and most of the endothelial-specific Cdk5 knockout<br />
mice did not survive past postnatal day 28. Analysis of the genotypes of embryos at<br />
different stages indicated that Cdk5fl/flTie2Cre embryos frequently died during late<br />
embryogenesis between E16.5 and E18.5. Endothelial-specific Cdk5 knockout leads to a<br />
non-separation phenotype between blood and lymphatic system including edema<br />
formation, blood-filled and dilated lymphatic vessels, defective lymphatic valve formation,<br />
and impaired draining function of the lymphatic system. Concerning blood vessels, we<br />
found a hypervascularization of endothelial-specific Cdk5 knockout mice. This was<br />
indicated by an increased number of branching points in the yolk sacs and skin of E16.5<br />
Cdk5fl/flTie2Cre embryos and increased branching and sprouting in the developing retina<br />
of d6 pups with Cdk5fl/flCdh5(PAC)-CreERT2 genotype after tamoxifen treatment as well<br />
as after pharmacological inhibition of Cdk5 with the small molecule roscovitine. Most<br />
importantly, we found a reduction of tumor growth of subcutaneous implanted wildtype<br />
B16F1 melanoma xenografts in endothelial-specific Cdk5 knockdown mice. Histological<br />
analysis of tumors showed an immature vascularization of tumors grown in<br />
Cdk5fl/flCdh5(PAC)-CreERT2 mice.<br />
To sum up, our results reveal an important vascular function of Cdk5 in angiogenesis and<br />
lymphangiogenesis, suggesting Cdk5 as druggable target for anti-angiogenic therapy.<br />
090<br />
A novel assay for the analysis of inhibitors of ergosterol biosynthesis<br />
Staudacher V; Müller C; Bracher F<br />
Ludwig-Maximilians-<strong>Universität</strong> München, Department für Pharmazie - Zentrum für<br />
Pharmaforschung, Butenandtstr. 5-13, 81377 München, Germany<br />
Systemic fungal infections are of increasing importance in intensive care, due to the rising<br />
portion of immunocompromised patients (cancer, AIDS, and organ transplantation patients)<br />
in clinical routine. These infections are characterized by a very high mortality, and very<br />
potent and fast-acting antifungal agents are needed for life-saving therapy. Moreover,<br />
resistance to established antifungal drugs (e.g. fluconazole) gains in clinical significance.<br />
Ergosterol is the central sterol in fungal metabolism, and inhibition of ergosterol<br />
biosynthesis represents a classical, but still highly effective strategy in antifungal therapy.<br />
Over the decades, inhibitors of several enzymes in ergosterol biosynthesis have been<br />
marketed, the most prominent ones being the azole-type inhibitors of the enzyme C14demethylase.<br />
The morpholine antifungal amorolfine inhibits two enzymes in ergosterol<br />
biosynthesis, but is not suitable for systemic application, and the squalene epoxidase<br />
inhibitors (naftifine, terbinafine) mainly target dermatophytes.<br />
Notably, the post-squalene part of ergosterol biosynthesis contains a significant number of<br />
additional enzymes which occur only in fungi, but not in mammals, and therefore are<br />
potential targets for new, selective antifungal drugs. In our ongoing research on the<br />
development of new antifungals [1] we focused on new inhibitors of enzymes in ergosterol<br />
biosynthesis. In order to perform effective optimization of first lead structures, there was a<br />
need to work out a screening system which enables us to (a) identify the target enzyme in<br />
ergosterol biosynthesis unambiguously, and (b) gain quantitative data on the effect of the<br />
inhibitors on overall ergosterol biosynthesis.<br />
For this purpose we developed a convenient whole-cell screening assay, which in a first<br />
step enables us to identify substances, which interfere with enzymes of the post-squalene<br />
part of ergosterol biosynthesis, and in a second step to determine IC50 values for inhibition<br />
114 Poster
of ergosterol biosynthesis.<br />
In this assay three different yeasts, Candida glabrata, Saccharomyces cerevisiae and<br />
Yarrowia lipolytica, are treated with test substances. After alkaline cell lysis the sterols are<br />
extracted with an organic solvent, and the crude extract is purified by dispersive solid phase<br />
extraction (dSPE). After silylation the sterols are analyzed by GC-MS.<br />
The obtained sterol patterns provide an indication of the inhibited enzyme(s), since<br />
inhibition of defined enzymes leads to the accumulation of specific sterols. By this way we<br />
can identify inhibitors of any enzyme in the post-squalene part of ergosterol biosynthesis in<br />
one single cellular assay, with no need for having the single purified enzymes in hands.<br />
Furthermore, we extended this qualitative screening method to a quantitative determination<br />
of the effect on total ergosterol biosynthesis. In analogy to a screening system worked out<br />
by us for inhibitors of cholesterol biosynthesis recently [2], incubation with the inhibitors in<br />
the presence of 13C-acetate, followed by GC-MS analysis of the labeled ergosterol isotopes<br />
allows for the convenient determination of IC50 values of the inhibitors.<br />
The system was calibrated with several known antifungals targeting ergosterol<br />
biosynthesis, and meanwhile applied to the identification of the target enzymes of a number<br />
of new inhibitors of ergosterol biosynthesis.<br />
References:<br />
1. Renard D, Perruchon J, Giera M, Müller J, Bracher F: Bioorg. Med. Chem. 2009, 17(23) 8123-8137.<br />
2. Giera M, Plössl F, Bracher F: Steroids 2007, 72(8): 633-642.<br />
091<br />
Cysteine-rich LIM-only protein 4 (CRP4) opposes angiotensin II induced<br />
pathological heart hypertrophy and fibrosis<br />
Straubinger, J.; Majer, M.; Ruth, P.; Lukowski, R.<br />
Institute of Pharmacy, Department of Pharmacology, Toxicology and Clinical Pharmacy, University<br />
of Tübingen, Germany<br />
Background: Cardiac hypertrophy is an adaptive response of the heart to many cardiovascular<br />
disorders including hypertension, infarction and defects of the valves. Although<br />
physiological heart hypertrophy, which refers to an adaption of the heart muscle to healthy<br />
exercise or pregnancy, and pathological hypertrophy share common features, only the<br />
pathological remodelling of the heart leads to fibrosis, increased diastolic stiffness, fatal<br />
failure and death. Clinical studies and functional analysis of genetically modified mice have<br />
established that natriuretic peptides (NP) like ANP and BNP and their cardiac receptors<br />
diminish development of pressure and volume induced heart hypertrophy and fibrosis via<br />
the second messenger cyclic guanosine-3´,5´-monophosphate (cGMP). However, the<br />
signaling events downstream of cGMP and its major effector cGMP kinase I (cGKI) are less<br />
clear. Here, we studied the role of the cysteine-rich LIM-only protein (CRP4) for the cardiac<br />
response to an increase in afterload and healthy exercise training. CRP4 is directly targeted<br />
by cGKI in the cardio-vascular system and a highly related homologue of the muscle LIM<br />
protein CRP3/MLP. CRP/MLP knockout mice develop a dilated cardiomyopathy and<br />
defects in CRP3/MLP are the cause for dilated and hypertrophic cardiomyopathies in<br />
human.<br />
Methods: Gene-targeted CRP4 knockout (KO) mice and their heterozygous (HET) and wild<br />
type (WT) littermates were subjected to a chronic pressure-dose of angiotensin II (AngII).<br />
Cardiac hypertrophy, as defined by the heart-to-body weight ratios (HW/BW), was<br />
determined in AngII treated mice and compared to littermate animals that received saline or<br />
were trained by a duration-controlled swimming exercise protocol to induce a physiological<br />
adaption of the heart muscle. Changes in hypertrophy marker genes, putative effects of<br />
AngII on components of the NP/cGMP pathway and the expression pattern of other<br />
members of the CRP protein family were analyzed in total mRNA and protein isolated from<br />
the ventricles. These experiments were corroborated by the localization of CRP4 in the<br />
myocardium and Sirius red stainings as a quantitative measure of fibrosis.<br />
Results: Under basal conditions and upon AngII infusion CRP4 mRNA and protein were<br />
detectable in the myocardium from WT mice, whereas CRP4 was absent from KO hearts<br />
and significantly reduced in HET mice. HW/BW ratios of all three genotypes were not<br />
different at baseline, but increased in HET and KO animals in response to infusion of AngII.<br />
Sirius red staining and quantitative RT-PCR experiments revealed an increase in interstitial<br />
fibrosis and a diminished production of anti-fibrotic factors such as BNP in HET and KO<br />
hearts. In contrast, neither the increase in cardiac mass nor the extent of fibrosis were<br />
altered upon physiological growth of the heart muscle between the different genotypes.<br />
Conclusion: Hearts of CRP4 KO mice are more susceptible to AngII-induced remodelling,<br />
whereas the lack of CRP4 does not alter physiological cardiac hypertrophy. Therefore, we<br />
conclude that CRP4 is a beneficial mediator in NP/cGMP/cGMP kinase I signaling to<br />
oppose pathological heart growth and fibrosis induced by cardiac Gαq-pathways.<br />
092<br />
Comparison of binding requirements in a series of benzimidazol-2-yl-aminosubstituted<br />
(L)-amino acids by simple QSAR models for the NMDAR glycineB<br />
site and the transporter LAT1<br />
Krauß, A. 1; Rotmann, A. 2 3; Martiné, U. 2; Closs, E. I. 2; Dannhardt, G. 1<br />
1 Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University, Staudingerweg 5,<br />
55128 Mainz, Germany<br />
2 Department of Pharmacology, Johannes Gutenberg-University, Obere Zahlbacher Straße 67,<br />
55101 Mainz, Germany<br />
3 Department of Parasitology, Ruprecht Karls-University, Im Neuenheimer Feld 324, 69120<br />
Heidelberg, Germany<br />
Central bioavailability is a major concern in the development of new ligands targeting the<br />
glycine binding site of the NMDA receptor, as potential applications comprise inter alia<br />
epilepsy, chronic pain, dementia (Alzheimer’s disease, Parkinson’s disease) or<br />
schizophrenia [1]. Uptake by amino acid transporters [2], especially large neutral amino<br />
acid transporters (system L), is therefore considered as a versatile strategy to cross the<br />
blood-brain barrier without disruption. The concept was proven for the low affinity glycineB<br />
site antagonist 4-chlorokynurenine as reviewed in [3], and recently applied to analogues of<br />
pregabalin [4] and analogues of melphalan [5].<br />
This work is based on a congeneric series of benzimidazol-2-yl-amino-substituted (L)amino<br />
acids obtained in a seven-step linear synthesis and assayed for NMDAR glycineB<br />
site binding (displacement of [ 3H]-MDL-105,519 from pig cortical membranes) as well as<br />
system L activity (competition with [ 3H]-L-Phe uptake in C6 rat glioblastoma cells). Two<br />
corresponding QSAR models have been derived, utilizing GraphPad Prism 3.00, GraphPad<br />
Instat 3.06 and NLREG 6.4. for stepwise multiple (non)linear regression. The NMDAR-<br />
QSAR suggests a parabolic relationship with STERIMOL parameter L and, likewise<br />
negative but linear, correlations with Swain and Lupton R and the hydrogen bond indicator<br />
variable HD. The LAT-QSAR discloses an increasing lipophilicity (ClogP) and a decreasing<br />
pka value (of the guanidino group, mainly experimental) as favourable for affinity. Taken<br />
together, within a restricted applicability domain, parallel steric resp. lipophilic demands<br />
dominate a certain opposite trend in the electronic effects. Semi-empirical quantum<br />
mechanical calculations with Spartan 04 (Wavefunction, Inc.) at the HF 3-21G(*) level [6]<br />
may explain these electronic prerequisites partly by =N---HN intramolecular hydrogen<br />
bonding, this proving advantageous for the NMDA receptor, but disadvantageous for LAT.<br />
However, the pronounced conformational flexibility of the scaffold allows for various binding<br />
modes, each with the free amino acid moiety as the most important and common motif.<br />
Moreover, compared to the rather specific and sterically demanding interactions at the<br />
glycineB site of the NMDAR [7], the system L-type transporters are known for their broad<br />
substrate profile, ranging from large neutral (LAT1) to large and small neutral (LAT2) amino<br />
acids [8]. A joint further development of compounds considering both targets is therefore<br />
regarded as possible.<br />
Acknowledgements: The VCCLAB (Virtual Computational Chemistry Laboratory, http://www.vcclab.org) is<br />
appreciated as a fast and user-friendly access to calculated log P values, enabling a comparison of different<br />
methods [9].<br />
References:<br />
1. Bauman, A. et al.: J. Label. Compd. Radiopharm. 2011, 54(10): 645–656.<br />
2. Closs, E. I. et al.: J. Nutr. 2004, 134(10): 2752S–2759S.<br />
3. Kohl, B. K.; Dannhardt, G.: Curr. Med. Chem. 2001, 8(11): 1275–1289.<br />
4. Belliotti, T. et al.: J. Med. Chem. 2005, 48(7): 2294–2307.<br />
5. Matharu, J. et al.: Bioorg. Med. Chem. Lett. 2010, 20(12): 3688–3691.<br />
6. Krauß, A.; Dannhardt, G.: Joint Meeting of the Czech, German and Hungarian Pharmaceutical Societies,<br />
2006, Marburg, Germany Abstract C045, p.115.<br />
7. Jansen, M.; Dannhardt, G.: Eur. J. Med. Chem. 2003, 38(10): 855–865.<br />
8. Verrey, F. et al.: Pflügers Arch. - Eur. J. Physiol. 2004, 447(5): 532–542.<br />
9. Tetko, I. et al.: J. Comput. Aid. Mol. Des. 2005, 19(6): 453–463.<br />
093<br />
A comparative study of in vitro methods analyzing endothelial barrier function:<br />
conventional macromolecular permeability measurements versus<br />
sophisticated impedance sensing<br />
Hornburger, M. C. 1; Mayer, B. A. 1; Vollmar, A. M. 1; Wegener, J. 2; Fürst, R. 1<br />
1 Department of Pharmacy, Center for Drug Research, Pharmaceutical Biology, University of<br />
Munich, Butenandtstr. 5-13, 81377 Munich, Germany<br />
2Institute for Analytical Chemistry, Chemo- & Biosensors, University of Regensburg, <strong>Universität</strong>sstr.<br />
31, 93053 Regensburg, Germany<br />
Endothelial barrier dysfunction, i.e. the opening of interendothelial junctions, and the<br />
consequent formation of edema are hallmarks of many severe diseases, such as sepsis,<br />
acute lung injury, or atherosclerosis. Drugs that specifically target endothelial barrierregulating<br />
systems are lacking and there is a strong demand to identify new<br />
pharmacological targets. For the validation of these targets, the barrier function of cultured<br />
endothelial cells is analyzed by two different approaches: 1) the classic measurement of<br />
macromolecular permeability and 2) the modern real-time cell impedance-sensing methods,<br />
which employ alternating currents to analyze changes of cellular morphology. Impedance is<br />
a complex physical quantity consisting of an imaginary capacitive (cell membrane<br />
properties) and a real resistive (intercellular junction stability) contribution.<br />
We aimed at deciphering the most appropriate method for the characterization of<br />
endothelial barrier-modulating agents. Therefore, we compared macromolecular<br />
permeability measurements (two-compartment Transwell ® system, membrane pore size 0.4<br />
μm, tracer: FITC-dextran 40 kDa) with three different commercially available impedance<br />
sensing devices: (i) ECIS ® (Applied Biophysics) and (ii) xCELLigence (Roche), with the<br />
cells directly grown on gold electrodes, and (iii) cellZscope ® (nanoAnalytics), where cells<br />
are placed on a permeable membrane (pore size 0.4 μm) between two electrodes.<br />
First, we tested for the correlation between macromolecular permeability and impedance<br />
measurements. Therefore, we employed the barrier-disruptive mediators TRAP (thrombin<br />
receptor-activating peptide), histamine, and TNF-α, as well as the barrier-stabilizing<br />
compound forskolin (adenylyl cyclase activator) and the Rho kinase (ROCK) inhibitor Y-<br />
27632. The impact of TRAP and forskolin on endothelial barrier function nicely correlated in<br />
all tested systems: forskolin elevated impedance values and reduced dextran flux, whereas<br />
TRAP evoked a fast and sustained decrease of impedance and a significant increase in<br />
macromolecular permeability. Interestingly, the histamine-induced barrier breakdown could<br />
not be detected by the Transwell ® assay, whereas a rapid and transient decrease of<br />
endothelial impedance occurred. The barrier-protecting ROCK inhibitor evoked a massive<br />
and long-lasting decline of impedance, whereas dextran flux was again not affected.<br />
Second, we compared the three impedance systems in order to disclose individual<br />
advantages or limitations. A key feature of the ECIS ® system is the opportunity to break<br />
down impedance in its resistive and capacitive contributions and, moreover, to discriminate<br />
betweenchanges of cell-cell and/or cell-matrix junctions. In contrast, xCELLigence only<br />
provides information on the overall impedance magnitude, internally referenced and<br />
normalized ascell index. Independent of the applied barrier-modulating stimulus, both<br />
systems returned essentially similar results with respect to the time course of the cell<br />
layers’ integral ionic permeability. The filter insert-using cellZscope ® device, however,<br />
reached its limit of sensitivity due to the low overall resistance of the endothelial cells under<br />
study (transendothelial electrical resistance< 5 Ω cm²).<br />
In conclusion, impedance sensing is a very sensitive, label-free and easy-to-use method to<br />
monitor changes in cellular morphology in real-time. Alterations in endothelial permeability<br />
are associated with these morphological changes, which are reflected by different ion fluxes<br />
in impedance sensing and by macromolecular fluxes in the Transwell ® assay. For the<br />
evaluation of new drug targets in endothelial barrier regulation, we therefore recommend to<br />
be aware of these differences and to combine impedance sensing with measurements of<br />
macromolecular permeability, since each method alone cannot fully determine all<br />
consequences of barrier-modulating agents.<br />
094<br />
A novel mouse model for studying the in vivo functions of the sodiumactivated<br />
potassium channel Slack (Slo2.2)<br />
Bausch A1; Hausmann M1; Baranski J1, Meyerdierks N1, Ruth P1 §, Lukowski R1 §,<br />
Poster 115
1 Institute of Pharmacy, Department of Pharmacology, Toxicology and Clinical Pharmacy,<br />
University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany<br />
§ These authors contributed equally to this work.<br />
Background: Sodium-activated potassium channels of the Slack (Slo2.2) subtype are<br />
abundantly present in many neurons [1]. On a cellular level Slack channels might be<br />
involved in action potential repolarisation, setting the resting membrane potential in some<br />
neurons, slow after hyperpolarisation, burst firing and adaptation of firing rate during high<br />
frequency firing thereby shaping neuronal activity and excitability [1]. Although the<br />
molecular properties of Slack channels have been investigated in detail [2] their exact in<br />
vivo-functions remain elusive. Evidence suggests that Slack may play an important role for<br />
physiological processes such as spatial hearing [3], olfaction [4] and nociception [5]. In<br />
addition to physiological functions Slack channels have been indicated to exert control over<br />
pathophysiological processes as cytoprotection following ischemic injury [2,6].<br />
Methods: To investigate the function of the sodium-activated potassium channel Slack<br />
(Slo2.2) in vivo we recently generated knockout mice (KO) with a targeted deletion of the<br />
pore forming exon. Additionally, using the Cre/loxP recombination system, we have<br />
established a mouse line that carries a “floxed” version of the pore forming exon and<br />
therefore enables a conditional (spatial and temporal) inactivation of the Slack gene.<br />
Reverse-Transcription-PCR (RT-PCR) and real-time-PCR were used to analyse the<br />
expression pattern and levels of Slack transcripts and transcripts of highly related<br />
potassium channels in Slack KO and littermate WT animals. Slack protein expression<br />
pattern was assessed by immunohistochemistry (IHC) and Western Blot using our novel<br />
anti-Slack-antibodies. Initial tests of the hypothesis that the Slack KO mice show a motor<br />
phenotype were performed by analysis of footprint patterns and the ability of Slack KO mice<br />
and their WT littermates to maintain balance in the “beam walk test”. Olfactory functions<br />
were investigated using food-finding (“buried pellet test”), innate preference and avoidance<br />
tests.<br />
Results: Slack KO mice are viable, fertile and do not show gross phenoptypical<br />
abnormalities under physiological conditions except for a delayed body-weight gain during<br />
the first 6 to 8 weeks after birth. Using RT-PCR we could confirm the absence of the pore<br />
region in Slack transcripts of Slack KO mice in all brain regions tested (e.g. cerebellum,<br />
neocortex and olfactory bulb). These results were confirmed by IHC analysis, which further<br />
demonstrated the absence of the Slack protein in 12-µm serial cross-sections of Slack KO<br />
brains. Although a high Slack-mRNA-level and Slack immunoreactivity were detected in<br />
brain regions that are involved in motor coordination Slack KO mice did not show an<br />
impaired motor function. All measures in the footprint pattern tests and performance results<br />
of the beam walk test were not different between Slack KOs and WT littermates.<br />
Furthermore, strong Slack-immunoreactivity was detected in the olfactory bulb with the<br />
highest protein density in the mitral cell layer and the outer plexiform layer containing the<br />
dendrites of the mitral cells. Together with the decrease in body weight of young Slack KO<br />
mice these findings raised the possibility that an olfactory disorder caused impaired<br />
suckling of the newborns. Indeed, the retrieval time for Slack KO mice to recover the buried<br />
pellet in the food-finding test was significantly prolonged compared to age- and littermatched<br />
WT mice.<br />
Conclusion: Our preliminary results indicate that Slack deficiency does not cause any<br />
gross abnormalities under physiological conditions. However, Slack ablation in sensory<br />
olfactory neurons and/or second-order neurons (e.g. periglomerular cells, mitral cells) of the<br />
olfactory bulb may be associated with mild olfactory dysfunctions. Together, we provide a<br />
powerful novel tool to study the functions and molecular details of Slack channels in a preclinical<br />
relevant model in vivo. Our ongoing and future investigations of these mice will help<br />
us to address the question whether Slack channels potentially constitute novel drug targets<br />
for stroke, epilepsy, ataxia or olfactory disorders.<br />
References:<br />
1. Bhattacharjee, A. and L.K. Kaczmarek: Trends Neurosci. 2005, 28(8): 422-8.<br />
2. Yuan, A. et al.: Neuron 2003, 37(5): 765-73.<br />
3. Yang, B., R. Desai, and L.K. Kaczmarek, J Neurosci. 2007, 27(10): 2617-27.<br />
4. Lu, S. et al.: J Neurophysiol. 2010, 103: 3311–3319.<br />
5. Tamsett, T.J., K.E. Picchione, and A. Bhattacharjee, J Neurosci, 2009, 29(16): p. 5127-34.<br />
6. Wojtovich, A.P. et al.: PLoS One 2011. 6(12): e28287.<br />
095<br />
Expression analysis of drug transporter proteins in RPMI 2650 cell line and<br />
excised human nasal mucosa<br />
Dolberg, A. M. 1; Reichl, S. 1<br />
1 Institut für Pharmazeutische Technologie, Technische <strong>Universität</strong> Carolo-Wilhelmina zu<br />
Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany<br />
The nasal mucosa is an interesting site for application of drugs with systemic action, since it<br />
has a number of advantages as a delivery route, including easy accessibility, extensive<br />
vascular supply and avoidance of gastrointestinal degradation as well as first pass<br />
metabolism. Therefore more and more applications for nasal systemic drug delivery have<br />
been developed during the last decades, and their number is still increasing [1].<br />
In the development of drugs for nasal application, identification and characterization of the<br />
different uptake and efflux systems are necessary. P-glycoprotein (P-gp) and multidrug<br />
resistance-associated proteins (MRP) are classified as ATP binding cassette (ABC)<br />
transporters based on their sequences, organisation of the ATP-binding domains and efflux<br />
function. ABC proteins represent a large family of integral membrane transporters that<br />
utilize the energy of ATP hydrolysis to carry specific substrates across membranes [2]. The<br />
solute carrier gene (SLC) superfamily encodes another large family of membrane-bound<br />
transporters, located in almost every cellular and organelle membranes. Proteins of the<br />
SLC family include passive transporters, ion-coupled transporters and exchangers [3].<br />
A comparison was performed between excised human nasal mucosa from turbinectomy<br />
surgeries and our in vitro model based on immortalized human nasal epithelial cells (RPMI<br />
2650) concerning the mRNA expression of different efflux and uptake transporter proteins.<br />
First investigations to prove functionality of different ABC transporters in RPMI 2650 cells<br />
was carried out using permeation studies and uptake assays.<br />
The mRNA expression of eight ABC transporters as well as 13 SLC transporters of excised<br />
human tissue and RPMI 2650 epithelial model were investigated and compared. The<br />
results showed promising similarity in the possible presence or absence of these efflux and<br />
influx transporters in human tissue and the in vitro model. A similarity between RPMI 2650<br />
models and human nasal mucosa has already been shown for barrier properties to<br />
evaluate passive drug permeation [4].<br />
To verify the functionality of the first transporter protein, P-gp, bidirectional transport studies<br />
using rhodamine 123 with RPMI 2650 cells grown on polyethylene terephthalate filter were<br />
performed. Indirect immunofluorescence staining for P-gp was also carried out. The<br />
findings confirmed the assumption that P-gp is not functionally expressed in these epithelial<br />
cells.<br />
An uptake assay using 5(6)-carboxy-2′,7′-dichlorofluorescein as a substrate for MRP1 and<br />
MRP5 was carried out. The result indicated functional expression of these efflux<br />
transporters in RPMI 2650 cells. Identification and localization of the responsible<br />
transporter proteins have to be proved by immunohistochemistry.<br />
Further investigations will be focused on western blot, immunohistochemical analysis and<br />
permeation as well as uptake studies using specific substrates for active transport to verify<br />
the protein expression and functionality of further drug transporters in human nasal mucosa<br />
and in vitro model based on RPMI 2650 cell line.<br />
Acknowledgments: We are grateful to the German Federal Institute for Risk Assessment (BfR), which<br />
funded this work under grant no. 3-1329-469. Furthermore, the authors thank Prof. Dr. Schroeder, Dr.<br />
Schmidt, Städtisches Klinikum Braunschweig and Dr. Reintjes, HNO-Praxis Schlosscarree Braunschweig for<br />
the supply of human nasal mucosa specimens.<br />
References:<br />
1. Dimova, S. et al.: Toxicology In Vitro 2005, 19: 107–122.<br />
2. Jones, P.M., George, A.M.: Cell. Mol. Life Sci. 2004, 61: 682–699.<br />
3. He, L., Vasiliou, K., Nebert, D.W.: Human Genomics 2009, 3: 195–206.<br />
4. Wengst, A., Reichl, S.: Eur. J. Pharm. Biopharm. 2010, 74: 290–297.<br />
096<br />
A new heterogeneous assay for measuring NAD + -dependent histone<br />
deacetylase (sirtuin) activity<br />
Schiedel, M. 1; Jung, M. 1 2<br />
1 Institute of Pharmaceutical Science, Albert-Ludwigs-<strong>Universität</strong>, Albertstr. 25, 79104 Freiburg,<br />
2 Freiburg Institute of Advanced Studies (FRIAS)<br />
Background<br />
Sirtuins represent a specific NAD +-dependent class of histone deacetylases (HDACs). By<br />
using NAD + as a cofactor, these enzymes cleave off the acetyl groups from the epsilonamino<br />
group of lysines in histones and other proteins, e.g. p53, FOXO proteins, p300 or<br />
HIV tat. The human family of sirtuins consists of seven members, which are distributed to<br />
different cell compartments and involved in the regulation of various physiological<br />
processes like apoptosis, cell differentiation, metabolism, DNA recombination and HIV tat<br />
transactivation. Thus, sirtuin inhibitors are interesting potential drugs for drug discovery [1].<br />
Inhibitor search is highly dependent on high throughput screening (HTS) which relies on<br />
suitable assay systems. We introduced a new heterogeneous assay based on fluorescence<br />
detection of Ac-p53-TAMRA, a known nonhistone substrate for sirtuins [2].<br />
Material and Methods<br />
Ac-p53-TAMRA is a fluorescence-labelled and biotinylated fragment of the<br />
tumorsuppressor p53. Because the immobilized Ac-p53-TAMRA, which is bound on<br />
streptavidin microplates, is not able to serve as Sirt-substrate, we set up a preincubation in<br />
1 mL Epi-tubes. After three hours at 37 °C the reaction is stopped and trypsin is added,<br />
which hydrolyzes only the deacetylated substrate. In the next step the test samples are<br />
dispensed on a streptavidin-coated microplate in order to immobilize the biotinylated<br />
substrate and to wash away the unbound components, including the fluorescence-tagged<br />
cleavage product of tryptic digestion. Finally the amount of remaining uncleaved but still<br />
biotinylated and TAMRA-labelled substrate is determined by fluorescence readout.<br />
Results<br />
We were able to establish a new nonisotopic, heterogeneous assay for NAD +-dependent<br />
histone deacetylases, which enables in-vitro-testing of library components with intrinsic<br />
fluorescence or quenching properties, that are prone to false-negative or –positive results in<br />
homogeneous fluorescence based assay systems. The presented detection protocol is<br />
suitable for HTS.<br />
References:<br />
1. Trapp, J., Jung, M.: Curr Drug Targets 2006, 7(11): 1553-1560.<br />
2. Milne, J.C. et al.: Nature 2007, 450(7170): 712-716.<br />
097<br />
Influence of different solvents on the applicability of a small intestinal<br />
microdialysis system in vivo<br />
Schönherr, D. 1; Hanke, U. 1; Siegmund, W. 2; Becker, D. 3; Weitschies, W. 1<br />
1University of <strong>Greifswald</strong>, Center of Drug Absorption and Transport, Institute of Pharmacy, Felix-<br />
Hausdorff-Straße 3, 17487 <strong>Greifswald</strong>, Germany<br />
2University of <strong>Greifswald</strong>, Center of Drug Absorption and Transport, Institute of Pharmacology,<br />
Felix-Hausdorff-Straße 3, 17487 <strong>Greifswald</strong>, Germany<br />
3Novartis Pharma AG, Technical Research & Development, Novartis Campus, WSJ-177.4.14,<br />
4056 Basel, Switzerland<br />
For better understanding the mechanisms of drug absorption after oral drug administration<br />
a new small intestinal microdialysis system was developed. To characterize the influence of<br />
different solvents on the applicability for in vivo studiesseveral experiments were<br />
performedin vitro.<br />
The small intestinal microdialysis system is made up of a minimodule which is built up of 30<br />
hollow fiber capillaries which consist of a polysulfone membrane (Fresenius Polysulfone ®)<br />
and a tubing system with an inflow, an outflow, and a tube for separating samples via air<br />
bubbles. The applicability of the system was tested with paracetamol (acetaminophen) as<br />
model substance using phosphate buffer USP pH 6.8, hydrochloric acid pH 1.2 and a<br />
biorelevant buffer pH 6.8 as solvent.For all experiments phosphate buffer USP pH 6.8<br />
served as perfusion medium as well. Two pumps were used to transport the samples<br />
(300 µL) after an intended equilibration time and to produce air bubbles separating the<br />
samples in the outflow tube. To examine the sensitivity of the system, the minimodule was<br />
inserted into a chamber filled with solvent. After pre-incubation, a solution of the drug<br />
substance was added (resulting theoretical concentration of 0.02 and 2.0 g/L).<br />
Subsequently, solvent was added to dilute the drug solution (resulting theoretical<br />
concentration of 0.01 and 1.0 g/L). Drug concentrations were calculated using UV<br />
spectroscopy and a previously determined minimodule scaling factor.<br />
Drug concentrations as well as concentration changes over time were successfully<br />
monitored for both drug concentrations and all investigated solvents. The functional<br />
applicability of the microdialysis system at both pH values was proven.<br />
The suitability of the new small intestinal microdialysis system to monitor concentration<br />
changes over time in vitro was demonstrated for different solvents. The applicability of the<br />
system in vivo will be examined in the near future.<br />
116 Poster
We thank Fresenius Medical Care GmbH for providing the hollow fiber capillaries. Financial support from<br />
Novartis Pharma AG is gratefully acknowledged.<br />
098<br />
Identification of benzimidazole derivatives as inhibitors of leukotriene<br />
biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein<br />
(FLAP)<br />
Luderer, S. 1, Banoglu, E. 2, Altenhofen, W. 3, Gerstmeier, J. 4, Pergola, C. 4, Werz, O. 4<br />
1 Department of Pharmaceutical Analytics, Pharmaceutical Institute, Eberhard-Karls-University<br />
Tuebingen, Auf der Morgenstelle 8, D-72076 Tuebingen, Germany<br />
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Etiler, 06330<br />
Yenimahalle, Ankara, Turkey<br />
3 Chemical Computing Group, AG, Kaiser-Wilhelm-Ring 11, 50676 Koeln, Germany<br />
4 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University<br />
Jena, Philosophenweg 14, D-07743 Jena, Germany<br />
Leukotrienes (LTs) are lipid mediators formed from arachidonic acid (AA) by the enzyme 5lipoxygenase<br />
(5-LO). 5-LO converts AA from membrane phospholipids to LTA4, aided by<br />
the 5-lipoxygenase-activating protein (FLAP). LTA4 is further metabolized to LTB4 and<br />
cysteinyl-LTs (cys-LTs) by LTA4 hydrolase or LTC4 synthase [1]. LTs are key mediators in<br />
inflammatory, allergic and cardiovascular diseases, but only Zileuton as direct 5-LO<br />
inhibitor (Zyflo CR®) and Cys-LT receptor antagonists (e.g. Montelukast, Singulair ©) are<br />
currently available on the market and used for the treatment of asthma.<br />
One promising target for pharmacological suppression of leukotriene biosynthesis is 5lipoxygenase-activating<br />
protein (FLAP). Here, we applied a virtual screening targeting<br />
FLAP based on a combined ligand- and structure-based pharmacophore model which led<br />
to the identification of 8 compounds (see below) as potential FLAP inhibitors [2].<br />
Compounds 1, 2 and 3 barely inhibited 5-LO product formation with IC50 values > 10 µM<br />
both in a cell-based and a cell-free system. Compounds 4, 5, 6 and 8 reduced 5-LO product<br />
synthesis in intact neutrophils, but also inhibited recombinant 5-LO suggesting a direct<br />
interference with 5-LO. In contrast, the benzimidazole derivative 7 potently suppressed LT<br />
formation in intact neutrophils (IC50=0.31μM) but essentially failed to inhibit 5-LO in cell-free<br />
systems suggesting an interaction with FLAP. Compound 7 was also active in a human<br />
whole blood assay, and acted anti-inflammatory in an in vivo model of acute inflammation,<br />
i.e. the carrageenan-induced pleurisy in rats. For structural optimization, a series of further<br />
benzimidazole-based derivatives were synthesized leading to more potent analogues with<br />
IC50 values between 0.12-0.19 μM. Our results disclose the benzimidazole scaffold bearing<br />
as a new chemotype for further development of anti-leukotriene agents.<br />
References:<br />
1. Radmark, O., et al., 5-Lipoxygenase: regulation of expression and enzyme activity. Trends Biochem Sci,<br />
2007. 32(7): p. 332-41.<br />
2. Banoglu, E., et al., Identification of novel benzimidazole derivatives as nhibitors of leukotriene<br />
biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein (FLAP). Bioorg Med Chem,<br />
<strong>2012</strong>. 20(12): p. 3728-41.<br />
099<br />
The fourth molybdenum enzyme mARC: Expression and N-reductive activity in<br />
different cell lines<br />
Jakobs H. 1; Reichmann D. 2; Bittner F. 2; Mendel R. 2; Clement B. 1;Havemeyer A. 1<br />
1Department of Pharmaceutical / Medicinal Chemistry, Pharmaceutical Institute, Christian-<br />
Albrechts-University of Kiel, Gutenbergstraße 76, 24118 Kiel, Germany<br />
2Department of Plant Biology, Technical University of Braunschweig, Humboldtstraße 1, 38106<br />
Braunschweig, Germany<br />
mARC (“mitochondrial amidoxime reducing component”) is a mammalian molybdenum<br />
enzyme that was discovered in our lab [1]. The enzyme is located in the outer mitochondrial<br />
membrane and together with the electron transport proteins cytochrome b5 and its NADHdependent<br />
reductase it is capable of reducing N-hydroxylated compounds such as the<br />
model substrate benzamidoxime [2,3].This is actually where the enzyme got its name from.<br />
Besides amidoximes, the enzyme system is also able to reduce other N-hydroxylated<br />
structures such as guanidines and sulphonamides and is thus involved in reductive drug<br />
metabolism[2]. For this reason it can be employed for the activation of<br />
N-hydroxylatedprodrugs using e.g. the prodrug principle of “amidoximes instead of<br />
amidines” [4]. Moreover, the mARC containing enzyme system is also able to reduce<br />
N-hydroxylated DNA-bases[2], which could be a mechanism of detoxification.However, the<br />
endogenous function of mARC is still unknown. The human genome codes for two mARC<br />
proteins, referred to as mARC1 and mARC2 [5].<br />
Our studies upon tissue distribution carried out with porcine material revealed highest<br />
expression levels for mARC in liver, thyroid and kidney [6].<br />
In continuation to our studies with primary hepatocytes, porcine hepatic and extrahepatic<br />
tissue fractions and recombinant human enzymes, we tested extrahepatic cell lines both on<br />
mARC1 and mARC2 expression and N-reductive activities. Cell lines included kidney cell<br />
lines HEK293, RC124, HMCL and Colo357, a pancreatic cancer cell line. Furthermore, we<br />
studied 3T3-L1, a fibroblast-like murine cell line that differentiates under appropriate<br />
conditions into adipocyte-like cells.<br />
N-reductive activity and expression of both mARC1 and mARC2 can be found in all studied<br />
cell lines. N-reduction in all human cell lines obey Michaelis-Menten-kinetics, but show<br />
differences in Vmax and Km. Conversion rates for benzamidoxime correlate with the<br />
expression of mARC1 but not with the expression of mARC2.<br />
All studied human cell lines can therefore be used to study both the enzyme system in<br />
general and the N-reduction of N-hydroxylated compounds.<br />
With regard to murine 3T3-L1,N-reductive activity is enhanced during differentiation into an<br />
adipocyte-like phenotype.<br />
Others found that enzymes and transporters that take part in the TCA cycle, fatty acid<br />
oxidation and ATP synthesis as well as mARC2 show changes in expression during<br />
differentiation of 3T3-L1 into an adipocyte-like cell type [7].Together with the fact that<br />
N-reductive activityis increased during differentiation of 3T3 L1 this could be a hint that<br />
mARC is involved in metabolic processes. Further studies on this topic will be necessary.<br />
References:<br />
1. Havemeyer, A. et al.:J. Bio. Chem.2006, 281(46): 34796–34802.<br />
2. Gruenewald, S. et al.: J. Med. Chem.2008, 51(24): 8173–8177.<br />
3. Neve, E. et al.: J. Biol. Chem.2011, 287(9): 6307–6317.<br />
4. Clement B.:Inventor. Methoden zur Behandlung und Prophylaxe der Pneumocystiscariniipneumonie<br />
(PCP) und anderen Erkrankungen sow e Verbindungen und Formulierungen zum Gebrauch bei besagten<br />
Methoden. 1993, German patent P4321444.4, PCT/DE 94/00756 (1994); U.S. patent 5,786,383 (1998 July<br />
28); European patent 0708640 (1998 September 16)<br />
5. Wahl, B.et al.: J. Biol. Chem.2011, 285(48): 37847–37859.<br />
6. Clement, B. et al.: Drug Metab. Rev.2010,42(S1): 1–323.<br />
7. Newton, B. et al.:J. Proteome Res. 2011,10(10): 4692–4702.<br />
100<br />
QSAR studies of the N-reduction of p-substituted benzamidoximes<br />
Treuer, E. 1; Reichmann, D. 2; Bittner, F. 2; Mendel, R. 2; Clement, B. 1;Havemeyer, A. 1<br />
1Pharmaceutical Institute, Christian-Albrechts-University, Gutenbergstraße 76, 24118 Kiel,<br />
Germany<br />
2 Department of Plant Biology, Technical University of Braunschweig, Humboldtstraße 1, 38106<br />
Braunschweig, Germany<br />
mARC (mitochondrial Amidoxime Reducing Component) is a molybdenum containing<br />
protein, which is localized in the outer mitochondrial membrane [1]. The genome of<br />
mammalians codes for two different forms of this protein (mARC 1 and mARC 2). Together<br />
with the electron transport proteins cytochrome b5 and cytochrome b5reductase, it catalyzes<br />
the N-reduction of different compounds. On the one hand, it transforms toxic and mutagenic<br />
N-hydroxylated base analogues [2]. On the other hand, it is involved in the activation of<br />
several prodrugs like N-hydroxypentamidine and guanoxabenz [3].<br />
For the development of new amidoximeprodrugs, we investigate structure-activity<br />
relationships of the N-reduction of p-substituted benzamidoximes. Therefore, several<br />
compounds were incubated with OMV (outer membrane vesicles) which was isolated from<br />
pig liver mitochondria. The resulting amidine was quantified with HPLC analysis and Km<br />
and Vmax were calculated. The influence of the electronic properties of the substituent in<br />
para-position on the N-reductive activity was measured. There are first hints that the<br />
conversion rates for the N-reduction of benzamidoximes with electron donating groups in<br />
para-position are higher than for compounds with electron withdrawing substituents.<br />
However, the differences are low and all para-substituted benzamidoximes were intensively<br />
reduced. Thus the prodrug principle amidoximes (N-hydroxyamidines) instead of amidines<br />
can be applied to allbenzamidine containing drug candidates irrespective of the<br />
substituents at the aromatic ring.<br />
References:<br />
1. Havemeyer, A. et al.: J. Biol. Chem.2006, 281(46): 34796-34802.<br />
2. Havemeyer, A., Lang, J., and Clement, B.: Drug.Metab.Rev. 2001, 43(4): 524-539.<br />
3. Gruenewald, S. et al.: J. Med.Chem. 2008, 51(24): 8173-8177<br />
101<br />
Development of an ELISA assay for screening of p38δ MAPK inhibitors<br />
Goettert M. a ,Bauer S. a , Shaalan N. b , Graeser, R. c , Laufer, S. a<br />
a Department of Pharmaceutcal and Medicinal Chemistry, Institute of Pharmacy, Eberhard Karls<br />
University of Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany<br />
b Department of Pharmaceutical Biology, Faculty of Pharmacy and Biotechnology, German<br />
University of Cairo, Egypt<br />
c ProQinase GmbH, Breisacher Str. 117, D-79106 Freiburg, Germany<br />
p38 mitogen activated protein kinases (MAPKs) are members of a larger group of serine/<br />
threonine protein kinases.<br />
They contribute in a variety of cellular processes such as gene expression, mitosis,<br />
differentiation, cell survival/apoptosis and biosynthesis/ release of pro- inflammatory<br />
cytokines [1].<br />
In many inflammatory diseases, e.g. rheumatoid arthritis (RA), the role of p38α isoform is<br />
widely investigated.<br />
Activated rheumatoid arthritis synovial fibroblasts (RASFs) are considered as key cells in<br />
the development of RA since they mediate the most relevant pathways of joint destruction<br />
n[2,3].<br />
The p38δ MAPK is activated in RASFs by a cytokine-independent pathway, leading to the<br />
expression of matrix metalloproteinases, e.g. MMP-1 and MMP-3, which contributes to the<br />
destruction of articular cartilage and bone [3,4].<br />
In the RA synovial tissue, all four isoforms- α, β, γ and δ- of the p38 MAPK have been<br />
detected. Particularly, p38δ MAPK occurs predominantly in the site of invasion and bone<br />
destruction. For this, the participation of p38δ MAPK becomes more and more evident<br />
[2,5].<br />
For the purpose of routine screening, we developed a direct 96 Well plate ELISA assay to<br />
identify inhibitors of p38δ MAPK. After incubation with a candidate inhibitor, the activity of<br />
p38δ MAPK is measured by the phosphorylation degree of activation transcription factor 2<br />
(ATF-2). The phosphorylated ATF-2 is directly detected by a monoclonal peroxidase<br />
conjugated antibody. Being a natural substrate of the p38 MAPKs, ATF- 2 phosphorylation<br />
is inversely correlated with the inhibitor potency.<br />
Poster 117
Based on already successfully established ELISA assays for p38α MAPK and JNK3, the<br />
advantages of this assay are its accuracy, easy handling, rapidness and the avoidance of<br />
using radioisotopes.<br />
References:<br />
1. Schindler, J.F., J.B. Monahan, and W.G. Smith, p38 pathway kinases as anti-inflammatory drug targets.<br />
Journal of Dental Research, 2007. 86(9): p. 800-811.<br />
2. Ospelt, C., et al., Toll-like receptors in rheumatoid arthritis joint destruction mediated by two dist nct<br />
pathways. Annals of the Rheumatic Diseases, 2004. 63: p. 90-91.<br />
3. Huber, L.C., et al., Synovial fibroblasts: key players in rheumatoid arthritis. Rheumatology, 2006. 45(6): p.<br />
669-675.<br />
4. Goettert, M., et al., Development of a p38δ mitogen activated protein kinase ELISA assay for the<br />
quantitative determination of inhibitor activity. Journal of Pharmaceutical and Biomedical Analysis, <strong>2012</strong>.<br />
5. Korb, A., et al., Differential tissue expression and activation of p38 MAPK alpha, beta, gamma, and delta<br />
isoforms n rheumatoid arthritis. Arthritis and Rheumatism, 2006. 54(9): p. 2745-2756.<br />
102<br />
Is Dimethylarginine-Dimethylaminohydrolase-2 (DDAH-2) involved in<br />
methylarginine metabolism?<br />
Altmann K. S. 1, Havemeyer A. 1, Beitz E. 1, Clement B. 1<br />
1Pharmaceutical Institute, Department of Pharmaceutical and Medicinal Chemistry, Christian-<br />
Albrechts-University of Kiel, Gutenbergstraße 76, 24118 Kiel, Germany<br />
DDAH isoenzymes, discovered in 1989 by Ogawa et al., catalyze the hydrolytic degradation<br />
of free endogenous methylarginines, Nω-monomethyl-L-arginine (L-NMMA) and<br />
Nω,Nω'-dimethyl-L-arginine (ADMA). [1,2] These methylarginines occur as endogenous nitric<br />
oxide synthase (NOS) inhibitors and reduce the formation of nitric oxide (NO).[3,4] By<br />
modulating the rates of L-NMMA and ADMA DDAH enzymes exhibit a therapeutic target to<br />
modulate the bioavailability of NO. [5,6]<br />
The postulated metabolism is evidenced for DDAH-1 but the involvement of DDAH-2 in the<br />
degradation of ADMA and L-NMMA is still a matter of debate.<br />
Experimental Procedures:<br />
Therefore, we determined the isoform specific DDAH protein expression profile in various<br />
porcine tissues by immunoblotting and correlated it with respective DDAH activity levels.<br />
Tissue DDAH activity was measured by the conversion of ADMA alone and in the presence<br />
of a DDAH inhibitor. Furthermore, metabolism of ADMA, L-NMMA and alternative DDAH<br />
substrates, S-methyl-L-thiocitrulline (SMTC) andNω-amino-L-arginine, was studied in<br />
porcine tissues each containing one DDAH isoform.<br />
Results:<br />
DDAH activity was detected in homogenates of porcine kidney, liver, and brain, i.e. tissues<br />
with high expression levels of DDAH-1, but not in those of spleen or thyroid, where DDAH-2<br />
predominates. By the same token neither methylarginines nor alternative DDAH substrates<br />
were converted in the DDAH-2-rich porcine thyroid cytosolic fractions, whereas all<br />
substrates were metabolized by the DDAH-1-rich kidney cytosol preparation.<br />
In addition, L-citrulline formation in porcine tissues was reduced to 50 % in the presence of<br />
1 mM of the DDAH inhibitor Nω-(2-methoxyethyl)-L-arginine (L-257), evidencing DDAH<br />
activity as a major source for the conversion. [7]<br />
Conclusion:<br />
Together, these data show that DDAH-1 and DDAH-2 have distinct tissue distributions as<br />
well as substrate selectivity indicating different roles in NO metabolism. While DDAH-1<br />
metabolizes the endogenous NOS inhibitors L-NMMA and ADMA, a physiological function<br />
of DDAH-2 has yet to be defined.<br />
References:<br />
1. Ogawa, T., Kimoto, M., and Sasaoka, K.: J. Biol. Chem. 1989, 264(17):10205–10209.<br />
2. Leiper, J. M. et al.: Biochem. J. 1999, 343Pt 1: 209–214.<br />
3. Anthony, S., Leiper, J., and Vallance, P.: Vasc Med. 2005, 10Suppl 1: S3-9.<br />
4. MacAllister, R. J. et al.: Br. J. Pharmacol. 1996, 119(8): 1533–1540.<br />
5. Dayoub, H. et al.: Circulation. 2003, 108(24): 3042–3047.<br />
6. Feng, M. et al.: Int. J. Cardiol. 2010, 144(2): 180–186.<br />
7. Rossiter, S. et al.: J. Med. Chem. 2005, 48(14): 4670–4678.<br />
103<br />
New heterocyclic sigma receptor ligands and their role in the activation of<br />
apoptotic pathways<br />
Korpis K1, Weber F2, Wünsch B2, Bednarski P J1 1 Institute of Pharmacy, University of <strong>Greifswald</strong>, F.-L.-Jahn-Straße 17, 17487 <strong>Greifswald</strong>, Germany<br />
2 Institute of Pharmacy, University of Münster, Hittorfstraße 58-62, 48149 Münster, Germany<br />
Sigma receptors are present in peripheral organs and in the central nervous system. They<br />
are divided into two distinct subtypes: σ1 and σ2. Both subtypes are involved in<br />
neuromodulatory processes and in particular the σ1 receptor is under evaluation for the<br />
treatment of a number of neurological disorders such as depression or schizophrenia. 1 In<br />
addition to the relevance of these receptors in neurological effects both subtypes are<br />
expressed in a high density in different human tumor cell lines. σ1 receptor antagonists and<br />
σ2 agonists have been shown to be involved in programmed cell death (apoptosis),<br />
whereas σ1 receptor agonists and σ2 receptor antagonists have antiapoptotic and<br />
neuroprotective activity. More specifically, Spruce et al. have shown that σ1 antagonists<br />
induce caspase-dependent apoptosis, which is in accord with recent observations that σ1<br />
agonists prevent caspase-3 activation. On the other hand, σ2 agonists have been shown to<br />
activate a caspase independent apoptotic pathway and have been proposed as putative<br />
anti-cancer drugs. 2<br />
The aim of this work is to determine the role of newly synthesized σ receptor ligands in the<br />
activation of apoptotic pathways.<br />
Flow cytometry with Annexin V and propidium iodide was performed to observe cell<br />
membrane alterations due to apoptosis. Experiments with human cancer cells treated with<br />
our compounds showed apoptosis levels increasing from 1 % in the control to 31 % in the<br />
treated cells. Another apoptosis assay was used to measure the expression of members of<br />
the caspase family to confirm apoptosis as the mechanism of cell death. Treatment of the<br />
multiple myeloma cell line RPMI 8226 caused dose-dependent activation of caspase-3,<br />
caspase-8 and caspase-9, indicating the activation of both the intrinsic and the extrinsic<br />
apoptotic pathway.<br />
However, Western blot analysis showed only cleaved products of caspase-8 and not<br />
caspase-9, evidence for activation of only the extrinsic pathway. These data suggest that<br />
our compounds do not suppress the expression of pro-survival Bcl-2 family proteins, such<br />
as Bcl-2.<br />
Further studies will address the crucial role in the extrinsic apoptotic pathway, which is<br />
initiated by activation of death receptors (i.e. TRAIL) and if there are other apoptosisindependent<br />
pathways of programmed cell death, for example autophagy.<br />
References:<br />
1. Abate, C. et al.: J. Med. Chem. 2011, 54(4): 1022-1032.<br />
2. Riganas, S. et al.: Bioorganic & Medicinal Chemistry <strong>2012</strong>, 20(10): 3323-3331.<br />
104<br />
A cell culture based assay for studies on inhibitors of the inducible nitric oxide<br />
synthase (iNOS) and effects of cold atmospheric pressure plasma treatment of<br />
keratinocytes and lymphocytes<br />
Schulz U1, Bundscherer L2, von Woedtke Th3, Masur K2, Morgenstern O1 1 Institut für Pharmazie, <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-<strong>Universität</strong> <strong>Greifswald</strong>, Friedrich-Ludwig-Jahn-Str. 17,<br />
17489 <strong>Greifswald</strong>, Germany<br />
2 Zentrum für Innovationskompetenz plasmatis, AG “Zelluläre Effekte” im INP <strong>Greifswald</strong> e.V.,<br />
Felix-Hausdorff-Str. 2, 17489 <strong>Greifswald</strong>, Germany<br />
3 Campus PlasmaMed im INP <strong>Greifswald</strong> e.V., Felix-Hausdorff-Str. 2, 17489 <strong>Greifswald</strong>, Germany<br />
The inducible nitric oxide synthase (iNOS) is a target of great interest for development of<br />
drugs for COPD and asthma [1], septic shock [2] and autoimmune diseases e.g. diabetes<br />
type 1 [3] and multiple sclerosis [4]. We synthesized some potential iNOS inhibitors with<br />
general structure 1 based on the fact that substances with thiourea or thiosemicarbazide<br />
structure elements showed iNOS-inhibitory properties before [5].<br />
To investigate the effect of our compounds on iNOS, we established a cell culture based<br />
assay using RINm5F cells that express the target under influence of interleukin-1β (IL-1β)<br />
and interferon-γ (IFN-γ) [6]. We optimized the produced amount of nitric oxide (measured<br />
as nitrite) using various combinations of the two cytokines with two cell counts. Additionally,<br />
we varied the incubation time pre and post addition of IL-1β and IFN-γ.<br />
Here we display the collected data that show that IFN-γ is, as expected, unable to induce<br />
iNOS on its own [7]. IL-1β alone increases nitrite concentration higher in short preincubation.<br />
IFN-γ in addition to IL-1β is able in very little concentrations to push the NO<br />
production to a maximum. We found a combination of low IL-1β and IFN-γ concentrations<br />
after a pre-incubation of 24 h and 48 h incubation after cytokine addition to be most<br />
effective.<br />
With these parameters we screened our derived substances for activity and found 7<br />
compounds to inhibit the target significantly higher than the reference aminoguanidine.<br />
Additionally to the screening of the chemical substances, we used the iNOS test model with<br />
modification to study effects of cold atmospheric pressure plasma treatment of<br />
keratinocytes (HaCaT cells) and lymphocytes (Jurkat cells). We searched for increased<br />
nitrite concentrations due to treatment of RINm5F cells with plasma or with supernatant of<br />
plasma-treated HaCaT and Jurkat cells. The aim was to investigate the influence of the<br />
plasma based secretion of the cytokines IL-1β and IFN-γ in order to induce NO production<br />
from the RINm5F cells. Possible inhibitory effects from produced cytokines e.g. TGF-β were<br />
investigated as well [8].<br />
Acknowledgments: Zentrum für Innovationskompetenz plasmatis, AG “Zelluläre Effekte” im INP <strong>Greifswald</strong><br />
e.V., Felix-Hausdorff-Str. 2, 17489 <strong>Greifswald</strong>, Germany<br />
The authors like to gratefully acknowledge the contribution of Liane Kantz to this work.<br />
References:<br />
1. Sugiura H, Ichinose M: Nitric Oxide 2011, 25(2): 138-144.<br />
2. Fortin C F, McDonald P P, Fulop T, Lasur O: Shock 2010, 33(4): 344-352.<br />
3. Cnop M, Welsh N, Jonas J C, Jörns A, Lenzen S, Eizirik D L: Diabetes 2005, 54(suppl 2): S97-S107.<br />
4. Steinert J R, Chernova T, Forsythe I D: Neuroscientist 2010, 16(4): 435-452.<br />
5. Al-Amiery A A, Al-Majedy Y K, Ibrahim H H, Al-Tamimi A A: Org. Med. Chem. Lett. <strong>2012</strong>, 2(1): 4-7.<br />
6. Ankarcrona M, Dypbukt J M, Brune B, Nicotera P: Exp. Cell Res. 1994, 213(1): 172-177.<br />
7. Cetkovic-Cvrlje M, Eizirik D L: Cytokine 1994, 6(4): 399-406.<br />
8. Mabley J G, Cunningham J M, John N, Di Matteo M A, Green I C: J. Endocrinol. 1997, 155(3): 567-575.<br />
105<br />
New Glutathione Peroxidase Inhibitors<br />
Lemmerhirt, H. 1; Schulz, R. 1; Emmrich, T. 1; Wilde, F. 1; Link, A. 1; Bednarski, P. J. 1<br />
1<strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-University, Institute of Pharmacy, Friedrich-Ludwig-Jahn-Straße 17, 17489<br />
<strong>Greifswald</strong>, Germany.<br />
Cancer cells have a variety of mechanisms to protect themselves from anti-cancer drugs.<br />
For example, they express drug efflux transporters like P-gp, increase repair of DNA<br />
damage or mutate targeted enzymes and receptors. A relatively new postulated<br />
mechanism of resistance is the over-expression of enzymes that deal with oxidative stress.<br />
It has been postulated that glutathione peroxidases (GPx, EC 1.11.1.9) is a key enzyme in<br />
the development of resistance. [1] The primary function of GPx is to prevent cells from<br />
oxidative stress by degrading peroxides like hydrogen peroxide to water or organic<br />
hydroperoxides to the corresponding alcohols.<br />
Hydrogen peroxide is postulated to be an important messenger of apoptosis. If cancer cells<br />
treated with anti-cancer drugs were to over-express GPx, then the signals for apoptosis<br />
would be weakened, leading to survival of more cancer cells [2].<br />
The aim of our work is to develop specific and reversible inhibitors of GPx to reverse drug<br />
resistance. Already some inhibitors of GPx are known, such as misonidazole and several<br />
118 Poster
thiol compounds but they have disadvantages; misonidazole shows neurotoxicity and thiols<br />
are unspecific and inhibit GPx irreversible. Furthermore thiols are instable with regard to<br />
oxidation.<br />
R. SCHULZ identified several promising lead structures by means of virtual screening with<br />
AutoDock and determined their GPx inhibitory activity by an enzyme assay [3]. One of them<br />
(1) was chosen for structural modification to improve inhibitory activity.<br />
The synthesized compounds have been tested for enzyme inhibitory activity in a microtiter<br />
based assay. The method is based on the oxidation of glutathione by GPx in the presence<br />
of hydroperoxides. The resulting glutathione disulfide (GSSG) is reduced back to GSH by<br />
glutathione reductase (GR) with consumption of NADPH. The rate of decrease of NADPH,<br />
monitored at λ = 340 nm, corresponds to the catalytic rate of GPx.<br />
We have now identified two new compounds that inhibit GPx with lower IC50 values than the<br />
lead compound. The substances will now be tested in vitro cell culture on human cancer<br />
cells resistant to common anti-cancer drugs. For example, ovarian carcinoma cell lines<br />
resistant to cisplatin will be used to assess whether anti-cancer-drug-resistance can be<br />
reversed with the new compounds.<br />
References:<br />
1. Saga, Y., et al.: Oncol Rep 2008, 20 (6): 1299-303.<br />
2. Gouaze, V., et al.: J Biol Chem 2002, 277 (45): 42867-74.<br />
3. Schulz, R.: Diploma Thesis 2008.<br />
106<br />
Stimulation and Inhibition of Angiogenesis by Non-Thermal Atmospheric-<br />
Pressure Plasma<br />
Haertel B. 1; Eiden K. 1; Deuter A. 1; Wende K. 1; von Woedtke T. 2; Lindequist U. 1<br />
1 Institute of Pharmacy, University of <strong>Greifswald</strong>, D-17489 <strong>Greifswald</strong>, Germany<br />
2 Leibniz-Institute for Plasma Science and Technology e.V. (INP), D-17489 <strong>Greifswald</strong>, Germany<br />
The study of angiogenesis has broad clinical implications e.g. in the fields of wound<br />
healing, oncology, hematology or ophthalmology. The formation of new blood vessels is an<br />
essential feature of tissue remodeling. Further, angiogenesis is a prerequisite for growth of<br />
solid tumors and development of metastasis. However, it is also involved in the<br />
pathophysiology of hematologic malignancies. For improving wound healing angiogenesis<br />
should be promoted, whereas in treating tumors angiogenesis should be inhibited. Inhibition<br />
of angiogenesis is also important in the treatment of macular edema or age-related macular<br />
degeneration. Angiogenesis depends on the expression of mediators initiating events<br />
leading to the formation of new microvessels. Non-thermal plasma induces a variety of<br />
effects on living cells in vitro, including production of fibroblast growth factor-2 (FGF-2) or<br />
reactive oxygen species by endothelial cells. Both FGF-2 and ROS belong to mediators of<br />
angiogenesis. However, up to now there are no quantitative data using non-thermal plasma<br />
in more complex angiogenesis models as the hen’s egg test on the chorioallantoic<br />
membrane (HET-CAM) or the aortic ring test (AOR). It is very important to know whether<br />
and how plasma influences angiogenesis in complex models.<br />
This study focused on the effects of plasma generated by the kINPen 09 [1] on<br />
angiogenesis using two different models: HET-CAM and rat aortic ring (AOR) test. In both<br />
models kINPen 09 treated medium (30 to 300s) was applied. ImageJ was used to analyze<br />
vessel area and fractal dimension after treating the CAM from embryonic day 11 to 13.<br />
Aortic rings were prepared from either LEW.1W or WOK.W rats. They were embedded in<br />
matrigel and treated daily for 4 days starting at day 4 after embedding. We developed a<br />
semi quantitative method to quantify outgrow of microvessels from aortic rings.<br />
In both models natural and spontaneous vessel formation was detected. In the HET-CAM<br />
assay vessel area and fractal dimension were significantly enhanced at embryonic day 14<br />
by the 120s-plasma treated medium compared to untreated controls. There was no effect of<br />
plasma (60 or 120s) on vessel growth of aortic rings prepared from LEW.1W rats. The<br />
angiogenic activity of rings from WOK.W rats was significantly (p
to its regulatory C2-like domain (C2ld). Though the crystal structure of 5-LO has been<br />
resolved, many of the distinct interaction sites and the involvement of the C2ld are still<br />
unclear. Therefore the C2ld (amino acids 1-115, fused to maltose binding protein) was<br />
expressed and purified in two steps via amylose affinity chromatography and, after<br />
cleavage, nickel chelating affinity chromatography. Gel filtration studies revealed a high<br />
tendency of the isolated domain to form soluble aggregates. But the single mutation of<br />
tryptophan 75 to alanine led to a dramatic improvement of its biophysical properties leading<br />
to mainly monomeric protein. The proper folding of the tryptophan mutant was successfully<br />
shown by its ability to bind Ca 2+ in a thermal shift assay. Crosslinking of 5-LO or the<br />
isolated C2ld with CLP revealed that the catalytic domain is also involved in the binding of<br />
CLP.<br />
Here we present the purification of a single-mutant that renders the 5-LO C2ld accessible<br />
for interaction studies elucidating the involvement of the regulatory domain to the binding of<br />
small molecules or proteins.<br />
References:<br />
1. Hammarberg T. et al. J Biol Chem. 2000, 275(49): 38787-93.<br />
2. Radmark O. et al. Trends Biochem Sci. 2007, 32(7): 332-41.<br />
3. Rakonjac M. et al. Proc Natl Acad Sci U S A. 2006, 103(35): 13150-5.<br />
4. Dincbas-Renqvist V. et al. Biochim Biophys Acta. 2009, 1789(2): 99-108.<br />
110<br />
Synthesis of annelated and substituted imidazolones and –thiones targeted at<br />
human NAD + -dependent histone deacetylases<br />
Beese K1; El Gaghlab K1; Rumpf T2; Jung M2; Link A1 1Institute of Pharmacy, <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-University <strong>Greifswald</strong>, Friedrich-Ludwig-Jahn-Str. 17,<br />
17489 <strong>Greifswald</strong>, Germany<br />
2Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstr. 25, 79104<br />
Freiburg, Germany<br />
Histones can be modified enzymatically by insertion and elimination of chemical groups<br />
mainly upon basic amino acids. A very significant histone modification is the acetylation<br />
forming lightly packed, active euchromatin that facilitates the accessibility of genes for<br />
transcription factors, whereas deacetylation leads to tightly packed, inactive<br />
heterochromatin, which results in a repression of transcription.<br />
NAD +-dependent histone deacetylases (HDACs), known as sirtuins, regulate epigenetic<br />
gene expression by deacetylation of histones and non-histone proteins. Thus, an increased<br />
enzyme activity is associated with the development of cancer, HIV and neurodegenerative<br />
diseases, and inhibitors of HDACs might represent promising drugs.<br />
In continuation of our efforts to identify modulators of HDACs with improved activity and<br />
selectivity we designed and synthesized analogs of a novel lead structure [1] reported by<br />
our group, recently, depicted below. Compounds such as 1-substituted1,3dihydroimidazo[4,5-c]quinolin-2-ones/thiones,1-substituted5-nitro-1,3-dihydronaphth[1,2d]imidazol-2-ones/thiones,<br />
and 1,5-disubstituted1,3-dihydrobenzimidazol-2-ones/thiones,<br />
that can be regarded as non-hydrolyzable analogs of the lactone splitomicin, were prepared<br />
and tested in a new nonisotopic, heterogeneous assay.<br />
introduction<br />
of<br />
hetero atoms<br />
or<br />
substituents<br />
annelation<br />
and /or<br />
substitution<br />
replacement<br />
by<br />
oxygen<br />
introduction<br />
of<br />
aliphatic or aromatic<br />
sidechains<br />
Acknowledgements: Technical assistance of Dr. A. Bodtke and K. Böheim is gratefully acknowledged.<br />
K.E.G. was recipient of grant DFG Li 765/4-2.<br />
References:<br />
1. Freitag, M. et al.: Bioorg Med Chem 2011, 19(12): 3669–3677.<br />
111<br />
ICAM-1 confers the TIMP-1-dependent anti-invasive action of cannabinoids on<br />
human lung cancer cells<br />
Ramer, R. 1; Bublitz, K. 1; Merkord, J. 2; Haustein, M. 1; Borchert, P. 1; Linnebacher, M. 2; Hinz,<br />
B. 1<br />
1 Institute of Toxicology and Pharmacology, University of Rostock, Schillingallee 70, D-18057<br />
Rostock, Germany<br />
2 Section of Molecular Oncology and Immunotherapy, Department of General Surgery, University<br />
of Rostock, Schillingallee 69, D-18057 Rostock, Germany<br />
Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix<br />
metalloproteinases-1 (TIMP-1). This study investigates the role of intercellular adhesion<br />
molecule-1 (ICAM-1) within this action. In the lung cancer cell lines A549, H358, and H460,<br />
cannabidiol (CBD; 0.001-3 μM) elicited concentration-dependent ICAM-1 up-regulation<br />
compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and<br />
p42/44 mitogen-activated protein kinase. Up-regulation of ICAM-1 mRNA by CBD in A549<br />
was 4-fold at 3 μM, with significant effects already evident at 0.01 μM. ICAM-1 induction<br />
became significant after 2 h, whereas significant TIMP-1 mRNA increases were observed<br />
only after 48 h. Inhibition of ICAM-1 by antibody or siRNA approaches reversed the antiinvasive<br />
and TIMP-1-upregulating action of CBD and the likewise ICAM-1-inducing<br />
cannabinoids Δ 9-tetrahydrocannabinol and r(+)-methanandamide when compared to<br />
isotype or nonsilencing siRNA controls. ICAM-1-dependent anti-invasive cannabinoid<br />
effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude<br />
mice, CBD elicited a 2.6- and 3.0-fold increase of ICAM-1 and TIMP-1 protein in A549<br />
xenografts, as compared to vehicle-treated animals, and an antimetastatic effect that was<br />
fully reversed by a neutralizing antibody against ICAM-1 [% metastatic lung nodules vs.<br />
isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM-1<br />
antibody]. Overall, our data indicate that cannabinoids induce ICAM-1, thereby conferring<br />
TIMP-1 induction and subsequent decreased cancer cell invasiveness.<br />
112<br />
Can Heparin influence the Cyr61 pathway for affecting cell adhesion<br />
processes?<br />
Schmitz, P. 1; Gerber, U. 1; Schütze, N. 2; Bendas, G. 1<br />
1 Department of Pharmacy, University Bonn, 53121 Bonn, Germany<br />
2 Orthopedic Center for Musculoskeletal Research, University Würzburg, 97074 Würzburg,<br />
Germany<br />
The cysteine-rich protein 61 (Cyr61, CCN1), a member of the CCN-family, is a matricellular<br />
protein expressed and secreted by various cells, which induce e.g. angiogenetic effects and<br />
enhance the tumorigenicity of solid tumors. Besides a range of functions Cyr61 mediates<br />
adhesion and migration of tumor cells by activating different members of the integrin<br />
adhesion receptor family. Integrins are crucial for different steps in course of the<br />
haematogenous metastasis. Heparin possesses antimetastatic effects by blocking<br />
adhesion receptors; selectins and selected integrins, e.g.VLA-4 (very late activation antigen<br />
4) [1,2], but heparin should also be able to bind Cyr61.<br />
However, neither the molecular mechanisms of Cyr61 affecting VLA-4 mediated adhesion<br />
nor interference by heparin in this pathway have been described. This study focused these<br />
aspects.<br />
To confirm direct binding of heparin to Cyr61, SAW biosensor studies were performed<br />
using a series of modified heparins and recombinant CCN1 proteins. To further focus on<br />
the Cyr61/VLA-4 binding pathway and potential impact on that by heparin, the impact of a<br />
Cyr61 downregulation in MV3 melanoma cells by shRNA technology on VLA-4 mediated<br />
cell adhesion was analysed by microscopic detection.<br />
Our data suggest a reduced VLA-4 mediated cell binding of Cyr61 knockdown MV3 clones<br />
in an adhesion assay, while exogenous recombinant Cyr61 restores the binding<br />
capacities.This confirms that VLA-4 is partly controlled by Cyr61 activity and make Cyr61<br />
as attractive target to interfere with metastasis. Concerning heparin interference, kinetic<br />
binding data suggest a direct interaction between heparin and Cyr61 for the first time.<br />
Binding affinities of fractionated heparin in the micromolar range were intensified by Nacetylation<br />
or 2-O-desulfation of heparin. Furthermore, Cyr61 accumulation at the cell<br />
surface glycosaminoglycan Syndecan-4 could be simulated demonstrating high binding<br />
affinities. However, this binding is non-reversible by adding high concentrations of heparin.<br />
Our data provide evidence for a certain impact of Cyr61 with VLA-4 mediated melanoma<br />
cell binding. The hypothesis of heparin interference in this pathway is strongly supported<br />
providing prospects for therapeutic interference in tumorigenicity and metastasis by<br />
blocking Cyr61 activity.<br />
We gratefully thank the “G.Ronzoni“ Institute, Milano for providing us the modified heparins.<br />
References:<br />
1. Schlesinger, M., et al., Thromb Res. 5 (<strong>2012</strong>): 603-10.<br />
2. Bendas, G., Borsig, L.; Int J Cell Biol. <strong>2012</strong>.<br />
113<br />
Cyclooxygenase-2 and peroxisome proliferator activated receptor gamma<br />
confer cannabidiol-induced apoptosis of human lung cancer cells<br />
Heinemann, K. 1; Ramer, R. 1; Merkord, J. 2; Salamon, A. 2; Linnebacher, M. 3; Hinz, B. 1<br />
1 Institute of Toxicology and Pharmacology, University of Rostock, Schillingallee 70, D-18057<br />
Rostock, Germany<br />
2 Department of Cell Biology, University of Rostock, Schillingallee 69, D-18057 Rostock, Germany<br />
3 Section of Molecular Oncology and Immunotherapy, Department of General Surgery, University of<br />
Rostock, Schillingallee 69, D-18057 Rostock, Germany<br />
The antitumorigenic mechanism of cannabidiol (CBD) is still controversial. This study<br />
investigates the role of cyclooxygenase-2 (COX-2) and peroxisome proliferator activated<br />
receptor γ (PPARγ) in CBD´s proapoptotic and tumor-regressive action. In lung cancer cell<br />
lines (A549, H460) and primary cells from a lung cancer patient CBD elicited decreased<br />
viability associated with apoptosis with the strongest toxic impact on primary tumor cells.<br />
Apoptotic cell death by CBD was suppressed by NS-398 (COX-2 inhibitor), GW-9662<br />
(PPARγ antagonist) and siRNA targeting COX-2 and PPARγ. CBD-induced apoptosis was<br />
paralleled by upregulation of COX-2 and PPARγ expression with a maximum induction of<br />
COX-2 mRNA after 8 h and continuous increases of PPARγ mRNA when compared to<br />
vehicle. In response to CBD tumor cell lines exhibited increased levels of COX-2dependent<br />
prostaglandins (PGs) among which PGD2 and 15-deoxy-Δ12 14-PGJ2 caused a<br />
translocation of PPARγ to the nucleus and induced a PPARγ-dependent apoptotic cell<br />
death. Moreover, in A549-xenografted nude mice CBD caused upregulation of COX-2 and<br />
PPARγ in tumor tissue, and tumor regression that was fully reversible by GW-9662.<br />
Together, our data demonstrate a novel proapoptotic mechanism of CBD involving initial<br />
upregulation of COX-2 and PPARγ and a subsequent nuclear translocation of PPARγ by<br />
COX-2-dependent PGs.<br />
114<br />
Synthesis of Zwitterionic Ligands for PET-Imaging in Melanoma<br />
Kriemen, E. 1; Maison, W. 1<br />
1 Pharmaceutical and Medicinal Chemistry, Bundesstraße 45, 20146 Hamburg, Germany<br />
Malignant melanoma, although it is far less prevalent than non-melanoma skin cancers, is<br />
the major cause of death from skin cancer.[1] This is because of the high aggressiveness of<br />
melanoma metastases and their resistance to conventional chemotherapy and external<br />
beam radiation therapy. Therefore an early diagnosis of melanoma is the key issue for<br />
increasing patient survival.[2]<br />
For our approach we focus on PET (positron emission tomography) as an imaging<br />
technique, which relies on decay characteristics of positron emitting (β+-decay)<br />
120 Poster
adionuclides.[3] Modern PET scanners have high sensitivities, but are still limited by<br />
specific binding/uptake to normal tissues and organs which lead to high background.<br />
Recent data showed that “zwitterionic” tracer molecules lower background binding<br />
dramatically by not interacting with serum proteins due to charge shielding.[4]<br />
With the mentioned principle in mind we designed a melanoma targeted radiotracer 1. It<br />
contains a DOTA moiety for proper chelating a radionuclide such as 89Zr and 68Ga.[5] The<br />
classical DOTA molecule is modified by geometrically balanced charge (positive charge<br />
with quaternary ammonium cations and negative charge with sulfonate groups).[4] This<br />
zwitterionic DOTA derivative is conjugated to CycMSH, a cyclic targeting peptide with high<br />
specificity to the melanocortin receptor MC1. The MC1 receptor is overexpressed in more<br />
than 80% of human metastatic melanoma tumor samples and is thus a valuable tumor<br />
marker.[6,7]<br />
We present the synthesis of a zwitterionic DOTA derivative and it’s conjugation to CycMSH<br />
by a solid phase protocol and evaluate the binding properties of this targeted imaging<br />
reagent to MC1.<br />
References:<br />
1. Siegel, R., Naishadham, D., Jemal, A.: CA Cancer J. Clin. <strong>2012</strong>, 62(1): 10–29.<br />
2. Anderson, C. M. et al.: Oncology 1995, 9(1): 1149–1158.<br />
3. Frangioni, J. V.: J. Clin. Oncol. 2008, 26(24): 4012–4021.<br />
4. Choi, H.S. et al.: Angew. Chem. Int. Ed. 2011, 50(28): 6258–6263.<br />
5. Verel, I., Visser, G. W. M., van Dongen, G. A.: J. Nucl. Med. 2005, 46(1): 164–171.<br />
6. Hadley, M. E.: Pigment Cell. Res. 1996, 9(5): 213–234.<br />
7. Guo, H.: J. Nucl. Med. 2011, 52(4): 608–616.<br />
115<br />
Is lysophosphatidylcholine (LysoPC) the key to explain the antimetastatic<br />
effects of empty liposomes?<br />
Ross, T. 1; Schlesinger, M. 1; Raynor, A. 2; Jantscheff, P. 2; Massing U. 2; Bendas, G. 1<br />
1 University of Bonn, Department of Pharmaceutical Chemistry II, An der Immenburg 4, 53121<br />
Bonn, Germany<br />
2 Tumor Biology Center Freiburg, Breisacher Str. 117, 79106 Freiburg, Germany<br />
Liposomes are extensive subject to current anticancer research. However, liposomal<br />
phospholipids (PL) are only regarded as passive carrier constituents. The findings of<br />
Graeser et al. [1] showing that empty liposomes composed of saturated PLs have<br />
antimetastatic effects in different mouse models suggest PL actively interfering in the<br />
metastatic course. Since cellular adhesion receptors are of key importance for the<br />
metastatic route of tumor cells, we proposed affected cell adhesion as the underlying<br />
mechanism. Recently we presumed lysophosphatidylcholine (LysoPC) as key molecule for<br />
this activity. One might assume that, due to the EPR-effect, liposomes accumulate in<br />
neoplastic tissue leading to an increased uptake and metabolisms of saturated PL. Since<br />
phospholipase A2 release is known to be often associated with tumor activity, increased<br />
levels of saturated LysoPC in tumor tissue might result. Jantscheff et al. [2] could show a<br />
reduction of melanoma cell adhesion in vitro as well as an inhibition of metastasis like lung<br />
invasion in vivo of cells pretreated with LysoPC. However, the underlying molecular<br />
mechanisms of LysoPC activity remain to be elucidated. Since any toxic activities of<br />
LysoPC or induction of apoptosis could be excluded, our current research focuses on the<br />
involvement of the plasma membrane affected by LysoPC and consequences for<br />
membrane associated signalling pathways.<br />
Using gas chromatography a rapid change in the lipid composition of the tumor cell<br />
membranes favouring the LysoPC derived fatty acids (FA) was shown under the influence<br />
of different LysoPC derivatives. In order to evaluate the impact of FA saturation on tumor<br />
membrane rigidification and underlying functional consequences, we compared the fully<br />
saturated Lyso-PC C18:0 and the unsaturated C18:1-derivative in MV3 melanoma cells.<br />
Using trimethylammoniumdiphenylhexatriene as a fluorescence anisotropy probe we could<br />
detect a significant rigidification of the cell membrane by C18:0 LysoPC, while C18:1<br />
LysoPC possessed only minor changes. The changes in membrane rigidity correspond with<br />
a diminished receptor-mediated migration capacity of the cells, as shown in microscopic<br />
wound healing assays. Thus, one might assume that LysoPC-induced membrane<br />
rigidification is involved in the reduced receptor mediated cell binding which might be one<br />
reason for the antimetastatic effects of LysoPC.<br />
To further focus on the molecular mechanisms of diminished receptor mediated cell binding<br />
we present preliminary data on the phosphorylation of the proteoglycan syndecan-4, which<br />
is an important element in the formation of focal adhesions complexes. Summarizing, we<br />
propose LysoPC as an active biological molecule affecting the plasma membrane fluidity<br />
with strong consequences for the formation of focal adhesion complexes. These data could<br />
also provide new insights and prospects for liposomal carriers in tumor therapy.<br />
References:<br />
1. Graeser, R. et al.: Pancreas 2009, 38(3): 330-337<br />
2. Jantscheff P. et al.: Mol Cancer Ther 2011, 10(1): 186-197<br />
116<br />
NEFA determination for functional lipidomics: comprehensive UPLC-MS/MS<br />
quantitation, qualification and parameter prediction [1]<br />
Hellmuth C, Weber M, Koletzko B, Peissner W<br />
Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children’s Hospital, Ludwig-<br />
Maximilians-University of Munich, 80337 Munich, Germany<br />
Objectives<br />
Metabolomics is an increasing field in pharmaceutical research and development.<br />
Metabolic profiling depicts abundance changes in endogenous metabolites [2]. E.g. levels<br />
of nonesterified fatty acids (NEFA) are highly associated with obesity, diabetes and<br />
metabolic syndrome. For instance, NEFA are elevated in T2DM adolescents in fasting state<br />
as well as after glucose challenge [3]. Though, straightforward quantitative methods for<br />
determination of NEFA species are still missing.<br />
Methods<br />
Serum samples were prepared by simple protein precipitation, avoiding cumbersome and<br />
solvent-consuming extraction or derivatization procedures. Short run time with high<br />
resolution was achieved by UPLC separation coupled to LC-MS/MS detection.<br />
Results<br />
Accurate quantification of 36 NEFA species in 20 µl of healthy human plasma was<br />
achieved, the highest numbers ever reported for a LC-MS application. The use of qualifier<br />
ions supports unequivocal analyte verification. Sample preparation is fast and nonexpensive.<br />
In combination with automated liquid handling, total assay time per sample is<br />
less than 15 minutes.<br />
Conclusion<br />
The presented method provides a rapid and comprehensive determination of NEFA in<br />
human plasma. This enables application in clinical trials with high sample number analyzed<br />
in short time and with low costs. Usage of 20 µl plasma facilitates application in studies<br />
with children or adolescents, when blood is obtained by finger sticks or heel pricks.<br />
References:<br />
1. Hellmuth C. et al.: Anal Chem. <strong>2012</strong> 84(3):1483-90<br />
2. Xu EY, Schaefer WH, Xu Q: Curr Opin Drug Discov Devel. 2009 12(1):40-52<br />
3. Nadeau KJ et al.: J Clin Endocrinol Metab. 2009 94(10):3687-95<br />
117<br />
The impact of DNA methylation on cellular differentiation of immune cells<br />
Flemming S. 1; Grützkau A. 2, Häupl T. 3; Günther S. 1<br />
1 Pharmaceutical Bioinformatics, Institute of Pharmaceutical Sciences, University of Freiburg<br />
2 German Arthritis Research Center, Berlin<br />
3 Department of Rheumatology and Clinical Immunology, Charité University Hospital, Berlin<br />
Epigenetic changes in DNA methylation are associated with regulation of gene expression<br />
and play an important role in cellular differentiation. Such changes might be of importance<br />
in chronic inflammatory diseases where autoreactive immune cells are thought to<br />
perpetuate inflammation and organ destruction. To explore these variations and its<br />
regulatory mechanism, we have gathered genome-wide DNA methylation data in human<br />
immune cells extracted from peripheral blood, specifically T-helper cells (CD4), Tsuppressor<br />
cells (CD8), macrophages (CD14), granulocytes (CD15), B-lymphocytes<br />
(CD19) and natural killer cells (CD56). Furthermore, we have analyzed methylation patterns<br />
associated to cell differentiation of T- and B-cells and the correlation with gene expression<br />
data.<br />
Cells from four healthy donors were sorted by FACS technology and the measurement was<br />
performed with the Illumina HumanMethylation450 BeadChip platform. These arrays<br />
provide a genome-wide coverage of more than 485,000 CpG methylation sites per sample<br />
at single-nucleotide resolution. To evaluate the correlation between methylation patterns<br />
and gene expression, we used the Affymetrix U133P platform.<br />
We observed that both, hyper- and hypomethylation of promoter regions of genes involved<br />
in cell differentiation correlate well with measured gene expression data. Additionally, we<br />
were able to identify a substantial number of common CpG methylation sites, which were<br />
differentially methylated between naive and memory states of T-(CD4, CD8) and B-cells<br />
(CD19).<br />
The impact of these alterations on inflammatory diseases will be a subject to further<br />
research which will include the analysis of autoreactive immune cells from rheumatoid<br />
arthritis patients.<br />
118<br />
Role of Histamine H4 receptors in eicosanoid biosynthesis.<br />
Dos Santos Capelo, R1; Kahnt, AS1; Steinhilber, D1 1 Institute of Pharmaceutical Chemistry, Max-von-Laue-Strasse. 9, 60438 Frankfurt am Main,<br />
Germany<br />
The H4 receptor is a G-protein-coupled receptor (GCPR) expressed in many cells of the<br />
immune system, including monocytes, mast cells, dendritic cells, natural killer cells,<br />
eosinophils and T cells. So far, not much is known about its physiological role, but the<br />
receptor is implicated in the pathology of various diseases such as allergy, autoimmune<br />
disorders, bronchial asthma, atopic dermatitis and pruritus. Upon activation the receptor<br />
couples to Gi/o proteins. This leads to a decrease in cAMP production [1,2,3,4], activates<br />
downstream mitogen-activated protein (MAP) kinase pathways [4] and mobilizes<br />
intracellular Ca2+ [1,5]. 5-lipoxygenase (5-LO) catalyzes the first two steps in leukotriene<br />
(LT) biosynthesis. Upon stimulation (e.g. MAP kinases and Ca2+ influx) the enzyme<br />
translocates to perinuclear membrane compartments where it is activated and starts the<br />
synthesis of LTA4 [6]. LTA4 can then be further metabolized by two terminal synthases.<br />
Soluble LTA4 hydrolase produces the potent chemoattractant LTB4, whereas membrane<br />
bound LTC4 synthase conjugates LTA4 with glutathione (GSH) to form the corresponding<br />
cysteinyl leukotrienes (CysLTs), LTC4, LTD4 and LTE4. Like the H4 receptor, 5-LO is<br />
primarily found in mature leukocytes such as granulocytes, mast cells, dendritic cells,<br />
monocytes / macrophages and B lymphocytes [7]. T cells and erythrocytes are 5-LO<br />
negative. 5-LO derived LTs are lipid mediators which were shown to primarily mediate<br />
inflammatory and allergic reactions. Besides their well studied role in asthma, 5-LO derived<br />
LTs have also been implicated to play a role in cardiovascular diseases and cancer [8].<br />
Recently, a connection between LT biosnythesis and histamine receptors has been drawn.<br />
Flamand et al. have published that histamine is able to inhibit LT formation in human PMN<br />
in a dose-dependent manner via Gs coupled H2 receptor activation. This inhibition was<br />
characterized by cAMP dependent protein kinase A (PKA) activation which in turn led to a<br />
decrease in arachidonic acid release and 5-LO translocation [9]. Based on this, we<br />
postulate that the human histamine H4 receptor might control LT biosynthesis in activated<br />
human leukocytes such as macrophages and eosinophils. Using western blot and<br />
polymerase chain reaction (PCR) technique we have investigated the H4 receptor<br />
expression in various cell lines and primary cells and have chosen suitable cell lines. Next,<br />
we plan to treat these cells with different cytokines plus histamine and monitor 5-LO<br />
activity. After this, we plan to elucidate the underlying mechanism to provide the basis for<br />
Poster 121
the development of potential new anti-leuktriene drugs.<br />
References:<br />
1. Oda, T. et al.: J. Biol. Chem. 2000, 275: 36781–36786.<br />
2. Nakamura, T. et al.: Biochem. Biophys. Res. Commun. 2000, 279: 615–620.<br />
3. Zhu, Y. et al.: Mol. Pharmacol. 2001, 59: 434–441.<br />
4. Liu, C. et al.: Mol. Pharmacol. 2001, 59: 420–426.<br />
5. Morse, K.L. et al.: J. Pharmacol. Exp. Ther. 2001, 296: 1058–1066.<br />
6. Rouzer C.A. et al.: J. Biol. Chem. 1988, 263(22): 10980-10988.<br />
7. Heaggstrom J.Z. et al.: Cell. Mol. Life. Sci. 2002, 59(5):742-753.<br />
8. Werz O. et al.: Pharmacol. Ther. 2006, 112(3):701-718.<br />
9. Flamand N. et al.: Br. J. Pharmacol. 2004, 141(4): 552-561.<br />
119<br />
Quantitative Analysis of Isolated EGFR Adaptor Protein Pattern by NSAF and<br />
SILAC Reveals Cellular Dynamics of Receptor Signaling and Internalization<br />
Foerster, S. 1, Hammer, E. 2; Kacprowski, T. 3, Albrecht, M. 3, Völker, U. 2, Ritter, C.A. 1<br />
1 Clinical Pharmacy, Institute of Pharmacy, <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-University, Friedrich-Ludwig-Jahn-<br />
Str. 17, 17487 <strong>Greifswald</strong>, Germany<br />
2 Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics,<br />
<strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-University, Friedrich-Ludwig-Jahn-Str. 15, 17487 <strong>Greifswald</strong>, Germany<br />
3 Department of Bioinformatics, Institute of Biometrics and Medical Informatics, University Medicine<br />
<strong>Greifswald</strong>, Ferdinand-Sauerbruch-Str. 1, 17475 <strong>Greifswald</strong>, Germany<br />
Overexpression, aberrant or enhanced activation of the epidermal growth factor receptor<br />
(EGFR) are frequent events in human cancers. By recruiting specific adaptor proteins to the<br />
activated intracellular receptor tyrosine kinase domain, EGFR signaling regulates<br />
proliferation, growth and differentiation [1]. We hypothesize that cellular responses are<br />
realized by characteristic patterns of adaptor proteins, which switch respective signal<br />
cascades and therefore developed a method to analyze the EGFR adaptor protein pattern<br />
in relation to its physical state.<br />
The EGFR-adaptor protein-complex was isolated by immunoprecipitation using the specific<br />
EGFR antibody cetuximab,which was biotinylated and bound to magnetic beads. A431 cells<br />
were stimulated with 100 ng/ml EGF for 30 minutes or left untreated and EGFR-associated<br />
protein complexes were precipitated from respective cell lysates. For stable isotope labeling<br />
in cell culture (SILAC), the stimulated cells were first adapted to heavy isotopes ( 13C) of Llysine<br />
and L-arginine and light cells were left untreated or incubated with 10 µM Tyrphostin<br />
AG1478 for 1 h before stimulation with EGF. Lysates of heavy ( 13C) and light ( 12C) cultures<br />
were mixed in a 1:1 ratio for precipitation. After pre-fractionation by 1D-SDS-PAGE and ingel-digestion,<br />
protein complexes were characterized by LC-ESI-tandem high resolution<br />
mass spectrometry (LC-MS/MS). SILAC provides a ratio of heavy/light peptides for<br />
quantification of proteins in stimulated and control samples while the label free complexes<br />
were analyzed with the normalized spectral abundance factor (NSAF), which overcomes<br />
sample to sample variation and enables a semi-quantitative determination of proteins from<br />
different samples [2].<br />
A set of 58 proteins was reproducibly identified in three LC-MS/MS replicates. The proteinprotein<br />
interactions within this set were obtained from the iRefIndex database and the<br />
corresponding protein interaction network was visualized in Cytoscape. The Gene Ontology<br />
annotations of the proteins were clustered and visualized in the network as well.. Most<br />
proteins are involved in intracellular localization and protein transport (27 proteins) or cell<br />
death (13 proteins). The first domain contained structural proteins such as actin and tubulin<br />
subunits as well as importins, exportins and transportins. The cell death regulating category<br />
included serine/threonine-protein phosphatase 2A and heat shock protein-ß1. Furthermore,<br />
several proteins were found which associations to the EGFR are yet unknown, such as<br />
Hook homolog 2 and cancerous inhibitor protein 2A. Quantification by SILAC identified<br />
subunits of the clathrin related adaptor protein complex, which were markedly pronounced<br />
upon EGF stimulation, suggesting increased receptor internalization. These data were<br />
confirmed by Western Blotting and immunofluorescence microscopy. EGFR signaling<br />
related proteins like growth factor receptor bound protein 2 (Grb2) and signal transducer<br />
and activator of transcription 3 (STAT3) were not subjected to stimulation dependent<br />
changes. Further dynamic changes revealed proteins which were affected by stimulation<br />
after inhibition of tyrosine kinase activity. This might indicate that alternative EGFR<br />
signaling pathways occurred. For instance, SHC-transforming protein 1 decreased upon<br />
stimulation and even more when EGFR activity was inhibited. Nevertheless, adaptor<br />
proteins are low abundant proteins, which is why we will work out a protein cross linking<br />
strategy to enrich for specific signaling molecules.<br />
As proof of principle, we were able to identify proteins that are part of the EGFR signaling<br />
pathway as well as proteins that relate to cellular localization and protein trafficking and<br />
whose abundances changed upon EGFR stimulation. Using this approach we will provide<br />
further insights into mechanisms of EGFR signal activation from a systems biology point of<br />
view.<br />
References:<br />
1. Oda, K. et al.: Mol.Syst.Biol. 2005, 1:2005.0010.<br />
2. Gokce, E. et al.: J.Am.Soc.Mass Spectrom. 2011, 22: 2199-2208.<br />
120<br />
Metabolism studies of Ifenprodil, a potent NR2B-selective NMDA receptor<br />
antagonist<br />
Falck, E.; Wünsch, B.<br />
Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-<strong>Universität</strong>,<br />
Hittorfstr. 58-62, 48153 Münster, Germany<br />
Ifenprodil is known as an antagonist for a special binding site at the NMDA receptor subunit<br />
NR2B, called ifenprodil binding site [1]. NMDA receptors change their concentration and<br />
composition with the development of the brain during the lifetime period [2]. Furthermore,<br />
NMDA receptors containing the NR2B subunit are restricted in specific sites of the human<br />
brain [3]. This makes the NR2B subunit an interesting target for the therapy of<br />
neurodegenerative diseases such as schizophrenia, epilepsy or Parkinson’s disease.<br />
Epilepsy for example causes neuronal cell death which leads to an increased glutamate<br />
concentration. This induces an overactivation of the NMDA receptor inducing further cell<br />
death. Ifenprodil as an antagonist can reduce NMDA receptor activation and protect the<br />
brain against further cell damage. The ifenprodil binding site is located on the surface of the<br />
NR2B subunit and can interact with other binding sites located at the receptor. Ifenprodil<br />
has a high affinity to the receptor but a rather low selectivity. The interaction with other<br />
receptors leads to undesired side effects, e. g. impaired motor function. The ifenprodil<br />
binding site at the NMDA receptor represents an interesting target and ifenprodil a<br />
promising lead compound for the development of innovative NMDA receptor antagonists<br />
with comparable affinity but increased selectivity.<br />
In addition to the poor receptor selectivity fast metabolic degradation and low bioavailability<br />
of ifenprodil have been reported in literature. The precise knowledge about the<br />
biotransformation of ifenprodil is important for the design of metabolically more stable<br />
analogues. However, the metabolism of this compound has not been studied in detail.<br />
Herein we report the metabolism of ifenprodil, regarding the phase I and phase II<br />
metabolites resulting from hydroxylation and conjugation reactions.<br />
Transformations were carried out with rat liver microsomes and the essential co-factors<br />
NADPH/H +, UDPGA, PAPS and SAM. The metabolites were identified by fragmentation<br />
steps carried out using HPLC-ESI-MS n. The supposed structures of the resulting fragments<br />
were confirmed by recording the exact mass yielding from time-of-flight detection. All<br />
metabolites were identified by comparing their fragmentation pattern with the typical<br />
fragmentation pattern observed with the parent compound ifenprodil.<br />
References:<br />
1. Williams, K.: Curr. Drug Targets 2001, 2: 285-298.<br />
2. Chazot, P. L.: Curr. Med. Chem. 2004, 11: 389-396.<br />
3. Childers, W. E., Baudy, R. B.: J. Med. Chem. 2007, 20: 2557-2562.<br />
122 Poster<br />
1<br />
121<br />
11-Keto-β-boswellic acids prevent insulitis in 2 animal models with<br />
autoimmune diabetes<br />
Ammon, H.P.T. 1, Shehata, A.M. 2, Jauch, J. 3, Bettio S. 4, Quintanilla-Fend, L. 4<br />
1Department of Pharmacology, Institute of Pharmaceutical Sciences, University of Tuebingen,<br />
2Department of Pharmacology, Faculty of Pharmacy, University of Beni-Sueif, Beni-Sueif, Egypt,<br />
3Institute of Organic Chemistry II, University of Saarland, Saarbrücken, Germany,<br />
4Institute of Pathology, University of Tuebingen, Germany<br />
Background and aims: Type 1 diabetes and Late onset Autoimmune Diabetes of the Adult<br />
(LADA) are autoimmune diseases where a chronic inflammatory process destroys insulin<br />
producing β-cells. Boswellic acids which derive from gum resin of Boswellia species<br />
(Olibanum) have been shown to possess immunmodulatory properties and suppress<br />
proinflammatory cytokines including NFκB (1). In the model of Multiple Low Dose-<br />
Streptozotocin ((MLD-STZ) diabetes and the genetic Non Obese Diabetic (NOD)mouse,<br />
which are thougt to correspond to human one type diabetes, it was tested whether or not<br />
an extract of the gum resin of boswellia serrata (BE), 11-Keto-β-boswellic acid (KBA) and<br />
Acetyl-O-11-keto-β-boswellic acid (AKBA) could prevent developement of insulitis and<br />
appearance of apoptotic cells in the pancreatic islets, increase of proinflammatory<br />
cytokines in the blood and increase of blood glucose level.<br />
Methods: BK +/+ male mice were i.p. injected with 40 mg/kg STZ for 5 days. A second<br />
group received 150 mg/kg BE, a third group 15 mg/kg AKBA and a fourth group 7.5 mg/kg<br />
KBA simultaneously over a period of 10 days. Infiltration of lymphocytes into pancreatic<br />
islets and apoptosis of islet cells were determined histochemically (CD-3 antibodies and<br />
anticaspase-3) after day 10. In addition, proinflammatory cytokines were determined in<br />
serum. Blood glucose was measured over a period of 35 days.<br />
Four weeks old NOD-mice received daily 7.5 mg/kg KBA i.p. over a period of three weeks.<br />
Thereafter the pancreatica have been taken for histochemical studies.<br />
Results: 10 days after first STZ injection there was infiltration of lymphocytes into<br />
pancreatic islets and appearance of apoptotic cells. Moreover, all tested proinflammatory<br />
cytokines in the serum were significantly increased. There was also a continuous increase<br />
of blood glucose. Simultaneous i.p. administration of BE, AKBA and KBA significantly<br />
reduced cytokines in the serum, infiltration of lymphocytes and apoptosis of islet cells. After<br />
21 days blood glucose levels showed no increase.<br />
In NOD-mice which develope insulitis and first apoptotic cells in the pancreas after four<br />
weeks of age administration of KBA prevented both to a large extent.<br />
Conclusion: An extract of the gum resin of Boswellia serrata (olibanum) and two of its<br />
constituents i. e. AKBA and KBA can prevent developement of insulitis probably through<br />
their inhibitory action on proinflammatory cytokines. 11-Keto-β-boswellic-acids seem to be<br />
promising compounds for the prevention and treatment of autoimmune diabetes.<br />
Supported by: The authors are grateful for support for the Dr. h. c. Bürger-Büsing Foundation and the<br />
German Diabetes Foundation.<br />
References:<br />
(1) Ammon, HPT: Phymed. 2010, 17: 862-867.<br />
122<br />
The Thermal Behavior of Histidine in Frozen Aqueous Solutions: The Effect of<br />
pH, Annealing, Histidine Concentration and Formulation Composition.<br />
Alhussein, A. 1 ;Gieseler, H. 1 2<br />
1Department of Pharmaceutics-University of Erlangen, Cauerstrasse 4, 91058 Erlangen, Germany<br />
2 GILYOS GmbH, Friedrich-Bergius-Ring 15, 97076 Wuerzburg, Germany<br />
Therapeutic proteins are a major focus of research and development activities in the<br />
pharmaceutical industry. Due to their limited stability in aqueous solutions, proteins often<br />
need to be converted into solid state to achieve an acceptable shelf life as pharmaceutical<br />
products [1]. The most commonly used method for manufacture of solid protein<br />
pharmaceuticals is freeze drying [1,2]. In turn, freeze drying process (lyophilization)<br />
generates many stresses during both freezing and drying which may cause the loss of<br />
protein bio-activity. Therefore many excipients are added to the protein formulation to<br />
overcome these stresses and to improve protein stability during freeze drying.<br />
Disaccharides, such as sucrose and trehalose, are widely used as protein stabilizers (cryoand<br />
lyoprotectant), and these sugars have been extensively studied in the literature to<br />
investigate thier stabilizing effect on proteins during the lyophilization. However, formulation<br />
development to achieve the best stabilizing effect on protein is still a great challenge for the<br />
experts of freeze drying.
In the last few years, amino acids are attracting increased attention in the field of the freeze<br />
drying of proteins, and many amino acids were used as protein stabilizer in the freeze dried<br />
products [3,4]. In contrast to the sugars, amino acids can also function as buffers in protein<br />
formulations. Therefore amino acids provide more choices for the design of proteins<br />
formulations. Histidine is one of the amino acids that can be added to the protein<br />
formulations to function as both buffer and protein stabilizer [5].<br />
It is well known that amorphism is an essential property for stabilization of proteins during<br />
freeze drying. Protein stabilizer should remain amorphous (in the same phase with protein)<br />
to be able to interact with the protein molecule by the formation of hydrogen bonds, leading<br />
thereby to the preservation of protein native structure [6]. Since the stabilizing effect of an<br />
amino acid (or other stabilizer) during lyophilization is directly correlated with its physical<br />
state, it is important to evaluate the stabilizing efficiency by studying the changes in the<br />
physical state of the amino acid upon cooling and heating.<br />
The aim of this study was to investigate the thermal behavior of histidine in frozen aqueous<br />
solutions at different conditions using DSC technique (Differential Scanning Calorimetry).<br />
This research focused on tow thermal events: Histidine crystallization and the glass<br />
transition of histidine. The effect of histidine concentration and pH of histidine solution on<br />
the physical state of histidine was investigated. Furthermore, some of popular excipients<br />
which are widely used in the freeze dried protein formulations (such as sucrose, mannitol,<br />
and Tween 80) were added into histidine solutions to evaluate thier effect on histidine<br />
during the DSC measurement. Additionally, the effect of annealing (which is a common<br />
practice applied during the freeze drying to enhance the crystallization of the bulking<br />
agents) was investigated, where all DSC scans of the studied formulations were performed<br />
with/without annealing to detect the possible effect of this step on the thermal behavior of<br />
histidine.<br />
The results showed that histidine crystallization may occur during the heating phase of the<br />
DSC-scan and this event depends strongly on both pH and histidine concentration.<br />
However, the enhancement of histidine crystallization by application of annealing seems<br />
also to be pH-dependent. Furthermore, the glass transition temperature of histidine (Tg )<br />
was greatly varied according to the pH of solution. The addition of sucrose to the histidine<br />
solution resulted in an inhibition of histidine crystallization, while the addition of mannitol or<br />
Tween 80 seems to have no inhibitory effect. The interpretation of histidine thermal<br />
behavior in the systems containing both mannitol and sucrose is more complex and<br />
depends on several factors including the weight ratio of mannitol to sucrose in the<br />
formulation.<br />
References:<br />
1. Wang, W.: Int. J. Pharm. 2000, 203(1): 1-60.<br />
2. Pikal, MJ.: Encyclopedia of Pharmaceutical Technology (Marcel Dekker Publishing Co. New York) 2002.<br />
3. Österberg, T.: Pharm. Res. 1997, 14(7): 892-898.<br />
4. Izutsu, K. et al.: Int. J. Pharm. 2005, 301(1-2): 161-169.<br />
5. Österberg, T.: Eur. J. Pharm. Sci. 1999, 8(4): 301-308.<br />
6. Carpenter, JF. et al.: J. Dairy. Sci. 1990, 73(12): 3627–3636.<br />
123<br />
Biomimetic catecholates for the chemical modification of metal surfaces:<br />
antifouling properties<br />
Khalil, F. 1; Maison, W. 1<br />
1 Pharmaceutical and Medicinal Chemistry, Bundesstraße 45, 20146 Hamburg, Germany<br />
Biofouling is the non-specific attachment of biological material (proteins, carbohydrates and<br />
prokaryotic cells) to surfaces upon their exposure to any biological milieu [1]. Biofouling is<br />
of great concern in many areas ranging from marine technology, biosensors, dentistry to<br />
biomedical implants and devices. The most common approach to prepare non-fouling<br />
surfaces is through the immobilization of polymers such as poly(ethylene glycol) (PEG),<br />
which are able to repel the adhesion of proteins and cells [2].<br />
Methods for the covalent immobilization of molecular monolayers rely on the use of<br />
functionalized anchor molecules, including thiols, silanols, phosphates or phosphonates. All<br />
of these immobilization techniques have drawbacks, such as limited stability, elaborate<br />
syntheses or a limited range of substrate materials. Catecholate, inspired by musseladhesive<br />
proteins (MAPs) [3] and bacterial siderophores [4,5], have received considerable<br />
interest because of their high binding affinity to various surfaces [6].<br />
By following a biomimetic approach, we have synthesized conjugates of catecholates and<br />
bifunctional tetravalent scaffolds based on a trisalkylmethyl core structure. Three<br />
catecholateamines have been conjugated to a central scaffold, resembling the tripodal<br />
metal binding motif of natural metal binders, such as MAPs and siderophores [7]. The<br />
resulting trimeric catecholates are easily coupled to different effector molecules for the<br />
stabile immobilization of effector molecules such as PEG on metal surfaces.<br />
Futher, the immobilization of these multivalent catecholate derivatives on TiO2 surface have<br />
been performed and showed promising antifouling properties.<br />
References:<br />
1. Hall-Stoodley, L., Costerton, J. W., Stoodley, P., Nat. Rev. 2004, 2(2), 95-108.<br />
2. Banerjee, I., Pangule, R. C., Kane, R. S., Adv. Mater. 2011, 23(26), 690-718.<br />
3. Waite, J. H., Tanzer, M. L., Science 1981, 212(4498), 1038-1040.<br />
4. Loomis, L. D., Raymond, K. N., Inorg. Chem. 1991, 30(5), 906-911.<br />
5. Gademann, K., Chimia 2007, 61(6), 373-377<br />
6. Lee, H., Dellatore, S. M., Miller, W. M., Messersmith, P. B., Science 2007, 318(5849), 426-430.<br />
7. Franzmann, E., Khalil, F., Weidmann, C., Schröder, M., Rohnke, M., Janek, J., Smarsly, B., Maison, W.,<br />
Chem. Eur. J. 2011, 17(31), 8596-8603.<br />
124<br />
Quantification of impurities with NMR spectroscopy: Comparison of different<br />
approaches to quantify small signals overlapped with large signals using the<br />
example topiramate.<br />
Wiest , J.; Ilko , D.; Holzgrabe, U.<br />
Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg,<br />
Germany<br />
Molecules without chromophores like topiramate (TOP) pose a challenge to analysts as<br />
they cannot easily be quantified via common photometric detection regimes like UV/VIS or<br />
fluorometry. Our aim was to develop an HPLC-CAD method for the determination of purity<br />
and content of TOP and to compare the results to the existing HPLC refractive index<br />
detection (HPLC-RI) method which has a poor sensitivity and a limited performance. The<br />
newly developed HPLC-CAD method obtained good results for quantification of TOP and<br />
the other related substances, however the main impurity diacetone fructose (DAF) could<br />
not be determined due to its high vapour pressure. To solve this problem, we tried to apply<br />
NMR spectroscopy. Prerequisite of qNMR is a clear separation of the signals of all<br />
components used for quantification. Variation of the solvent resulted in pyridine-d5 which<br />
gave the best signal separation.The spectra were acquired with inverse gated 13C<br />
decoupling using globally optimized alternating phase rectangular pulse (GARP) to remove<br />
disturbing 13C satellites. To avoid rotation sidebands the spectra were measured without<br />
spinning. Processing the spectra has a great influence on the results. Thus we compared<br />
the results of four Topiramate batches with an internal standard using a standard addition<br />
method. The results were obtained with three different approaches: (i) Deconvolution of the<br />
overlapping signals executed with TopSpin 3.0; (ii) Integration after baseline adjustment (iii)<br />
Quantification by signal height. An experimental protocol was found which is particularly<br />
suitable for quantification of DAF residues in TOP samples.<br />
References:<br />
Almeling, S.; Ilko, D.; Holzgrabe, U.: J. Pharm. Biomed. Anal. <strong>2012</strong>, Epub ahead of print<br />
www.elsevier.com/locate/jpba , 23.05.<strong>2012</strong><br />
Pauli, G.F.: Phytochem. Anal. 2001, 12: 28-42<br />
125<br />
Impact of drug-drug interactions on the INR values of patients treated with<br />
Marcumar ® in the ambulatory care setting<br />
Bertram, L. 1; Krämer, I. 1<br />
1 Department of Pharmacy, University Medical Center of Johannes Gutenberg-University,<br />
Langenbeckstr.1, 55131 Mainz, Germany<br />
PURPOSE: Treatment with the oral vitamin K antagonist phenprocoumon (Marcumar ®) is<br />
associated with a high risk of interactions with additionally prescribed medication and selfmedication.<br />
In consequence the efficacy of Marcumar ® can be increased or reduced and<br />
the resulting INR-values may drift out of the therapeutic range. Thus the risk of thromboembolic<br />
events or bleeding increases.<br />
The aim of this study was to investigate whether patients’ INR-values in the ambulatory<br />
health-care sector in Germany are influenced by additionally prescribed potentially<br />
interacting drugs [1].<br />
METHODS: 50 patients treated with Marcumar ® were evaluated in a retrospective study<br />
over three years (1800 months in total). The INR-values and type of comedication of ten<br />
patients each recruited in five physician offices in Rhineland-Palatinate were retrieved from<br />
the patients’ medical records. The percentage rate of INR values lying in the target range<br />
(e.g. INR 2-3) and extended target range (target range +/- 0.2) was evaluated for individual<br />
patients. Time in therapeutic range (TTR) was calculated by using the Rosendaal-Method<br />
[2]. Potentially interacting drugs associated with serious interactions were chosen from the<br />
literature [1]. The Mann-Whitney-U-test was used to determine any significant relationship.<br />
RESULTS: Patients taking potentially interacting drugs in addition to a permanent<br />
phenprocoumon therapy achieved lower rates of adequate INR values than patients not<br />
taking potentially interacting drugs. The median percentage rate of INR values within the<br />
therapeutic range amounted to 69% without interacting comedication and 53% with using<br />
interacting comedication (p= 0.008) as well as 83% versus 67% (p= 0.076) for the median<br />
extended therapeutic range, and 75% versus 61% (p= 0.002) for median TTR respectively.<br />
CONCLUSION: Results document that the concomitant intake of interacting drugs<br />
significantly affects the rate of INR-values in therapeutic range and TTR. With regard to the<br />
extended target INR range the difference is not significant. However study results clearly<br />
show that the efficacy of phenprocoumon in routine clinical practice is depending on the<br />
potentially interacting concomitant drug therapy. These combinations should be avoided<br />
whenever possible because additional monitoring is necessary and safety of the<br />
anticoagulant therapy is compromised.<br />
References:<br />
1. S. Isstas, „Pharmakovigilanz bei Patienten mit Phenprocoumon-Therapie- Untersuchungen zu<br />
potentiellen Interaktionen und Betreuungsqualität“, dissertation, Johannes Gutenberg-UniversityMainz,<br />
2011.<br />
2. Rosendaal, F.R. et al.: Thromb. Haemost.1993, 69: 236-239.<br />
126<br />
A novel combinative particle size reduction technology for drug nanocrystals<br />
production and further nanosuspension transfer to tablets<br />
Poster 123
Salazar J1, Müller R H1, Möschwitzer J P1 2<br />
1 Institute of Pharmacy, Dept. of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Freie<br />
<strong>Universität</strong> Berlin, Kelchstrasse 31, 12169 Berlin, Germany<br />
2 Pharmaceutical Development, Abbott GmbH & Co. KG, Knollstrasse 50, 67061 Ludwigshafen am<br />
Rhein, Germany<br />
The aim of this research was to systematically analyze the process parameters affecting<br />
the comminution effectiveness of a novel particle size reduction method. This combinative<br />
method employs spray-drying (SD) as bottom-up step and drug pre-treatment followed by<br />
high pressure homogenization (HPH) as top-down process for nanosuspension production<br />
1 2. The liquid nanosuspensions were then transferred to immediate release (IR) tablets by<br />
applying downstream techniques such as wet granulation (WG), freeze-drying (FD) or SD.<br />
Finally, a dissolution test was performed for drug release assessment.<br />
The poorly water-soluble compound glibenclamide (BCS Class II) was used as model drug.<br />
The influence of drug and surfactant concentrations during SD on the particle size reduction<br />
effectiveness of the HPH was analyzed. The drug was dissolved in ethanol to prepare<br />
solution concentrations of 1%, 2% or 3% (w/v). The amounts of docusate sodium salt<br />
(DSS) in the ethanolic solution were 0%, 0.1%, 0.2%, 0.3% or 0.4% (w/v). The ethanolic<br />
solutions were then spray-dried using a Mini Spray Dryer B-290 coupled to an Inert Loop B-<br />
295 (Büchi Labortechnik AG, Switzerland). The morphology and solid state of the spraydried<br />
powders were analyzed by SEM, DSC and PXRD. Suspensions were produced by<br />
dispersing 1% (w/w) drug (treated/untreated) in demineralized water using an Ultra-Turrax<br />
T-25 (IKA Werke GmbH & Co. KG, Germany). In the case of the drug powders processed<br />
without surfactant DSS 0.2% (w/v) was added as dispersant. The different suspensions<br />
were then homogenized by employing the HPH technique (1500 bar, 20 cycles) using a<br />
Micron LAB 40 homogenizer (APV Systems, Germany). The particle size of the<br />
nanosuspensions was analyzed using photon correlation spectroscopy and laser<br />
diffractometry. The liquid nanosuspensions were down-streamed by WG, FD or SD and<br />
then processed to tablets using lactose monohydrate as filler. Mannitol was used as matrix<br />
former for FD and SD (0%-6% w/v). A dissolution test was performed to determine the<br />
immediate drug release performance of the tablets.<br />
It was found that high drug concentrations and middle DSS concentration resulted in<br />
porous, flowable and amorphous drug powders after SD. These characteristics resulted in<br />
smaller particle sizes after reduction. The drug pre-treatment can significantly improve the<br />
particle size reduction effectiveness of the HPH. The best nanosuspension produced with<br />
the spray-dried powders (2% drug, 0.2% DSS during SD) showed a very small particle size<br />
(236 nm mean particle size, d50% of 0.131 µm and d90% of 0.285 µm) and a narrow size<br />
distribution. The best drug release was 90% after 10 min and it was delivered by SD of the<br />
best nanosuspension with 2% mannitol before tableting. This drug release was markedly<br />
better compared to the one delivered by the tablets processed with micronized drug (26%<br />
drug release after 10 min). The rank order of the tablet release rate employing the<br />
downstream techniques was: SD tablets > FD tablets > WG tablets. The surfactant and<br />
drug concentrations used for the SD process are critical and need to be selected carefully.<br />
The transfer of the drug nanocrystals to tablets was successfully achieved.<br />
References:<br />
[1]: Möschwitzer, J., Method for producing ultrafine submicronic suspensions, WO/002006094808A3, 2006<br />
[2]: Möschwitzer, J., Müller R.H., New method for the effective production of ultrafine drug nanocrystals,<br />
Journal of Nanoscience and Nanotechnology 6(9-10): 3145-3153, 2006<br />
127<br />
Differential scanning calorimetry as a supportive tool for the analysis of<br />
nanomilled fenofibrate suspensions<br />
Komoß, C. 1; Bitterlich, A. 2; Kwade, A. 2; Bunjes, H. 1<br />
1Institute of Pharmaceutical Technology, TU Braunschweig, Mendelssohnstraße 1, 38106<br />
Braunschweig, Germany<br />
2Institute for Particle Technology, TU Braunschweig, Volkmaroder Straße 5, 38104 Braunschweig,<br />
Germany<br />
An important approach to overcome poor dissolution properties and the often resulting bad<br />
biopharmaceutical profile of active pharmaceutical ingredients (API) is to reduce the API´s<br />
particle size, a process also known as “nanosizing” [1]. This leads to a strongly increased<br />
surface-to-volume ratio and, thus, to an increased dissolution rate. However, the large<br />
specific surface area creates a positive gain of free energy. Hence, there is a tendency<br />
towards crystal growth, (re-)agglomeration of the particles and, thus, instability of the<br />
nanosuspensions.<br />
A reduction of particle size can also lead to altered solid state properties like a reduced<br />
melting point [2]. Therefore, in this study, differential scanning calorimetry (DSC) analysis of<br />
nanosuspensions of the lipid lowering agent fenofibrate (kindly provided by Novartis<br />
Pharma AG) was conducted. The suspensions were produced by wet media milling using a<br />
modified planetary ball mill [3] (PM400, Retsch GmbH) or a stirred media mill (Dispermat<br />
SL 5 C nano, VMA Getzmann GmbH) and different polymers as stabilizers.<br />
Nanosuspensions were additionally analyzed using dynamic light scattering (DLS) and<br />
optical methods like transmission electron microscopy (TEM) and polarization microscopy.<br />
The evolution of onset and peak maximum temperature of PVA-stabilized fenofibrate<br />
nanosuspensions over grinding time was investigated via DSC indicating that both values<br />
decreased with longer grinding times [fig.1]. However, particle size determined via dynamic<br />
light scattering passed through a minimum and increased again with extended grinding<br />
times. TEM micrographs revealed that this phenomenon resulted from the formation of<br />
agglomerates containing extremly small particles after very long grinding times. Thus, the<br />
decrease of onset and peak maximum temperature was correlated with decreasing primary<br />
particle size.<br />
Furthermore, DSC was applied to differentiate between different types of instability of the<br />
fenofibrate nanosuspensions: particle growth or agglomeration of very small crystals,<br />
respectively. For this purpose, the onset and peak maximum temperatures of a HPMCstabilized<br />
nanosuspension at day 0 and after 1 week of storage under different conditions<br />
were compared. In combination with TEM investigations, the data clearly showed an<br />
increase in melting temperature for the samples with a pronounced crystal growth. DSC<br />
analysis can thus be a helpful approach not only for getting information about the real<br />
primary particle size but also for differentiating between different kinds of instabilities.<br />
124 Poster<br />
Temperature [ C]<br />
78<br />
77<br />
76<br />
75<br />
74<br />
73<br />
72<br />
71<br />
70<br />
69<br />
68<br />
Onset<br />
Peak<br />
z-avg<br />
4 6 8 10 20 40 60 100 200 400 800<br />
Grinding time [min]<br />
Fig. 1: Onset and peak maximum temperature determined via DSC and particle size (zaverage)<br />
determined via DLS of a PVA-stabilized fenofibrate nanosuspension at different<br />
points of the milling process<br />
References:<br />
1. Merisko-Liversidge, E., Liversidge, G.G.: Adv. Drug Deliv. Rev. 2011, 63(6): 427-440.<br />
2. Bunjes, H., Koch, M. H. J., Westesen, K.: Langmuir 2000, 16(12): 5234–5241.<br />
3. Juhnke, M., Berghausen, J., Timpe, C.: Chem. Eng. Technol. 2010, 33(9): 1412-1418.<br />
128<br />
Hydrogels for drug delivery: hydrolytically cleavable carbamate linkers for time<br />
controlled protein release<br />
Hammer N. 1,Brandl F.P. 1, Kirchhof S. 1 , Meßmann V. 1, Teßmar J.K.V. 1, Göpferich A. 1<br />
1 University of Regensburg, Department of Pharmaceutical Technology, <strong>Universität</strong>sstr.31<br />
Regensburg, 93053, Germany<br />
In the past decades, the significance of macromolecular drugs of biological origin has<br />
increased enormously. However, their application is still hampered by short biological halflives.<br />
Hydrogels are one possibility to protect these delicate molecules against<br />
physicochemical instability and proteolytic degradation. Furthermore, hydrogels are<br />
considered biocompatible due to their physicochemical similarity to native extracellular<br />
matrix. Despite these advantages, one frequently observed problem is the rapid release of<br />
incorporated proteins due to the high water content of the hydrogels. To overcome this<br />
problem, proteins can be reversibly attached to the hydrogel backbone by suitable linker<br />
molecules.<br />
We used non-degradable hydrogels to characterize carbamate-mediated protein release.<br />
The gels were formed from star-shaped poly(ethylene glycol) (PEG) alkyne and starshaped<br />
PEG azide; the cross-linking reaction was catalyzed by 0.5 µmol CuSO4 and 2.5<br />
µmol ascorbic acid. For the attachment of proteins to the hydrogel structure, we<br />
synthesized different star-shaped PEG linkers. Two to three of the four end-groups were<br />
modified with an alkynyl groups to allow cross-linking with star-shaped PEG azides. The<br />
remaining end groups were modified with hydrolytically cleavable carbamates for reversible<br />
protein binding.<br />
Gel properties were studied by oscillatory rheology. Swelling and stability of the gel<br />
cylinders in phosphate buffered saline at 37 °C were analyzed by weighing the cylinders at<br />
regular time points. We compared protein release from hydrogels with and without<br />
carbamate linkers. For protein binding, lysozyme was incubated with the carbamate linker<br />
for 2h at room temperature and mixed with additional polymer to form hydrogels. The gel<br />
cylinders were incubated in phosphate buffered saline at 37 °C. The amount of released<br />
protein was determined at regular time points by Bradford assay.<br />
Gelation started after about 16 min depending on the copper concentration.Oscillatory<br />
rheology showed a gel strength of 7600 Pa, which indicates an almost complete<br />
crosslinking reaction. Hydrogels with attached lysozyme still had a gel strength of 5400 Pa.<br />
We determined swelling ratios of about 1.2 (without lysozyme) and 1.28 (with lysozyme),<br />
respectively. The gel cylinders were stable for more than 2 weeks. Without binding,<br />
incorporated lysozyme was released within the first 24 h. Attachment of the protein to the<br />
hydrogel backbone decelerated the release significantly. After cleavage of the carbamate<br />
linker, 22 % of lysozyme was released within 14 days. The study is not completed yet;<br />
further time points will follow.<br />
lysozyme conc. [%]<br />
20%<br />
release of attached lysozyme<br />
0%<br />
0 5 10 15<br />
time [d]<br />
Altogether, covalent attachment of proteins to the gel structure by degradable carbamate<br />
linkers seems to be a promising strategy to control protein release.<br />
Acknowledgments: DFG, grant GO 565/16-1<br />
lysozyme conc. [%]<br />
50%<br />
1000<br />
900<br />
800<br />
700<br />
600<br />
500<br />
400<br />
300<br />
200<br />
100<br />
0<br />
Particle size [nm]<br />
release of lysoyzme without linker<br />
100%<br />
0%<br />
0 20<br />
time [h]<br />
40
129<br />
Production of Resveratrol Nanocrystals by a Novel Combinative Process (H 42<br />
Technology)<br />
Liu, T. 1; Müller, R. H. 1; Möschwitzer, J. P. 1 2<br />
1 Institute of Pharmacy, Dept. of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Freie<br />
<strong>Universität</strong> Berlin, Kelchstr.31 Berlin, 12169,Germany<br />
2 Pharmaceutical Development, Abbott GmbH & Co. KG, Knollstr. 50 Ludwigshafen am Rhein,<br />
67061, Germany<br />
In order to improve the manufacturing efficiency of drug nanocrystal, a new combinative<br />
particle size reduction technology (H 42) has been developed recently. Spray-drying as a<br />
bottom-up step is applied to modify the properties (e.g. crystallinity and morphology) of the<br />
raw drug particles. The subsequent high pressure homogenization step is used to obtain<br />
the final nanosuspensions. According to the design of experiment (DoE) methodology,<br />
different compositions of resveratrol (API) and sodium cholate (surfactant) were dissolved<br />
in ethanol and the solutions were spray-dried using a mini spray dryer B-290 with inert loop<br />
B-295 (Büchi Labortechnik AG, Switzerland). The crystallinity of spray-dried powders was<br />
analyzed by differential scanning calorimetry (DSC, Mettler Toledo AG, Germany) and<br />
powder X-ray diffraction (PXRD) with Philips PW 1710 diffractometer control unit (Philips<br />
Industrial & Electro-Acoustic Systems Division, The Netherlands). High pressure<br />
homogenization (HPH) for 20 cycles at 1500 bar using a Micron Lab 40 homogenizer (APV<br />
Deutschland GmbH, Germany) was employed to produce nanosuspensions. Laser<br />
diffractometry (LD, Malvern Instruments, UK) and photon correlation spectroscopy (PCS,<br />
Malvern Instruments, UK) were applied to determine the particle size. The PXRD and DSC<br />
results showed that 6 DoE points of spray-dried powder resulted in amorphous drug, which<br />
evidently indicated a relationship between formulation of the feed and final API crystallinity.<br />
After only 1 homogenization cycle at 1500 bar, a particle size of around 200nm (d 0.5) was<br />
achieved by using modified API, which was comparable with the result of unmodified API<br />
after 20 cycles. The production time for the nanocrystals had been significantly reduced. On<br />
the other hand, the largest particle size (d 0.9) obtained from modified API (736 nm) was<br />
much smaller than the unmodified drug (2.2 µm) after 20 cycles. However, no direct<br />
correlation was found between the degree of crystallinity and the particle size of the drug<br />
nanocrystals after HPH. To sum up, due to the short production time and low energy input<br />
the new combination technology (H 42) was confirmed to be an effective method to<br />
produce resveratrol nanocrystals. Further more detailed study will be focused on the<br />
mechanism of this process and influencing factors of particle size reduction.<br />
130<br />
Differentiation of several identically prepared batches of tablets using near<br />
infrared spectroscopy<br />
Kaminski, D; Baumann, K<br />
1 Technische <strong>Universität</strong> Braunschweig, Institute for Medical and Pharmaceutical Chemistry,<br />
Beethovenstr. 55, 38106 Braunschweig, Germany<br />
Near infrared spectroscopy (NIR) is the first choice method in pharmaceutical industry for<br />
rapid inspection of raw materials. It is also suitable for online process control of the<br />
products as the NIR is a nondestructive analytical technique. Chemical composition as well<br />
as physical properties can be monitored. Even small variations, which are unavoidable<br />
during the production process, lead to significant changes in NIR spectra. This allows for a<br />
close monitoring of the physical properties, such as compaction pressure [1], humidity [2]<br />
and the homogeneity of active pharmaceutical ingredient (API) distribution [3] during the<br />
production process and the storage of tablets.<br />
This leads to the question whether it is possible to differentiate several batches of directtabletted<br />
ibuprofen with exactly the same manufacturing process by means of chemometric<br />
methods. Different methods of data pre-treatment are compared and an outlier detection is<br />
performed by principal component analysis (PCA). Those samples that show outlying<br />
spectra are scrutinized with respect to their content of the AIP which is independently<br />
determined by titration. The tabletting material and the production process are deliberately<br />
varied in order to study the sensitivity of the developed chemometric methods.<br />
The approach presented here attempts to separate the random error and deterministic<br />
variations in the spectral signal. The goal of this work is to define confidence limits based<br />
on a comprehensive characterization of all sources of spectral variations in order to detect<br />
tablets which are likely out of specification for further analysis by orthogonal analytical<br />
techniques.<br />
References:<br />
1. Kirsch JD, Drennen JK: J. Pharmaceut. Biomed. 1999, 19: 351-362<br />
2. Last IR, Prebble KA: J. Pharm. Biomed. Anal. 1993, 11: 1071-1076<br />
3. Plugge W, Van der Vlies C: J. Pharm. Biomed. Anal. 1993, 11: 435-442<br />
131<br />
Development of a medicated chewing gum containing riboflavin phosphate<br />
Wilde L. 1, Wollatz U. 1, Kordaß B. 2, Glöckl G. 1, Weitschies W. 1<br />
1Institute of Pharmacy, <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong>-University, Department of Biopharmaceutics &<br />
Pharmaceutical Technology, Center of Drug Absorption and Transport (C_DAT), Felix-Hausdorff-<br />
Straße 3, D-17487 <strong>Greifswald</strong>, Germany<br />
2<strong>Greifswald</strong> University Hospital, D-17475 <strong>Greifswald</strong>, Germany<br />
Medicated chewing gums are most often used for the treatment of travel-sickness or for the<br />
dehabituation of smokers. It was our idea to test, whether they might also be applicable to<br />
characterise the chewing motion of different patients by dentists. Therefore, a medicated<br />
chewing gum was developed, which contains an active agent, Riboflavin-5’-phosphate<br />
sodium, that can be dissolved by the saliva. Two different chewing gum matrices were<br />
investigated: Cafosa Gum S/A (Spain) and Gumlink A/S (Denmark). Both products are<br />
lipophilic mixtures consisting of elastomers for elasticity, resins for a cohesive body, waxes<br />
as softening agents and fats as plasticizers. Both gum bases can be compressed directly<br />
with the help of an eccentric tablet punch. For the preparation of the Gumlink gums, 19.9 g<br />
gum base and 100 mg lecithin were mixed in a cube blender for 10 minutes. After addition<br />
of 4 mg riboflavin phosphate and again blending for 10 minutes, the punches had to be<br />
lubricated with magnesium stearate to prevent sticking. The Cafosa gums were prepared in<br />
the same way, but with 19.8 g gum base, 200 mg lecithin and 20 mg riboflavin phosphate.<br />
In this way, we produced biconvex gums with a diameter of 12 mm and a weight of 500 mg.<br />
For the determination of content, a gum was given into an Erlenmeyer flask and 6 mL of<br />
heptane was added. After complete dissolution of the gum 20.0 mL purified water was<br />
added, the two phases were mixed intensely for 2 minutes and then separated by standing<br />
for 1 minute without stirring. For the Cafosa gums, two millilitres of the water phase were<br />
diluted and analysed at a wavelength of 267 and 600 nm (background correction) using a<br />
Cary 50 Scan (Varian). The water phase of the Gumlink gums was analysed without<br />
dilution. The determination of content show a content uniformity for the Gumlink chewing<br />
gums of 0.091 mg ± 0.0006 mg (n = 7) and for the Cafosa gums of 0.886 mg ± 0.070 mg (n<br />
= 7).<br />
For the chewing trials, the gums were chewed 5, 7 or 10 times (n = 6) by one volunteer,<br />
removed with a forceps and the saliva was rinsed off with purified water. Afterwards the<br />
determination of content followed as mentioned above.<br />
The results for the first chewing tests (figure 1) showed that chewing five times dissolved<br />
about 25 % of active agent, while seven chews increased this value to nearly 35 %. Ten<br />
chews released even more than 50 % of riboflavin phosphate out of the chewing gum<br />
matrix.<br />
Figure 1: Amount of riboflavin phosphate (Gumlink) after chewing 5, 7, 10 times; nominal<br />
quantity in each gum: 0.1 mg riboflavin phosphate (n = 6, mean ± SD).<br />
The Cafosa gum base was less suitable for spectrometric analysis because of the opacity<br />
of the solution even after filtration and centrifugation. That was the reason for higher<br />
absorption and thus incorrect content readings.<br />
We succeeded in producing uniform chewing gums with a small amount of hydrophilic<br />
active agent by direct compression and developed a method for the determination of<br />
content. We showed that the number of chews affects the dissolution of this agent, so that<br />
conclusions can be drawn from the chewing motion.<br />
Acknowledgments: The gum bases were a gift donated by Cafosa Gum S/A (Spain) and Gumlink A/S<br />
(Denmark). Financial support by the Federal Ministry of Education and Research (FKZ: 03IP612) is<br />
gratefully acknowledged.<br />
132<br />
Test purchase of slimming products containing unapproved active ingredients<br />
that may cause harmful side effects<br />
Parr MK1 2; Geyer H1; Paul M3, Smolka K4, Schänzer W1 1 Freie <strong>Universität</strong> Berlin, Pharmaceutical Chemistry, Königin-Luise-Str. 2+4, 14195 Berlin,<br />
Germany<br />
2 German Sport University Cologne, Biochemistry, Am Sportpark Müngersdorf, 50933 Cologne,<br />
Germany<br />
3 Stadt Köln, Gesundheitsamt, Neumarkt 15-21, 50667 Cologne, Germany<br />
4 Zollkriminalamt, Bergisch-Gladbacher-Str. 837, 51069 Cologne, Germany<br />
An increasing trend for attractive body appearance, stronger muscles and slender shape,<br />
coupled with a maximum of convenience is recognized in broad parts of society.<br />
Suggestions from advertisements and the easy access of image enhancing products,<br />
combined with the implication that such products cannot be harmful, make people believe<br />
that they can simply take “pills for everything” [1].<br />
Thus a huge market of dietary supplements offers numerous products containing a variety<br />
of unapproved and partially also harmful ingredients.<br />
Herein we present the results of products obtained by test purchases on the Internet. All<br />
products promised to cause extreme weight losses in a very short time period. The<br />
products were delivered by regular mail after credit card payment. No cautionary note was<br />
found on the label of the products.<br />
They were tested by GC-MS for their active ingredients. One of the products, named DNP<br />
Burn, brand name Body Advance Performance, was found to contain high amounts of 2,4dinitrophenol<br />
(Figure 1(1)). Identity was confirmed by comparison with authentic reference<br />
material. Semi-quantitation yielded an amount of about 200 mg. DNP uncouples the<br />
oxidative phosphorylation in the mitochondria resulting in a drastically increased metabolic<br />
rate. Several fatalities have been recorded after its intake [2].<br />
Other products were tested positive for the presence of methylhexanamine (Figure 1(2)).<br />
Due to its indirect sympathomimetic effects it was tested as locally administered<br />
vasocontrictor for the relief of hyperplasic nasal mucosa. Currently no pharmaceutical<br />
containing methylhexanamine is approved. Nowadays lots of preparations with<br />
methylhexanamine are marketed as supplements for slimming purposes. Increasing<br />
numbers of adverse analytical findings with methylhexanamine in doping control [3] indicate<br />
a growing market. Recently two fatal casualties in physical stress under the influence of<br />
methylhexanamine are investigated. This resulted in a recall of such products by the US<br />
FDA [4].<br />
Labelling of weight loss supplements uses several substitutes such as geranium root<br />
extract, geranium oil extract, Geranamin(e), 4-methyl-2-hexanamin(e), Forthan(e), 4methyl-2-hexylamin(e),<br />
2-hexanamine-4-methyl-, 2-methyl-4-methylhexan, Floradrene, 1,3dimethylamylamin(e),<br />
DMAA, 1,3-dimethylpentylamin(e), 2-amino-4-methylhexan(e), or<br />
pentylamine-1, 3-dimethyl- making the inference almost impossible for the consumers.<br />
Poster 125<br />
OH<br />
NO 2<br />
NO 2<br />
NH 2<br />
(1) (2)<br />
Figure 1: Chemical structure of ingredients of weight loss supplements<br />
(1) 2,4-dinitrophenol, DNP, (2) 4-methylhexan-2-amine<br />
References:<br />
1 Graham MR, Ryan P, Baker JS, Davies B, Thomas NE, Cooper SM, Evans P, Easmon S, Walker CJ,<br />
Cowan D, Kicman AT. Counterfeiting in performance- and image-enhancing drugs. Drug Test Anal,<br />
2009;1(3):135-42.
2 U.S. Dept Health & Human Services/Agency for Toxic Substances & Disease Registry. Hazardous<br />
Substances Data Bank HSDB. Toxicological Profile for Dinitrophenols, 1995, online at<br />
http://www.atsdr.cdc.gov/ToxProfiles/tp.asp?id=729&tid=132, accessed 16.05.<strong>2012</strong><br />
3 World Anti-Doping Agency. Adverse analytical findings reported by accredited laboratories, online at<br />
http://www.wada-ama.org/en/Science-Medicine/Anti-Doping-Laboratories/Laboratory-Statistics, accessed<br />
16.05.<strong>2012</strong><br />
4 DMAA News: FDA challenges marketing of DMAA products for lack of safety evidence, <strong>2012</strong>, online at<br />
http://fdarecalls.wordpress.com, accessed 16.05.<strong>2012</strong><br />
133<br />
Coating of collars via fluidised-bed technology: Influence of process<br />
parameters on coating homogeneity<br />
Wentzlaff, M. 1; Seidlitz, A. 1;Nagel, S. 1; Harder, C. 2; Schnittker, C. 3; Trip, E. 3; Bock, R. 3;<br />
Grabow, N. 4; Sternberg, K. 4;Weitschies, W. 1<br />
1Biopharmaceutics and Pharmaceutical Technology, University of <strong>Greifswald</strong>, Felix-Hausdorff-<br />
Str.3, 17487 <strong>Greifswald</strong>, Germany<br />
2Biotronik SE & Co.KG, Hartmannstr. 65, 91052 Erlangen, Germany<br />
3Biotronik SE & Co.KG, Woermannkehre 1, 12359 Berlin, Germany<br />
4Institute for Biomedical Engineering, University of Rostock, Friedrich-Barnewitz-Str.4, 18119<br />
Rostock, Germany<br />
Introduction<br />
Collars are polymeric tubes mounted on cardiac pacemaker leads containing an active<br />
substance to reduce inflammatory and proliferative reaction of myocardia after implantation<br />
[1,2]. Currently the active pharmaceutical ingredient is incorporated in collars during its<br />
manufacturing via injection moulding technology. However, this often results in high drug<br />
content variability. The fluidised-bed process is an established method used in galenics for<br />
coating of a large amount of small dosage forms with high uniformity.<br />
The purpose of this research was to adapt the process parameters of fluidized-bed process<br />
for coating of collars.<br />
Materials and Methods<br />
The fluidised-bed apparatus Mini-Glatt (Glatt GmbH, Germany) was used for coating of<br />
placebo-collars made of polyurethane. An aqueous dispersion of Eudragit RS ® 30D<br />
containing the water-soluble model substance fluorescein sodium was usedas the coating<br />
lacquer. Different coating processes parameters like spraying pressure, varied nozzle<br />
inserts and spray rate were investigated for optimisation coating homogeneity. Coating<br />
homogeneity was studied by scanning electron microscopy.<br />
Results<br />
By varying the spraying pressure it is possible to influence the surface morphology. While a<br />
spraying pressure of 0.7 bar created a rough surface morphology the surface became<br />
smoother when increasing the spraying pressure to 1.1 bar. A smoother surface was also<br />
created by reducing the nozzle orifice. It was shown that a nozzle orifice with a diameter of<br />
0.3 mm reduced the surface roughness compared to an orifice with a diameter of 0.5 mm.<br />
A reduction of the spray rate from 2.35 g/min to 0.77 g/min also resulted in a smoother<br />
surface.<br />
Conclusion<br />
Our findings provide a basis for coating of collars via fluidized-bed technology. By adapting<br />
the process parameters it was possible to produce coatings with desired morphologic<br />
characteristics.In order to obtain a sufficient coating thickness with a uniformdrug content,<br />
coating conditions have to be defined precisely. Therefore, further trials with optimization of<br />
coating dispersion concentrations and composition are necessary to ensure minimum<br />
mechanical stress acting on the collars and coatings.<br />
Acknowledgments: This project was partially funded by the Federal Ministry of Education and Research<br />
(BMBF) within the research project REMEDIS<br />
References:<br />
1. A. Wilson et al.: PACE1990, 13: 1876-1878.<br />
2. G. Brewer et al.: PACE 1988, 11: 1760-1769.<br />
134<br />
Evaluation of Interactions between Extractables of Elastomeric Closures and<br />
Protein Therapeutics<br />
Richter, C. 1 2, Lipke, U. 1, Zapf, T. 1, Lamprecht, A. 2, Lipperheide, C. 1<br />
1 Federal Institute for Drugs and Medical Devices, Kurt-Georg-Kiesinger-Allee 3, 53175 Bonn,<br />
Germany<br />
2 Institute of Pharmacy, University of Bonn, Gerhard-Domagk-Str.3, 53121 Bonn, Germany<br />
Parenteral liquid medicinal products are commonly offered in glass containers sealed with<br />
stoppers, which are made of elastomeric materials by vulcanization with the utilisation of<br />
various additives. These additives are predominantly small molecules. As they remain<br />
unlinked in the rubber material after the production process they are capable to migrate into<br />
the product formulation (“leachables”) during product shelf life and to react with the<br />
ingredients of the formulation. Particularly, medicinal products containing proteins as active<br />
ingredients are susceptible to react with substances released from the elastomeric material.<br />
In order to confirm compatibility between container and the product migration studies are<br />
requested prior to marketing authorisation. They should give evidence that quality, safety<br />
and efficacy of the protein containing medicinal product remains unaffected by leachables<br />
until end of shelf-life.<br />
The aim of the present study is to establish an appropriate method for evaluating the impact<br />
of leachables from elastomeric closures on protein stability without time-consuming<br />
migration studies.<br />
For the study various rubber stoppers were purchased from different vendors from the<br />
market. These rubber stoppers were extracted by applying worst case conditions<br />
(extraction with 2-propanol for 3 hours under reflux conditions) in order to receive the<br />
maximum amount of extractable substances (“extractables”) which is equivalent to the<br />
maximum amount of potential leachables. The extraction profiles of the different rubber<br />
stoppers were examined by RP-HPLC-DAD with a method, especially developed for the<br />
simultaneous analysis of polar and rather non-polar extractables. For further investigations,<br />
a chlorobutyl rubber stopper was chosen which showed the highest amount of extractables.<br />
Extractables stock solution was prepared as follows: 16 samples of the chlorobutyl rubber<br />
stopper were extracted in 200ml of 2-propanol for 6h under reflux conditions. After filtration<br />
(PE filter 0.45 µm) and solvent evaporation an oily residue was obtained. The residue was<br />
dissolved in 20ml of medium (20% (w/w) 1.2-propanediol and 0.5% (w/v) polysorbate 80 in<br />
water) for 1h in water bath (60-70°C). The resulting stock solution was filtrated and the<br />
amount of extractables was analyzed by RP-HPLC-DAD to confirm all extractables solved<br />
in the stock solution.<br />
The extractables stock solution was added to solutions of human serum albumin and<br />
different protein therapeutics and stored at 5°C. Samples were taken at several time points<br />
and examined by size-exclusion chromatography and capillary electrophoresis in order to<br />
detect physical and chemical alterations in protein structure as a result of the contact with<br />
the extractables.<br />
The proposed test approach turned out to be a suitable alternative to time consuming<br />
migration studies to investigate potential interactions between rubber stoppers and protein<br />
therapeutics.<br />
135<br />
In vitro dissolution testing of drug-eluting stents: Impact of implantation<br />
procedure<br />
Seidlitz, A. 1; Nagel, S. 1; Semmling, B. 1; Grabow, N. 2; Sternberg, K. 2; Weitschies, W. 1<br />
1 Institute of Pharmacy, EMA University of <strong>Greifswald</strong>, Felix-Hausdorff-Straße 3, 17487 <strong>Greifswald</strong>,<br />
Germany<br />
2 Institute for Biomedical Engineering, University of Rostock, Friedrich-Barnewitz-Straße 4, 18119<br />
Rostock, Germany<br />
Drug-eluting stents (DES) are drug/device combination products that are used to physically<br />
and pharmacologically prevent the renarrowing of a previously occluded natural passage.<br />
In the case of coronary DES the drug is intended to suppress intimal hyperplasia which<br />
often occurs after balloon angioplasty and stenting. Due to the placement of the DES in<br />
contact with the vessel wall, which is the site of action for the released drug, and the flowing<br />
blood, not only drug release but also its distribution between these compartments is of<br />
greatest importance. In vitro drug release and distribution can be examined using the<br />
vessel-simulating flow-through cell [1], a method adapted from the compendial flow-through<br />
cell (USP 4) for DES. In this setup the DES is implanted into a hydrogel compartment<br />
simulating the vessel wall and is perfused by PBS pH 7.4 representing the blood which is<br />
recirculated through the system (closed loop). For the studies presented here a<br />
haemostatic valve (Y-connector with rotating adapter and Tuohy-Borst valve) was added to<br />
the setup allowing for the placement of the DES in the gel via balloon catheter under flow<br />
conditions. Release and distribution of fluorescent model substances from coated stents<br />
(hydrophilic fluorescein sodium or hydrophobic triamterene embedded in a<br />
polymethacrylate blend, Eudragit RL/RS ratio 3/7) were examined using this setup. Stents<br />
were introduced via the haemostatic valve and the placement in the gel lumen under media<br />
flow (flow rate 35 mL/min, perfusion time prior to stent expansion 1 min) was compared to<br />
expansion into the air-filled gel lumen and subsequent perfusion. The experiments were<br />
terminated after different perfusion times up to 60 min and drug content in the perfusion<br />
media, in the hydrogel and the amount remaining in the stent coating were determined<br />
fluorimetrically. The results are presented in figure 1.<br />
Figure 1: Normalized amount of fluorescein sodium (A) and triamterene (B) in the<br />
compartment media, hydrogel or stent coating over time as a function of implantation<br />
procedure (without media present in the lumen or with media flowing through hydrogel<br />
during stent placement at 35 mL/min), n = 3 for each data point, mean ± SD.<br />
The initial distribution of released fluorescein sodium between the gel and media was<br />
greatly influenced by the implantation procedure. A much lower concentration in the<br />
hydrogel was observed when implanting under media perfusion of the lumen. This must be<br />
attributed to the fact that a certain amount of the fast released model substance is released<br />
exclusively into the media prior to contacting the hydrogel. When implanting without flow on<br />
the other hand, the initial release occurs solely into the gel for the first seconds. For<br />
triamterene, which is released much slower from the stent coating, this initial difference<br />
concerning the available acceptor compartments does not seem to have an impact on<br />
model substance release and distribution. Based on these results, the implantation<br />
procedure can be expected to have a great influence on release and distribution from fast<br />
releasing stent systems. The methods is also promising for other drug/device combination<br />
products applied in the vascular system such as drug-eluting balloons, which are intended<br />
to deliver their drug load to the vessel wall within very short contact times of approximately<br />
1 min.<br />
Acknowledgments: Financial support by the European Regional Development Fund (ERDF) and the<br />
European Social Fund (ESF) within the collaborative research between economy and science of the state<br />
Mecklenburg-Vorpommern is gratefully acknowledged. The authors thank Biotronik SE & Co. KG, B. Braun<br />
Melsungen AG and Rhoem GmbH for generously supplying raw materials.<br />
References:<br />
1. Seidlitz, A. et al.: J. Control. Release 2008, 130(1): 2-8.<br />
136<br />
A Novel Method to Characterize Screw-Elements in Pharmaceutical Twin-<br />
Screw-Extrusion<br />
Kiene, F. 1; Eeckman, L. 2; Thommes, M. 1<br />
1 Institut für Pharmazeutische Technologieund Biopharmazie, Heinrich-Heine-<strong>Universität</strong><br />
Düsseldorf, Germany<br />
2Department of PharmaceuticsLaboratory of Pharmaceutical Technology, Ghent University,<br />
Belgium<br />
Twin screw-extrusion is a commonly used technique in pharmaceutical applications. One of<br />
the relevant parameters in pharmaceutical extrusion is the screw configuration, because it<br />
can be adjusted to achieve desired product properties[1].The aim of this study was to<br />
characterize different types of conveying and kneading elements by their specific mechanic<br />
power and their transportation capacity, using high viscous silicon oil as model compound.<br />
For this, a part (4D) of the barrel of the lab scale extruder Micro 27GL-28D (Leistritz,<br />
Nuremberg, Germany) was separated with a front- and back-plate in order to investigate a<br />
smaller section of the barrel. A pressure sensor,I31-S-6-M-B01C-1-4-D (Gefran,<br />
126 Poster
Provagliod’Iseo Brescia, Italy),was attached to the front-plate. The torque was measured by<br />
a torque-gaugeT22/50 (HBM Messtechnik, Darmstadt, Germany)[2].The barrel load was set<br />
to 75% (Wacker ® AK 100000 Silicon oil, WackerChemie AG, Burghausen, Germany) and<br />
the rotation speed of the screws was varied from 50 to 200 rpm.<br />
Figure 1: Schematic drawing of the extruder setup; A: Back-plate with assembled screw; B:<br />
Isolated Extruder-Barrel<br />
Depending on the screw geometry different torqueand pressure were obtained and showed<br />
a linear correlation to rotation speed. This could be explained by the newtonian behaviour<br />
of silicon oil. Furthermore the specific mechanic power as a function of rotation speed (f)<br />
and torque (τ) for different loadings (m) was calculated as:<br />
. Equation 1<br />
Kneading blocks and conveying elements applied similar specific mechanic power to the<br />
material while differences within the subtypes can be observed. For example a high<br />
staggering angle of the kneading blocks led to lower values for the specific mechanic<br />
power. This could be explained by a higher transportation capacity of elements with lower<br />
staggering angle and can be determined by the pressure. Considering the conveying<br />
elements, a lower pitch resulted in a higher pressure andconsequently in a higher<br />
transportation capacity.<br />
In conclusion the setup was able to measure the specific mechanic power and the<br />
transportation capacity of different screw elements. Based on this, it will be possible to get<br />
deeper insight into the extrusion process.<br />
1. Djuric, D. and P. Kleinebudde, J Pharm Sci 2008, 97(11): 4934-4942.<br />
2. Köster, M. and M. Thommes, Chemical Engineering Journal 2010, 164(2-3): 371-375.<br />
137<br />
Rational Design of Nanoparticle Dissolution Testing for Parenterals: The<br />
Challenge of In Vitro Drug Release<br />
Janas,C. 1; Wacker, M. 1<br />
1Institute of Pharmaceutical Technology, Goethe-University, D-60438 Frankfurt, Germany<br />
Over the past decade significant research efforts have focused on a new generation of<br />
nanocarrier systems. These parenteral drug delivery devices are well-suited for the delivery<br />
of drugs with poor aqueous solubility, unfavourable body distribution and/or toxicity after<br />
intravenous injection. Due to the modified drug release of the active moieties from the<br />
nanocarrier matrix, physiology-based drug release models are needed in order to assure<br />
the quality and safety of these products. Moreover, they provide information about the drug<br />
release under in vivo conditions and are therefore an important tool in formulation<br />
development.<br />
There are several approaches which aimed to adopt standard methods from the European<br />
Pharmacopeia to the dissolution testing of parenteral nanoparticles and liposomes. Most of<br />
these studies showed a number of limitations due to the special requirements of the<br />
nanosized dosage form [1].<br />
In particular the isolation of the dissolved drug from particle bound drug molecules in the<br />
polymeric matrix is one major challenge in the development of an efficient dissolution<br />
method. Further aspects are the selection of dissolution media with regards to physiological<br />
parameters such as a solubility enhancement by human plasma, the colloid stability under<br />
in vivo conditions and the highly specific changes in surface properties by degradation and<br />
decoration with different plasma proteins. In order to ascertain anexperimental setup, that<br />
covers a wide range of different drug substances, the developed model has to be validated<br />
by using drugs with various physico-chemical properties.<br />
In our survey PLGA nanoparticles (NPs) loaded with model compounds intended for<br />
parenteral application are investigated in different release studies using dialysis-based<br />
methods. Buffers for dissolution media are prepared with various additives in order to not<br />
only bridge the gap between drug transport through the dialysis membrane but also to<br />
create conditions related to the in vivo situation.<br />
Initial experiments with the poorly soluble photosensitizer mTHPP were run with several<br />
membrane types to identify the transport rate of the API assuring that the dissolution of the<br />
drug is followed by direct passage through the membrane. The analysis shows acceptable<br />
transport rates in hydro-alcoholic solution and in complex with β-Methylcyclodextrin (0,1%<br />
in phosphate buffer saline, PBS). Based on these results the dissolution behaviour of<br />
mTHPP incorporated into PLGA NPs in PBS pH 7,2 is studied and compared with<br />
dissolution in buffer solutions composed of increasing amounts of foetal calf serum (FCS)<br />
and cyclodextrins (CD) respectively [2].<br />
Furthermore the colloid stability of investigated nanocarriers in phosphate buffer in the<br />
presence and absence of FCS is analysed as a function of time. There was no effect on<br />
particle size and polydispersity index (measured with photon correlation spectroscopy)<br />
during 24 h of incubation at 37°C which might predict no/delayed aggregation in vivo. The<br />
results of dissolution testing of mTHPP demonstrate a release of 100 % in both media (see<br />
Fig.1) and hence the sustained release of the NP formulation depending on media<br />
composition is investigatedin our experiments.<br />
Fig.1: Particle size, polydispersity index, and dissolution of mTHPPNP (n=3)<br />
Acknowledgments: Biolitec research GmbH, Dr. Arno Wiehe and Dr. Volker Albrecht<br />
References:<br />
1. D Souza S, DeLuca P,:Pharm. Res.2006, 23 (3): 460-474.<br />
2. DesRoches M-C et al.: J.Photochem.Photobiol. B. 2006, 85 (1): 56-64.<br />
138<br />
Chemically selective imaging of structured surfaces – a novel complementary<br />
analytical approach for pharmaceutical investigations<br />
Kann, B. 1;Gantzsch, S. 1; Lehr, C.M. 1 2 3; Schaefer, U. F. 1; Windbergs, M. 1 2 3<br />
1Saarland University, Department of Biopharmaceutics and Pharmaceutical Technology, Campus<br />
A4.1, 66123 Saarbruecken, Germany<br />
2PharmBioTec GmbH, Campus C2.2, 66123 Saarbruecken, Germany<br />
3Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123 Saarbruecken,<br />
Germany<br />
Confocal Raman microscopy is an upcoming analytical technique in the field of<br />
pharmaceutics. It is well suited for the contactless and chemically selective characterization<br />
of drug delivery systems and in vitro test systems.However, confocal Raman microscopy<br />
requires a smooth sample surface as spectra are exclusively recorded from the focal plane.<br />
As pharmaceutical samples often exhibit a structured surface such as lyophilisates and<br />
porous materials, confocal Raman analysis is impaired.<br />
We overcome the pitfalls of a confocal setup by combining Raman microscopy with optical<br />
profilometry as complementary analytical tools gaining a full-fledged characterization of the<br />
sample surface independent of its structure.<br />
To evaluate our novel approach, we analyze a protein-loaded lyophilisate. The therapeutic<br />
efficiency of proteins highly depends on their physical structure and molecular<br />
conformation, thus changes can have severe side effects on drug dosing for the patient.<br />
However, maintaining their therapeutically active conformation throughout manufacturing<br />
and within the final dosage form during shelf-life is still a major challenge. We freeze dry an<br />
aqueous solution containing a mixture of native and heat denaturated bovine serum<br />
albumin (BSA) for 48 h with a final drying step for 1 h gaining a solid lyophilisate. Optical<br />
profilometry of the surface is performed prior to Raman analysis (WITec alpha 300/500) as<br />
visualized in Figure 1a. As structural changes within the protein get along with a change in<br />
their Raman spectra, Raman microscopy is a valuable method to detect protein<br />
deconformation. The shift of the amide I peak in the Raman spectrum of the protein<br />
occurring between 1500-1800 cm-1 is used to discriminate between active and heat<br />
denaturated BSA within the dosage form. By overlaying the topography map with the<br />
respective Raman spectra at each measuring point we gain a spatially resolved false color<br />
image of the lyophilisate indicating each protein conformation in a different color.<br />
Furthermore, we investigate a Transwell-based permeation model. Polymeric membranes<br />
provide a suitable alternative to cell culture models overcoming the drawbacks of timeconsuming<br />
preparation and high expenses. For simulation of the intestinal mucosa, the<br />
apical side of the membrane is coated with lipids. Due to its non-destructive and chemically<br />
selective nature, Raman microscopy is a well suited technique for the analysis of chemical<br />
composition and three dimensional morphology of these systems.Raman spectra are<br />
recorded and subsequently converted into chemically resolved images (WITec alpha<br />
300/500). A comprehensive physical characterization of the transwell-based intestinal<br />
model is performed of the pure membrane as well as of the coated membrane before and<br />
after experiments. The pore morphology of the pure membrane is investigated with Raman<br />
spectroscopy in xz and xy-direction resulting in virtual cross-section images and three<br />
dimensional presentations (Figure 1b), respectively. After the coating, the pore morphology<br />
is verified again. Coating itself is achieved by centrifuging the lipid onto the membrane and<br />
a subsequent drying step. To monitor the coating formation on the membrane, a<br />
topography map is created after each coating step followed by subsequent Raman<br />
analysis.<br />
Figure 1. a) Topography map of a lyophilisate. b) Three dimensional image of the pore<br />
morphology of a polymeric membrane.<br />
We successfully introduce chemically selective imaging of structured surfaces for<br />
pharmaceutical applications by combining confocal Raman microscopy and optical<br />
profilometry as complementary analytical techniques.<br />
139<br />
Custom-tailoring microsystems for production intensification of lipid based<br />
colloidal drug delivery systems<br />
Finke, J. H. 1, Niemann, S. 1,Richter, C. 2, Gothsch, T. 3, Kwade, A. 3, Büttgenbach, S. 2, Müller-<br />
Goymann, C. C. 2<br />
1 Technische <strong>Universität</strong> Braunschweig, Institut für Pharmazeutische Technologie, Mendelssohnstr.<br />
1, 38106 Braunschweig, Germany<br />
2 Technische <strong>Universität</strong> Braunschweig, Institut für Mikrotechnik, 3 Technische <strong>Universität</strong><br />
Braunschweig, Institut für Partikeltechnik<br />
The manufacturing of colloidal drug delivery systems poses big challenges to the<br />
Poster 127
production process. It comprises a variety of different process steps, especially if solid lipid<br />
nanoparticles (SLN) are produced. Commonly, the whole process is broken down to<br />
separate process steps in different devices and performed batch-wise. In terms of process<br />
intensification, a change towards substantially smaller, cleaner and more efficient<br />
equipment or methods is required. Therefore, a continuously working microsystem (MS)<br />
approach was applied for SLN manufacturing. It comprises: (i) dispersion of solid active in<br />
the lipid phase, (ii) initial formation of lipid droplets in the aqueous phase, (iii)<br />
homogenization of these to colloidal, narrowly distributed droplets and (iv) crystallization of<br />
the latter in a defined way (Fig. 1). Additionally, this approach reduces educt batch sizes<br />
and product loss, thus facilitating its application as an instrument for formulation screening.<br />
In this study, the progress of process intensification is presented with regard to the<br />
homogenization step.<br />
MSs for high pressure homogenization were primarily fabricated in silicon/glass [1].<br />
Different geometric design approaches were tested and ranked regarding their droplet<br />
breakup efficiency in emulsions: straight channels < Z-channels < Y-channel ≈ orifice.<br />
Furthermore, various minor structural changes within the main geometric principles were<br />
analysed [2].<br />
According to silicone MS results, orifice channels fabricated in stainless steel [3] were<br />
investigated in detail as the most promising design principle. In general, steel MS supply<br />
lower fragility, higher process pressures and elevated temperatures, as necessary for SLN<br />
production. To elucidate the design parameters which influence the colloidal dispersion<br />
efficiency, orifice widths, number of consecutive orifices, and the distance between orifices<br />
were varied. It was shown that double orifices yield lower particle sizes compared to single<br />
orifices. Nevertheless, triple orifices do not provide further decrease in particle size, but<br />
rank between double and single orifices. These phenomena are attributed to the<br />
development of counter pressure towards the first orifice by a second one causing a more<br />
efficient breakup, on the one hand. On the other hand, the pressure drop is distributed over<br />
a higher number of orifices reducing the stress intensity at each orifice and, thus, the<br />
overall efficiency in case of three orifices. A longer distance between orifices appears more<br />
efficient in droplet breakup likely due to a reduction of counter pressure effects because a<br />
stable flow pattern is not able to recover due to the proximity of two orifices at a low<br />
distance (300 µm, equal to orifice length). For the combination of 80 and 120 µm wide<br />
orifices in one MS, it has been demonstrated more efficient if the smaller orifice is exposed<br />
to the product flow first, yielding the higher pressure drop over the size determining, first<br />
distinct energy input. These findings were confirmed by applying a pressure regulating<br />
valve, facilitating the arbitrary variation of counter pressure within the experimental setup. It<br />
was proved that a moderate counter pressure drastically decreases resulting particle sizes<br />
for a single orifice MS. In double orifices no further decrease in particle size was detected<br />
due to external counter pressure.<br />
Regarding the producibility of SLN, it was shown that particles can be produced in custom<br />
tailored MS with comparable or even better properties (particle size and distribution, melting<br />
behaviour) within one cycle instead of 20 cycles in a conventional homogenization setup.<br />
Conclusively, the effectiveness of MSs for high pressure homogenization in a continuously<br />
perfused microsystem for SLN production was systematically improved and crucial<br />
parameters were identified. This demonstrates a successful process intensification<br />
compared to conventional colloidal dispersion production.<br />
Fig. 1. Schematic representation of SLN production process in an microsystem approach<br />
Acknowledgments: The present study was performed in the context of the Research Group 856<br />
funded by the German Research Foundation (DFG). One of the authors (S. B.) gratefully<br />
acknowledges the financial support of the Volkswagen Foundation.<br />
References:<br />
1. Lesche, C., Büttgenbach, S.: MST Kongress 2009<br />
2. Finke, J. H. et al.: 8th CESPT, Austria, Graz 2010<br />
3. Richter, C., Krah, T., Büttgenbach, S.: Microsyst. Technol. <strong>2012</strong>: in press<br />
140<br />
Air drying of drug nanosuspensions as a simple small scale process for<br />
screening and feasibility studies<br />
Laabs, F.; Bunjes, H.<br />
Institute of Pharmaceutical Technology, Mendelssohnstraße 1, 38106 Braunschweig, Germany<br />
The conversion of poorly water soluble pharmaceutical drug substances into<br />
nanosuspensions is a common approach to overcome their poor dissolution and absorption<br />
properties. However, solid dosage forms are often preferred with regard to improved<br />
physicochemical stability and better patient convenience. Established unit operations like<br />
spray drying or freeze drying can be applied to convert nanosuspensions into solid dosage<br />
forms. However, preservation of the particle size during the drying process can be<br />
challenging [1]. Furthermore, the conversion step is often time consuming (e. g. freeze<br />
drying) and/or only a single sample can be processed at a time (e. g. spray drying). This is<br />
especially impedimental for screening studies where processing of many samples is<br />
required.<br />
In order to circumvent the above mentioned issues, a simple small scale air drying process<br />
was established. In this study, the applicability of the air drying process on screening and<br />
feasibility studies was investigated. In particular, the influence of the type and concentration<br />
of different carrier materials and the transferability of the air drying results onto those<br />
obtained by spray drying were assessed for two different API nanosuspensions.<br />
A naproxen nanosuspension (5 %) stabilized with Kollidon VA64 (2.5 %) and a griseofulvin<br />
nanosuspension (5 %) stabilized with Kollidon VA64 (2.5 %) and sodium dodecyl sulfate<br />
(0.25 %) were prepared by wet media milling for 4 h using a stirred media mill (Dispermat<br />
SL 5 C nano). Air drying of the nanosuspensions was performed on a polystyrene well plate<br />
or using an evaporator (EVA EC S) and samples were compared to those obtained by<br />
spray drying (Büchi B-191). After processing, the dried samples were resuspended and the<br />
particle size was determined via photon correlation spectroscopy.<br />
As exemplified in Figure 1 and 2, air drying of the nanosuspensions could successfully be<br />
performed and the results were similar to those obtained by spray drying. As already<br />
observed in previous spray drying studies [2], drying of the griseofulvin nanosuspension<br />
was shown to be less critical compared to the naproxen nanosuspension.<br />
In conclusion, the air drying process seems promising for screening and feasibility studies<br />
and can provide first estimates of results that can be achieved using other drying<br />
processes.<br />
Figure 1: Particle size values<br />
(PCS) of a naproxen<br />
nanosuspension before and after<br />
spray and air drying with Kollidon<br />
VA64 as carrier material at<br />
different carrier:API ratios<br />
Figure 2: Particle size values (PCS)<br />
of a griseofulvin nanosuspension<br />
before and after spray and air<br />
drying with different carrier<br />
materials at a carrier:API ratio of<br />
0.5:1<br />
Acknowledgments: We would like to thank A. Bitterlich and Prof. Dr. A. Kwade (TU Braunschweig, Institute<br />
for Particle Technology) for the cooperation concerning the development and the production of the<br />
nanosuspensions.<br />
References:<br />
Van Eerdenbrugh, B. et al.: Eur. J. Pharm. Sci. 2008, 35(1-2): 127-135<br />
Laabs, F., Komoß, C., Bunjes, H.: 8 th PBP World Meeting <strong>2012</strong>, Istanbul, Turkey<br />
141<br />
Tablet disintegration – design of an apparatus to measure the disintegration<br />
force and water uptake of tablets simultaneously<br />
Quodbach J1, Kleinebudde P1 1 Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitaetsstrasse<br />
1, 40225 Duesseldorf, Germany<br />
The underlying mechanisms of action of tablet disintegrants are still mostly unknown. Even<br />
though tablet disintegration is often the first step in the bioavailability cascade, only few<br />
attempts have been made recently to understand how disintegration takes place or is<br />
influenced [1, 2, 3]. An apparatus was built to gain a more profound knowledge about the<br />
disintegration force kinetics, water uptake kinetics, and differences of tablets containing<br />
various super disintegrants. The measuring principle is based on a design from the<br />
University of Pavia [4] and was altered to allow for a higher sample throughput and more<br />
precise results. The experimental setup is schematically shown in Figure .<br />
One beaker is placed on a balance (Sartorius CP224s) another one is placed under the<br />
measuring probe of a Texture Analyser (TA.XTplus). The beakers are connected with a<br />
tube allowing water to flow from one beaker to the other. A tablet holder made of aluminium<br />
with 19 holes (r = 0.5 mm) is placed in the beaker on the Texture Analyser. An additional<br />
filter paper is placed on the tablet holder. The tablet itself is attached to the measuring<br />
probe with adhesive tape. Water is filled into the beakers until the tablet holder is in contact<br />
with the surface. Then the measuring probe with the tablets is slowly moved downwards<br />
until a trigger threshold is reached (0.25 N) when the tablet touches the tablet holder and<br />
the data acquisition begins. The Texture Analyser registers the force the tablet exerts onto<br />
the measuring probe, whereas the balance records the water uptake of the tablets as the<br />
negative mass change. Adhesive tape is fixed around the circumference of the tablet to<br />
ensure that the complete amount of force is transmitted to the Texture Analyser.<br />
As example, three measurements are presented in Figure . The measured tablets consist<br />
of 2 % Kyron T-314 (Corel Pharma Chem), a polacrilin potassium ion-exchange resin also<br />
used as super disintegrant, 97.5 % dibasic calcium phosphate (Chemische Fabrik<br />
Budenheim KG) and 0.5 % magnesium stearate (Merck AG). Initially, little water uptake<br />
results in a strong force development. Afterwards, the water uptake and force behave<br />
almost linear, which is followed by a decrease of absolute force. This can be due to a<br />
collapse of the internal structure after the maximum force is reached.<br />
Further improvements of the apparatus are still necessary to get a more precise resolution<br />
of the mass change, especially at the beginning. Yet, it proves to be an easy to use tool<br />
which allows insights into the relationship between water uptake and force development but<br />
also into kinetic processes during tablet disintegration.<br />
References:<br />
1. Desai P M, Liew C V, Heng P W S, J. Pharm. Sci. <strong>2012</strong>, 101(6): 2155-2164.<br />
2. Krausbauer E, et. al. J. Pharm. Sci. 2007, 97(1): 529-541.<br />
3. Massimo G, et. al.: Pharm. Dev. Technol. 2000, 5(2): 163-169.<br />
4. Caramella C: Pharm. Tech. Int. September 1990, (9):30-42.<br />
128 Poster<br />
z-average diame er (nm)<br />
1950<br />
1900<br />
1850<br />
250<br />
200<br />
150<br />
100<br />
50<br />
0<br />
be ore drying<br />
after spray drying<br />
after air drying (well plate)<br />
PdI<br />
10 1 5 1 2:1 1:1 0.5:1 0 1<br />
carrier:API ra io<br />
1.0<br />
0.9<br />
0.35<br />
0.30<br />
0.25<br />
0.20<br />
0.15<br />
0.10<br />
0.05<br />
0.00<br />
PdI<br />
z average d ameter (nm)<br />
300<br />
250<br />
200<br />
150<br />
100<br />
50<br />
0<br />
before drying<br />
after sp ay drying<br />
after air drying (well plate)<br />
after air drying (evaporator)<br />
PdI<br />
mann tol lac ose KVA 6 K30<br />
carr er material<br />
Figure 1: Experimental setup Figure 2: Water uptake versus force<br />
development<br />
0. 0<br />
0.35<br />
0.30<br />
0.25<br />
0.20<br />
0.15<br />
0.10<br />
0.05<br />
0.00<br />
PdI
142<br />
Selection of solvent type affects nanocrystal particle size during precipitation<br />
combined with high pressure homogenization process<br />
Sinha, B. 1; Möschwitzer, J. P. 2; Müller, R. H. 1<br />
1 Institute of Pharmacy, Dept. Pharmaceutics, Biopharmaceutics&Nutricosmetics, Freie University<br />
of Berlin, Berlin, 12169, Germany;<br />
2 Pharmaceutical Development, Abbott GmbH &Co.KG, Ludwigshafen, 67061, Germany<br />
Precipitation combined with high pressure homogenization could be a feasible technique for<br />
production of small size drug nanocrystal. Solvent type plays a critical role in nucleation,<br />
stabilization and particle growth during precipitation process. The aim of this study was to<br />
investigate how various solvents affect the final particle size of drug nanocrystal during this<br />
combination technique.<br />
Precipitation step was combined with high pressure homogenization process to prepare<br />
drug nanocrystal in this study. Ibuprofen (IBP, BASF, Germany) and resveratrol<br />
(E.denkFineChemie GmbH, Germany) were used as two model compounds. Solvent phase<br />
(S) was prepared by solubilizing drug in a water miscible solvent such as alcohol (Ethanol,<br />
Methanol, Isopropanol), acetone, tertrahydrofuran (THF), dimethylformamide (DMF),<br />
dimethylsulfoxide (DMSO) and ethylacetate. Aqueous solution of SDS (0.2%) was mixed<br />
with either 0.1% HPMC (viscosity 5 cps) or with 0.5% PVP (K40) to constitute the antisolvent<br />
phase (AS) for Ibuprofen and Resveratrol, respectively. Using a needle and a pump<br />
system, S phase was injected in the AS phase just before homogenization zone in<br />
Emulsiflex C5 (Avestin Europe GmbH, Mannheim, Germany) operated at 1000-1200 bar.<br />
Particle size of the nanocrystal was measured by laser diffractometry using Mastersizer<br />
2000 (Malvern Instruments, UK). Solvents were rank ordered based on the minimum<br />
particle size obtained using a solvent.<br />
Depending on the solvent type, particle size of drug nanocrystals was found to change.<br />
When isopropanol was used for making ibuprofen nanocrystal, the smallest particle size<br />
obtained was 306 nm [d(0.5)]. It was only possible to reduce particle size further to less<br />
than 200 nm when solvent was changed to acetone, methanol, ethanol, THF or DMSO.<br />
Mean particle size was increased when ethyl acetate and DMF was used. Therefore, the<br />
solvent preference rank order for preparing IBP nanocrystal was found to be<br />
THF~Ethanol>Methanol~DMSO>Acetone>IPA>Ethyl acetate. On the other hand, use of<br />
DMSO produced the smallest size resveratrol nanocrystal, i.e. 153 nm [LD (50%)]. Hence,<br />
the solvent preference rank order for resveratrol was found to be<br />
DMSO~Ethanol~IPA~Acetone>Methanol>DMF>THF. Other factors such as<br />
solvent/antisolvent ratio, drug concentration in the solvent phase and stabilizer composition<br />
was also found to be critical to control the particle size.<br />
Beside other factors, selection of the right solvent for precipitation study is necessary to<br />
achieve smaller particle size. Further optimization of other parameters leads to the smallest<br />
size drug nanocrystal in precipitation coupled with homogenization process.<br />
143<br />
Identification of factors affecting the sorption isotherms of spray dried binary<br />
carbohydrate mixtures containing maltodextrin and sucrose<br />
Tackenberg, M. W. 1 2; Horvat, M. 1;Thommes, M. 2; Kleinebudde, P. 2; Schuchmann, H. P. 1<br />
1 KIT – Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Food<br />
Process Engineering (LVT), Kaiserstr. 12, 76131 Karlsruhe<br />
2 Heinrich-Heine-University, Institute of Pharmaceutics and Biopharmaceutics, Universitaetsstr. 1,<br />
40225 Duesseldorf, Germany<br />
Maltodextrins, amorphous starch degradation products, are widely used as excipients for<br />
solid drug formulations. The different types of Maltodextrins are characterized by their<br />
dextrose equivalent (DE), which is correlated to the average molecular size (DE 0: ~ starch;<br />
DE 100: glucose). The DE of common maltodextrins varies between 2 and 20.<br />
Furthermore, maltodextrins can be distinguished by their ratio of amylose to amylopectin<br />
content and carbohydrate composition with respect to the origin. These differences can be<br />
used to tailor-made new matrix formulations with outstanding properties for drug<br />
administration like inclusion compounds and storage stability. This study elucidates the<br />
effect of type and content of the maltodextrins on the sorption isotherms of spray dried<br />
binary carbohydrate matrices.<br />
Various maltodextrins (Glucidex ® with DE 6, Waxy Maize based; Glucidex ® with DE 12 and<br />
17, Maize based, Roquette Frères; Kleptose ® Linecaps, Pea based with DE 12 and 17,<br />
Roquette Frères) were coprocessed with sucrose (Compri-Zucker O, Südzucker AG) using<br />
spray drying (Büchi Minispray Dryer B-191, Büchi Labortechnik AG). Afterwards the<br />
sorption isotherms were determined at 25.0 °C (SPS 11, PMT Analytical GmbH & Co. KG).<br />
Therefore 0.5 g sample were dried to a water activity (aW) < 0.005, followed by a increasing<br />
of the water activity in steps of 0.1 to aW 0.9. The obtained experimental data points<br />
between aW 0.1 and 0.8 were fitted with the Guggenheim-Anderson-de Boer Equation<br />
(GAB) (1). For all isotherms R² > 0.99 was observed.<br />
m����K C a� m=<br />
(1−Ka� )(1 − K a� +CKa�) (1)<br />
• m = water content; aW = water activity<br />
• C = Guggenheim constant; K = correcting factor<br />
• mmono = water content of the saturated molecule monolayer of the surface<br />
The sucrose content increases the water adsorption at aW ≥ 0.4, as shown for the<br />
Glucidex ® DE 17 formulations (Fig. 1). This could be correlated to the highly hygroscopic<br />
amorphous sucrose molecules. At aW ≤ 0.3 the water uptake decreases with increasing<br />
sucrose content, which could be caused by the dominant properties of the high molecular<br />
weight fraction at low aW [1].<br />
Furthermore an effect of the DE to the water adsorption was found, as shown for the<br />
Glucidex ® DE 6 and 17 formulations with 20 % sucrose (Fig. 2). At low aW (< 0.5) the water<br />
uptake decreases with increasing DE. At aW > 0.5 it increases with increasing DE. This<br />
could be explained with the dominant properties of the high molecular weight compounds at<br />
low aW and the increasing lower molecular weight compounds (higher DE) at higher aW, too.<br />
Finally, an influence of the starch source (Maize or Pea) on the sorption isotherm of the<br />
maltodextrin sucrose mixtures was not detected, as shown for the Glucidex ® DE 17 and<br />
Kleptose ® Linecaps (KL) DE 17 (Fig. 2).<br />
Fig. 1: Influence of sucrose content on<br />
sorption behaviour.<br />
Fig. 2: Influence of DE and starch basis on<br />
sorption behaviour.<br />
From the results, it can be assumed that the average molecular size is the most important<br />
factor regarding the sorption behaviour of carbohydrates. Hence, this behaviour can be<br />
customized by a combination of maltodextrin and sucrose.<br />
Acknowledgments: Roquette Frères; Südzucker AG, Max-Buchner-Forschungsstiftung; AIF-ZIM Project<br />
KF2256805<br />
References:<br />
1. Radosta, S. et al.: Starch/Stärke 1989, 41(10): 395-401<br />
144<br />
Improved test on emulsifying properties of lanolin forcommunity pharmacies<br />
Koeck J; Kortmann C; Pein M; Breitkreutz J<br />
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitaetsstraße 1,<br />
40225 Duesseldorf, Germany<br />
Lanolin is a commonly used ointment base in dermal preparations. However, several<br />
problems are known like its sensitization potential, discussed controversially [1, 2],<br />
insufficient emulsification and stability properties [3]. As of its natural origin the emulsifying<br />
properties (EP) change depending on batch and age.<br />
For evaluating EP there have been two tests established: Addition of water and coldemulsifying<br />
according to German Pharmacopoeia [4] and titration, which was introduced by<br />
Reimannusing a solution containing Octoxynol-10 [5]. The first test only differentiates<br />
between “pass” and “fail”, the latter provides the possibility to check the EP more precisely<br />
and faster.As Octoxynol-10 is rarely used in community pharmacies, there is a need to<br />
improve the Reimann method.<br />
Lanolin of two qualities was used: Eucerinumanhydricum (EA) (Beiersdorf, D-<br />
Hamburg,Release date: 2004) and UnguentumAlcoholumLanae (UAL) (Caesar &Loretz, D-<br />
Hilden, Release date: 2010).<br />
Test samples were prepared of cold-emulsifying with stirring by hand 10 g of lanolin with<br />
15 g of water.<br />
Titration solutions (TS) were prepared, containing 4 g Methylene Blue, 1 g Octoxynol-10<br />
and water to 100 ml as prescribed by Reimann (TS1). The other test was performed<br />
exchanging Octoxynol-10 (HLB=13.6, Triton® X-100, Roth, D-Karlsruhe) byPolysorbate 80<br />
(HLB=15.0, Casear&Loretz, D-Hilden) (TS2).<br />
After titration in small steps (approximately 0.1 ml) test sample was stirred by hand until a<br />
blue coloured homogenous semisolid substance resulted. Endpoint was specified when TS<br />
was not further incorporable when stirring.<br />
It was possible to formulate a titration solution with Polysorbate 80 (TS2) which can be<br />
used with comparable results to Octoxynol-10 (Amount of TS to instability of aqueous<br />
lanolin (UAL): TS1 0.9 ml and TS2 1.0 ml). With this optimized method it is possible to use<br />
the proposed test on EP in community pharmacies with conventional and available<br />
excipients.<br />
Further, it was possible to differentiate between lanolin of different qualities and ages (EA<br />
and UAL) with TS1, according to its EP(Amount of TS1 to instability of EA: 0.4 ml and UAL:<br />
0.9 ml). Reimann specified an amount of at least 0.5 ml TS1 to use lanolin for dermal<br />
preparations.<br />
Concluding, it becomes feasible for community pharmacies to easily test EP of lanolin<br />
before preparing drug formulations. Other water-in-oil emulsions will be investigated by this<br />
test in future.<br />
References<br />
1. Kligman, A.M.: Contact Dermatitis.1998, 39(3): 103-107.<br />
2. Aberer, W.: Dermatologie in Beruf und Umwelt.2006, 54(4): 135-139.<br />
3. Eifler-Bollen, R.: Pharmazeutische Zeitung.2002, 147(17): 1898.<br />
4. Deutsches Arzneibuch: Wollwachsalkoholsalbe(Deutscher Apotheker Verlag).1999.<br />
5. Reimann, H.: PharmazeutischeZeitung.1993, 138(2): 114-115.<br />
145<br />
Tailor-made production of quercetin and rutin nanocrystals for investigating<br />
the correlation between the size and effect to cell<br />
Chen,R. 1, Wszelaki, N. 2, Melzig, M. F. 2, Müller, R. H. 1, Keck, C. M. 1 3<br />
1Institute of Pharmacy, Free University of Berlin, Kelchstrasse 31, 12169, Berlin<br />
2Institute of Pharmacy, Free University of Berlin, Königin-Luise-Strasse 2+4, 14195 Berlin<br />
3Applied Pharmacy Division,University of Applied Sciences Kaiserslautern, Campus Pirmasens,<br />
Carl-Schurz-Strasse 10-16, 66953 Pirmasens<br />
Flavonoids are believed having potential to protect neuro cell from neurogenerative<br />
damages caused by endogenous toxins. However, most flavonoids are poorly soluble,<br />
which lead to a low bioavailability and/or cell uptake when they are applied in aqueous<br />
solution form. To formulate the flavonoids into nanocrystals is an effective method to<br />
increase the solubility and to enable the cell uptake. As the solubility enhancing effect and<br />
the ability to be taken up by cells is size dependent, tailor-made production of nanocrystals<br />
is the first required step for investigating the correlation between size and effect to cell in<br />
future.<br />
In this study, quercetin and rutin nanocrystals were produced by high pressure<br />
homogenization (HPH) (LAB 40, APV Deutschland) in sterile condition. For the further<br />
investigation about the influence of size on the performance of nanocrystals in cell culture,<br />
a series of nanocrystals with different sizes were tailor-made with Poloxamer 188 as a nontoxic<br />
surfactant (0.2%, w/w) and glycerol as a osmotic adjuster (2.5%, w/w) by applying<br />
different HPH pressure (up to 1500 bar) and cycles (up to 20 cycles) and choosing different<br />
active concentrations (2%, 0.2%, 0.02%). Particles size was characterized by photon<br />
Poster 129
correlation spectroscopy (PCS), laser diffractometry (LD) and light microscopy. Zetapotential<br />
and short-term stability were examined. For correct size analysis the<br />
measurement method of PCS was optimized by choosing different measuring cuvettes and<br />
using different dilution media and sample concentrations. The ways to adding glycerol in<br />
nanosuspension was also compared.<br />
Via controlling production condition, a series of nanocrystals (from 172 nm to 1500 nm)<br />
were produced. Results showed that an increase in cycles of HPH could lead to a<br />
significant smaller size for quercetin but not for rutin. Lower active concentration in<br />
quercetin had no influence on size but in rutin it provided a smaller size. Zeta-potential in<br />
50μS/cm NaCl solution and original dispersion medium of quercetin nanocrystals were both<br />
above 30 mV, which indicated a better stability compared to rutin nanocrystals holding a<br />
zeta-potential of around 25 mV. The short-term stability also confirmed that the quercetin<br />
nanocrystals in 4 oC had a good stability. Through investigating PCS cuvettes and dilution<br />
methods, measurement condition was fixed as using a low volume cuvette, active powder<br />
saturated diluting medium and 0.02% measuring concentration. Adding glycerol in<br />
suspension before HPH gave a slightly smaller size compared to HPH without glycerol.<br />
However, for precise controlling the concentration of glycerol in final product, it was added<br />
in after HPH.<br />
In conclusion, different sizes of quercetin and rutin nanocrystals for cell culture test can be<br />
tailor-made by applying different pressures and cycles as well as using different active<br />
concentrations. The preliminary requirement for further cell culture research was achieved.<br />
Acknowledgements: The authors would like to thank China Scholarship Council for financial support.<br />
146<br />
Hydrogels for drug delivery: influence of macromer branching on<br />
characteristics of poly(ethylene glycol) based hydrogels<br />
Kirchhof, S. 1, Brandl F. 1, Hammer N. 1, Meßmann V. 1, Teßmar J. 1 and Göpferich A. 1<br />
1 University of Regensburg, Department of Pharmaceutical Technology, <strong>Universität</strong>sstr.31, 93053<br />
Regensburg, Germany<br />
Poly(ethylene glycol) (PEG) based hydrogels are interesting materials for a variety of<br />
biomedical applications. In the field of controlled drug delivery, the use of PEG gels has<br />
been promoted due to their excellent biocompatibility. Nevertheless, most of the existing<br />
cross-linking mechanisms are associated with disadvantages, such as the formation of<br />
potentially harmful radicals. A relatively unknown cross-linking mechanism for hydrogels is<br />
the Diels-Alder click reaction. Previously, we have shown the suitability of this reaction for<br />
the formation of suitable PEG based hydrogels. Here, we report the influences of the<br />
macromer branching factor on the characteristics of the resulting hydrogels.<br />
Gelation time / min<br />
250<br />
200<br />
150<br />
100<br />
50<br />
0<br />
4armPEG-hydrogel<br />
8armPEG-hydrogel<br />
5 10 15<br />
Total polymer concentration / w/v<br />
Figure 1: Gelation times of different PEGhydrogels;<br />
gelation time depends on<br />
branching factor and total polymer<br />
concentration<br />
Rea tive mass increase Mt/M0<br />
18<br />
16<br />
14<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
15% 4armPEG-hydrogel<br />
5% 8armPEG-hydrogel<br />
0<br />
0 1 2 3 4 15 16 17 18 19 20 21<br />
Time / days<br />
Figure 2: Swelling behaviour of different<br />
PEG-hydrogels; 4armPEG-hydrogels were<br />
dissolved within 4 days, whereas<br />
8armPEG-hydrogels swelled on a lower<br />
level even after 3 weeks<br />
PEG with branching factors of four and eight (4armPEG10k-OH and 8armPEG10k-OH)<br />
were used as raw materials. 4armPEG10k-furan, 8armPEG10k-furan and 4armPEG10kmaleimide<br />
were synthesized according to previously established protocols [1]. For the<br />
synthesis of 8armPEG10k-maleimide, 8armPEG10k-amine and Nmethoxycarbonylmaleimide<br />
were reacted to yield the desired product. All compounds were<br />
characterized by 1H-NMR spectroscopy. For hydrogel preperation, defined amounts of<br />
4armPEG10k-maleimide and 4armPEG10k-furan or 8armPEG10k-maleimide and<br />
8armPEG10k-furan were separately dissolved in Millipore water. The two precursor<br />
solutions were mixed to initiate gel formation. Gelation kinetics and gel strength were<br />
studied by performing oscillatory shear experiments on a TA Instruments AR 2000<br />
rheometer with parallel plate geometry. For swelling studies, gel cylinders (Ø 7 mm) were<br />
incubated with 10 ml of phosphate buffer solution (pH 7.4).<br />
Rheological studies showed decreasing gelation times with increasing polymer<br />
concentrations (Fig. 1). This was observed for both degrees of branching. Moreover, the<br />
gelation time dramatically decreased by increasing the branching factor from four to eight.<br />
This is explained by the higher cross-linking density of the resulting hydrogels. 4armPEGhydrogels<br />
(15 % polymer) showed very high swelling and dissolved within one week (Fig.<br />
2). This could be explained by the reversibility of the Diels-Alder reaction. In contrast to<br />
that, 8armPEG-hydrogels showed almost no swelling even at lower polymer concentrations<br />
(5 % polymer). Until now, it is not completely clear why 8armPEG-hydrogels show this<br />
extraordinary stability. Either the retro-Diels-Alder reaction could be unfavorable or the gels<br />
could show self-healing properties, a phenomenon which has been described for other<br />
materials cross-linked by Diels-Alder reactions. Further studies are under way.<br />
In summary, our studies showed a clear influence of the branching factor on gelation time,<br />
gel strength, swelling and degradation. Given their favorable gelation time and long-term<br />
stability, 8armPEG-hydrogels seem to be a promising biomaterial for biomedical<br />
applications.<br />
Acknowledgments: This work has been supported by Deutsche Forschungsgemeinschaft (DFG), grant<br />
number GO565/16-1.<br />
References:<br />
1. Kirchhof, S. et al.: Application of the Diels-Alder Reaction as a New Cross-Linking Mechanism for<br />
Poly(ethylene glycol) Based Hydrogels, ACS Meeting Philadelphia, <strong>2012</strong><br />
147<br />
Chemical stability studies on the antileishmanial drug candidate KuRei300 in<br />
lipid based nanocarrier formulations as important prerequisite for in vivo<br />
experiments<br />
Kupetz, E. 1; Preu, L. 2; Bunjes, H. 1<br />
1 Institute of Pharmaceutical Technology, Technische <strong>Universität</strong> Braunschweig,<br />
Mendelssohnstraße 1, 38106 Braunschweig, Germany<br />
2 Institute of Medicinal and Pharmaceutical Chemistry, Technische <strong>Universität</strong> Braunschweig,<br />
Beethovenstraße 55, 38106 Braunschweig, Germany<br />
Visceral leishmaniasis is an infection caused by Leishmania donovani parasites. The WHO<br />
assigned it the status of a neglected tropical disease thus encouraging research for new<br />
active compounds. In a directed synthesis program the paullon chalcon derivative<br />
KuRei300 emerged as a promising drug candidate. It exhibited good antileishmanial activity<br />
in experiments with axenic amastigotes and human macrophages [1]. In vivo studies in<br />
mice are intended and a new parenteral formulation for KuRei300 was developed for this<br />
purpose [2]. This formulation is a mixed micellar solution based on phospholipids and a bile<br />
salt. It meets important quality criteria for parenteral preparations such as the use of<br />
approved excipients, physical stability and the possibility for sterile filtration. Another<br />
fundamental aspect that is, however, often overlooked is the possible decomposition of a<br />
formulated agent during storage. The present study therefore addressed the chemical<br />
stability of KuRei300 in liquid formulations.<br />
Since DMSO is a common solvent for in vivo / in vitro tests, a preliminary experiment was<br />
performed in which KuRei300 containing solutions were stored either under the influence or<br />
protection from light and monitored for signs of decay by HPLC. In the light exposed<br />
solution the original drug content declined rapidly and one predominant new peak<br />
appeared. The corresponding compound was identified as the Z-isomer of KuRei300 by 1H<br />
NMR spectroscopy. As a result of these findings all samples were stored protected from<br />
light in subsequent experiments. In a second set of experiments the stability of KuRei300 in<br />
the mixed micellar carrier solution was assessed under different conditions. To obtain the<br />
mixed micellar solution Lipoid S100 (13.4% w/w) was dispersed in an aqueous phase that<br />
contained sodium glycocholate (7.4% w/w overall) and was buffered to pH 7.4 to avoid<br />
phospholipid hydrolysis. This turbid phospholipid dispersion was then vigorously shaken<br />
until a translucent solution had formed. A KuRei300 solution in ethanol was pipetted into a<br />
vial and the ethanol was evaporated thus forming a substance coat on the walls of the vial.<br />
The micellar solution was then added and the vial placed on a rotator. During this passive<br />
loading procedure the major part of the substance film dissolved in the carrier. After<br />
loading, excess KuRei300 was filtered off and the loaded carrier was transferred into fresh<br />
vials which were crimped [2]. The samples were produced without and with 0.1% EDTA in<br />
the aqueous phase and were or were not flushed with nitrogen. At regular time intervals the<br />
chemical stability of KuRei300 was evaluated by HPLC. In the non-stabilized preparation<br />
(no N2, no EDTA) the substance underwent drastic and quick decomposition. The signals in<br />
the chromatogram differed, however, very much from those in the DMSO solution. Z-<br />
Kurei300 was not formed in significant amounts. Nitrogen flushing of the vial´s head space<br />
considerably slowed down the decay and after 10 weeks additional EDTA demonstrated a<br />
further benefit.<br />
In conclusion, the chemical stability of new drug candidates needs to be given the<br />
appropriate attention from the earliest stages of formulation. Photoisomerization and its<br />
impact on pharmacodynamics have been reported for anti-tumorigenic chalcones<br />
previously [3]. The relevance for the biological activity of KuRei300 has not been studied<br />
yet. Our study demonstrated that simple techniques such as light exclusion, storage under<br />
an inert gas and addition of a metal complexing agent (EDTA) strongly improved the<br />
chemical stability of the drug candidate. Such measures can prevent hard-to-interpret<br />
results from in vitro / in vivo experiments that may result if a potential chemical<br />
decomposition is disregarded.<br />
Acknowledgements: We would like to thank J. Ryczak (Institute of Medicinal and Pharmaceutical Chemistry,<br />
Beethovenstraße 55, 38106 Braunschweig) for the synthesis of KuRei300 and assistance in the<br />
interpretation of NMR spectra.<br />
References:<br />
1. Reichwald, C. et al.: J. Med. Chem. 2008, 51(3): 659-665<br />
2. Kupetz, E. Et al.: 8 th PBP World Meeting <strong>2012</strong>, Istanbul, Turkey<br />
3. Iwata, S. et al.: Biol. Pharm. Bull. 1997, 20(12): 1266-1270<br />
148<br />
Development of a microfluidic system for screening of colloidal drug<br />
formulations under flow conditions<br />
Pretor, S. 1; Finke, J.H. 1; Al-Halhouli, A.T. 2; Schmolke, H. 3; Busker, M. 4;Dietzel,<br />
A. 2;Büttgenbach, S. 2;Klages, C.-P. 3;Behrends, S. 4; Reichl, S. 1;Müller-Goymann, C.C. 1<br />
1Institut für Pharmazeutische Technologie, TU Braunschweig, Mendelssohnstraße 1, 38106<br />
Braunschweig, Deutschland<br />
2Institut für Mikrotechnik, TU Braunschweig,<br />
3Institut für Oberflächentechnik, TU Braunschweig;<br />
4Institut für Pharmakologie und Toxikologie, TU Braunschweig<br />
In recent years, research and drug development have focused on microfluidic systems<br />
because they offer miniaturization, parallelization and automation of screening processes<br />
[1]. In biotechnology, these systems are used as microbioreactors, whichhave already<br />
shown to be versatile tools for screening of microorganisms and optimal production<br />
parameters in small scale [2]. Additionally, cultivation of mammalian cell lines is<br />
alsopossible in microscale under flow conditions[3].<br />
Amicrofluidic systemconsisting of apolydimethylsiloxane (PDMS) fluidic channel bonded on<br />
a glass surface, a piezo driven micropump (Bartels Mikrosysteme) and a culture medium<br />
reservoir has been developed for the cultivation of mammalian cells. In a first series of<br />
experiments, immortalized human corneal epithelial cells (HCE-T) and primary human<br />
dermal fibroblasts (HDF)were cultivated in themicrochannel of the microfluidic system and<br />
examined according to their attachment and viability on surface-treated PDMS.<br />
In a second approach, the cellular uptake of fluorescence-labeled solid lipid nanoparticles<br />
was investigated. The aim was to checkthe suitability ofthe microfluidic system for tracing of<br />
nanoparticles, because it maysimulate physiological conditions by applying defined<br />
amounts of shear stress to the cells.<br />
Methods:<br />
Theattachment behavior of HDF on hydrophilizedPDMS plates with positively and<br />
negatively charged surfaces was estimated. The surface modification was done by layerby-layer<br />
deposition of polyelectrolytes.Positively charged surfaces were coated with<br />
130 Poster
fibronectin in addition. Cells were seeded on these plates and the ratio of attached cells<br />
was calculated by counting suspended cells after certain periods of time. Afterwards, the<br />
remaining cells were microscopically observed. Untreated PDMS served as negative<br />
control.<br />
For flow experiments, a constant medium flow of 0.2-1.0 ml/min was applied to the cells for<br />
24 hours. Cell staining was performed with Hoechst 33342, Annexin V and<br />
Ethidiumhomodimer to indicate viability, apoptosis and necrosis, respectively.<br />
Fluorescence labeled solid lipid nanoparticleswere prepared by using a microchannel<br />
emulsification method according to [4].HCE-T and HDF-cells were incubated with<br />
nanoparticles bothon a standard glass well plate and in the flow system.The<br />
attachednanoparticleswerevisualizedwith a confocal laser scanning microscope equipped<br />
with a 60x oil immersion objective.<br />
Results:<br />
Optimal cell growth wasachieved via treatment of PDMS surfaces with a positively charged<br />
coating and an additional fibronectindeposition.This coatingresulted in short attachment<br />
times after cell seeding tomicrochannel surfaces.<br />
Neither PDMS and glass, nor shear stress of 10 dyn/cm² affected growth of HCE-T and<br />
HDF cells in microchannels negatively with respect to cell viability.<br />
Solid lipid nanoparticles were able to transport the fluorescent dyecoumarin-6 as a model<br />
drug to epithelial cells.Its fluorescence intensity was investigated by CLSM both on special<br />
culture plates and in themicrochannels.<br />
Acknowledgement:The present study was performed in the context of the research group 856 mikroPART<br />
funded by the German Research Foundation (DFG). One of the authors (S. B.) gratefully acknowledges the<br />
financial support of the Volkswagen Foundation.<br />
References:<br />
[1] Dittrich, P.S.; Manz, A.: Nature20065: 210-218<br />
[2] Demming et al.: Biomicrofluidics2011 5: 014104<br />
[3] Kim et al.: Lab on a Chip20077: 681-694<br />
[4] Finke et al.:2nd Symposium on Phospholipids in Pharmaceutical Research Heidelberg2011(poster)<br />
149<br />
Optimizing the PCS measurement methods for nanoemulsions to see minimal<br />
changes in the z-average<br />
Harden, D. 1; Müller, R. H. 1; Keck, C. M. 1 2<br />
1 Institute of Pharmacy, Department of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Freie<br />
<strong>Universität</strong> Berlin, Kelchstraße 31, 12169 Berlin, Germany<br />
2 Applied Pharmacy Division, University of Applied Sciences Kaiserslautern, Campus Pirmasens,<br />
Carl-Schurz-Straße 10-16, 66953 Pirmasens, Germany<br />
The first instrument for the photon correlation spectroscopy (PCS) was introduced to<br />
market in the end of 1970 (Malvern Instruments). Today the PCS is one of the most<br />
frequently used instruments to measure the mean particle size (z-average) of<br />
nanoparticles. PCS is known as a very accurate method with high reproducibility. However,<br />
reproducibility not only depends on the method, but can also be influenced by the sample.<br />
An example is particle interaction, i.e. repulsive or attractive forces, which can lead to faster<br />
or slower diffusion and thus to smaller or larger size results. Particle interaction depends on<br />
the particle concentration within the sample. Hence, if the concentration varies between<br />
different measurements of one sample, the size results might not be reproducible. The aim<br />
of this study was to investigate the reproducibility of PCS measurements (Zetasizer Nano<br />
ZS, Malvern Instruments, UK) using different concentrations of a nanoemulsion (Lipofundin<br />
MCT 20%, B.Braun Melsungen AG, Germany) for the measurements.<br />
Today, modern instruments can analyze samples containing high concentrations of<br />
nanoparticles. In this method (1), the distance of the laser beam in the measuring cell is<br />
automatically adjusted to the particle concentration of the sample. This is done to avoid<br />
multiple scattering effects and to obtain an optimal intensity of the scattered light on the<br />
detector. The results of the PCS measurements using this method did not lead to similar<br />
results. Results show that there was a variation of 8 nm for a particle concentration of<br />
0.005% to 1.0% (228 nm to 236 nm). The variation was random, i.e. with increasing the zaverage<br />
went up and down.<br />
In case of the older PCS instruments, there was no changing in the distance of the laser<br />
beam of measuring point to the cuvette wall, therefore for these measurements a lower<br />
particle concentration must be chosen/ selected, if not there would be multi scattering and<br />
this will cause a smaller z-average. This method (2) was transferred to the Zetasizer Nano<br />
ZS with a constant distance of 4.65 mm from the wall to the measurement point. The same<br />
particle concentrations as in method 1 (1% to 0.005%) were chosen. The results showed<br />
that there was an concentration dependent increase in the z-average from 184 nm (1%) to<br />
236 nm (0.005%), however, the differences in the size when analyzing low concentrations<br />
(0.05% to 0.005%) was only 2nm, whereas it was 8nm with method 1.<br />
The results indicate that the reproducibility the PCS analysis should be investigated for<br />
each sample, which could possess particle interactions. For this different concentrations<br />
should be analysed using either the automatic method 1 or method 2 with the fixed laser<br />
beam position. In case of the nanoemulsion it is best to analyze the samples with method 2,<br />
e.g. using lower concentrations of the nanoemulsion and no automatic adjustment to obtain<br />
reproducible size results. The concentration range for good reproducibility was found be<br />
0.02% to 0.005%.<br />
150<br />
Efficiency increase by transfer of a two-pot to a single-pot fluid bed<br />
granulation<br />
Germer, K. 1; Rockmann, Th. 2; Wolf, B. 1<br />
1 Anhalt University of Applied Sciences, Pharmaceutical Engineering, Strenzfelder Allee 28, 06406<br />
Bernburg, Germany<br />
2 Salutas Pharma Corp., Teamleader Galenics, Otto-von-Guericke Allee 1, 39197 Barleben,<br />
Germany<br />
Traditionally, pharmaceutical granulates are manufactured by spray agglomeration in a<br />
mixing apparatus. In the past, the wet granulates were dried by time consuming tray drying.<br />
Nowadays, fluid bed drying is the preferred procedure. The application of two consecutive<br />
steps in different plants is still connected with material loss and contamination risk for which<br />
reason both agglomeration and drying step should be performed in consecutive steps in<br />
one and the same apparatus [1, 2].<br />
The aim of the investigation was the transfer of a two-step process consisting of spray<br />
mixing agglomeration followed by fluid bed drying of a commercial drug product to a singlepot<br />
procedure in a batch laboratory fluid bed apparatus. Therefore, a fluid bed granulator<br />
and dryer GPCG 1.1 (Glatt Corp., Binzen, Germany) was used (Figure 1). To optimize the<br />
process parameters of fluid bed agglomeration, the spray rate was varied between 20 and<br />
40 g/min, all other process parameters were kept constant. Parameters of the laboratory<br />
fluid bed drying process referred to the production process. Dry granulates were<br />
compressed to tablets. Evaluation criteria were the maintenance of stable and reproducible<br />
fluid bed process, furthermore acceptable yields and high granulate and tablet quality<br />
according to market product specification and pharmacopoeia.<br />
Figure1: Fluid bed granulator and dryer GPCG 1.1<br />
Best products were received with high inlet air temperature (80°C), high spray rate (30-40<br />
g/min) and additional water spraying. Without interruption of the fluid bed process the wet<br />
agglomerates were dried. The whole granulation process was stable and reproducible.<br />
Product yield was above 90%. Granulate and tablet properties fulfilled the specified quality<br />
criteria. Particle size was slightly increased with increasing spray rate compared to the<br />
production process, but there was no significant influence neither on granulate nor on tablet<br />
properties. The two-step granulation process with two different plants was successful<br />
transferred to a single-pot fluid bed process saving process time and equipment and<br />
reducing material loss and contamination risk.<br />
References:<br />
1. K. Giry et al.: Chemical Engineerig and Processing 2009, 48: 1293-1301.<br />
2. J.Z. Gao et al.: International Journal of Pharmaceutics 2002, 237: 1-14.<br />
151<br />
Determination of residence time distribution in hot melt extrusion via image<br />
analysis<br />
Reitz E; Brock D; Thommes M<br />
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine University, Universitaetsstrasse<br />
1, 40225 Duesseldorf, Germany<br />
Hot melt extrusion is an important and modern technology in pharmaceutical applications<br />
and is gaining more and more interest because of its versatile capabilities. One<br />
fundamental parameter for process understanding and process control is the residence<br />
time (RT). This parameter plays an important role especially in hot melt extrusion<br />
processes. The knowledge of the RT is important in critical hot melt extrusion processes,<br />
e.g. in processing of heat sensitive products. For such processes a short RT is desired to<br />
prevent degradation. The knowledge of the RT is also important for those types of solid<br />
dispersions, where a drug shall be grinded or dispersed and for which a long RT is<br />
beneficial.<br />
The aim of this study was to develop a method for an on-line RT measurement. The idea<br />
was to determine the grey value of the extrudates by adding carbon black as tracer to white<br />
sugar alcohol extrudates (figure 1).<br />
Figure 1: colouring of the white sugar alcohol extrudate by carbon black<br />
0 200 00 600 800<br />
time [s]<br />
1000 1200 1 00<br />
Poster 131<br />
black<br />
grey<br />
white<br />
Xy itol 0. mm<br />
Xy itol 0. mm<br />
Manni ol 0. mm<br />
Manni ol 0. mm<br />
Figure 2: residence time distribution for xylitol and mannitol, measured by taking pictures of<br />
the extrudates leaving the die (n=2)
Hot melt extrusion (Mikro 27GL-28D, Leistritz, Nuremberg) was performed with xylitol and<br />
mannitol (n=2). Carbon black in a defined amount (0.5 g carbon black as bolus by 40 g/min<br />
powder feed rate) was added and a series of pictures (Nikon D300, Nikon Corp. Japan)<br />
was taken from the extrudate in the die region in two-second intervals. A software algorithm<br />
(Matlab version R2009a, Mathworks) was developed which enabled the calculation of a<br />
grey value of a defined piece of the extrudate over the time resulting in a residence time<br />
distribution (RTD).<br />
It was possible to calculate the grey value for the extrudate emerging from the die plate at<br />
each time point, describing the load of the tracer. An adjustment of the grey values to a<br />
black and white standard was necessary to exclude any light effects affecting the colour of<br />
the extrudate on the photograph. Therefore on each photograph black and white standards<br />
were recorded as well. The camera software cut a piece of the extrudate as well as of the<br />
black and white standard out of the photographs for each time point. For these three parts<br />
of the photographs, the grey values were calculated and the value for the extrudates was<br />
adjusted by the standard values.<br />
Table 1: important parameters of the RTD<br />
onset [s] midpoint [s] offset [s]<br />
xylitol 317 435<br />
690<br />
328 430<br />
706<br />
mannitol 233 324<br />
523<br />
249 325<br />
525<br />
With this calculation, the RT as well as the RTD were determined (figure 2). This process<br />
was reproducible for both carriers used in this study (table 1). Due to the high sampling rate<br />
of the photos, the residence time could be determined very precisely.<br />
In conclusion, a tool for residence time measurement was developed, which helps to<br />
understand and to control hot melt extrusion processes.<br />
152<br />
Systematic studies of process parameters for wet granulation in a high-shear<br />
mixer<br />
Hoffmann, B. 1; Mosig, J. 1; Kleinebudde, P. 1<br />
1 Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitaetsstr.1,<br />
40225 Duesseldorf, Germany<br />
The aim of this study was to investigate the influence of different process parameters on<br />
wet granulation processes in a high-shear mixer. The statistical evaluation was arranged by<br />
the programme MODDE 9.0 (Umetrics, Sweden). The DOE had a full factorial design of 4<br />
factors with an interaction model and three repetitions of the center point.<br />
Lactose monohydrate (Granulac 200, Meggle, Germany) was used as excipient. For the<br />
granulation polyvinylpyrrolidone (Kollidon 30, BASF, Germany) was added as solid binder<br />
and water as granulation liquid.<br />
It was granulated in a 10 L-bowl of a high-shear mixer (Diosna, Germany). Water addition<br />
time was kept constant at 200 s, the chopper was started every minute for 10 s. Varied<br />
parameters of the process were the impeller speed (imp; 150 rpm to 300 rpm), the liquid<br />
content (liq; 10 % to 12 % (w/w) related to solid mass), the binder content (bin; 4 % to 6 %<br />
(w/w) of total solid mass) and the wet massing time (wmt; 0 to 300 s). Response<br />
parameters were the particle size distribution (x50, x25 and x75), the interquartil range, the<br />
yield (particles < 1250 µm) and the particle size distribution of the yield (x50 of the yield<br />
y50). Further response parameters were bulk density, tapped density and the Hausner factor<br />
(according to Ph. Eur. 2.9.34).<br />
The mean particle size (x50) ranged between 150 µm – 1700 µm. Although most granules<br />
had an x50 about 200 - 300 µm, there were coarse granules obtained in every batch. This<br />
became apparent in the results of the yield, as 94 % in the best and only 10 % in the worst<br />
case could be obtained. Decreasing binder content had the biggest influence on the<br />
increase of the yield. Contrary to expectations a lower influence of the impeller speed in<br />
contrast to the other factors was observed. After backward regression, it became only<br />
significant for the x50 and the y50. This should be exemplified by the coefficient plot of the y50<br />
(fig. 1). The effect of the liquid content and the wet massing time was much higher (imp<br />
106.5 µm; liq 236.7 µm; bin 177.6 µm; wmt 240.2 µm). In contrast to literature1. a<br />
significant effect of the impeller speed on the interquartil range as parameter for the width<br />
of particle size distribution could not be found. For densities and the Hausner factor no<br />
parameter seemed to have a significant effect.<br />
In conclusion, liquid content, wet massing time and binder content had the greatest effect<br />
on analyzed granule properties. Further studies shall investigate if these findings can be<br />
transferred to high-shear granulation processes with further filler-binder combinations.<br />
[µm]<br />
140<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
-20<br />
-40<br />
imp<br />
iq<br />
Figure 1 – Coefficient plot of y50 (R² Adj. = 0.936; Q² = 0.828)<br />
References:<br />
1. Oulahna, D. et al., Powder Technology 2003 130: 238-246.<br />
bin<br />
wmt<br />
imp*bin<br />
imp*wmt<br />
liq*wmt<br />
bin*wmt<br />
153<br />
Fluorescent Nanoparticles capped with Octreotide for Targeting of<br />
Somatostatin Receptors<br />
Abd-Ellatif. A, Hennig. R, Pollinger. K, Tessmar. J .and Goepferich. A<br />
Department of Pharmaceutical Technology, University of Regensburg, Regensburg, Germany<br />
Somatostatin receptor subtype 2 (SSTR2) is widely expressed in normal and tumor cells,<br />
which is clinically used for diagnostic purposes [1].Targeting therapeutic nanoparticles to<br />
the SSTR2 using somatostatin analogue octreotide (OCT), would be a promising approach<br />
for certain cancer cells, since this would reduce side effects and increase drug delivery<br />
efficacy to the target site, highly over-expressing the respective receptors.<br />
To attach OCT to nanoparticles, it was succinylated with succinic anhydride at low pH<br />
selectively at its N-terminus [2]. Succinylated OCT was then conjugated using a watersoluble<br />
carbodiimide (EDC) and N-hydroxy succinimide to photo-stable fluorescent<br />
Quantum dot nanoparticles. These have an emission wavelength of 655 nm and are<br />
functionalized with amine-derivatized polyethylene glycol (PEG).These are known to have a<br />
very low non-specific binding to cellular membranes.<br />
Succinylated OCT, was characterized by mass spectrometry and the formulated<br />
nanoparticles were characterized by size exclusion chromatography (SEC), fluorescence<br />
spectrometry, 1H-NMR,FTIR spectroscopy and for the resulting zeta-potential using<br />
dynamic light scattering. Finally, cellular uptake was studied for SSTR2-positive pancreatic<br />
carcinoid tumor cells (BON1) using confocal microscopy and flow cytometry.<br />
Mass spectrometry indicated mono-succinylation of OCT by succinic anhydride in aqueous<br />
media, as the result showed an increase in molecular mass of about 100 Da. A slight peak<br />
shift was observed in SEC for OCT-Qdots compared to the native nanoparticles. In addition<br />
a slight red shift was observed in the fluorescence emission spectra of Qdots after<br />
conjugation to OCT. The zeta-potentials showed an inversion to positive charge of Qdots<br />
after the conjugation to OCT. FTIR spectroscopy and 1H-NMR confirmed the attachment of<br />
OCT to Qdots, which served as proof for the binding of the peptide to Qdots.<br />
When BON1 cells incubated with the ligand modified particles, confocal microscopy and<br />
flow cytometry showed high binding to BON1cells. Flow cytometry revealed the<br />
displacement of Qdots-OCT, when high concentrations of free OCT were used to displace<br />
it.<br />
We concluded that nanoparticles modified with OCT may serve as promising tool to target<br />
different cells that over-express SSTR2 receptors.<br />
This work has been financially supported by a scholarsh p from the Arab Republic of Egypt for Ahmed<br />
Abdellatif.<br />
References:<br />
1. Reubi, J.C. et al., J. Clin. Endocrinol. Metab. 2010, 95(5): 2343-50.<br />
2. Kinstler, O.B. et al., Pharm Res. 1996, 13(7): 996-1002.<br />
3. Updegrove, T.B. et al., RNA 2011, 17(3): 489-500.<br />
4. Na, D.H. et al., AAPS Pharm. Sci.Tech. 2003, 4(4): E72.<br />
154<br />
An accelerated release method for fast assessment of real time release from<br />
an intravaginal ring<br />
Externbrink A1, Klein S1 1 <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong> University <strong>Greifswald</strong>, Department of Pharmacy, Institute of Biopharmacy and<br />
Pharmaceutical Technology, C_DAT, Felix-Hausdorff.Str. 3, 17489 <strong>Greifswald</strong><br />
Purpose<br />
Intravaginal rings (IVR’s) are usually designed to deliver the active drug over long periods<br />
of time. Accelerated drug release testing would allow for fast assessment of real time<br />
release during product development and quality control. Therefore, the objective of the<br />
present study was to develop a discriminative accelerated release method for an IVR and<br />
to establish a relationship between real time and accelerated release profiles.<br />
Methods<br />
NuvaRing ®, an approved hormonal contraceptive IVR which releases 120 µg etonogestrel<br />
(ENG) and 15 µg ethinylestradiol per day over 21 days, was selected as a model<br />
formulation. Release experiments were performed with ring segments and the release from<br />
one IVR was calculated with respect to the mass ratio. Prior to the release experiments,<br />
rings were cut into 10 segments which were endcapped with acrylate glue to prevent<br />
uncontrolled diffusion which would affect the overall release. USP apparatus 7 (400-DS)<br />
was used to determine the daily etonogestrel release. 10 mL of a vaginal fluid simulant<br />
were used as the release medium. The first set of experiments was performed at 37 °C<br />
(real time conditions) and automated sampling as well as media replacement was<br />
performed every 12 hours. Subsequent experiments were performed at elevated<br />
temperatures, i.e. 44, 50 and 55 °C (accelerated conditions). Sampling and media<br />
replacement was performed in appropriate intervals.<br />
Results<br />
For all temperatures the zero order release constants were calculated from the slopes of<br />
the cumulative release versus time graph and increased with increase in temperature. The<br />
elevated temperature release constants were plotted according to the arrhenius equation<br />
and a linear correlation between rate constants and temperature was found. The predicted<br />
rate constant at 37 °C was in agreement with the experimental value.<br />
The real time and accelerated release profiles at 50 °C were directly compared by plotting<br />
the cumulative real time release versus the accelerated release and a strong linear<br />
correlation was obtained with a correlation coefficient of 0.999. Additionally, the cumulative<br />
real time and accelerated release profiles at 50 °C were plotted on the same axis after time<br />
scaling. Applying the scaling factor which was determined as the ratio between the release<br />
constants at elevated temperature and 37 °C real time and accelerated release profiles at<br />
50 °C were superimposable.<br />
Reservoir type IVR’s like NuvaRing ® typically show an initial burst release phase followed<br />
by near zero order release kinetics with a slight decrease in daily drug release over time.<br />
Drug release is usually expressed as daily release versus time. In order to be able to<br />
monitor the changes in the daily real time release profile over time with accelerated test<br />
conditions the sampling frequency was adjusted to reflect daily real time release. The<br />
required sampling interval at 50 °C was determined from the ratio between zero order<br />
release constants at 37 °C and 50 °C. A good agreement between the daily real time<br />
release profiles and the elevated temperature release profiles with adjusted sampling<br />
intervals was observed.<br />
Conclusion<br />
An accelerated release method that is predictive of real time release was developed.<br />
132 Poster
Temperature appears to be a valid parameter to accelerate ENG release from NuvaRing ®.<br />
An Arrhenius relationship between temperature and release constants indicates that the<br />
integrity of the dosage form and the release medium is maintained under accelerated<br />
conditions. Comparison of real time and elevated temperature release profiles at 50 °C<br />
revealed a strong correlation between real time and accelerated release. In order to assess<br />
the changes in the daily release profiles over time the sampling intervals at elevated<br />
temperatures can be adjusted to reflect daily real time release. With the proposed method<br />
and a temperature of 50 °C the overall duration of drug release from Nuvaring segments<br />
could be reduced from 21 to less than five days.<br />
155<br />
Sartan-modified quantum dots targeting the Angiotensin II receptor type 1<br />
Hennig R1; Pollinger K1; Wenzel R1; Breunig M1; Tessmar J1; Göpferich A1 1 University of Regensburg, Dept. of Pharmaceutical Technology, <strong>Universität</strong>sstr. 31, 93053<br />
Regensburg, Germany<br />
The angiotensin II type 1 (AT1) receptor, which belongs to the class of G protein-coupled<br />
receptors, is a pharmacological target of great interest. Although it is expressed<br />
ubiquitously throughout the body, it is known that this receptor is overexpressed in several<br />
tissues due to pathological conditions and, therefore, represents a promising target for<br />
nanoparticulate drug delivery [1, 2].<br />
To address nanoparticles to AT1 receptors we labelled commercially available fluorescent<br />
quantum dots (Qdots) with a metabolite of losartan, EXP3174, which is largely responsible<br />
for the actions of losartan in vivo [3]. In addition to the increased receptor affinity, EXP3174<br />
has a carboxylic function that allows relatively easy conjugation to nanoparticles.<br />
Furthermore, amides of that carboxylic group are known to retain a high receptor blocking<br />
efficacy in similar derivatives [4]. First, quantitative real-time PCR showed that human<br />
adrenal corticocarcinoma NCI-H295R cells, which served as receptor-positive model cells,<br />
highly expressed the AT1 receptor. Flow cytometry (FACS) experiments with these ontarget<br />
and HeLa cells as off-target cells revealed a strong binding of EXP3174-modified<br />
Qdots to AT1-positive but not to AT1-negative HeLa cells. To further investigate the<br />
distribution pattern of the modified Qdots we performed binding experiments with a confocal<br />
laser scanning microscope (CLSM). The EXP3174-Qdots showed a membrane-associated<br />
staining of the NCI-H295R cells, indicating a binding to the cellular surface. This was<br />
expected for antagonist-modified nanoparticles which do not provoke receptor<br />
internalization upon ligand binding. In competitive displacement experiments a great excess<br />
of free ligand was needed to displace the EXP3174-modified Qdots from the cells, which is<br />
characteristic for nanoparticles that exhibit multivalent binding [5].<br />
In conclusion, we could show that a non-peptide antagonist for the AT1 receptor can be<br />
bound to quantum dots in order to target AT1-positive cells in a multivalent manner.<br />
Although the chemical character is significantly altered by conjugation, a sufficient receptor<br />
affinity remains. This allows for potential targeting of AT1 receptor overexpressing sites<br />
within the body.<br />
Acknowledgements: This work was supported by the Deutsche Forschungsgemeinschaft: DFG grant No<br />
GO565/17-1.<br />
References:<br />
1. Dvir, T. et al.: Nano Lett. 2011, 11(10): 4411–4414.<br />
2. Suzuki, K. et al.: Am. J. Pathol. 2007, 170(6): 1841-1853.<br />
3. Fabiani, M. et al.: Am. J. Hypertens. 2000, 13(9): 1005–1013.<br />
4. Santella, J. B. et al.: Bioorg. Med. Chem. Lett. 1994, 4(18): 2235–2240.<br />
5. Montet, X. et al.: J. Med. Chem. 2006, 49(20): 6087-6093.<br />
156<br />
Biphasic drug release from coated pellets<br />
Priese, F., Wolf, B.<br />
Anhalt University of Applied Sciences, Pharmaceutical Engineering, Neues Labor, Strenzfelder<br />
Allee 28, D-06406 Bernburg, Germany<br />
Pellets used for multiparticulate drug delivery systems may contain the API (active<br />
pharmaceutical ingredient) incorporated into the pellet core or as a coating layer on the<br />
pellet surface. Rapid release occurs when the API is readily soluble. With the help of further<br />
excipients the API release can be varied in a very broad range. Retarded release is<br />
achieved with functional polymers showing swelling capacity but no solubility under<br />
physiological conditions [1]. Additional to the advantage of homogeneous drug release of<br />
multiparticulate systems a biphasic release may be achieved by incorporation of a fast as<br />
well as a slow or retarded released dosage of the API. The therapeutic effect is reached<br />
immediately after drug administration and the patient is supplied with drug over a long time<br />
period by the slow released maintenance dosage.<br />
Inert microcrystalline cellulose pellets (Cellets ® 200, IPC GmbH, Dresden, Germany) were<br />
coated with the model API sodium benzoate as maintenance dosage (Figure) in a first step.<br />
The coating liquid consists of sodium benzoate and furthermore of polyvinylpyrrolidone for<br />
film stability increase and talcum to prevent the pellets from sticking during the coating<br />
process. For retarded release of that dosage the pellets were coated with Surelease ®<br />
dispersion (ethylcellulose, Colorcon Corp.) in a second step. At least the pellets were<br />
coated with the initial sodium benzoate dosage. The coating was performed in a batch<br />
laboratory fluid bed apparatus with Wurster technique (GPCG 1.1, Glatt Corp., Binzen,<br />
Germany). The weight ratio of initial to maintenance dosage was varied (1:2.2 and 1:6).<br />
Evaluation criteria were the coating process stability, the product yield and the pellet<br />
quality. Mean pellet size, sphericity, bulk density and drug release were measured.<br />
Figure: Scheme of threefold coated pellets<br />
The fluid bed coating processes were stable. At the end of the first and the second coating<br />
step the coating liquids were changed so that the process was performed without<br />
interruption. Yield was above 90%, significant material loss at the inner wall of the chamber<br />
or at the partition was never observed. The pellets were received as particles with smooth<br />
surface and sphericity of 0.95-0.96 indicating homogeneous growth of the several coating<br />
layers. The initial sodium benzoate dosage from the external layer was released<br />
immediately after 1 minute. The maintenance dosage was retarded released due to<br />
ethylcellulose film swelling (90% release after 17-25 minutes). In comparison, the release of<br />
sodium benzoate coated pellets without polymer film occurs completely within 1 minute due<br />
to readily solubility and high dissolution rate. The retarding effect of Surelease is improved<br />
with increasing layer thickness. By variation of the ratio of initial to maintenance dosage<br />
and polymer layer thickness the release kinetics can be varied in a wide range. The final<br />
pellets are filled into hard gelatin capsules or compressed into tablets. The influence of the<br />
compression process on the integrity of the pellets and the maintenance of the release<br />
kinetics is under investigation.<br />
Acknowledgement: The technical assistance of the student Markus Seifert in the experimental trials is<br />
grateful acknowledged.<br />
References:<br />
[1] Siepmann, F., Siepmann J., Walther, M., MacRae, R.J., Bodmeier, R.: Polymer blends for controlled<br />
release coatings, Journal of Controlled Release 2008, 125: 1–15.<br />
157<br />
Influence of fluid bed rotor pelletization parameters on pellet properties<br />
Wolf B, Ren X<br />
Anhalt University of Applied Sciences, Pharmaceutical Engineering, Neues Labor, Strenzfelder<br />
Allee 28, D-06406 Bernburg, Germany<br />
API (active pharmaceutical ingredient) loaded pellets are used for the preparation of<br />
multiparticulate drug delivery systems. Actual market products are pellet filled hard gelatine<br />
capsules, in some cases pellets are compressed into tablets. The API may be incorporated<br />
into the pellet or sprayed onto the surface of inert pellets forming an external layer. Enteric<br />
resistance or controlled release can be achieved in both cases with further coating layers.<br />
Pellets are predominantly manufactured by extrusion/spheronization, but fluid bed rotor<br />
agglomeration [1, 2] is of increasing interest because of the advantage of a single pot<br />
process, i.e. mixing of the initial solid components, agglomeration and drying are performed<br />
in one and the same apparatus.<br />
Preliminary studies were performed with a simple binary mixture of microcrystalline<br />
cellulose and lactose monohydrate 1:1 to evaluate the possible process parameter range.<br />
In a second trial, the highest possible content of ternary model substances (benzocaine,<br />
calcium hydrogen phosphate, talcum and maize starch) in the mixture was investigated.<br />
Evaluation criteria were process stability and pellet quality. Finally, by use of a 23 design of<br />
experiments (DoE) the influence of the process parameters spray rate, process air<br />
temperature and rotor disc rate and the influence of the model substances (content 10% in<br />
the formulation) on the pellet formation process and on the mean particle size d50 was<br />
investigated. The pellet manufacturing process was performed in a laboratory fluid bed<br />
granulator and dryer (GPCG 1.1 with rotor disc equipment, Glatt Corp., Binzen, Germany).<br />
Purified water was used as granulation liquid. At the end of the agglomeration process the<br />
product was dried to residual moisture below 5%.<br />
With the binary mixture, the pellet fluid bed manufacturing was possible in a broad range of<br />
spray rate, process air temperature and rotor disk rate. The pellets were received in<br />
satisfactory spherical shape. The maximum content of maize starch was 40%, higher<br />
amount led to overwetting and formation of inacceptable large agglomerates. The content<br />
of insoluble and not swellable calcium hydrogen phosphate was limited to 40%. Above this<br />
value agglomeration did not occur anymore, the fluid bed process broke down and the<br />
product precipitated onto the wall and at the spray nozzle. This was also the case with<br />
more than 15% of coarse-grained benzocaine as well as fine-grained talcum what is<br />
sometimes used in low concentration as anti-sticking agent in coating processes.<br />
Factor and interaction Effect Tendency<br />
Factor Pellet size<br />
A = Spray rate high positive A ↑ ↑<br />
B = Process air temperature high negative B ↑ ↓<br />
C = Rotor disk rate weak negative C ↑ ↓<br />
Interaction A/B and A/C negative A/B, A/C ↑ ↓<br />
Interaction B/C and A/B/C weak positive B/C, A/B/C ↑<br />
Table: Evaluation of DoE, factors, effects and tendency on pellet size<br />
Low and high level of the process parameters were spray rate 15/25 g/min, process air<br />
temperature 50/70°C and rotor disc rate 450/750 rpm. The evaluation of the DoE<br />
experiments is summarized in the table. The influence of spray rate on particle size is high<br />
positive and that of process air temperature high negative as expected. The rotor disk rate<br />
does not significant effect the particle size. Effects by interaction are proved but not<br />
significant. Fine-grained and insoluble calcium hydrogen phosphate does not influence the<br />
particle size. Vice versa coarse-grained benzocaine gives high effect. The flow properties of<br />
the products are influenced by the particle size and the shape. Angle of slope is diminished<br />
with increasing pellet size indicating improved flow properties. On the other hand flow time<br />
is raised with increasing particle size. Large pellets deviate from spherical shape more than<br />
smaller ones deteriorating the flow property. Fluid bed rotor granulation may be used for<br />
pellet manufacturing in a wide range of process parameters but the drug substance<br />
properties i.e. initial particle size, solubility and hydrophilic-lipophilic properties should be<br />
taken into consideration.<br />
References:<br />
[1] Parikh, D.M., Mogavero, M.: Batch fluid bed granulation. In: Parikh, D.M. (editor): Handbook of<br />
pharmaceutical granulation technology (Taylor&Francis) 2005.<br />
[2] Kristensen, J.,Hansen, V.W.: AAPS PharmSciTech 2006,7(1): E153-E162<br />
Poster 133
158<br />
Stability and cellular uptake of a model vaccine containing Resovist ® as<br />
marker for MRI tracking<br />
K. Thom1, K. Schulz1, K. Aurich2, G. Glöckl1, J.-P. Kühn3 and W. Weitschies1 1Institute of Pharmacy, EMA University of <strong>Greifswald</strong>, <strong>Greifswald</strong>, D-17489, Germany<br />
2Institute of Immunology and Transfusion Medicine, EMA University of <strong>Greifswald</strong>, <strong>Greifswald</strong>, D-<br />
17489, Germany<br />
3Institute of Diagnostic Radiology and Neuroradiology, EMA University of <strong>Greifswald</strong>, <strong>Greifswald</strong>,<br />
D-17489, Germany<br />
Aluminum containing adjuvants in vaccines applied for immunopotentiation have been<br />
considered as save and efficacious for decades. However, their mode of action could not<br />
be ascertained in detail to date. Tracking the adjuvant after injection using magnetic<br />
resonance imaging (MRI) may be a new method to investigate the fate of aluminum<br />
hydroxide (AH) adjuvant in vivo. For this purpose the MRI contrast agent Resovist ® (Bayer<br />
Schering Pharma AG) containing ferucarbotran nanoparticles, was combined with<br />
Aluminum hydroxide Gel (Sigma). Prior to MRI analysis this combination was investigated<br />
concerning stability and cellular interaction.<br />
Opposite surface charges (zeta potential Resovist ® -38 mV, Aluminum hydroxide Gel<br />
+13 mV) lead to electrostatic attraction and hence to adsorption by the adjuvant within<br />
30 minutes. Tracking the AH adjuvant requires a strong adsorption of the ferucarbotran<br />
nanoparticles even under in vivo conditions. Therefore the degree of adsorption was<br />
determined in simulated interstitial fluid (sIF) [1] and in plasma for a period of 24 h. At<br />
different timepoints unbound iron content was determined using atomic absorption<br />
spectrometry (AAS).<br />
Investigating the uptake of ferucarbotran and ferucarbotran adsorbed to AH adjuvant into<br />
peripheral mononuclear blood cells (PBMC) gave an indication of the influence of the<br />
nanoparticles on the adjuvants behavior after injection of the model vaccine. For isolating<br />
PMBC a density gradient centrifugation was used (OptiPrep ®, Sigma). Cells were incubated<br />
either with Resovist ®, Aluminum hydroxide Gel or the combination thereof at 37 °C and<br />
300 rpm. One fraction was incubated without additives as negative control. For cell dilution<br />
different media were used (RPMI with 10 %, 50 % or without fetal calf serum = FCS, 100 %<br />
FCS or sIF) to investigate media influence on the uptake into cells. After one hour cells with<br />
adsorbed or incorporated nanoparticles were separated utilizing their magnetic behaviour.<br />
Subsequently magnetic and non-magnetic cells were counted. The iron content of each<br />
separated fraction with sIF as dilution medium was determined using AAS.<br />
In our experiments sIF eluted twice as much iron than plasma during the first hour but after<br />
6 and 24 hours iron content in all samples was nearly the same. Agitation of the samples<br />
during incubation did not have any effect on the elution. Iron amounts in the supernatant<br />
below 7 % of the input indicate high adsorption strength after injection of the model vaccine.<br />
Resovist ® was used in this study to ensure visibility of the AH adjuvant for MRI. The<br />
contrast agent leads to signal loss on MR images if it is accumulated in tissues. Incubation<br />
with ferucarbotran particles led to an iron uptake into cells which allowed separation in a<br />
magnetic field. During this process the dilution medium had an obvious influence. With<br />
increasing FCS concentration in the medium the number of magnetic cells decreased due<br />
to the rising amount of adherent proteins which block the cell surface. The amount of<br />
ferucarbotran which was taken up within sIF medium was similar to the values for RPMI<br />
with 50 % FCS. This correlates with the protein content of 2.5 % in sIF which corresponds<br />
to 5 % proteins in serum respectively. When the Resovist ® particles were adsorbed to AH<br />
adjuvant prior to incubation the magnetic cell fraction in each dilution medium exceeded the<br />
one reached after pure Resovist ® treatment. The enhancement of the iron uptake may<br />
facilitate signal loss resulting in visibility.<br />
The non-magnetic fraction of Resovist ® treated cells contained more iron than the magnetic<br />
fraction propably due to an excess of unbound Resovist ®. Cells incubated with Resovist ®<br />
adsorbed to AH gave opposite results. Here the AH-ferucarbotran aggregates showed<br />
magnetic behaviour by themselves and thus increased the iron content of the magnetic cell<br />
fraction.<br />
In conclusion, adsorption of Resovist ® is most probably stable enough for tracking AH<br />
adjuvant in vivo. AH adjuvant enhanced the iron uptake into PMBC. This might facilitate the<br />
detection on MR images<br />
References:<br />
1. Wolff, L. et al.: Vaccine 2009, 27: 1834-1840.<br />
159<br />
Effects of non-thermal plasma on HaCaT keratinocytes is not mediated by<br />
ozone<br />
Blackert S1, Haertel B1, Talmann L2, Oehmigen K2, von Woedtke T2, Lindequist U1 1 Institute of Pharmacy, University of <strong>Greifswald</strong>, D-17489, Germany<br />
2 Leibniz-Institute for Plasma Science and Technology e.V. (INP), D-17489 <strong>Greifswald</strong>, Germany<br />
Non-thermal atmospheric-pressure plasma has been shown to influence as first target the<br />
cell membrane with its embedded proteins. Such cell surface molecules as integrins,<br />
cadherins or the epidermal growth factor receptor (EGFR) are of importance in wound<br />
healing and also for development of cancer metastasis. Cold plasma comprises of<br />
electrons, positive or negative ions, free radicals [e.g. reactive oxygen species (ROS),<br />
ozone] and other excited atoms and molecules. Further, plasma has an optical emission in<br />
the UV-region, especially UVB. Each of these components can affect the cells during<br />
treatment.<br />
Since it is known that during plasma treatment ozone is generated, we wanted to know as<br />
to whether this ozone concentration affects HaCaT keratinocytes.<br />
Adherent HaCaT keratinocytes were treated with plasma by a surface dielectric barrier<br />
discharge in air and argon (1 to 5 min) or with ozone (5 min). Ozone was generated by an<br />
Ozonisator and monitored by FT-IR (100, 400, 900 and 1800 ppm). Intracellular ROS,<br />
apoptosis (annexin V/propidium iodide staining) and cell surface molecules (α2-, α3-, α4-,<br />
α6-, αV-, β1-integrin, E-cadherin, EGFR) were analyzed by flow cytometry 24h after<br />
treatment.<br />
Besides a reduction of cell viability significant intracellular changes were observed. DBD/air<br />
plasma for 5 min caused an increased expression of α2- and β1-integrin whereas Ecadherin<br />
and EGFR expression was not influenced. The effects of DBD/argon plasma were<br />
less pronounced. Apoptosis was only increased by DBD/air plasma (5 min) although the<br />
proportion of apoptotic cells was rather low. Intracellular ROS detected by the fluorescent<br />
dye CM-H2DC-FDA increased from 6.6 ± 1.1% (untreated control cells) to 18.0 ± 3.2% (5<br />
min DBD/air) and 10.9 ± 1.3% (5 min DBD/argon). A concentration of about 100 ppm<br />
ozone was measured above the solid phase during a 5 min DBD/air treatment cycle which<br />
had no influence on integrin, E-cadherin or EGFR expression. 1800 ppm ozone caused an<br />
increase of α2- and β1-integrin whereas all other molecules measured were not affected.<br />
Taken together, the extent of effects depended on the nature of plasma (air vs. argon) and<br />
the exposure time of cells to the plasma. Short (≤ 1 min) treatment cycles did neither<br />
change cell surface protein expression nor induced apoptosis or intracellular ROS. The<br />
lowest concentration of ozone tested (100 ppm = about 200 mg/m 3), which is 1000 fold<br />
higher than the MAC had marginal effect on viability and no influence on integrin<br />
expression of HaCaT keratinocytes. The effects of plasma on cell membrane proteins<br />
observed are rather attributed to induction of intracellular ROS than to generation of ozone.<br />
* Work supported by the German Federal Ministry of Education and Research within the joint research<br />
project Campus PlasmaMed” (grant no. 13N11182)<br />
160<br />
Evaluation of German and International drug interaction databases<br />
Pauly A. 1; Wolf C. 1; Busse M. 2; Strauss A-C. 2; Krebs S. 2; Dörje, F. 2; Leuner, K. 1<br />
1 Professur für Molekulare und Klinische Pharmazie, Cauerstraße 4, 91058 Erlangen, Germany<br />
2 Apotheke des <strong>Universität</strong>sklinikums Erlangen, Palmsanlage 3, 91054 Erlangen, Germany<br />
Background<br />
Drug interactions are a common problem in drug therapy, especially if polypharmacy is<br />
needed [1]. There are a number of databases available helping to identify clinically relevant<br />
drug interactions. They vary in scope, accuracy, comprehensiveness, user-friendliness and<br />
cost. So far, there does not exist an evaluation which includes German databases.<br />
Therefore, we compared seven databases in German and English to determine the best<br />
database for daily clinical practice.<br />
Methods<br />
We identified more than 100 drug interaction pairs out of our daily routine and literature<br />
research. These were first validated using three reference databases (Stockley’s, DrugDex,<br />
iFacts). Out of the 100 selected pairs 50 were classified as clinically relevant and 50 as<br />
clinically non-relevant.<br />
A predefined scoring system was used to assess the scope, accuracy and<br />
comprehensiveness of the selected drug interaction databases. ABDA-database, MediQ,<br />
Pharmavista and MMI Pharmindex were selected as German drug interactions<br />
programmes. Lexi-Interact, Epocrates and drugs.com represented the international test<br />
databases.<br />
Results<br />
With its reliable monographs and high accuracy, Lexi-Interact scored 509 of 600 possible<br />
points and ranked first in our evaluation. Lexi-Interact was the most comprehensive<br />
database (180 of 200 possible points) with detailed monographs containing not only<br />
mechanism, severity and management of the drug interaction, but also onset, level of<br />
evidence and discussion of evidence, and literature references. These monograph<br />
components were not provided in all databases.<br />
The free of charge Swiss database Pharmavista (495 of 600 points) provided detailed and<br />
accurate monographs, but only recognized Swiss trade names. The German ABDA-<br />
Database ranked third in the overall score (453 of 600 possible points), because it omitted<br />
the level of evidence and discussion of evidence. The ABDA-Database scored best in<br />
accuracy with 335 out of 400 possible points.<br />
MMI Pharmindex (441 of 600 possible points), MediQ (413 of 600), drugs.com (382 of 600)<br />
and Epocrates (357 of 600) ranked fourth to seventh. These databases failed to distinguish<br />
between clinically relevant and clinically non-relevant interactions in a higher percentage,<br />
revealed more problems identifying clinically non-relevant interactions and suffer from<br />
limitations in the quality and comprehensiveness of their monographs.<br />
Discussion<br />
For the daily clinical routine, an easy to use and accurate system is needed, which<br />
identifies drug interactions without the tendency of overalerting. Whereas most databases<br />
were self-explanatory, MMI pharmindex was more complex to use. Drugs.com displayed<br />
the highest rate of excessive alerts. As frequent alerts of clinically irrelevant drug<br />
interactions lead users to ignore the warnings [2], this might be a major caveat to use this<br />
database in the daily clinical routine.<br />
Conclusion<br />
When used appropriately, drug interaction database can help to identify clinically relevant<br />
drug interactions and thereby increase patient safety. However, we should be aware of the<br />
limitations of drug interaction databases. In our evaluation Lexi-Interact proved to be the<br />
most reliable and comprehensive source of drug interaction information.<br />
References:<br />
1. Storka, A. et al, Wiener Medizinische Wochenschrift, 2009. 159(17-18): 462-469.<br />
2. Payne, T.H., et al., Proceedings / AMIA . Annual Symposium. 2002: 602-606.<br />
161<br />
Applicability of different omeprazole formulations via enteral feeding tubes<br />
Hanke, U. 1, Iber, S. 1, Wedemeyer, R.-S. 2, Blume, H. 2, Weitschies, W. 1<br />
1 University of <strong>Greifswald</strong>, Center of Drug Absorption and Transport, Institute of Pharmacy,<br />
Department of Biopharmaceutics and Pharmaceutical Technology, Felix-Hausdorff-Street 3, 17487<br />
<strong>Greifswald</strong>, Germany<br />
2 SocraTec R&D Concepts in Drug Research and Development GmbH, Im Setzling 35, 61440<br />
Oberursel, Germany<br />
For patients with swallowing disorders or other diseases that preclude the oral route of food<br />
intake the use of enteral feeding tubes is necessary. Often the administration of protonpump<br />
inhibitors (PPI) like omeprazole is required for these patients to avoid stress-related<br />
mucosal bleeding or acid pneumonitis [1]. Unfortunately, there is still a lack of knowledge<br />
regarding the handling of drug application via feeding tubes. The aim of this study was to<br />
investigate the ability of three different omeprazole formulations (Nexium ® mups 40 mg,<br />
enteric coated tablets containing micropellets (Astra Zeneca GmbH), Omeprazol Hennig ®<br />
40 mg (Hennig Arzneimittel GmbH & Co. KG) and OMEP ® 40 mg (Hexal AG), both<br />
capsules with enteric coated pellets) to pass enteral feeding tubes in order to obtain<br />
recommendations for an appropriate application of these formulations via this route of<br />
administration.<br />
The feasibility of application was tested using two different feeding tubes (representatively<br />
for percutaneous endoscopic gastrostomy (PEG): Freka ® PEG-Set gastral CH 9, length:<br />
30 cm, 1 terminal port; representatively for nasogastric tubes: Freka ® Intestinale Sonde<br />
134 Poster
CH 9 for PEG CH 15, length: 120 cm, 2 lateral, 1 terminal port; both Fresenius Kabi AG).<br />
As application media served tap water, boiled tap water and different commercially<br />
available still mineral waters. The used media were analysed regarding pH, buffer capacity,<br />
and electrical conductivity. Diameter, swelling behaviour, and stability of the pellets over<br />
480 min were also investigated. To study the application of the pellet formulations via<br />
feeding tubes an appropriate application protocol had to be established. Briefly, the tubes<br />
were washed with 30 mL test fluid before and after the application. Pellets from one opened<br />
capsule or one tablet were administered together with 30 mL of the test fluid by using<br />
50 mL catheter syringes with included Luer adaptors. The contents were slowly injected<br />
through the tubes while gently shaking the syringe to avoid obstructions. For a complete<br />
removal of the Omeprazol Hennig ® and OMEP ® pellets out of the syringe the use of further<br />
60 mL of additional test fluid was necessary. The contents of the syringes were injected into<br />
the tubes either directly or after 1 h of rest. After tube passage the administered<br />
suspensions were collected, decanted, solved in 100 mL 0.1 M NaOH and further diluted.<br />
The analyses of the diluted probes (1:20) and calibration measurements were performed at<br />
304 nm using an UV spectrophotometer (Varian Cary 50, Varian Inc., Palo Alto, USA).<br />
Pellets of Nexium ® mups were small enough to pass all tubes without any problems. Pellets<br />
of OMEP ® only were able to pass the PEG without using the Luer adaptor. The application<br />
through the nasogastric tube immediately resulted in obstructions. Omeprazol Hennig ®<br />
pellets passed the PEG without any difficulties, the nasogastric tube could only be passed<br />
immediately after preparation of the suspension without using the Luer adaptor. This may<br />
be attributed to the swelling of the pellets during 1 h of rest. The swollen pellets had a<br />
higher tendency to agglomerate. For all media comparable results were obtained. For the<br />
latter two formulations nasogastric tubes with CH > 9 need to be tested.<br />
In conclusion, the ability of omeprazole formulations to pass through a feeding tube is<br />
extremely dependent on the properties of the formulation and the used liquid as well as on<br />
the type of feeding tube. Micropellets as used in Nexium ® mups are small enough to pass<br />
even small feeding tubes like nasogastric tubes without any problems. The bigger the pellet<br />
diameter, the higher is the risk of obstruction. Also the shape of the pellets has an impact<br />
on the administration as non spheric formed pellets more often tend to agglomerate inside<br />
the tube than spheric ones. Short tubes with only on terminal port like PEGs showed less<br />
obstructions compared to long tubes with lateral ports like nasogastric tubes. Generally, all<br />
omeprazole formulations should be administered slowly and under continuous shaking of<br />
the syringe immediately after preparation of the solution. Tube washing should be<br />
performed before and after the administration of drugs.<br />
References:<br />
1. Johnson, J. L. et al.: Am. J. Health-Syst. Pharm. 2008, 65: 2324-2325.<br />
2. Devlin, J. W. et al.: Ann. Pharmacother 200, 39: 1667-1677.<br />
162<br />
Nanostructured Lipid Carriers (NLC): The optimum concentration and<br />
production condition<br />
Junmahasathien T. 1; Müller R. H. 1; Keck, C. M. 1 2<br />
1 Institute of Pharmacy, Department of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Freie<br />
<strong>Universität</strong> Berlin, Kelchstrasse 31, 12169, Berlin, Germany<br />
2 Applied Pharmacy Division, University of Applied Sciences Kaiserslautern, Campus Pirmasens,<br />
Carl-Schurz-Strasse 10-16, 66953 Pirmasens, Germany<br />
It is interesting that there is a finished product (lipid matrix) which is the mixture of oils, fatty<br />
alcohol and vegetable waxes (Polyglyceryl-2 Dipolyhydroxystearate, Octyldodecanol,<br />
Carnauba Wax, Candellila Wax, Beeswax, Cetearyl Glucoside and Cetearyl Alcohol) It can<br />
be used as NLC.<br />
Nanostructured lipid carriers (NLC) have been developed to overcome the disadvantages<br />
of solid lipid nanoparticles (SLN). Solid lipid mixed with liquid lipid, to decrease the degree<br />
of perfect crystal of lipid matrix.[1,2] The aim of this study was to develop a new<br />
formulation, e.g. the NLC-preconcentrate, containing high amounts of small sized NLC. For<br />
this propose, a new lipid matrix was used and the influence of the lipid concentration (10-<br />
30%) including the number of high pressure homogenization cycles (3, 5 and 10 cycles) on<br />
the particle size and physical stability were investigated. The formulation consisted of a new<br />
lipid matrix, Plantacare® 2000UP 2% as a stabilizer and water. Each formulation was<br />
premixed by an Ultra-Turrax at 8,000 rpm for 30 seconds and produced at 80°C using high<br />
pressure homogenization (HPH) at 500 bar. The results showed that the increase in the<br />
lipid concentration led to a larger particle size. More cycles led to smaller particles. The<br />
smallest particle size (145 nm) was obtained from the formulation containing 10% lipid<br />
which produced by applying 5 cycles of HPH. The maximum lipid concentration was 25%.<br />
Samples containing higher concentration, e.g. 30% lipid content, solidified during the<br />
production. The physical stability was affected by the lipid content, e.g. a higher lipid<br />
content led to a faster increase in particle size over time. Formulations containing 10% lipid<br />
remained physically stable over time. In conclusion, a new NLC formulation was<br />
successfully developed and optimized in this study. The optimum lipid concentration for<br />
producing this NLC is 10%.The optimal production conditions are 5 cycles of HPH, 500 bar<br />
at 80°C.<br />
Acknowledgments: UPM Handelsgesellschaft mbH, Haupstrasse 47 D-21447 Handorf Germany, PharmaSol<br />
GmbH, Blohmstrasse 66A 12307 Berlin Germany<br />
References:<br />
1. Teeranachaideekul V., et al.: Eur. J. Pharm. Biopharm. 2007, 67: 141-148.<br />
2. Müller R. H., et al.: Adv Drug Deliv Rev. 2007, 59: 522-530.<br />
163<br />
Regulations of Preclinical Studies and Clinical Trials in the Former GDR<br />
Retzar A., Friedrich C. 1<br />
1 Institute for the History of Pharmacy, Philipps-<strong>Universität</strong> Marburg, Roter Graben 10, 35032<br />
Marburg, Germany<br />
The poster deals with the legal conditions of preclinical studies and clinical trials in the<br />
former German Democratic Republic.<br />
Since 1949 it was determined that introducing pharmaceuticals was not permitted without<br />
registration. The decision on registration was made by the Ministry of Health based on the<br />
recommendation of the so-called ‘Zentraler Gutachterausschuss für Arzneimittelverkehr<br />
(ZGA)’, a commission of clinicians, pharmacologists, pharmacists and representatives from<br />
industry.<br />
In 1960 a directive regulated the procedure of registration, which required the submission of<br />
pharmacological and clinical surveys.<br />
In 1964 the Medicinal Products Act was established containing the obligation to prove the<br />
effectiveness and inoffensiveness of new drugs in preclinical studies and clinical trials<br />
before they were launched. Under the new regulations approval by the ZGA was necessary<br />
to conduct clinical trials. Therefore, an application had to be filed, which provided<br />
information about extent, methods and responsibilities of the trial.<br />
Due to increasing requirements in the field of drug studies the regulations had been<br />
revised. Consequently, the 12th implementing rule of the Medicinal Products Act came into<br />
effect in 1976. From now on drug studies were subdivided into 4 levels. Measures of postmarketing<br />
surveillance in level no. 4 were determined to detect adverse drug reactions.<br />
Acknowledgments: Institute for the History of Pharmacy, Ariane Retzar.<br />
References:<br />
Richter, J., Keune, H. G.: Arzneimittelrecht der DDR. Kommentar Teil I (Akademie-Verlag Berlin) 1972.<br />
Hackenberger, F., Koch H.: medicamentum 1977, 18(5): 130–135.<br />
164<br />
Pharmacy under the sea – The medical supply of the German „U-Boot-Waffe“<br />
during the World Wars<br />
Vongehr, F. 1; Friedrich, C. 1;<br />
1 Institut für Geschichte der Pharmazie der Philipps-<strong>Universität</strong> Marburg, Roter Graben 10, 35032<br />
Marburg, Germany<br />
Despite the fact that German submarines have been a common subject of military-historical<br />
research and literature, the medical service aboard which was determined by the special<br />
environmental conditions is a rather neglected aspect. However, it is known that<br />
complicated surgery such as amputations could be performed even in dived submarines,<br />
although the crews had to deal with severe problems like climate, humidity and narrowness.<br />
The question arises how the medical supply was organized.<br />
Beginning with the first German submarine „U 1“ commissioned in 1906 the new boats<br />
were only equipped with a few remedies, such as disinfecting ointment and acetylsalicylic<br />
acid, but during Wolrd War I the experience lead to the conclusion to extend the selection<br />
of drugs on the boats. Therefore, the navy issued a compendium, the so-called „Ärztlicher<br />
Ratgeber für Unterseeboote“, containing a more detailed medical supply and several<br />
instructions for the commanders, who mostly had to operate their boat without a physician.<br />
During World War II the „Kriegsmarine“ had learned from the experiences of the<br />
„Kaiserliche Marine“ and published revised editions of the „Ärztlicher Ratgeber“ in the<br />
1940s. The medical supply was now significantly augmented and several boats even had<br />
physicians among the crew. Especially submarines designed for tactical operations in<br />
remote areas carried an extensive supply of drugs, including anti-malaria drugs and<br />
sulfonamides.<br />
The allies confiscated important documents regarding the German „U-Boot-Waffe“ including<br />
logfiles and medical data for their own use after victory. It had lasted several decades until<br />
the documents were returned and opened for research. The historical sources regarding<br />
pharmaceutical questions aboard the German submarines are now an object of detailed<br />
research.<br />
165<br />
Market surveillance study to medical products with pyridostigmine:<br />
tetramethylurea contamination in tablets and injections relevant to regulatory<br />
legislation for toxicological concerns<br />
Bogan R; Zimmermann T<br />
Central Institute of the Bundeswehr Medical Service, Ingolstädter Landstraße 102, 85748 Garching<br />
Hochbrück<br />
Pyridostigmine is a well known Active Pharmaceutical Ingredient (API) described in<br />
monographs of the European, the British and the United States Pharmacopeia.<br />
Nevertheless in previous studies we elucidated a hitherto new degradation pathway of<br />
pyridostigmine leading to formation of tetramethylurea (TMU) [1,2]. Based on these data we<br />
analysed marketed medical products with API pyridostigmine for the occurrence of TMU.<br />
The samples tested were within and after expiry date. Dosage forms of tablets and<br />
injections were considered. As shown before the compendial method of the European<br />
Pharmacopeia could be used for detection and quantification of TMU.<br />
We examined four batches of tablets in two dosage strengths and two batches of injections<br />
in one dosage strength from two marketing authorization holders. Samples were analysed<br />
two times in a distance of 24 months; in between they were stored at the recommended<br />
condition below 25°C. At the start of the study the remaining time to the expiry date of 5<br />
batches was between 5 and 48 months. For comparison reason one batch 7 months after<br />
expiry date was included. At the retest time 5 batches exceeded the expiry date for 4 to 31<br />
months whereas 1 batch was still 24 months before expiry date. At the first examination<br />
TMU was detected in one batch of tablets 9 months before expiry date and in the one batch<br />
of injections which had exceeded the expiry date for 7 months. The second examination<br />
revealed an additional batch with TMU and a marked increase of TMU content in those<br />
batches which were already contaminated. The highest contamination was found in one<br />
batch of tablets with 0.36 % (area percent related to API) 9 months before expiry date and<br />
0.51 % resp. 15 months after expiry date.<br />
The study proved that the predicted degradation pathway also occurs in market authorized<br />
medical products. TMU contamination seems to be in particular a problem at the end or<br />
after expiry date. Nevertheless the occurrence of TMU even within expiry date was shown<br />
and has to be considered. The values measured exceed the threshold values for<br />
degradation products of the ICH [3]. Toxicological evaluation does not suggest acute<br />
toxicity in the relevant dose range [4,5], but in terms of chronic toxicity the risk of a<br />
teratogenic effect of TMU can not be excluded [6,7].<br />
As a conclusion of our findings efforts on a revision of the relevant monograph<br />
pyridostigmine in the European Pharmacopeia as well as regulatory provisions are in<br />
process.<br />
References:<br />
1. Bogan, R. Loch, M.: Wehrmed. Mschr. 2010, 54: 242-247<br />
2. Bogan, R.: Joint meeting of the austrian and german pharmaceutical societies, abstract volume, 2011, PS<br />
001: 86<br />
3. International conference on harmonization of technical requirements for registration of pharmaceuticals<br />
for human use, ICH Harmonised Tripartite Guideline Q3B, Impurities in new drug products, 2006<br />
4. Dixon, R.L. et al.: Arch. Int. Pharmacodyn. Ther. 1966, 160: 333<br />
5. Edler, L. et al.: Food and chemical toxicology. 2002, 40: 283-326<br />
Poster 135
6. Munley, S.M. et al.: Drug. Chem. Toxicol. 2001, 24: 259-271<br />
7. Teramoto, S. et al.: Teratology. 1981, 23: 335-342<br />
166<br />
Improved taste evaluation of pharmaceutics using food sensors?<br />
Eckert, C., Pein, M.<br />
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine University, Duesseldorf,<br />
Germany<br />
Introduction: Two electronic taste sensing systems are commercially available. Whereas<br />
one sensor set with seven sensors related to specific gustatory stimuli is offered for the<br />
Japanese taste sensing system (Insent), Alphamos (France) offers two sensor sets<br />
containing seven cross-selective sensors for the αAstree. The two sensor sets are<br />
recommended for different applications. While sensor set 2 is marketed for pharmaceutical<br />
applications, sensor set 5 is recommended for taste evaluation of food. So far, this<br />
differentiation has not been questioned. Therefore, two active pharmaceutical ingredients<br />
(APIs) and two different lozenges were characterised using these two sensor sets.<br />
Materials and methods: Caffeine citrate (DAC) was purchased from Fagron (Barsbuettel,<br />
Germany), quinine hydrochloride (Ph. Eur.) from Caesar&Loretz (Hilden, Germany). Both<br />
APIs were determined in concentration ranges from 0.05 mM to 50 mM in 12 levels. Both<br />
lozenges were evaluated using different sample weights (0.5-12 g/100 ml).The samples<br />
were dissolved in aqua purificata and measured using the αAstree equipped with sensor<br />
sets 2 and 5. The measurement setup was used according to Pein [1].<br />
Results and discussion: Each sensor of both sensor sets fulfils the repeatability<br />
requirements [2] with RSD values
34 patients (BMI < 20: 4, 20-25: 17, > 25: 13). In 18 patient charts the documentation of<br />
bodyweight and height was missing. As an index for renal function the GFR was evaluated<br />
from serum creatinine. 25 (48.1%) patients showed a GFR > 60 ml/min/1.73 m², 21 (40.4%)<br />
patients between 30-59 ml/min/1.73 m² and 6 (11.5%) patients < 30 ml/min/1.73 m².<br />
Potassium levels as an important electrolyte for cardiac functions were for almost all<br />
patients in a normal range (3.5-5.1 mmol/l).<br />
CONCLUSION: Our results confirm the study of Groth-Tonberge et al. concerning fallpromoting<br />
drugs on a cardiological ward [2]. Average age, medication per patient and most<br />
prescribed medication groups (diuretics, beta blockers, angiotensin-converting-enzyme<br />
(ACE) inhibitors, antibiotics) were in good agreement and a potential relation between falls,<br />
polypharmacy and higher age could be shown. Additionally, considering the fact that more<br />
than 50% of the patients also had an impaired kidney function (GFR < 60 ml/min/1.73 m²)<br />
the importance of pharmaceutical care focused on both polypharmacy and adequate drug<br />
dosing is evident and could help to prevent fall events.<br />
References:<br />
[1] Pierobon, A., Funk, M. Sturzprävention bei älteren Menschen. Risiken–Folgen–Maßnahmen (Georg<br />
Thieme Verl.) 2007.<br />
[2] Groth-Tonberge, C., Feuchtinger, J., Strehl, E. Krankenhauspharmazie 2011, 32(11): 565.<br />
[3] Levey, A. S. et al. Ann Intern Med 2009, 150(9): 604.<br />
170<br />
Development of lipid micropellets with metformin hydrochloride using solventfree<br />
cold extrusion/spheronization<br />
Petrovick, G.F.; Breitkreutz, J.<br />
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, 40225 Duesseldorf,<br />
Universitaetsstrasse 1, Germany<br />
The diabetes mellitus type 2 (DM2) constitutes 85 to 95% of all diabetes cases in<br />
developed countries and has reached epidemic proportions in several nations [1, 2].<br />
Metformin is the drug of choice to treat DM2 in many countries [3, 4]. But the relatively large<br />
size of available tablets (doses form 500 to 1000 mg) can cause difficulty to swallow<br />
(especially in elderly patients) which may lead to discontinuation of treatment. Considering<br />
this problem, microparticulate systems have advantages when compared to tablets or<br />
capsules [5]. Solid lipid extrusion in a twin-screw extruder is an innovative preparation<br />
method for matrices in which lipids are treated below their melting ranges in a thermomechanical<br />
process. The drug is dispersed in a lipid matrix in which the lipid does not melt<br />
but softens [5, 6].<br />
A hard fat (Witocan ® 42/44) was used as lipidic binder. The extrusion was performed at 33<br />
°C in a Micro 27GL-28D extruder (Leistritz, Nuremberg, Germany) with co-rotating twin<br />
screws, using a screw plate with 91 dies of 0.5 mm and 1.35 mm length, with a powder<br />
feed rate of 40 g/min. The extrudates were spheronized (Schlueter spheronizator RM 300,<br />
Schlueter, Neustadt am Ruebenberge, Germany) for 15 min at 1500 rpm and 33 ± 1 °C.<br />
Metformin hydrochloride lipid micropellets (figure 1) with a high drug load of 80% presenting<br />
narrow particle size distribution and aspect ratio under 1.2 could be obtained. The process<br />
parameters such as pressure and product final temperature were also evaluated (figure 2).<br />
Spheronization was difficult as the process temperature range was difficult to control.<br />
Friction and heat conveying were major issues as they influence processability and product<br />
quality.<br />
Solvent-free cold extrusion followed by spheronization present itself as a robust method to<br />
obtain high dosage micropellets of metformin with adequate size and shape characteristics<br />
which are mainly impacted by the spheronization step.<br />
Figure 1: Micropellets with 80%<br />
metformin hydrochloride<br />
Figure 2: Process parameters<br />
References:<br />
1. Zimmet, P.Z. et al., J Diabetes Complicat 1997, 11(2): 60-68.<br />
2. Giannella-Neto, D.; Gomes, M.B. Diabetol Metab Syndr 2009, 1(1): 1-3.<br />
3. Gregorio, F. et al., Arch Gerontol Geriat 1996, 22(1): 161-170.<br />
4. Soto, A. et al., Endocrinol Nutri 2008, 55(2): 39-52.<br />
5. Breitkreutz, J.; Boos, J., Eur J Pharm Biopharm 2007, 56, 255-260.<br />
6. Witzleb, R. et al., Powder Technol 2011, 207, 407-413.<br />
171<br />
Development of a simple, rapid and reliable assay to count enterococcus<br />
faecium with a CASY coulter counter to assess antibiotic therapy<br />
Goebgen E; Wicha SG; Kloft C<br />
Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin,<br />
Kelchstraße 31, 12169 Berlin, Germany<br />
OBJECTIVES: Determination of bacterial counts plays a key role when the interaction<br />
between bacteria and antibiotic are investigated, i.e. to assess the potency of a drug,<br />
evaluate drug combinations or improve dosing schedules. Cell counts of bacteria are<br />
usually determined by densitometry or plate counting methods. As densitometry is highly<br />
imprecise and plate counting methods are time-consuming, cost intense and require long<br />
preparation times, there is a clear need for an easier method to quantify bacteria. In 1953,<br />
W. Coulter developed the technique of electronic cell counting by measuring the change in<br />
resistance for blood cell counting. About 50 years ago, Kubitschek [1] and Swanton et al.<br />
[2] demonstrated the utility of the Coulter Counter in cell counting of bacterial cells and the<br />
determination of cell volumes in growing cultures of bacteria. In comparison to the standard<br />
methods, electronic cell counting promises to save time, to reduce costs whilst providing a<br />
higher content of information (e.g. cell counts, cell volume and distribution of cell volume)<br />
[1,2]. The objective of the current analysis was to establish an assay on a CASY Coulter<br />
Counter as a fast and simple electronic cell counting method for E. faecium to assess<br />
antibacterial effects in vitro.<br />
METHODS: The Coulter Counter (CASY 1, Roche Diagnostics) included a pump, an<br />
aperture tube (60 µm) and the electronic components: one electrode inside the tube, one<br />
outside and the voltage pulse measuring unit. The number of bacteria is determined by<br />
measuring the change in resistance when a particle passed the aperture. Additionally, the<br />
amplitude of the pulse is proportional to the size and the volume of the particle. To<br />
decrease background-counts, all fluid media were filtered through a 0.2 µm filter. An overnight<br />
subculture of E. faecium (ATCC 700221) was used as stock suspension with an initial<br />
inoculum size equal to McFarland 3 in 0.9% NaCl. Serial dilutions in phosphate buffered<br />
saline with peptone (PBSP) were prepared. Dependent on the bacterial concentration of the<br />
suspension, samples of 10, 100 or 1000 µL were measured in 10 mL filtered, ready-to-use<br />
measuring liquid (Casy ton, Roche Diagnostics). The electronic counting method was<br />
compared to the standard plate count method [3]: 100 µL of each suspension was diluted<br />
and poured on Columbia agar plates. Plates containing 30 – 500 colony forming units<br />
(CFU) were read.<br />
RESULTS: The mean diameter range of E. faecium correlated well with the physical<br />
measuring range of the capillary from 1.2 µm to 45 µm. McFarland standard 3 was<br />
equivalent to 2.2x10 8 colony forming units (CFU)/mL. Analogues to a bioanalytical method,<br />
the performance of this electronic cell count assay was evaluated according to the EMA<br />
guideline [4]. Current investigations suggest agreement with the guideline with respect to<br />
linearity, accuracy and precision of the bacterial cell counts with a lower limit of<br />
quantification of 2.2x10 4 CFU/mL.<br />
CONCLUSIONS: A rapid, precise and simple assay for electronic cell counting was<br />
established and allows for bacterial cell counting of E. faecium in PBSP with a sample<br />
volume up to 1 mL. Thus further studies using the Coulter Counter with a smaller aperture<br />
tube for other bacterial species such as E. coli or P. aeruginosa are indicated. Bacterial<br />
growth experiments, in-process controls and the measurement of postantibiotic effects<br />
could be possible applications for the more efficient Coulter Counter principle so as to<br />
streamline the clinical and preclinical development of an antibacterial agent and to facilitate<br />
decision taking in the development of dosing schemes.<br />
References:<br />
1. Swanton EM, Curby WA, Lind HE: Appl. Environ. Microbiol. 1962, 10(5): 480-485.<br />
2. Kubitschek HE: Nature. 1958, 182: 234-235.<br />
3. Schwalbe R, Steele-Moore L, Goodwin AC: Antimicrobial susceptibility testing protocols (CRC Press)<br />
2007.<br />
4. EMEA/CHMP/EWP/192217/2009<br />
172<br />
Cost of drugs in the ambulatory health care sector – how much do we really<br />
spend?<br />
Aßmann, G. 1 2; Fiß, T. 1; Marschall, P. 2; Fleßa, S. 2; Hoffmann, W. 3<br />
1 German Center for Neurodegenerative Diseases (DZNE), site Rostock/<strong>Greifswald</strong>; Ellernholzstr.<br />
1/2; 17487 <strong>Greifswald</strong>, Germany<br />
2 University of <strong>Greifswald</strong>, Faculty of Law and Economics, Friedrich-Loeffler-Str, 70, 17489<br />
<strong>Greifswald</strong>, Germany<br />
3 University of <strong>Greifswald</strong>, Institute for Community Medicine; Ellernholzstr. 1/2 , 17487 <strong>Greifswald</strong>,<br />
Germany<br />
Introduction: As a result of demographic change the prevalence of age-related diseases<br />
increases. Due to the associated intensive drug therapy even in this part the German health<br />
system faces increasing costs and major challenges. Currently there is a lack of knowledge<br />
about daily drug costs in the ambulatory setting. Our aim was the calculation of daily costs<br />
for regularly taken drugs (prescribed and over-the-counter) in the AGnES-home (AGnES:<br />
GP-supporting, community-based, e-health-assisted systemic intervention) visit cohort.<br />
Additionally we focussed on associations between medication costs and sociodemographic<br />
parameters.<br />
Methods: Basis of our analysis was a subsample of the AGnES-home visit cohort who<br />
received a home medication review between 2006 and 2008 (n=778 patients with n=4985<br />
regularly taken drugs). Current drug costs were added automatically at the time of data<br />
recording based on the German drug index. Missing prices were completed by mean DDDcosts<br />
which were extracted from the German Drug Report. Sociodemographic criteria were<br />
taken from the initial proband interview.<br />
Results: Mean age of all participants was 78.8 years, women were significantly older<br />
(women n=555; 71.3%, 79.9 years, p
Herbal medicinal products containing ethanol are widely used in children [1], since ethanol<br />
it is an excellent extraction solvent for the phytochemical components of herbal drugs and<br />
contributes to the stability of fluid preparations. Toxicological and pharmacokinetic<br />
evaluations [2] have shown that the small amounts of ethanol applied with therapeutic<br />
doses of these medicines are safe even in this age group. Despite these medicines have<br />
been used safely since many decades, they occasionally are subject of discussion in the<br />
public, triggered by the increasing problem of recreational misuse of alcoholic beverages by<br />
children and young persons [3, 4]. Therefore, there is a growing need of a systematic<br />
evaluation of epidemiological data on these medicines.<br />
For an evaluation of the experience gained from the therapeutic use of these medicines, 17<br />
pro- and retrospective studies with 10 different herbal medicinal products containing<br />
ethanol at doses of 40 to 240 mg per single application, depending on the age group, have<br />
been analyzed. These studies cover 50,425 patients between 0 and 12 years of age. In<br />
these studies, altogether 15 adverse drug effects have been described, none of which was<br />
attributable to the ethanol content of the medicines.<br />
In a worldwide survey including these and further 6 herbal medicinal products it was shown<br />
that during the past few years, more than 764 millions daily doses have been sold,<br />
corresponding to more than 33 millions patients (according to data obtained from<br />
manufacturers; figures are available partly from 1993 onwards, partly from 2003/4<br />
onwards). From the packages sold in Germany in the years between 2005 and 2009, 48.1<br />
millions were attributable to self-medication, and 10.8 millions to prescriptions reimbursed<br />
by health insurance (IMS, Frankfurt). As non prescription medicines are reimbursed in<br />
Germany only in children, at least the latter part of the prescriptions can be attributed to<br />
children to an extent of at least 90 %. All of these medicines are registered or licensed by<br />
regulatory authorities, so that adverse effects are covered by the pharmacovigilance<br />
system. No adverse effects attributable to the ethanol content have been reported.<br />
This set of pharmacovigilance data supports the conclusion drawn from the experience of a<br />
safe use over decades, i.e. that the ethanol content of herbal medicinal products does not<br />
give any causes for concern regarding their safety even in children.<br />
Dedication in memoriam: This contribution is dedicated to Prof. Dr. Hilke Winterhoff, Institute for<br />
Pharmacology and Toxicology, University of Münster, Germany, who died on 9 May 2010. She has initiated<br />
and supported this work.<br />
References:<br />
1. Gundermann, K.-J. et al., Neurogastroenterol Motil 2010, 22(S1): 69.<br />
2. Kelber, O. et al., Pharm Ind 2008, 70: 1124-1128.<br />
3. Gemeinsamer Bundesausschuss (G-BA), Bundesanzeiger 21 vom 08. Februar 2011.<br />
4. HMPC, 2010. Reflection paper. EMA/HMPC/85114/2008.<br />
174<br />
Oral mesalazine treatment of ulcerative colitis – a review of current dosage<br />
forms<br />
Kersten E1, Klein S1 1<strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong> University <strong>Greifswald</strong>, Department of Pharmacy, Institute of Biopharmacy and<br />
Pharmaceutical Technology, C_DAT, Felix-Hausdorff.Str. 3, 17489 <strong>Greifswald</strong><br />
Ulcerative colitis (UC) is a chronic and debilitating illness prevalent in the Western<br />
population characterized by chronic inflammation of the colon that is continuously<br />
distributed in the colonic mucosa. Most of the currently available agents used in UC<br />
treatment, e.g. mesalazine, are aimed to downregulate the chronic inflammation in the<br />
intestinal mucosa. The main principle of mesalazine action is a topical reduction of<br />
inflammation in the mucosa. Thus, oral mesalazine dosage forms should release the active<br />
substance selectively at the inflamed areas in the GI tract. For a long time marketed enteric<br />
coated formulations containing 250 – 500 mg of the active drug represented the<br />
formulations of choice for this purpose. However, since average daily mesalazine doses are<br />
typically > 3g/ multiple daily dosing is required. Thus, more recently, several new<br />
formulations including coated multiparticulates and an extended release tablet formulation<br />
containing much higher drug loads were released to the market to improve both<br />
effectiveness and patient compliance.<br />
To get an idea, if the newer types of formulations do represent a benefit with regard to<br />
improving UC therapy, we studied drug release of the various formulations with a pHgradient<br />
method that has been established as a predictive in vitro model in earlier studies<br />
(Klein 2005, 2008). Experiments were performed with a BioDis ® Release Rate Tester. In<br />
contrast to simulating only the recommended dosing conditions, i.e. fasted gastrointestinal<br />
(GI) pH-conditions, as has been done in previous studies, in the present study both fasted<br />
and fed state dosing conditions were addressed. Experiments were also performed with the<br />
official mesalazine test methods described in the United States Pharmacopoeia (USP) to<br />
screen, if the could also come along with a in vivo predictivity.<br />
Results obtained in our experiments clearly show that the site and extent of drug release<br />
are strongly dependent on the type of formulation whereas the drug load has a minor<br />
impact on the release pattern. Unexpectedly, drug release from all enteric dosage forms<br />
was not much affected by the different pH-conditions in the fasted and fed GI lumen.<br />
However, as expected, results from the compendial test setup were different from those<br />
obtained with the biorelevant pH-gradients and therefore will not really be predictive for in<br />
vivo drug release.<br />
Results indicate that none of the marketed dosage forms releases the active drug<br />
selectively in the colon. Moreover, as the marketed formulations show significant<br />
differences in their release profiles, the choice of the dosage form should be based on an<br />
individual basis. The enteric formulations were not prone to the pH-conditions used to<br />
simulate fed state gastric residence. However, it should be kept in mind that beside the pHconditions,<br />
other physicochemical and mechanical aspects can affect drug release,<br />
particularly when comparing fasted vs. fed state dosing conditions. As particularly, the<br />
tablet formulations are expected to be sensitive to a variety of these factors, this will be<br />
addressed future studies.<br />
References:<br />
Klein, S., J. Stein, and J. Dressman, J Pharm Pharmacol, 2005. 57(6): 709-19.<br />
Klein, S., et al. J Contr Release, 2008. 130(3): 216-219.<br />
175<br />
ATR-IR-Imaging of Irbesartan Generic Tablets - Comparison with the Originator<br />
Norwig, J.; Jansen, R.<br />
Bundesinstitut für Arzneimittel und Medizinprodukte (BfArM), Kurt-Georg-Kiesinger-Allee 3, D-<br />
53175 Bonn, Germany<br />
According to the CTD format applicants for a marketing authorisation are requested to<br />
declare the exact composition of the dosage form and of the whole product.[1] Modern<br />
ATR-IR-Imaging allows a full identification of the constituents without any sample<br />
preparation for a chromatographic method in the conventional sense.[2]<br />
Neither an extraction, nor a separation, nor any chromatographic method is required. In<br />
case of Irbesartan tablets manufacturers from Far East could be checked whether the<br />
composition complies. Moreover it is estimated which content and which particle size the<br />
active substance shows in comparison with the originator.<br />
The imaging is an impressive powerful method to check the compliance with the<br />
declaration. The time consumption is estimated to be 1 h.<br />
However impurities in the 0.1 percentage range could not be identified. Therefore a trace<br />
analytical procedure should be combined with the method for full compliance with the<br />
Pharmacopoeia.[3, 4]<br />
Further images show the whole range of excipients.<br />
References:<br />
1. Notice to Applicants, Presentation and format of the dossier CTD in EudraLex, VOLUME 2B, Medicinal<br />
products for human use, Editor: European Commission Directorate General III, Brussels, July 2003.<br />
2. ATR Imaging of Pharmaceutical Tablets, PerkinElmer Life and Analytical Sciences.<br />
3. European Pharmacopoeia 7th Edition 2010, Monograph on Irbesartan.<br />
4. Irbesartan tablets, Monograph US Pharmacopoeia 35, Rockville (<strong>2012</strong>).<br />
176<br />
Lipopeptide-modified carrier systems – the composition determines cell<br />
selectivity<br />
Sydow, K1, Nikolenko, H1, Taraschewski, A1, Dathe, M1 1 Leibniz Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125 Berlin,<br />
Germany<br />
Liposomal und micellar delivery systems have become one of the most promising options<br />
for the improvement of transport efficiency of poorly soluble drugs and for drug targeting.<br />
To increase selective uptake, target recognizing and transmembrane transport-mediating<br />
motifs are used. To achieve these requirements we developed lipopeptides, among them<br />
P2A2[1]. Because of their amphiphilic character, the peptides self-assemble to micelles and<br />
integrate into the lipid bilayer of liposomes. Interestingly, P2A2-micelles and -liposomes<br />
differ in cell selectivity and activate different modes of cellular uptake. To investigate the<br />
role of particle size and surface charge/charge distribution as potential determinants of<br />
particular uptake mechanisms, we synthesized lipopeptides of varying amino acid<br />
composition. Zeta potential measurement and dynamic light scattering were used to<br />
characterize the different micellar and liposomal systems. Cell selectivity was investigated<br />
by uptake studies with endothelial cells of the human aorta (HAoEC) and human brain<br />
microvessels (HBMEC).<br />
All lipopeptides (P2A2, LP1, LP2) differ in amino acid composition but are identical in the<br />
number charged residues. P2A2 and LP1 have both 12 cationic residues, LP2 bears 12<br />
anionic amino acids. All lipopeptides form micelles with an hydrodynamic diameter (d[Vol])<br />
of about 10 nm. The zeta potential of the micelles correlates with the charge properties of<br />
the lipopeptide: P2A2/ LP 1 – positive; LP 2 - negative surface potential. Additionally, three<br />
other peptide-bearing carrier-systems, different in particle size and their molar lipid to<br />
peptide ratio (c l/p) were prepared. Large unilamellar vesicles (LUV, d[Vol] about 90 nm, c<br />
l/p = 1000) and small unilamellar vesicles (SUV d[Vol] about 25 nm, c l/p = 1000 or 20)<br />
have a distinct size distribution. At cl/p = 1000, the zeta potential does poorly distinguish<br />
from the potential of control liposomes. With higher lipopeptide concentration, c l/p = 20, the<br />
zeta potential changes in accordance with the amino acid motif. Uptake studies were done<br />
by using Laser Scanning Microscopy (LSM) and Fluorescence Activated Cell Sorting<br />
(FACS).<br />
All positively charged lipopeptide carriers were internalized into HAoEC and HBMEC.<br />
Negatively charged LP 2 particles were preferentially adsorbed on the cell surface. The<br />
overall interaction efficacy followed the order: micelles > SUV (c l/p = 20) > LUV (c l/p =<br />
1000) > SUV (c l/p = 1000). The uptake of P2A2-micelles into HBMEC was two times<br />
enhanced compared to HAoEC. In comparison to micelles (no cell specificity), LP 1<br />
incorporation into SUV (c l/p = 20) seems to improve cell specificity to HAoEC. A lower lipid<br />
to peptide ratio could enhance uptake of P2A2- and LP 1 - SUV.<br />
Self-aggregation of lipopeptides and binding to liposomes results in efficient cellrecognizing<br />
and uptake mediating systems. Cell-selectivity depends on both the molecular<br />
138 Poster
structure of peptides, the particle size and on the lipid to peptide ratio in liposomal<br />
formulations. The investigated lipopeptides provide a keytool for the development of drug<br />
delivery systems.<br />
References:<br />
1. Leupold, E., Nikolenko, H. & Dathe, M., Biochim. Biophys. 2009, 1788(2): 442-449.<br />
177<br />
Standard doses of intravenous and enteral moxifloxacin provide insufficient<br />
drug exposure for nosocomial pneumonia in intensive care unit patients<br />
Schaeftlein, A. 1 2; Kees, M. 3; Heininger,A. 4; Kloft, C. 1<br />
1Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, FreieUniversitaet Berlin,<br />
Kelchstraße 31, 12169 Berlin,Germany<br />
2and Graduate Research Training program PharMetrX, Germany,<br />
3 Dept. of Anesthesiology and Intensive Care, Charité University Hospital – Campus Benjamin<br />
Franklin, Hindenburgdamm 30, 12200 Berlin, Germany<br />
4 Dept. of Anesthesiology and Intensive Care, University Hospital of Tuebingen, Hoppe-Seyler-<br />
Str.3, 72076 Tübingen, Germany<br />
Background: Moxifloxacin (MOX), a fourth-generation fluoroquinolone,is approved for<br />
treatment of community acquired pneumonia. Due to a broad spectrum and high enteral<br />
bioavailability (>91%) of MOX in healthy volunteers (HV) [1], intravenous/enteral sequential<br />
therapy may also be a suitable treatment option for hospital acquired pneumonia (HAP),<br />
possibly resulting in reduced costs and complications [2].In the current population<br />
pharmacokinetic analysisi.v. and enteral administration of MOX in intensive care unit<br />
patients (ICUP) were compared to determine the clinical feasibility of sequential therapy in<br />
ICUP on a pharmacokinetic basis.Additionally,patient specific characteristics (covariates)<br />
explaining interindividual variability (IIV) on pharmacokinetic (PK) parameters were<br />
investigated, and the simulated exposure of MOX was linked to the efficacybreakpoint of<br />
fluoroquinolonesfor HAP.<br />
Methods: Based on MOX concentration measurements after multiple i.v. and enteral<br />
administrations of 400 mg MOX in 25 ICUP,population PK analysis was performed using<br />
the nonlinear mixed-effect modelling approach (NOMEMTM).Covariates[demographics, coadministered<br />
drugs, state of disease markers] were tested for relevanteffectson the IIV of<br />
absorption rate constant (ka), enteral bioavailabilty, volumes of central and peripheral<br />
distribution and clearance (CL).Based on the final population PK model, 1000<br />
concentration-timecourses were simulated and the AUCtau were usedforprobability of target<br />
attainment functions(fAUC/MICHAP>75[3]), which is indicative for the efficacy and treatment<br />
response of MOX for ICUP with HAP.<br />
Results: Measured MOX concentrations in ICUP were best described and predicted by a<br />
first-order absorption and eliminationtwo compartment PK model. Estimates of CL (11.3<br />
L/h) and volume of distribution(114 L) were in accordance with the literature [4] butF (76%,<br />
95% confidence interval [59%; 94%]) was reduced in ICUP in comparison to HV [1]. IIV on<br />
ka (1.09 1/h) was high (135%) due to a lagging increase of MOX concentrations after<br />
enteral dosing observed in some ICUP which could not be integrated into the final model.<br />
Body weight, age, height, sex, administration of opioids or catecholamines, plasma<br />
proteins, presence of shock, application via gastric tube and sequential organ failure<br />
assessment score [5] had no significant effect on the IIV of MOX PK in ICUP. Only<br />
creatinine clearance (CLCR) effects the elimination of MOX and explained 1/4 of the IIVin<br />
CL, reducing CL by 0.43% for every 1 mL/min decline of CLCR. An acceptable probability<br />
(>0.9) to attain the PKPDtarget for HAP was achieved for only lowMICvalues of0.125<br />
µg/mL or less.<br />
Conclusions: Based on our PK/PD analysis, enteral bioavailability of MOX was little reliable,<br />
limiting the suitability of a switch to enteral therapy in ICUP. In addition, a favourable<br />
outcome of MOX treatment for HAP cannot generally be expected in the investigated<br />
setting, since MIC90values for typical pathogens of HAP (e.g E. coli) exceed 0.125 mg/L.<br />
Hence, unless a highly susceptible HAP pathogen is confirmed, an antibiotic regimen in<br />
ICUP with additional or alternative agents or a dose escalation might be considered.<br />
References:<br />
1. Burkhardt O, Stass H, Thuss U et al.:ClinPharmacokinet 2005, 44(9):969-76.<br />
2. Ramirez JA, Vargas S, Ritter GW et al.:Arch Intern Med1999; 159(20):2449-2454.<br />
3. Ambrose PG,Bhavnani SM, Rubino C et al.:Clin Infect Dis2007, 44(1):79-86.<br />
4. Jeffry AF, Tornøe CW, Brundage R et al.:J ClinPharmacol2011, 51(8):1152-1162.<br />
5. Vincent JL, Moreno R, Takala J et al.: Intensive Care Med1996; 22(7):707-710.<br />
178<br />
Antistatic effect of different silica types on free flowing direct compression<br />
excipients<br />
Knop K.; Martens A.;Sölter V.<br />
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, <strong>Universität</strong>sstr. 1,<br />
40225 Düsseldorf, Germany<br />
When powders are handled, e.g. during sieving, mixing, or powder flow through a<br />
hopper,electrostatic charging may occur. Problems in the process of capsule filling or<br />
tableting can be a result of this electrostatic charge [1]. Antistatic agents such as<br />
magnesium stearate, talc, polyethylene glycolsor silicon dioxide are added to prevent these<br />
problems. The aim of the present study was to evaluate the influence of different types of<br />
silicon dioxide on the electrostatic charging of free flowing powders which are used for<br />
direct compression.<br />
Three free flowing direct compression excipientsFlowlac ® 100 (lactose monohydrate),<br />
Parteck ® M 100 (mannitol), and Ludiflash ® (co-processed mannitol with crospovidone,<br />
polyvinylacetate andpovidone) were investigated using the flowability test apparatus no. 1<br />
equipped with nozzleno. 3 (25 mm opening) described in Ph.Eur. 2.9.16 “Flowability”.The<br />
excipients were equilibrated to environmental conditions (21°C, 45% rel. humidity) for at<br />
least 48 h. Six different types of silicon dioxide were added in concentration of 0.5 and 1%:<br />
Hydrophilic (Aerosil ® 200 and 300), hydrophobic (Aerosil ® R972), and granulated fumed<br />
silica (Aeroperl ® 300) as well as porous silica (Syloid ® 244FP and Sylysia ® 350FCP).All<br />
types were tested in undried and dried status (105°C) to take the different water content<br />
into account. The powders were mixed in a Turbula ® mixer for 5 min. The flow rate was<br />
registered by the weight gain per time on a balance and the electrostatic potential was<br />
measured by an electrostatic sensor, focussed on the powder-stream in a well defined<br />
distance. All measurements were carried out in fivefold in an air-conditioned room at 21°C<br />
and 45% relative humidity.<br />
Pure Flowlac ® showed a high flowability and a negative electrostatic charge of about -0.5<br />
kV. The flowability was only slightly improved by the addition of silica. The electrostatic<br />
charge could not be reduced by the addition of the powdered fumed silica but by the<br />
granulated fumed silica and the porous silica types.<br />
Parteck ® M without additives was negatively charged to -1.5 kV during flow. This high<br />
voltage could be lowered to about -0.3 kV by the fumed silica types. The addition of the<br />
porous silica types led to a change in polarity and a charge of about +0.3 kV. The flowability<br />
of Parteck ® was worse than that of Flowlac ® but could be improved by the addition of each<br />
type of silica.<br />
Ludiflash ® was positively charged during flow up to +0.5 kV. All silica types with the<br />
exception of the granulated type reduced the charging to values around zero. Ludiflash ®had<br />
a good flowability like Flowlac ®. The addition of fumed silica led to a decline in flow whereas<br />
the porous silica had no influence.<br />
Neither the concentration of the silica in the powder mixture (0.5 or 1%) nor the water<br />
content of the silicon dioxides (dried or undried) showed a consistent influence onthe<br />
electrostatic charge and the flowability of the powder mixtures. In the investigated rage,<br />
both factors were only of minor influence.<br />
The choice of the best antistatic agent from the group of silicon dioxides has to be made<br />
individually for every powder or powder mixture. The porous silica types showed a charge<br />
reduction for all three investigated systems whereas the powdered fumed silica failed in the<br />
case of Flowlac ®.<br />
References:<br />
1. Szendeleit S.M., Elektrostatische Aufladung im Prozess der Tablettierung, Diplomathesis,<br />
Halle/Düsseldorf 2010.<br />
179<br />
Taste sensing system for the selection of drug candidates in early<br />
pharmaceutical development stage and rational development of taste masked<br />
orodispersible dosage forms<br />
Guhmann M1 2, Gerber F2, Poellinger N2, Breitkreutz J3, Weitschies W1 1 Department of Biopharmaceutics and Pharmaceutical Technology, University of <strong>Greifswald</strong>,<br />
17489 <strong>Greifswald</strong>, Germany<br />
2 Glatt Pharmaceutical Services, Technology Centre, Glatt GmbH, 79589 Binzen, Germany<br />
3 Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University of Düsseldorf, 40225<br />
Düsseldorf, Germany<br />
The ability of an electronic taste sensing system to guide the selection of drug candidates<br />
and rationalize the development of taste masked orodispersible formulations was<br />
evaluated.<br />
Diclofenac was used as drug model investigating the free acid, the sodium and the<br />
potassium salt forms. Calibrations curves were established with different concentration<br />
ranges. Measurements were performed using the electronic taste sensing system TS-<br />
5000Z (Insent Inc., Japan) qualified based on ICH guideline Q2 [1]. The system was<br />
equipped with 7 lipid membrane sensors representing bitterness (1,2 and 3), sourness,<br />
saltiness, umami, and astringency and corresponding aftertaste qualities. Sensor<br />
responses (mV) of three consecutive measurements were analyzed by univariate data<br />
evaluation. Taste masked Orally Disintegrating Tablets (ODTs) were developed using the<br />
most suitable diclofenac drug form considering taste modalities, and evaluated by the<br />
electronic tongue versus placebos using Principal Component Analysis (PCA).<br />
Clear differences between the free acid form of diclofenac and its salt counterparts were<br />
detected by the electronic tongue. Diclofenac salts induced bitter (taste and aftertaste) and<br />
astringent (taste) responses with different detection intensities; saltiness stimuli was<br />
additionally recorded for the potassium salt. In contrast, only a bitter aftertaste from sensor<br />
3 was detected for the acid form. Based on these results, diclofenac acid was selected for<br />
manufacturing taste masked ODTs. Taste masking strategies used granules produced by<br />
coating of single drug crystals or wet granulation, further compressed into ODTs. The PCA<br />
map showed that intermediated products (granules) and finished products (ODTs) could be<br />
distinguished by the electronic tongue. All formulations differed from the pure diclofenac<br />
acid. Using PC-1 (77% of the information), the taste reduction was 33% and 54% for drug<br />
coated granules and matrix granules respectively; 67% and 71% for their corresponding<br />
ODTs.<br />
Investigating diclofenac as a drug model, the electronic tongue proved to be a valuable tool<br />
for the selection of different drug candidates in the early pharmaceutical development<br />
phase and the rational development of taste masked orally disintegrating dosage forms<br />
Acknowledgments: The authors gratefully acknowledge the Institute of Pharmaceutics and<br />
Biopharmaceutics of the Heinrich-Heine University Düsseldorf, especially Maren Preis, for providing support<br />
and advice.<br />
Reference:<br />
1. Woertz, K et al.: J. Pharm. Biomed. Anal. 2010, 51: 497-506.<br />
180<br />
Complexation of Itraconazole with various cyclodextrin derivatives: Solubility<br />
and 1H-NMR studies<br />
Taupitz, T. 1; Bodtke; A. 2; Böheim, K. 2; Link, A. 2; Klein, S. 2<br />
1 Goethe University Frankfurt am Main, Institute of Pharmaceutical Technology, Max-von-Laue-Str.<br />
9, 60438 Frankfurt (Main)<br />
2 <strong>Ernst</strong>-<strong>Moritz</strong>-<strong>Arndt</strong> University <strong>Greifswald</strong>, Department of Pharmacy, Institute of Biopharmacy and<br />
Pharmaceutical Technology, C_DAT, Felix-Hausdorff.Str. 3, 17489 <strong>Greifswald</strong><br />
In the present study we wanted to investigate the complexation behaviour of different<br />
cyclodextrin derivatives with itraconazole and moreover elucidate the impact of these<br />
derivatives on the solubility of this poorly soluble compound. For this purpose binary<br />
cyclodextrin complexes of itraconazole, a weakly basic BCS class II drug, were prepared<br />
using the following cyclodextrin derivatives: I) 2-hydroxypropyl-β-cyclodextrin (HP-β-CD),<br />
II) sulfobutylether-β-cyclodextrin (SBE-β-CD) and the recently developed hydroxybutenylβ-cyclodextrin<br />
(HBen-β-CD). The main objective was to obtain formulations that show an<br />
improved solubility superior to that of the pure drug and further to investigate the<br />
interactions in the different itraconazole cyclodextrin complexes in detail.<br />
A freeze drying procedure was used to prepare all inclusion complexes of itraconazole. The<br />
pure drug and all formulations were then subject to pX-RD analysis and solubility tests. To<br />
elucidate the complex formation of each cyclodextrin derivative with the compound, 1H-<br />
NMR studies were performed. Finally, solubility tests were performed in compendial and<br />
biorelevant media simulating conditions in the stomach and the upper small intestine.<br />
Poster 139
PX-RD diagrams of the complex formulations indicated that amorphous inclusion<br />
complexes were obtained. Spectra obtained from the 1H-NMR studies revealed chemicals<br />
shifts of the triazol- and the aromatic protons indicating interactions between each<br />
cyclodextrin derivative and these areas of the drug molecule. Results of the solubility<br />
experiments showed a significant increase of itraconazole aqueous solubility. Particularly,<br />
the recently developed HBen-β-CD derivative could tremendously improve the solubility<br />
under gastric and small intestinal conditions.<br />
Based on the results of the present study, we assume that the interactions between each of<br />
the cyclodextrin derivatives and itraconazole lead to an improvement of the compounds<br />
solubility. However, the extent of this effect varied as a result of the derivative used for<br />
complex formation. Overall, it can be said, that the investigated cyclodextrin derivates were<br />
very useful to increase the solubility of itraconazole in compendial and biorelevant test<br />
media and therefore represent a promising group of excipients for future use in solubility<br />
(and bioavailability) enhancement of poorly soluble compounds.<br />
140 Poster
Autorenverzeichnis<br />
Abdel-Aziz H 98, 103, 137<br />
Abd-Ellatif A 132<br />
Abrahmsén-Alami S 32<br />
Abrahmsson B 32<br />
Abromeit H 102<br />
Abstiens K 80<br />
AbulAzm S 104<br />
Achenbach J 56<br />
Adams R H 114<br />
Adler M 137<br />
Ahmad K 113<br />
Ahmed M 82<br />
Alban S 58<br />
Albrecht M 122<br />
Al-Halhouli A T 130<br />
Alhazmi H 33<br />
Alhussein A 122<br />
Ali H 69<br />
Alresly Z 106<br />
Altenhofen W 117<br />
Altmann K S 118<br />
Ammon H P T 122<br />
Andermark V 111<br />
Anschütz M 32<br />
Arnold N 106<br />
Aßmann G 137<br />
Astner I 91<br />
Aurich K 134<br />
Baaske P 80<br />
Bäcker C 106<br />
Bakowsky U 70<br />
Banoglu E 117<br />
Bantscheff M 25<br />
Baranski J 115<br />
Bartusch A 136<br />
Barzen S 105<br />
Bauer J 102<br />
Bauer K 103<br />
Bauer S 103, 117<br />
Baumann K 79, 104, 125<br />
Bausch A 115<br />
Becker D 116<br />
Bednarski P J 65, 106, 118, 119<br />
Beese K 120<br />
Behrends S 130<br />
Behrendt C T 99<br />
Beißner N 101<br />
Beitz E 118<br />
Below A 110<br />
Bendas G 120, 121<br />
Berg S 43<br />
Bernat V 23, 105<br />
Bernhardt G 28, 98<br />
Bertram L 123<br />
Bertram N 35<br />
Bertsche T 87<br />
Besheer A 80<br />
Bettio S 122<br />
Bibb J A 114<br />
Bickmann D 110<br />
Biller A 137<br />
Bischoff F 100<br />
Bitterlich A 78, 124<br />
Bittner F 117<br />
Blackert S 134<br />
Block S 60<br />
Bloßfeld M 112<br />
Blume H 32, 134<br />
Bock R 126<br />
Bodem J 95, 96, 97<br />
Bodtke A 106, 110, 139<br />
Boeckler F M 55<br />
Bogan R 135<br />
Böheim K 139<br />
Borchert P 120<br />
Böttger S 107<br />
Bracher F 114<br />
Braig S 100<br />
Brand T 113<br />
Brandl F 86, 130<br />
Brandl F P 124<br />
Brandt S 60<br />
Brandt W 109<br />
Breitkreutz J 83, 110, 129, 137, 139<br />
Breunig M 133<br />
Brock D 131<br />
Brückner A 111<br />
Brückner C 102<br />
Brüßler J 70<br />
Bublitz K 120<br />
Bui T H 105<br />
Bundscherer L 118<br />
Bunjes H 75, 78, 124, 128, 130<br />
Buschauer A 28, 98<br />
Busker M 130<br />
Busse M 134<br />
Butterweck V 111<br />
Büttgenbach S 127, 130<br />
Chahar M 101<br />
Chamseddin C 105, 106<br />
Chbeib M 107<br />
Chen R 129<br />
Cheng X 63<br />
Cianciulli C 33, 110<br />
Claes D 108<br />
Clement B 117, 118<br />
Closs E I 115<br />
Collnot E M 69<br />
Crüsemann M 106<br />
Daily A 38<br />
Damm G 42<br />
Dannhardt G 115<br />
Dathe M 138<br />
Dayyoub E 70<br />
de Jong J C 100, 114<br />
Deally A 114<br />
Decker M 36<br />
Dehm F 101<br />
Delves M 98<br />
Dengler M 78<br />
Deuter A 119<br />
Dhein S 94<br />
Diel P 112<br />
Dietzel A 130<br />
Dolberg A M 116<br />
Donath F 32<br />
Dörje F 134<br />
Dorn A 101, 104<br />
Dos Santos Capelo R 121<br />
Dötsch V 119<br />
Duhr S 80<br />
Dzikowski R 61<br />
Eckert C 136<br />
Eeckman L 126<br />
Efferth T 37, 45<br />
Eiden K 119<br />
El Gaghlab K 120<br />
Elz S 102<br />
Emmrich T 65, 106, 118<br />
Engel A 43<br />
Engels B 95, 96<br />
Enzensperger C 103, 104<br />
Erdmann D 28<br />
Externbrink A 132<br />
Fahrmayr C 40<br />
Falck E 34, 122<br />
Ferandin Y 61<br />
Fersht A R 55<br />
Fey P 95<br />
Finke J H 127, 130<br />
Fischer D 73<br />
Fischer M 99<br />
Fischer S 104<br />
Fiß T 89, 137<br />
Flemming S 121<br />
Fleßa S 137<br />
Foerster S 122<br />
Förster F 100<br />
Fossen T 105<br />
Franzmann E 27<br />
Fréchet J M J 72<br />
Freichel M 48<br />
Freitag A 107<br />
Frentsch M 71<br />
Freyse E-J 43<br />
Friedrich C 135<br />
Frieß W 20, 81<br />
Fromm M F 40<br />
Fuchs S 97<br />
Fugel W 61<br />
Funke A 90<br />
Fürst R 97, 114, 115<br />
Gantzsch S 127<br />
Garbacz G 30, 109<br />
Gehringer M 95<br />
Gerber F 139<br />
Gerber U 120<br />
Germer K 131<br />
Gerstmeier J 117<br />
Geyer H 125<br />
Gieré R 74<br />
Gieseler H 122<br />
Glöckl G 113, 125, 134<br />
Gminski R 74<br />
Gobleder S 102<br />
Goebgen E 137<br />
Goettert M 103, 117<br />
Gohlke H 22<br />
Goos K-H 105<br />
Autorenverzeichnis 141
Göpferich A 86, 124, 130, 132, 133<br />
Görke K 109<br />
Gothsch T 127<br />
Grabow N 113, 126<br />
Graeser R 117<br />
Greinacher A 50, 59, 60<br />
Grez M 22<br />
Groll M 99<br />
Grotefend S 94<br />
Grün J 96<br />
Grüning B A 107<br />
Grützkau A 121<br />
Guhmann M 139<br />
Gündisch D 103<br />
Günther S 54, 107, 121<br />
Gust R 52<br />
Guthoff R 94<br />
Ha K 101<br />
Hackbarth C 60<br />
Hackenberg F 68<br />
Haertel B 119, 134<br />
Häfner A-K 119<br />
Hager B 114<br />
Hähn S 99<br />
Hahne T 33, 110<br />
Hamacher A 66<br />
Hamburger M 97<br />
Hammer E 122<br />
Hammer N 86, 124, 130<br />
Hammerschmidt S 60<br />
Hanke T 101<br />
Hanke U 116, 134<br />
Hansen F K 101<br />
Hansen S 31<br />
Harden D 131<br />
Harder C 126<br />
Hare E 69<br />
Härter A 111<br />
Hartmann R W 65, 100, 114<br />
Hartung E 97<br />
Hasenpusch D 107<br />
Haug K G 111<br />
Häupl T 121<br />
Haupt O 112<br />
Hausmann M 115<br />
Häussler S 100<br />
Haustein M 120<br />
Havemeyer A 117, 118<br />
Heilmann J 98<br />
Heinemann K 120<br />
Heininger A 139<br />
Held J 98, 99<br />
Heller E 25<br />
Hellmuth C 121<br />
Henn C 100<br />
Hennig L 111<br />
Hennig R 132, 133<br />
Hentschel-Humeida U 97<br />
Hessler N 73<br />
Hiddemann L 109<br />
Hinz B 111, 120<br />
Hipler U-C 73<br />
Hirt C 65<br />
Hochhaus G 111<br />
Hoffmann B 132<br />
Hoffmann C 25<br />
Hoffmann W 89, 137<br />
Hofmann B 105, 119<br />
Holl K 99<br />
Holloway S 95<br />
Holzgrabe U 25, 96, 103, 123<br />
Hornburger M C 115<br />
Hörold M 136<br />
Horvat M 129<br />
Hoser S 103, 137<br />
Humar M 54<br />
Iber S 134<br />
Ihling C 106<br />
Ihling C H 47<br />
Ilko D 123<br />
Illarionov B 99<br />
Jaax M 60<br />
Jaehde U 87<br />
Jäger C 54<br />
Jain A K 32<br />
Jakobs H 117<br />
Janas C 127<br />
Jansen R 138<br />
Jantscheff P 121<br />
Jauch J 122<br />
Jedlitschky G 50<br />
Jenett-Siems K 106<br />
Jenner D 96<br />
Jensen A A 103<br />
Jira T 105<br />
Joerger A C 55<br />
Juchum M 96<br />
Juli C 96<br />
Jung M 53, 112, 116, 120<br />
Junmahasathien T 135<br />
Kabisch J 109<br />
Kacprowski T 122<br />
Kage H 95<br />
Kahnt A S 121<br />
Kaminski D 125<br />
Kann B 127<br />
Karcher S 104<br />
Kaske M 28<br />
Kassack M U 66<br />
Katritzky A R 101<br />
Kaufeld A M 98<br />
Keck C M 108, 129, 131, 135<br />
Keck P R W E F 100<br />
Kees M 139<br />
Keiser M 42, 43<br />
Kelber O 98, 103, 137<br />
Keller M 28<br />
Kersten E 138<br />
Kesselring J 95<br />
Kessner S 136<br />
Khalil F 27, 123<br />
Kiefer T 65<br />
Kiefer W 96<br />
Kiene F 126<br />
Kipping T 136<br />
Kirchhof S 86, 124, 130<br />
Kirsch B 100<br />
Klages C-P 130<br />
Klein C 95<br />
Klein S 30, 132, 138, 139<br />
Kleinebudde P 128, 129, 132<br />
Kloft C 109, 136, 137, 139<br />
Klos S 103<br />
Knop K 139<br />
Knopke C 32<br />
Kobilka B K 23<br />
Koeberle A 100, 102<br />
Koeck J 129<br />
Kolb P 23, 105<br />
Kölbel K 47, 106<br />
Koletzko B 121<br />
Kölln C 112<br />
Kolodziej H 98, 99<br />
Komoß C 78, 124<br />
Könczöl M 74<br />
König J 40<br />
Konzuch S 99<br />
Kordaß B 125<br />
Korpis K 118<br />
Kortmann C 129<br />
Köster R 114<br />
Kovacevic A 108<br />
Koziolek M 30, 109<br />
Kraft K 137<br />
Kralisch D 73<br />
Krämer I 123<br />
Krauel K 60<br />
Kraus B 36<br />
Krauß A 115<br />
Krauth F 136<br />
Krebs S 134<br />
Kressirer C 100<br />
Kreutzer M F 95<br />
Kriemen E 120<br />
Kroemer H K 50<br />
Kruggel S 61, 109<br />
Kuehnl S 102<br />
Kühn J-P 134<br />
Kummer J 94<br />
Kumpfmüller J 109<br />
Kunfermann A 99<br />
Kunick C 61, 109<br />
Kupetz E 130<br />
Kurz T 98, 99<br />
Kwade A 78, 124, 127<br />
Laabs F 128<br />
Lalk M 106, 109<br />
Lally G 68<br />
Lamers C 56<br />
Lämmerhofer M 101<br />
Lamprecht A 126<br />
Lang C 26<br />
Lange C 106<br />
Laufer S 95, 103, 104, 117<br />
Laufer S A 100, 101, 107<br />
Lehmann J 103, 104<br />
Lehr C M 69, 127<br />
Lehr T 29<br />
Lemcke T 61, 107, 108, 109, 110<br />
Lemmerhirt H 65, 106, 118<br />
Lendlein A 71<br />
Leonard F 69<br />
Lestari M L A D 104<br />
Leuner K 134<br />
Leven M 98<br />
Liebeke M 46<br />
Liebl J 114<br />
Liedl K 21<br />
Liedtke A J 100<br />
Lienau C 99<br />
Liening S 101<br />
Lill A 105<br />
Lindequist U 106, 119, 134<br />
Link A 65, 106, 118, 120, 139<br />
Linnebacher M 120<br />
Lipke U 126<br />
Lipperheide C 126<br />
142 Autorenverzeichnis
Liu T 125<br />
Loch C 94<br />
Löhr J H 110<br />
Lohse M J 25<br />
Lu C 100<br />
Lucas X 107<br />
Luderer S 117<br />
Lukowski R 49, 115<br />
Lütnant I 98<br />
Maedler K 19<br />
Mai A 63<br />
Maison W 27, 108, 120, 123<br />
Majer M 115<br />
Manna C M 114<br />
Marschalek R 113<br />
Marschall P 137<br />
Martens A 139<br />
Martiné U 115<br />
Martz K E 101<br />
Marxer E 70<br />
Masch A 64<br />
Massing U 121<br />
Masur K 118<br />
Mathew S 71<br />
Matz F 97<br />
Matz M 79<br />
Maurer C K 100, 114<br />
Mayer B A 115<br />
Meijer L 61, 100, 109<br />
Mell N 69<br />
Melzig M F 94, 107, 129<br />
Mendel R 117<br />
Menrath C 97<br />
Menzen T 81<br />
Merfort I 54, 74<br />
Merkenschlager A 112<br />
Merkord J 120<br />
Mersch-Sundermann V 74<br />
Messmann V 86<br />
Meßmann V 124, 130<br />
Metz A 22<br />
Meyer A 111<br />
Meyerdierks N 115<br />
Michael S 98<br />
Michel A 119<br />
Minichmayr I 109<br />
Moll H 95<br />
Monbaliu J-C M 101<br />
Mordmüller B 98, 99<br />
Morgenstern O 118<br />
<strong>Moritz</strong> S 73<br />
Möschwitzer J P 104, 124, 125, 129<br />
Moscow J A 38<br />
Moser M 114<br />
Mosig J 132<br />
Müller A 73<br />
Müller C 114<br />
Müller F A 73<br />
Müller R 18, 100, 109<br />
Müller R H 104, 108, 124, 125, 129,<br />
131, 135<br />
Müller-Goymann C C 127, 130<br />
Mundt S 105, 110<br />
Münsterberg M 108<br />
Munteanu M 73<br />
Müsken M 100<br />
Muth F M 103<br />
Nagaraj M 82<br />
Nagel S 94, 109, 113, 126<br />
Nagel-Steger L 111<br />
Nauert C 137<br />
Neidhardt I 110<br />
Nett M 95<br />
Neumann M 84<br />
Nieber K 98, 103, 105, 112, 137<br />
Niedermeyer T H J 38, 110<br />
Niemann S 127<br />
Nikolenko H 138<br />
Nimtz M 105<br />
Northoff H 102<br />
Norville I H 96<br />
Norwig J 138<br />
Nowotny B 97<br />
Nüssler A 42<br />
Oak P 37<br />
Oberholzer A E 61<br />
Oehmigen K 134<br />
Oetjen E 107<br />
Okpanyi S N 137<br />
Olausson B E S 106<br />
Oli S 97<br />
Oliferenko A A 101<br />
Oswald S 41, 43<br />
Ott I 67, 111, 114<br />
Parr M K 112, 125<br />
Patel H 54, 107<br />
Patil S 68<br />
Paul M 125<br />
Pauly A 134<br />
Pein M 129, 136<br />
Peissner W 121<br />
Perfahl S 119<br />
Pergola C 117<br />
Pertz H H 98<br />
Peters K 111<br />
Petrovick G F 137<br />
Pettelkau J 106<br />
Pfeifer B 109<br />
Pham T L H 110<br />
Plouffe D 98<br />
Pluym N 28<br />
Poellinger N 139<br />
Pohl J 42<br />
Poll B 109<br />
Pollinger K 132, 133<br />
Popella S-D 101<br />
Porzel A 106<br />
Potterat O 97<br />
Pradel G 96<br />
Preisitsch M 110<br />
Pressburger N 61<br />
Pretor S 130<br />
Preu L 130<br />
Priese F 133<br />
Proschak E 56<br />
Puerstinger J 110<br />
Quintanilla-Fend L 122<br />
Quodbach J 128<br />
Quosdorf S J 99<br />
Radebold J 42<br />
Rakow T 119<br />
Ramer R 111, 120<br />
Raszek M 105<br />
Ratin M 61<br />
Rau O 56<br />
Rauch B 50<br />
Rauh D 62, 64<br />
Rauwald H J 111<br />
Rauwald H W 94<br />
Raynor A 121<br />
Redweik S 33, 94<br />
Reichl S 51, 101, 112, 116, 130<br />
Reichmann D 117<br />
Rein H 77, 136<br />
Reitz E 131<br />
Ren X 133<br />
Rethwilm A 97<br />
Retzar A 135<br />
Richter C 126, 127<br />
Rieger M 96<br />
Rischer M 85<br />
Ritter C 82<br />
Ritter C A 122<br />
Robaa D 103, 104<br />
Roch T 71<br />
Rockmann T 131<br />
Rödl C B 105<br />
Roessler C 64<br />
Rollinger J M 102<br />
Rösch P 96<br />
Rosenbaum D M 23<br />
Ross T 121<br />
Rothenhöfer M 98<br />
Rotili D 63<br />
Rotmann A 115<br />
Rumpf T 120<br />
Runge D 42<br />
Rustenbeck I 79<br />
Ruth P 115<br />
Salamon A 120<br />
Salazar J 124<br />
Sarkar-Tyson M 96<br />
Savtschenko A 94<br />
Schad C 95<br />
Schaefer U 31<br />
Schaefer U F 127<br />
Schaeftlein A 139<br />
Schäfer J 70<br />
Schaible A M 102<br />
Schanda J 22<br />
Schänzer W 125<br />
Scherer O 102<br />
Scherübl R 98<br />
Scheuch E 43<br />
Schiedel M 116<br />
Schirmeister T 95, 96, 97, 107<br />
Schlesinger M 121<br />
Schmidt D 23, 105<br />
Schmidt H 136<br />
Schmidt I 96<br />
Schmitz P 120<br />
Schmolke H 130<br />
Schmuhl E 111<br />
Schneider F 84<br />
Schneider G 56<br />
Schneider T 95, 96<br />
Schnittker C 126<br />
Schollmeyer D 100<br />
Schönherr D 116<br />
Schramek N 92<br />
Schröder G 105<br />
Schröder M 119<br />
Schröder T 106<br />
Schubeis T 82<br />
Schubert-Zsilavecz M 56, 101<br />
Schuchmann H P 129<br />
Schug B 32<br />
Autorenverzeichnis 143
Schulz K 134<br />
Schulz R 65, 106, 118<br />
Schulz S 37<br />
Schulz U 118<br />
Schulze M 103<br />
Schumacher K 79<br />
Schümmelfeder J 70<br />
Schur J 114<br />
Schurigt U 95, 96<br />
Schutkowski M 64<br />
Schütt R 113<br />
Schütze N 120<br />
Schwarz R 47<br />
Schweder T 109<br />
Schweimer K 96<br />
Scriba G K E 102<br />
Seeling A 110<br />
Seibel J 96<br />
Seidlitz A 94, 113, 126<br />
Semmling B 113, 126<br />
Seufert F 96<br />
Shaalan N 117<br />
Shehata A M 122<br />
Shoichet B K 23<br />
Siegmund W 42, 43, 116<br />
Siems K 106<br />
Sinha B 129<br />
Sinz A 34, 47, 106<br />
Smolka K 125<br />
Snitko M 95<br />
Söderlind E 32<br />
Sölter V 139<br />
Sotriffer C 95, 96, 97<br />
Sotriffer C A 107<br />
Spitzer G 21<br />
Stark H 105<br />
Staudacher V 114<br />
Steinbach A 100, 114<br />
Steinhilber D 56, 57, 105, 113, 119, 121<br />
Steinhoff B 137<br />
Steri R 56<br />
Sternberg K 113, 126<br />
Stintzing F C 39<br />
Storz M P 114<br />
Straßer A 24, 102<br />
Straubinger J 115<br />
Strauss A-C 134<br />
Streciwilk W 68<br />
Strunz A K 99<br />
Stuppner H 102<br />
Sutter A 90<br />
Swiatecka-Hagenbruch M 38<br />
Sydow K 138<br />
Tabares P 97<br />
Tacke M 68, 114<br />
Tackenberg M W 129<br />
Tajarobi F 32<br />
Talmann L 134<br />
Tänzler D 47<br />
Taraschewski A 138<br />
Taupitz T 139<br />
Tchoukouegno Ngueu S 112<br />
Tessmar J 132, 133<br />
Teßmar J 130<br />
Teßmar J K V 124<br />
Thiessen A 35<br />
Thom K 134<br />
Thommes M 76, 126, 129, 131<br />
Thürmann P A 88<br />
Tränkle C 103<br />
Trauner D 37<br />
Treuer E 117<br />
Trip E 126<br />
Tschammer N 23, 105<br />
Tschan S 98<br />
Tshuva E Y 114<br />
Ullrich A 42<br />
Venema K 113<br />
Viridé A 32<br />
Vissiennon C 105<br />
Vogel S 107<br />
Völker U 44, 122<br />
Vollmar A 100<br />
Vollmar A M 37, 97, 100, 114, 115<br />
von Schwarzenberg K 37, 100<br />
von Woedtke T 118, 119, 134<br />
Vongehr F 135<br />
Voß U 98<br />
Wacker M 127<br />
Wagner S 107<br />
Wagner T 112<br />
Waltenberger C 107<br />
Wanner G 37<br />
Wätzig H 33, 94, 110, 119<br />
Weber B 111<br />
Weber C 60<br />
Weber F 118<br />
Weber M 121<br />
Wedemeyer R-S 134<br />
Wegener J 115<br />
Weidel E 114<br />
Weiser D 98, 103<br />
Weitschies W 30, 32, 43, 84, 94,<br />
109, 113, 116, 125, 126, 134, 139<br />
Weiwad M 96<br />
Welinder A 32<br />
Welker A 95, 96, 97<br />
Wende K 105, 119<br />
Wendling F 97<br />
Wentsch H 104<br />
Wentzlaff M 126<br />
Wenzel R 133<br />
Wenzel S 109<br />
Werz O 100, 101, 102, 117<br />
Wesar F 73<br />
Wessjohann L A 106<br />
Wich P R 72<br />
Wicha S G 136, 137<br />
Wichmann C 22<br />
Wiedmann R M 37<br />
Wiegand C 73<br />
Wiest J 123<br />
Wilcken R 55<br />
Wilde F 106, 118<br />
Wilde L 125<br />
Willbold D 111<br />
Willer E A 97<br />
Wilson C G 113<br />
Windbergs M 127<br />
Winkelmann V 103<br />
Winter G 80<br />
Wischke C 71<br />
Witt S 136<br />
Wittmann H-J 24, 102<br />
Wolber G 21<br />
Wolf B 131, 133<br />
Wolf C 134<br />
Wolff H 36<br />
Wolff M 111<br />
Wollatz U 125<br />
Wollenhaupt S 104<br />
Wray V 105<br />
Wszelaki N 129<br />
Wu H 95<br />
Wünsch B 34, 97, 98, 99, 118, 122<br />
Wurster M 106<br />
Xu Y 33<br />
Zahler S 100, 114<br />
Zapf T 126<br />
Zhang S 114<br />
Zimmer A 82<br />
Zimmer C 100, 114<br />
Zimmermann T 135<br />
Zischka H 37<br />
Zlotos D P 103<br />
Zwernemann G 136<br />
144 Autorenverzeichnis
Notizen<br />
145
Notizen<br />
146
Notizen<br />
147
Notizen<br />
148
Notizen<br />
149
Notizen<br />
150
151
152<br />
<strong>Greifswald</strong> Innenstadt<br />
1 Tierpark<br />
2 Botanischer Garten<br />
3 Credneranlagen<br />
4 Amtsgericht/Finanzgericht<br />
5 Bahnhof<br />
6 Einrichtungen der <strong>Universität</strong><br />
7 Stadtbefestigung<br />
8 Kirche St. Jacobi<br />
9 Historische Sternwarte<br />
10 Rubenowplatz und –denkmal<br />
11 Einkaufszentrum Dompassage<br />
12 Hauptgebäude der <strong>Universität</strong><br />
13 Sozio-kulturelles Zentrum St. Spiritus<br />
14 Audimax<br />
15 Caspar-David-Friedrich-Zentrum<br />
16 Musikschule<br />
17 Alte <strong>Universität</strong>sbibliothek<br />
18 Dom St. Nikolai<br />
19 Museumshafen<br />
20 Kirche St. Joseph<br />
21 Fußgängerbrücke<br />
Geschäftführer und Leiter der Geschäftsstelle Apotheker Dr. Michael Stein<br />
<strong>DPhG</strong> Geschäftsstelle<br />
Hamburger Allee 26 - 28<br />
60486 Frankfurt/Main<br />
Tel.: 069-7191596-0<br />
Fax: 069-7191596-29<br />
Email: info@dphg.de<br />
http://www.dphg.de<br />
25.09.<strong>2012</strong><br />
22 Caspar-David-Friedrich-Denkmal<br />
23 Alfried Krupp Wissenschaftskolleg <strong>Greifswald</strong><br />
24 Volkshochschule<br />
25 Fischmarkt<br />
26 Rathaus<br />
27 Kunstwerkstätten<br />
28 Literaturzentrum „Wolfgang Koeppen“<br />
29 Stadtbibliothek<br />
30 Stadthaus (im Bau)<br />
31 Gotische Giebelhäuser<br />
32 Fangenturm<br />
33 Internationales Begegnungszentrum<br />
34 Kirche St. Marien<br />
35 Museumswerft<br />
36 Pommersches Landesmuseum<br />
37 Stadtarchiv<br />
38 Mensa<br />
39 Fallada-Geburtshaus<br />
40 Stadthalle/Theatercafé<br />
41 Landesarchiv Vorpommern<br />
42 Theater Vorpommern
Kartenmaterial zur Verfügung gestellt vom Amt für Wirtschaftsförderung
Kartenmaterial zur Verfügung gestellt vom Tourismusverband Vorpommern e.V., Layout von: Grafikagentur Progress 4<br />
1 Biotechnikum (Targets 11./12.10.)<br />
2 Center of Drug Absorption and Transport C_DAT<br />
3 Mathematisch-Naturwissenschaftliche Fakultät mit<br />
Institut für Pharmazie<br />
4 Leibniz-Institut für Plasmaforschung und Technologie<br />
(INP <strong>Greifswald</strong> e.V.)<br />
5 Zentrum für Zahn-, Mund- und Kieferheilkunde<br />
6 Institut für Biochemie (Moleküle 11./12.10.)<br />
7 Klinik und Poliklinik für Hals-, Nasen-, Ohrenkrankheiten,<br />
Kopf- und Halschirurgie<br />
8 Institut für Physik (Tabletten 11./12.10.)<br />
9 Arboretum des Botanischen Gartens<br />
10 <strong>Universität</strong>sbibliothek<br />
11 Klinik und Poliklinik für Hautkrankheiten<br />
12 Johannes-Kirche<br />
13 BDH-Klinik <strong>Greifswald</strong> GmbH<br />
14 Volksstadion<br />
15 Kleine Mensa<br />
16 <strong>Universität</strong>sklinikum <strong>Greifswald</strong><br />
mit Hörsaal Nord (13.10.)<br />
A EUROPA HOTEL <strong>Greifswald</strong>