DPhG Jahrestagung 2012 - Ernst-Moritz-Arndt-Universität Greifswald

DPhG-Jahrestagung 2012
Moleküle, Targets und Tabletten -
Translationale Forschung für Arzneimittel der Zukunft
11. – 13. Oktober in Greifswald
Tagungsband
www.dphg.de
ISBN: 978-3-00-039318-1
Verlag: Universitätsbibliothek Greifswald

Sponsoren der DPhG-Jahrestagung 2012

Förderer der DPhG-Jahrestagung 2012

DPhG
Deutsche Pharmazeutische Gesellschaft e. V.
Jahrestagung 2012
Programm
&
Tagungsband
Institut für Pharmazie
der
Mathematisch-Naturwissenschaftlichen Fakultät
Ernst-Moritz-Arndt-Universität
Greifswald
11.–13. Oktober

4
Moleküle, Targets und Tabletten –
Translationale Forschung für Arzneimittel der Zukunft
Wissenschaftliches Komitee Organisationskomitee
Prof. Dr. Patrick J. Bednarski Prof. Dr. Werner Weitschies
Prof. Dr. Thomas Jira Prof. Dr. Christoph Ritter
Prof. Dr. Heyo K. Kroemer Prof. Dr. Andreas Link
Prof. Dr. Ulrike Lindequist PD Dr. Gregor Radau
Prof. Dr. Sandra Klein Dr. Winfried Stahlkopf
Prof. Dr. Andreas Link Dr. Thomas Emmrich
Prof. Dr. Bernhard H. Rauch Dipl.-Pharm. Lisa Wilde
Prof. Dr. Christoph Ritter Dipl.-Pharm. Felix Wilde
Prof. Dr. Thomas Schweder
Prof. Dr. Werner Siegmund
Prof. Dr. Werner Weitschies
Dipl.-Pharm. Heidi Lemmerhirt

Inhaltsverzeichnis
Allgemeine Informationen 6
Programm 7
Vorprogramm – Mittwoch, 10. Oktober 2012 8
Pharmaziehistorische Veranstaltung Pharmazie in Greifswald 8
Hauptsymposium Arzneimittelkontrolle 8
Hauptprogramm – Donnerstag, 11. Oktober 2012 9
Diskussionsveranstaltungen Pharmazie 2020 – Perspektiven in Forschung und Lehre 9
Eröffnung der Jahrestagung 2012 9
Hauptprogramm – Freitag, 12. Oktober 2012 11
Hauptprogramm – Sonnabend, 13. Oktober 2012 15
Vorträge 17
Plenarvortrag Moleküle 18
Plenarvortrag Targets 19
Plenarvortrag Tabletten 20
Sessionvorträge Moleküle 21
Session Struktur-basiertes Design 21
Session Molekulare Werkzeuge 25
Session IVIVC: Modelle und Modellierungen 29
Session Moderne Trenntechniken 33
Session Naturstoffe 36
Session Klinische Wirkstoffentwicklung 40
Sessionvorträge Targets 44
Session Proteomics und Metabolomics 44
Session Transporter und Ionenkanäle 48
Session Nukleäre Rezeptoren und Transkriptionsfaktoren als Wirkstofftargets 52
Session Thrombozytenfunktion und Gerinnung 58
Session Neue Targets und Testsysteme 61
Session Tumortherapie und Resistenzmechanismen 65
Sessionvorträge Tabletten 69
Session Nanomedizin 69
Session Pharmaverfahrenstechnik 75
Session Bioanalytik: Testsysteme und Datenqualität 79
Session Spezielle Darreichungsformen 83
Session Optimierte Patientenversorgung 87
Session Arzneimittelüberwachung/Regulatory Affairs 90
Poster 93
Autorenverzeichnis 141
5

Allgemeine Informationen
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Internetzugang
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Tagung in Greifswald nutzen zu können. Die allgemeinen, von Ihrer Hardware (Laptop, PC, Mobiltelefone)
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äußeres Identifizierungsverfahren TTLS
inneres Identifizierungsverfahren PAP
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Alternativ dazu kann man sich über den Tagungsaccount in das Netzwerk der Uni Greifswald einloggen:
Benutzer
Passwort
DPhG_2012
Die Universität übernimmt für Schäden, die aus der Anwendung des Funknetzes (Wireless-LAN) entstehen, keinerlei
Haftung.
Auf Seite 151 findet sich ein Linienplan des öffentlichen Nahverkehrs Greifswald (Bus).
6

Programm

Vorprogramm – Mittwoch, 10. Oktober 2012
► Die Veranstaltungen des Vorprogramms finden im Hörsaal des C_DAT statt.
Pharmaziehistorische Veranstaltung
Pharmazie in Greifswald
14:00–14:15 Begrüßung und Einführung
Prof. Dr. Christoph Friedrich (Marburg)
14:15–15:00
15:00–15:45
15:30–15:45 Kaffeepause
15:45–16:30
16:30–17:15
Zur Entwicklung des Pharmaziestudiums an der Universität Greifswald vor 1945
Prof. Dr. Christoph Friedrich (Marburg)
Zur Entwicklung der Pharmazie an der Universität Greifswald nach 1945
Prof. Dr. Thorsten Beyrich (Greifswald)
Das Apothekenwesen der Stadt Greifswald von den Anfängen bis zum 20.
Jahrhundert
Dr. Hartmut Bettin (Greifswald)
Die Homöopathie an der Universität Greifswald
Dr. Ulrich Meyer (Berlin)
Vorprogramm
Abend für Offizinapotheker
18:00–18:45 Chair: Prof. Dr. Andreas Link
Michael Jackson – DieSehnSUCHT nach Schlaf
Prof. Dr. Dieter Steinhilber/Prof. Dr. Theo Dingermann (Frankfurt am Main)
19:00–20:00
Individualisierte Medizin
Podiumsgespräch Dr. Hildegard Kaulen (FAZ), Prof. Dr. Theo Dingermann (Frankfurt am
Main), Prof. Dr. Wolfgang Lieb (Greifswald), Dr. Christian Ude (Darmstadt), u.a.
Ganztägiges
Hauptsymposium Arzneimittelkontrolle
Eigenverantwortlich organisiert und selbständig durchgeführt von der Fachgruppe Arzneimittelkontrolle –
Pharmazeutische Analytik.
► Ort: Biotechnikum, Raum 0.09
Das Programm publiziert die Fachgruppe unter:
http://www.pharmchem.tu-bs.de/forschung/waetzig/#veranstaltungen
ab 17:00 Uhr
Hörsaal
Pharmazie
Sitzung des Verbandes der Professoren an Pharmazeutischen Hochschulen der
Bundesrepublik Deutschland e. V. (VdPPHI)
8 Programm – 10.10.

Hauptprogramm – Donnerstag, 11. Oktober 2012
Diskussionsveranstaltungen
Pharmazie 2020 – Perspektiven in Forschung und Lehre
08:30–10:00 5 Parallelveranstaltungen der einzelnen Prüfungsfächer
Audimax HS 1 Audimax HS 2 Audimax HS 3 Audimax HS 4 Audimax HS 5
09:45
Uni Hauptgebäude
Pressekonferenz
im Beratungsraum des Rektorats, Domstr. 11, Eingang 2
10:00–10:30 Pause (Fußweg in die Stadthalle)
10:30–12:00
Stadthalle, Kaisersaal
13:00–13:30
Stadthalle, Kaisersaal
13:30–14:15
Stadthalle, Kaisersaal
14:15–15:00
Stadthalle, Kaisersaal
Abschlussdiskussion Plenarveranstaltung
Chair: Prof. Dr. Angelika M. Vollmar /Prof. Dr. Dieter Steinhilber
Eröffnung der Jahrestagung 2012
Moleküle, Targets und Tabletten –
Translationale Forschung für Arzneimittel der Zukunft
Dipl.-Ing. Jörg Hochheim, Leiter des Dezernats für Bauwesen und Umwelt und
1. Stellvertreter des Oberbürgermeisters der Hansestadt Greifswald
Prof. Dr. Rainer Westermann, Rektor der Ernst-Moritz-Arndt-Universität Greifswald
Plenarvortrag Moleküle
Prof. Dr. Rolf Müller (Saarbrücken)
Genom-basierte mikrobielle Naturstoff-Forschung für die
Entwicklung von anti-Infektiva
Plenarvortrag Targets
Prof. Dr. Kathrin Mädler (Bremen)
Anti-entzündliche Therapien für Diabetes – Rettung für die
Beta-Zelle
15:00–15:30 Fußweg zum neuen Campus
15:30–16:00 Kaffeepause
Programm – 11.10. 9

Moleküle ► Hörsaal Biochemie
16:00–17:30 Struktur-basiertes Design
Chair: Prof. Dr. Wolfgang Sippl & Prof. Dr. Christoph Sotriffer
Prof. Dr. Gerhard Wolber (Berlin): „New algorithms for 3D pharmacophore-based virtual screening“
Prof. Dr. Holger Gohlke (Düsseldorf): „From Determinants of RUNX1/ETO Tetramerization to Small-Molecule
Protein-Protein Interaction Inhibitors Targeting Acute Myeloid Leukemia“
Dr. Peter Kolb (Marburg): „Docking to GPCRs: ligand diversity, efficacy and selectivity“
Dr. Andrea Straßer (Regensburg): „Molecular dynamic simulation of a symmetric active state hβ2R-Gαβγhomodimer
complex - structural and energetical analysis“
Targets ► Hörsaal Biotechnikum
16:00–17:30 Proteomics und Metabolomics
Chair: Prof. Dr. Irmgard Merfort & Prof. Dr. Thomas Schweder
Prof. Dr. Uwe Völker (Greifswald): „OMICs-Analysen von Populations-basierten Kohorten – Mögliche Ansätze
für das Biomarkerscreening“
Prof. Dr. Thomas Efferth (Mainz): „Drug target fishing in silico and in vitro“
Dr. Manuel Liebeke (London): „From whole-organism to sub-cellular localization of metabolites by mass
spectrometry imaging“
Rico Schwarz (Halle): „Incorporation of unnatural amino acids to monitor conformational changes in peroxisome
proliferator-activated receptor alpha (PPARα)“
Tabletten ► Hörsaal Physik
16:00–17:30 Nanomedizin
Chair: Prof. Dr. Udo Bakowsky & Prof. Dr. Claus-Michael Lehr
Prof. Dr. Claus-Michael Lehr (Saarbrücken): „Nanomedicines for targeted drug delivery to the inflamed intestinal
mucosa“
Prof. Dr. Udo Bakowsky (Marburg): „Selective gene vehicles for enhanced DNA and siRNA delivery“
Dr. Christian Wischke (Teltow): „Modulating immune responses of human cells by adjuvant loaded particulate
carriers“
Jun.-Prof. Dr. Peter R. Wich (Mainz): „Acid-Degradable Dextran Particles for the Delivery of Biotherapeutics“
Astrid Müller (Jena): „Characterization of Bacterial Nanocellulose as Drug Delivery System for Low and High
Molecular Weight Drugs“
Dr. Mathias Könczöl (Freiburg): „Biomedical applications of magnetite in the respiratory system - Toxicological
considerations and mechanistic studies in A549 lung cells“
17:30–19:00 Posterpräsentationen (Institut für Biochemie)
19:00–22:00 Begrüßungsabend, Posterpräsentationen (Institut für Biochemie)
10 Programm – 11.10.

Hauptprogramm – Freitag, 12. Oktober 2012
Moleküle ► Hörsaal Biochemie
09:00–10:30 Molekulare Werkzeuge
Chair: Prof. Dr. Ulrike Holzgrabe & Prof. Dr. Andrea Sinz
Dr. Marcus Bantscheff (Cellzome AG): „Chemoproteomic target deconvolution of bioactive molecules“
Christopher Lang (Erlangen): „Molecular tools for positron emission tomography (PET): Development of a highly
selective radiotracer for the detection of neurotensin receptor 1 positive tumors“
Prof. Dr. Wolfgang Maison (Hamburg): „Analogs of siderophores and mussel adhesion proteins for applications
in clinical hygiene and implant medicine“
Dr. Eberhard Heller (Würzburg): „Microwave-Enhanced Synthesis of Fluorescent Dyes for Pharmacological
Purposes Using Real-Time Observation by UV/Vis Spectroscopy“
Prof. Dr. Armin Buschauer (Regensburg): „Fluorescent and radiolabelled ligands as molecular tools for NPY
receptors“
10:30–11:00 Kaffeepause
11:00–12:30 IVIVC: Modelle und Modellierungen
Chair: Prof. Dr. Charlotte Kloft & Prof. Dr. Werner Weitschies
Dr. Thorsten Lehr (Saarbrücken): „From Mice to Men: Application and Impact of in vivo Modeling and Simulation
in Drug Research and Development“
Dr. Grzegorz Garbacz (Greifswald): „Estimating the In Vivo Release Behaviour of Extended- and Immediate-
Release Products using biorelevant bissolution stress tests“
Dr. Steffi Hansen (Saarbrücken): „Improved input parameters for diffusion models of skin absorption“
Arun Jain (Greifswald): „Interindividual gastrointestinal erosion and inter- as well as intraindividual
gastrointestinal transit time variability of hydrophilic matrix tablets“
12:30–13:30 Mittagspause (C_DAT Seminarraum)
13:30–15:00 Moderne Trenntechniken
Chair: Prof. Dr. Thomas Jira & Prof. Dr. Michael Lämmerhofer
Prof. Dr. Hermann Wätzig (Braunschweig): „Continuous Improvement in Pharmazeutischer Analytik: schneller,
präziser, selektiver“
Prof. Dr. Andrea Sinz (Halle): „Untersuchung von Proteinen und Metaboliten -- Was können moderne
Massenspektrometer leisten?
Evamaria Falck (Münster): „Metabolism studies of Ifenprodil, a potent NR2B-selective NMDA receptor
antagonist“
Dr. Anette Thiessen (Anton Paar GmbH): „Macroscopic Questions and Molecular Answers: Measuring Optical
Rotation in Latest Chiral Separation Techniques“
15:00–15:30 Kaffeepause
15:30–17:00 Naturstoffe
Chair: Prof. Dr. Ulrike Lindequist & Prof. Dr. Angelika M. Vollmar
Dr. Birgit Kraus (Regensburg): „Hepatic bioactivity of phenolic natural compounds“
Dr. Karin von Schwarzenberg (München): „Mode of cell death induction by pharmacological V-ATPase
inhibition“
Dr. Timo Niedermeyer (Berlin/Greifswald): „Cyanobacteria in Anticancer Natural Products Research“
Prof. Dr. Florian C. Stintzing (WALA Heilmittel GmbH): „Applied phytochemical research – an industry’s
perspective“
Programm – 12.10. 11

17:15–18:45 Klinische Wirkstoffentwicklung
Chair: Prof. Dr. Klaus Mohr & Prof. Dr. Werner Siegmund
Prof. Dr. Jörg König (Erlangen): „Single- and multiple-transfected cell lines as tools for the analysis of drug
transport and of the interplay of drug transport and drug metabolism“
Dr. Stefan Oswald (Greifswald): „Methods to investigate the clinical relevance of drug transporters“
Dr. Dieter Runge (PRIMACYT GmbH): „The functionality of uptake transporters in primary hepatocytes of
different species can be verified by estrone-3-sulfate”
Anett Engel (Greifswald): „Solubilizing agents influence the pharmacokinetics of probe drugs by transporter
interaction“
20:00–23:00 Gesellschaftsabend (Stadthalle, Theatercafé)
Targets ► Hörsaal Biotechnikum
09:00–10:30 Transporter und Ionenkanäle
Chair: Prof. Dr. Bernhard H. Rauch & Prof. Dr. Peter Ruth
Prof. Dr. Marc Freichel (Heidelberg): „TRP channels as regulators of blood pressure and fertility“
Dr. Robert Lukowski (Tübingen): „Role of Ca2+ -activated K + Channels in Breast Cancer Proliferation“
PD Dr. Gabriele Jedlitschky (Greifswald): „Transporters in human platelets: physiological function and impact for
pharmacotherapy“
PD Dr. Stephan Reichl (Braunschweig): „Expression analysis of drug transporter proteins in RPMI 2650 cell line
and excised human nasal mucosa“
10:30–11:00 Kaffeepause
11:00–12:30 Nukleäre Rezeptoren und Transkriptionsfaktoren als
Wirkstofftargets
Chair: Prof. Dr. Manfred Schubert-Zsilavecz & Prof. Dr. Dieter Steinhilber
Prof. Dr. Ronald Gust (Innsbruck): „Investigation on the estrogen receptor alpha selectivity of 2,3,5-triaryl-1Hpyrroles“
Prof. Dr. Manfred Jung (Freiburg): „Chemical epigenetics: histone modifying enzymes as targets to regulate
transcription“
Prof. Dr. Irmgard Merfort (Freiburg): „Helenalin derivatives and their impact on transcriptional control of
mediators involved in rheumatic diseases“
Prof. Dr. Frank Böckler (Tübingen): „Rescuing mutant p53 using halogen-enriched fragment libraries (HEFLibs)“
Jun. Prof. Dr. Eugen Proschak (Frankfurt): „Novel leads for PPARs and FXR: a computer-aided drug design
approach“
Prof. Dr. Dieter Steinhilber (Frankfurt): „Inhibitors of class I histone deacetylases induce 5-lipoxygenase
expression“
12:30–13:30 Mittagspause (C_DAT Seminarraum)
13:30–15:00 Thrombozytenfunktion und Gerinnung
Chair: Prof. Dr. Susanne Alban & Prof. Dr. Andreas Greinacher
Prof. Dr. Susanne Alban (Kiel): „Novel oral anticoagulants – State of the art and aspects of their application“
Prof. Dr. Andreas Greinacher (Greifswald): „Novel platelet inhibitors – State of the art and aspects of their
application”
Dr. Krystin Krauel (Greifswald): „Naturally occurring PF4/polyanion complexes prime for heparin-induced
thrombocytopenia as a misdirected host defense mechanism“
12 Programm – 12.10.

15:00–15:30 Kaffeepause
15:30–17:00 Neue Targets und Testsysteme
Chair: Prof. Dr. Manfred Jung & Prof. Dr. Stefan Laufer
Prof. Dr. Conrad Kunick (Braunschweig): „PfGSK-3: A target for new antimalarial drugs?“
Prof. Dr. Daniel Rauh (Dortmund): „Chemical Oncology – Converging Cancer Genetics, Structural Biology and
Medicinal Chemistry“
Dr. Dante Rotili (Rom): „Development of lysine mimic quinazoline-based selective inhibitors of a Jmj-C histone
demethylase family“
Prof. Dr. Mike Schutkowski (Halle): „Profiling of epigenetic targets using peptide microarrays“
17:15–18:45 Tumortherapie und Resistenzmechanismen
Chair: Prof. Dr. Patrick J. Bednarski & Prof. Dr. Conrad Kunick
Prof. Dr. Rolf W. Hartmann (Saarbrücken): „Anti-infectives research to combat drug resistant tumors?“
Prof. Dr. Matthias Kassack (Düsseldorf): „Chemoresistance of ovarian cancers against platinum complexes“
Prof. Dr. Ingo Ott (Braunschweig): „Medicinal Chemistry with Organometallics - From Catalysts to Anticancer
Drugs?“
Dr. Matthias Tacke (Dublin): „NHC-Silver(I) and NHC-gold(I) complexes as bioorganometallic anticancer and
antibacterial drugs“
Riad Schulz (Greifswald): „A new class of glutathione peroxidase inhibitors is able to reverse resistance to
chemotherapeutics in human B-cell lymphoma cell lines“
20:00–23:00 Gesellschaftsabend (Stadthalle, Theatercafé)
Tabletten ► Hörsaal Physik
09:00–10:30 Pharmaverfahrenstechnik
Chair: Prof. Dr. Peter Kleinebudde & Prof. Dr. Christel Müller-Goymann
Prof. Dr. Heike Bunjes (Braunschweig): „Formulierungs-Screening mit kolloidalen Wirkstoffträgersystemen“
Dr. Markus Thommes (Düsseldorf): „Pharmaceutical Extrusion technology – Recent developments“
PD Dr. Hubert Rein (Bonn): „Hot melt extruded dosage forms“
André Bitterlich (Braunschweig): „Nanogrinding of naproxen: influence of process parameters on product
quality“
10:30–11:00 Kaffeepause
11:00–12:30 Bioanalytik: Testsysteme und Datenqualität
Chair: Prof. Dr. Hermann Wätzig & Prof. Dr. Gerhard Winter
Prof. Dr. Knut Baumann (Braunschweig): „Bildverarbeitung für biomedizinisch eingesetzte Mikroskopie“
Dr. Achmed Besheer (München): „The use of microscale thermophoresis (MST) for protein characterization and
formulation“
Tim Menzen (München): „High-Throughput Protein Melting Temperature Analysis by Differential Scanning
Fluorimetry“
Prof. Dr. Christiane Ritter (Braunschweig): „NMR Spektroskopie von hochmolekularen Proteinkomplexen“
12:30–13:30 Mittagspause (C_DAT Seminarraum)
Programm – 12.10. 13

13:30–15:00 Spezielle Darreichungsformen
Chair: Prof. Dr. Jörg Breitkreutz & Prof. Dr. Sandra Klein
Prof. Dr. Jörg Breitkreutz (Düsseldorf): „Orodispersible dosage forms – novel concepts, innovative products,
new therapeutic options“
Marco Neumann (Greifswald): „Challenges in developing gastroretentive dosage forms“
Matthias Rischer (Losan Pharma GmbH): „Innovative nanoparticular dosage forms – advantages in
pharmacokinetics and administration“
Susanne Kirchhof (Regensburg): „Development and pharmaceutical technological applications of Diels-Alder
hydrogels”
15:00–15:30 Kaffeepause
15:30–17:00 Optimierte Patientenversorgung
Chair: Prof. Dr. Ulrich Jaehde & Prof. Dr. Christoph Ritter
Prof. Dr. Ulrich Jaehde (Bonn): „Medication safety research as challenge for pharmaceutical scientists“
Prof. Dr. Thilo Bertsche (Leipzig): „Klinische Pharmazeuten zur Verbesserung der Patientensicherheit in der
Arzneimitteltherapie“
Prof. Dr. Petra A. Thürmann (Witten/Herdecke): „Arzneimitteltherapiesicherheit in Alten- und Pflegeheimen“
Dr. Thomas Fiß (Greifswald): „Identifikation von Risikofaktoren für das Auftreten von Arzneimittelbezogenen
Problemen im Rahmen von häuslichen Medikationsreviews“
17:15–18:45 Arzneimittelüberwachung/Regulatory Affairs
Chair: Dr. Olaf Queckenberg & PD Dr. Klaus Raith
Dr. Andreas Sutter (Bayer AG): „Analyzing the use of (Q)SAR models for the evaluation of potential genotoxic
impurities: A cookbook in support of ICH M7“
Dr. Adrian Funke (Bayer AG): „Control strategy for an active coating process based on systematic process
development (DoE) and in-line Raman spectroscopy (PAT)“
Dr. Isabel Astner (Braunschweig): „Gewebe und Zellen – Arzneimittel für neuartige Therapien“
Dr. Nicholas Schramek (Oberschleißheim): „Analytik von illegalen Arzneimitteln“
20:00–23:00 Gesellschaftsabend (Stadthalle, Theatercafé)
14 Programm – 12.10.

09:00–09:45
HS Nord Med.
10:00–11:30
HS Nord Med.
11:30–11:45
HS Nord Med.
Hauptprogramm – Sonnabend, 13. Oktober 2012
11:45–12:15 Kaffeepause
12:15–13:00
HS Nord Med.
Plenarvortrag Tabletten
Prof. Dr. Wolfgang Frieß (München)
Analyse von Aggregaten und Partikeln in Proteinarzneimitteln
– was ist eigentlich das Problem?
Kurzvorstellung von Leuchtturmprojekten
• Helmholtz Institute for Pharmaceutical Research (HIPS)
Saarland
• Center of Drug Absorption and Transport (C_DAT)
Greifswald
• Zentrum für Pharmaverfahrenstechnik (PVZ)
Braunschweig
• Fraunhofer Projektgruppe – Translationale Medizin und Pharmakologie (TMP) Frankfurt
am Main
Pharmazie 2020 – Perspektiven in Forschung und Lehre
Chair: Prof. Dr. Dieter Steinhilber
Abschlussveranstaltung
Verleihung von Ehrungen und Preisen
Programm – 13.10. 15

Vorträge

Plenarvortrag Moleküle
Genom-basierte mikrobielle Naturstoff-Forschung für die Entwicklung von anti-Infektiva
Müller R
Helmholtz-Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Pharmaceutical Biotechnology,
Saarland University, Campus, Building C2 3, 66123 Saarbrücken, Germany
Microorganisms continue to be one of the most important resources of bioactive molecules exhibiting potential for
pharmaceutical application (1). This is especially true for urgently needed antibacterials which almost exclusively are
derived from bacteria or fungi. Aiming at the identification of novel hit and lead structures ideally addressing new
targets in pathogens, genome analysis of microbial producer organisms has become very useful for the in silico
prediction of the hypothetical potential of a given organism for the production of secondary metabolites (2,3).
Although revealing tremendous potential in terms of the presence of typical natural product biosynthetic genes, the
translation of this knowledge into "real compounds in the flask" clearly represents the bottleneck in this approach. In
the presentation I will discuss our recent efforts to develop and employ genome mining to a) identify (4,5), b)
heterologously express (6,7) and c) modify secondary metabolite biosynthesis (8,9). In addition, I will present two
success stories where novel natural product scaffolds were isolated and have been found to indeed address new
targets in pseudomonads and staphylococci (10-12).
Reference List:
1. Newman, D. J., Cragg, G. M.: J.Nat.Prod. 2012, 75(3): 311–335.
2. Bode, H. B., Müller, R.: Angew.Chem.Int.Ed. 2005, 44(42): 6828–6846.
3. Wenzel, S. C., Müller, R.: Natural Product Reports 2011,26(11):1385-407.
4. Schneiker, S. et al.: Nat.Biotechnol. 2007, 25(11): 1281–1289.
5. Cortina, N. S. et al.: Angew.Chem.Int.Ed. 2012, 51(3): 811–816.
6. Wenzel, S. C. et al.: Chem.Biol. 2005, 12(3): 349–356.
7. Fu, J. et al.: Nat.Biotechnol. 2012, 30 440–446.
8. Rachid, S. et al.: ChemBioChem 2011, 12(6): 922–931.
9. Quade, N. et al.: Nat Chem Biol Nat Chem Biol 2012, 8(1): 117–124.
10. Bielecki, P. et al.: ChemBioChem 2012, in press.
11. Steinmetz, H. et al.: Angew.Chem.Int.Ed. 2011, 50(2): 532–536.
12. Dehn, R. et al.: Angew.Chem.Int.Ed. 2011, 50(17): 3882–3887.
18 Plenarvorträge

Plenarvortrag Targets
Anti-inflammatory compounds as novel tools to rescue the pancreatic β-cell in diabetes
Maedler K
Islet Biology Laboratory, Centre for Biomolecular Interactions Bremen, University of Bremen, Germany
In both, type 1 and type 2 diabetes mellitus the main processes leading to β-cell failure are apoptosis and loss of
function. Although the β-cell has an enormous capacity to adapt to conditions of higher insulin demand, e.g. in
obesity and pregnancy, its continuous overstimulation leads to a β-cell overwork, subsequent loss of function and
apoptosis and diabetes progression. This is a result from a complex interplay of environmental factors together with
genetic predisposition.
Many studies demonstrate how cytokines and chemokines have an active role in triggering the immune-response
against the β-cell population. In a recent study we have identified 2 classical inflammatory mediators, the cytokine IL-
1β and the chemokine CXCL10, which both play an active role in triggering β-cell destruction.
We have identified the Toll like receptor 4 as the receptor for CXCL10 and as new pathway for the induction of β-cell
apoptosis.
In the lecture, I will give an overview on mechanisms of destruction of the β-cell in diabetes, the influence of obesity,
the crosstalk between fat and pancreas and resulting new therapeutic approaches to fight onset and progression of
diabetes.
Key words: diabetes, β-cells, apoptosis, proliferation, inflammation, IL-1β, CXCL10
Plenarvorträge 19

Plenarvortrag Tabletten
Analysis of aggregates and particles in protein drug products – what is the challenge?
Frieß W
Department of Pharmacy; Pharmaceutical Technology and Biopharmaceutics; Ludwig-Maximilians Universitaet Muenchen, Butenandtstr. 5,
D-81377 Muenchen
Protein aggregates and particles are currently a hot topic in pharmaceutical industry as well as for the regulatory
agencies. FDA sees a strong scientific basis for aggregates, including sub-visible particulates, to induce immune
responses but insufficient data to determine the likelihood of immune response induction given the levels/types of
aggregates in any given product. Therefore, aggregates and sub-visible particulates should be assumed critical to
the observed safety and efficacy profile and controlled commensurate with risk [1]. In order to adequately define the
actual risk, on the one hand more detailed studies on the causes and correlation of protein aggregates and particles
with immunogenicity are deemed necessary. On the other hand the detailed characterization of protein particles is
mandatory. The latter includes both quantity and quality such as size, shape, morphology, bond (covalent or
noncovalent) or protein conformation. This implies that no single method can cover the entire range but instead a
multitude of orthogonal methods is applied for characterization [2,3]. All methods come with limitations which need to
be considered and new methods are developed. For example classical size exclusion chromatography (SEC) may
provide inaccurate results in the analysis of protein aggregates due to protein adsorption to column media as well as
dissociation or formation of aggregate species at the higher salt concentration typically applied in the eluent.
Orthogonal methods like analytical ultracentrifugation or asymmetrical flow field flow fractionation should always be
used in the development of SEC methods and to corroborate the accuracy of SEC results [4]. In order to obtain
additional information, methods can be combined, for example a combination of SEC with addition of fluorescent dye
enables to trace unfolding of the individual separate protein species [5]. Another option is to combine SEC with the
determination of individual second virial coefficients of monomer and oligomers in protein samples [6]. For
characterization of subvisible particles several new methods have been established. Nanoparticle tracking analysis
(NTA) is an innovative system for sizing particles from about 30 to 1,000 nm based on laser light scattering
microscopy to track individual nanoparticles moving under Brownian motion [7]. Another system called Archimedes is
based on a fluidic microchannel embedded inside a resonator through which the sample is transported and as
particles flow through the resonators the change in resonant frequency gives the individual particle mass and size
with high resolution [8]. But also established methods like the classic Coulter Counter experience a renaissance as
the results of the standard optic based methods to analyse subvisible particles like light obscuration of micro flow
imaging depend on refractive index differences between particle and formulation [9]. Ultimately these developments
also give rise to new guidelines to direct the industry and USP has just published a chapter “Subvisible Particulate
Matter in Therapeutic Protein Injections” for comment in the Pharmacopia Forum. The critical evaluation of
protein aggregates and particles is not only challenging for standard formulations but also presents a major
roadblock for drug delivery systems [10].
References:
1. Rosenberg A.: Workshop on protein aggregation and immunogenicity, July 2012 Breckenridge, CO.
2. Mahler H.-E. et al.: J. Pharm. Sci. 2009, 98(9): 2909–2934.
3. Zölls S. et al.: J. Pharm. Sci. 2012, 101(3): 914-935.
4. Carpenter J. et al.: J. Pharm. Sci. 2010, 99(5), 2200–2208.
5. Printz M., Friess W.: J. Pharm. Sci. 2012, 101(2): 826-837.
6. Printz M., Kalonia D., Friess W.: J. Pharm. Sci. 2012, 101(1): 363-372.
7. Filipe V., Hawe A., Jiskoot W.: Pharm. Res. 2010, 27(5): 796-810.
8. Burg T.P. et al.: Nature 2007, 446(7139), 1066-1069.
9. Hawe A.: Workshop on protein aggregation and immunogenicity, July 2012 Breckenridge, CO.
10. Jiskoot W. et al.: J. Pharm. Sci. 2012, 101(3): 946-954.
20 Plenarvorträge

Sessionvorträge Moleküle
Session Struktur-basiertes Design
New algorithms for 3D pharmacophore-based virtual screening
Spitzer G1 , Liedl K1 , Wolber G2 1Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria
2Institute of Pharmacy/Pharmaceutical Chemistry, Computer-Aided Drug Design, Freie Universität Berlin, Königin-Luisestr. 2+4, 14195 Berlin,
Germany
Virtual screening using three-dimensional arrangements of chemical features (3D pharmacophores) has become a
highly relevant method for virtual hit identification and lead optimization. Although frequently used, considerable
differences exist in the interpretation of chemical features and the implementation of their corresponding 3D overlay
algorithms [1].
We have recently continued the development of our 3D alignment algorithm relying on pattern recognition. Based on
this method, a workflow for high-performance multi-conformational database screening has been created using new
fingerprint filtering techniques that delivers a higher degree of geometric accuracy than previously published or
commercially available approaches. Despite the considerable gain in geometric subsampling and the resulting
increased alignment accuracy, this technique allows for computationally highly efficient virtual pharmacophore
screening allowing to process millions of molecules in hours on a desktop computer. The impact of increased
geometric accuracy on virtual screening will be presented and discussed by means of benchmarks and prospective
screening studies.
References:
1. Spitzer G et al., J. Chem Inf. Model 2010, 50(7): 1241-1247.
Sessionvorträge Moleküle 21

From Determinants of RUNX1/ETO Tetramerization to Small-Molecule Protein-Protein Interaction
Inhibitors Targeting Acute Myeloid Leukemia
Metz A1 ; Schanda J2 ; Wichmann C2 ; Grez M2 ; Gohlke H1 1 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University, Universitätsstr. 1, 40591 Düsseldorf, Germany
2 Georg-Speyer-Haus, Institute for Biomedical Research, Paul-Ehrlich-Str. 42-44, 60596 Frankfurt, Germany
A promising way to interfere with biological processes is through the control of protein-protein interactions by means
of small molecules. Many of these small-molecule modulators known to date have not been found by rational design
approaches. In large part this is due to the challenges that one faces in dealing with protein binding epitopes.
However, recent advances in the understanding of the energetics and dynamics of protein binding interfaces open up
a way to apply rational design approaches also for finding protein-protein interaction modulators. Here, we target the
homotetramerization of the NHR2 domain within the RUNX1/ETO fusion protein using computational methods
developed in our group [1, 2, 3]. Homotetramerization of the NHR2 domain leads to the onset of acute myeloid
leukemia.
Initially, energetic contributions of individual amino acids within the NHR2 homotetramer were analyzed. Ala
substitution of amino acids with strong energetic contributions abolished tetramer formation. The resulting NHR2
dimers fail to induce leukemia in mice. These studies revealed the existence of a hot spot at the NHR2 dimertetramer
interface, suitable for a molecular intervention.[4] Based on the hot spot information, an 18-mer peptide was
designed that inhibits NHR2 tetramerization. This peptide was used as a model compound to establish in vitro
assays. Using the hot spot and peptide information, small-molecule inhibitors of the NHR2 tetramerization were
identified by virtual screening and validated by several in vitro assays. Some of these compounds led to the induction
of differentiation markers and apoptosis in NHR2-dependent cell lines. These compounds may thus be potential
chemotherapeutics in the case of acute myeloid leukemia.
References:
1. Kruger DM, Gohlke H, Nucl. Acids Res. 2010, 38: W480-W486.
2. Gohlke H, Kiel C, Case DA, J. Mol. Biol. 2003, 330: 891-913.
3. Metz A et al., J. Chem. Inf. Model. 2012, 52, 120-133.
4. Wichmann C et al., Blood 2010, 116: 603-613.
22 Sessionvorträge Moleküle

Docking to GPCRs: ligand diversity, efficacy and selectivity
Kolb P 1 ; Rosenbaum D M 2 ; Schmidt D 1 ; Bernat V 3 ; Tschammer N 3 ; Kobilka B K 2 ; Shoichet B K 4
1 Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35039 Marburg, Germany
2 Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305, USA
3 Chemistry and Pharmacy, Friedrich Alexander University Erlangen, Schuhstraße 19, 91052 Erlangen, Germany
4 Pharmaceutical Chemistry, University of California, 1700 4th Street, Box 2550, San Francisco, CA 94158, USA
With GPCRs becoming amenable to crystallization in the recent past [1], structure-based computational approaches
to ligand discovery have become possible. As only about 1% of the GPCRome has been solved, however, receptor
structure prediction is equally important. I will discuss the usage of x-ray structures and homology models for docking
and highlight some of the conclusions that can be drawn from three recent docking campaigns.
Campaign 1 used the recently determined X-ray structure of the β2-adrenergic receptor [2] to investigate the
advantages and limitations inherent in a structure-based approach to ligand discovery against this and related GPCR
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 –
structural and energetical analysis
Straßer A1 ; Wittmann H-J2 1 Department of Pharmaceutical/Medicinal Chemistry II, University of Regensburg, 93040 Regensburg, Germany
2 Faculty of Chemistry and Pharmacy, University of Regensburg, 93040 Regensburg, Germany
Homo- and heterodimers of several GPCRs are suggested by a large number of experimental studies [1]. Most
experimental studies give hint for asymmetric dimers, consisting of two receptor protomers, coupling to only one
heterotrimeric G protein [1]. However, there is only hint, but no clear experimental evidence for a symmetric
homodimer, consisting of two receptor protomers, each coupling to one heterotrimeric G protein [1].
Based on the crystal structure of the κ-opiod receptor [2] and bovine rhodopsin [3], we manually aligned two active
hβ2R-Gαβγ-complexes [4] in a symmetric manner. For refinement of the resulting hβ2R-Gαβγ-homodimer, we
performed an extensive scan of the potential energy surface in an analogous manner, as already described [5].
Thereby, one hβ2R-Gαβγ-complex was translated along and rotated around the x-, y- and z-axis. Subsequently, the
resulting hβ2R-Gαβγ-homodimers were energetically minimized and the complex with smallest potential energy was
embedded into a POPC lipid bilayer and molecular dynamic simulation of the hβ2R-Gαβγ-homodimer (Fig. 1A) was
performed, including lipid, sodium and chloride ions, extra- and intracellular water.
The MD simulation revealed strong van-der-Waals interactions between both hβ2 receptors. POPC molecules placed
between both hβ2 receptors stabilize the hβ2R-hβ2R-interaction (Fig. 1B). Additionally, the symmetric active state
hβ2R-Gαβγ-homodimer is strongly stabilized by electrostatic and hydrogen bond interaction between the Gα- and
Gβ-subunits of complex 1 and 2 (Table 1). Furthermore, hydrogen bond and electrostatic interactions were detected
between the Gβ subunits of different monomers (Table 1).
Table 1: Interactions within the hβ2R-Gαβγ-homodimer
hβ2R (1) –
hβ2R (2)
Gα (1) –
Gβ (2)
Gβ (1) –
Gβ (2)
hβ2R (1)–
Gαβγ (2)
no.
of H
bonds
dist.
don-acc
< 0.35
nm
Eelec.(SR)
[kJ/mol]
EvdW
[kJ/mol]
3 3 -288 ± 36 -329 ± 15
6 7 -543 ± 46 -142 ± 15
3 2 -135 ± 44 -105 ± 12
0 0 -5 ± 4 -4 ±1
The molecular dynamic simulation revealed a stable symmetric active state hβ2R-Gαβγ-homodimer. Thus, the
suggested homodimerization between two hβ2 receptors, based on experimental studies [6], could be also found in
silico. The amino acids, determined in silico to be responsible for the interaction between both hβ2R-Gαβγcomplexes
represent a good starting point for further experimental studies.
References:
1. Kamal, M.; Maurice, P.; Jockers, R.: Pharmaceuticals 2011, 4 273-284.
2. Wu, H. et al.: Nature 2012, doi:10.1038/nature10939.
3. Park, J.H. et al.: Nature 2008, 454 183-187.
4. Rasmussen, S.G.F. et al.: Nature 2011, doi:10.1038/nature10361.
5. Strasser, A.; Wittmann, H.-J.: J Comput Aided Mol Des 2010, 24 759-769.
6. Angers, S. et al.: PNAS 2000, 97(7)3684-3689.
24 Sessionvorträge Moleküle

Session Molekulare Werkzeuge
Chemoproteomic target deconvolution of bioactive molecules
Bantscheff M
Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany
Chemoproteomics represents an emerging research discipline at the interface of medicinal chemistry, biochemistry,
and cell biology focused on studying the molecular mechanisms of action of drugs and other bioactive small
molecules. In this presentation, experimental strategies in current chemical proteomics research will be described
and illustrated using recent examples of successful applications (e.g. [1,2]). Finally, areas in drug discovery where
chemical proteomics-based assays using native endogenous proteins are expected to have substantial impact will be
highlighted.
References:
1. Bantscheff et al. Nat Biotechnol. 2011, 29(3): 255-65.
2. Dawson et al., Nature 2011, 478(7370): 529-33.
Microwave-Enhanced Synthesis of Fluorescent Dyes for Pharmacological Purposes Using Real-
Time Observation by UV/Vis Spectroscopy
Heller E1 ; Lohse M J2 ; Hoffmann C2 ; Holzgrabe U1 1 Institute for Pharmacy and Food Chemistry 1, Am Hubland, D-97074 Würzburg, Germany
2 Institute for Pharmacology and Toxicology 2, Versbacher Straße 9, D-97078 Würzburg, Germany
Fluorescent dyes play an important role as markers to investigate biological signalling pathways or to study binding
of active agents to receptors. It is possible to label proteins with 2 dyes (FRET technology, e.g. FlAsH and ReAsH
[1,2]) or to mark the active agent with one dye [3]. Tagging of active agents with fluorescent dyes is an alternative
method for radioactive marking.
Here we aimed to synthesize several dyes of different structural classes, e.g. substituted fluoresceins, pyrilium and
pyridine dyes, by means of microwave irradiation. Microwave-enhanced reactions are rather fast in comparison to
conventional heating and hence, the reaction time can be shortened from hours or days to minutes. Real time
monitoring of the reaction progress is often difficult, because classical monitoring (e.g. HPLC, TLC and NMR) can
take up to 30 minutes analysis time. This can cause overreaction or decomposition of the product. We created a
novel method which allows a real-time observation of the reaction progress in the microwave cavity. This allows
detecting the exact end-point of the reaction [4] and avoids the formation of side products. The knowledge was used
to speed up the synthesis of FlAsH and analogues. Additionally the dyes were obtained with excellent purity and high
yields.
References:
1. Hoffmann, C. Lohse M. J.: BIOspektrum 2006, 12(5): 495-497.
2. Lohse, M. J. et al.: Curr. Opin. Pharm. 2007, 7, 547-553.
3. Schneider, E. et al.: Chembiochem. 2007, 8(16), 1981-1988.
4. Heller, E. et al.: Eur. J. Org. Chem. 2010, 19, 3569-3573.
Sessionvorträge Moleküle 25

Molecular tools for positron emission tomography (PET): Development of a highly selective
radiotracer for the detection of neurotensin receptor 1 positive tumors
Lang C
Department of Chemistry and Pharmacy, Friedrich Alexander University Erlangen-Nürnberg (FAU), Schuhstr.19, D-91052 Erlangen, Germany.
Neurotensin (NT) is an endogenous, 13 amino-acid peptide that mediates a number of pharmacological effects
involving dopamine transmission, analgesia, and hypothermia [1]. In addition to these attributes NT acts as a key
player in multiple steps of cancer progression in numerous types of cancer cells.[2] Those carcinogenic effects are
presumably evoked by an abnormal expression of the neurotensin receptor 1 (NTS 1). Based on this overexpression
we aimed for a specific labeling of tumors by molecular imaging techniques such as positron emission tomography
(PET).
In collaboration with the Clinic of Nuclear Medicine of FAU, we have reported on the design and synthesis of a
radiolabeled derivative of the hexapeptide NT 8-13, the active fragment of the endogenous agonist neurotensin [3]. As
an extension of these investigations, we planned to construct a 18F-labeled non-peptidic NTS 1 selective ligand based
on the potent NTS antagonist SR142948A [4]. We herein report on the novel PET-Tracer 18F-LC40, which could be
synthesized by taking advantage of our click chemistry based ligation of 2-deoxy-2-[ 18F]fluoroglucosyl azide (azido-
[ 18F]FDG) to the alkyne functionalized analog of the lead structure. Receptor binding experiments using NTS 1
expressing CHO cells indicated a Ki value for F-LC40 of 0.5 nM with 70 fold selectivity towards NTS 2. The precursor for
18F-labeling was obtained by a palladium-catalyzed aminocarbonylation [5] of the respective bromoarene derivative with
an alkynylamine using Mo(CO)6 as carbon monoxide source. The CuAAC using azido-[ 18F]FDG afforded the glycosyl
ligand 18F-LC40 in a radiochemical yield of 20% in a total synthesis time of 75 min.
The µPET studies using HT29 (human colon carcinoma cell line) xenografted nude mice demonstrated specific binding
of 18F-LC40 in vivo. Furthermore, the tracer displayed a good tumor to blood ratio and excellent metabolic stability both
in vitro and in vivo.
O
O
O
N N
O
NH COOH
N
18 F-LC40
N
N
N
N
O OH
OH
References:
1. for review, see: Myers, R et al., ACS Chem. Biol. 2009, 4(7): 503–525
2. for review, see: Dupouy, S et al., Biochimie. 2011, 93: 1369–1378
3. Maschauer, S et al., Angew. Chem. Int. Ed., 2010, 49(5): 976–979
4. Labeeuw, B et. Al, 1996, Patent WO 96/3282.
5. Appukkuttan, P et al., Tetrahedron Lett., 2008, 49 : 5625–5628
18 F
OH
26 Sessionvorträge Moleküle

Analogs of siderophores and mussel adhesion proteins for applications in clinical hygiene and
implant medicine
Maison W1 ; Franzmann E2 ; Khalil F1 1 Universität Hamburg, Pharmaceutical and Medicinal Chemistry, Bundesstr. 45, 20146 Hamburg, Germany
2 Justus-Liebig-Universität Giessen, Organic Chemistry, Heinrich-Buff-Ring 58, 35390 Giessen, Germany
The chemical modification of material surfaces by molecular self-assembly of functionalized anchor molecules is an
attractive concept for various applications in implant medicine, bone engineering and clinical hygiene. The major
advantages of this principle for the generation of functional surfaces are a) the availability of efficient and scalable
coating techniques (compatible with large surface areas and three dimensional objects), b) the generation of
chemically well-defined surfaces and c) the need of relatively low amounts of anchor molecules.
Recently, catecholates have received considerable interest as reagents for the mild modification of metal and metal
oxide surfaces by convenient dip-and-rinse protocols.[1-3] This approach may be considered biomimetic, because
catecholate moieties are key components of natural metal-binders such as mussel adhesion proteins. In addition,
catecholates are abundant motifs in many siderophores like enterobactin.
Inspired by the intriguing molecular symmetry of siderophores such as enterobactin, we present the design and
synthesis of biomimetic triscatecholates with a tripodal binding motif based on adamantane scaffolds. These
triscatecholates form extremely stable molecular monolayers on metal surfaces and may be covalently conjugated to
any effector molecule of choice. We describe the immobilization properties of these multimeric catecholates on
implant materials such as TiO2 and demonstrate antifouling properties and toxicity data for PEG (effector =
polyethylene glycol) conjugates.
References:
1. J. H. Waite, J. H.; Tanzer, M. L.: Science 1981, 212(4498): 1038-1040.
2. Brubaker, C. E., Messersmith, P. B.: Langmuir 2012, 28(4): 2200-2205.
3. E. Franzmann, F. et al.: Chem.-- Eur. J. 2011, 17(31): 8596-8603.
Sessionvorträge Moleküle 27

Fluorescent and radiolabelled ligands as molecular tools for NPY receptors
Buschauer A1 ; Keller M1 , Pluym N1 ; Kaske M1 , Erdmann D1 , Bernhardt G1 1 Lehrstuhl für Pharmazeutische/Medizinische Chemie II, Universität Regensburg, 93040 Regensburg, Germany
In search for pharmacological tools for various G-protein coupled receptors (GPCRs) we explored the synthesis of
fluorescent ligands and radioligands as and the applicability of the labelled compounds to cellular studies of
neuropeptide Y (NPY) receptors as examples of peptidergic GPCRs. Four NPY receptor subtypes (Y1R, Y2R, Y4R,
Y5R) are functionally expressed in humans. Whereas Y1R, Y2R and Y5R are more sensitive to NPY and peptide YY
(PYY), the Y4R prefers the pancreatic polypeptide (PP) as the endogenous agonist.
Two complementary approaches to labelled NPY receptor ligands were considered:
(1) Preparation of “universal” ligands, addressing more than one receptor subtype. For instance, fluorescent
derivatives of the endogenous peptides are useful molecular tools for confocal microscopy or flow cytometry [1]. The
latter enables the simultaneous multiparametric determination of receptor subtype selectivities of investigational
compounds under equilibrium conditions, using mixtures of cells expressing the respective receptors [1].
(2) Synthesis of selective radiolabelled or fluorescent tracers, preferably nonpeptides, for the identification of NPY
receptor subtypes on cells and in tissues, and for the determination of binding data. Special attention was paid to
labelling strategies based on side chain modifications at the ε-amino group of lysine in peptides such as NPY (Y1,2,5R
agonist) or [K4 ]hPP (Y4R agonist), and, in particular, at the guanidino group of arginine, both in peptides and
nonpeptides [1-7].
So far, the functionalisation of arginine at NG has been largely neglected in the design of GPCR ligands. However,
previous studies from our laboratory demonstrated that the acylation of strongly basic guanidine residues, which are
characteristic of numerous biologically active compounds, is a general, very effective biosisosteric approach to retain
(or even increase) the pharmacological activities and to substantially improve the drug-like properties by lowering the
basicity of GPCR ligands. This also paved the way to radioactive ( 3H, 18F) tracers and fluorescent ligands.
Tritiated high affinity Y1R [5, 6] and Y2R antagonists were accessible by coupling of amino-functionalized guanidine
derivatives with succinimidyl [2,3-3H]propanoate. By analogy, prototypical [ 18F]-PET ligands for the detection of Y1Rs,
e.g. on breast cancer cells, were obtained. Interestingly, space-filling fluorophores were tolerated as well. To improve
the signal-to-noise ratio, red-emitting fluorophores, e. g. cyanine or pyrylium dyes, or dyes emitting in the near
infrared were preferred for coupling with argininamide-type Y1R [2, 7] and Y2R antagonist core structure by linkers
attached to the guanidine group. This approach resulted in receptor affinities in the one- to two-digit nanomolar
range. Labelling of short C-terminal peptides, modified, e.g., with cyclic β-amino acids, gave selective fluorescent tool
for the Y4R. Green emitting ligands were designed with respect to colocalization studies.
The presented strategies resulted in valuable pharmacological tools for the detection of the receptor of interest in
vitro and/or in vivo as well as for the characterisation of receptors and ligands in radiochemical and fluorescencebased
assays, including flow cytometry and confocal microscopy.
References:
1. Schneider, E. et al.: Chembiochem 2006, 7: 1400-1409.
2. Schneider, E. et al.: Chembiochem 2007, 8: 1981-1988.
3. Pluym, N. et al.: ChemMedChem 2011, 6: 1727–1738.
4. Ziemek et al.: J. Recept. Signal Transduct. 2007, 27: 217.
5. Keller, M. et al.: J. Med. Chem. 2008, 51: 8168-8172.
6. Keller, M. et al.: ChemMedChem 2011, 6: 1566-1571.
7. Keller, M. et al.: Bioorg. Med. Chem. 2011, 19: 2859-2878.
28 Sessionvorträge Moleküle

Session IVIVC: Modelle und Modellierungen
From Mice to Men: Application and Impact of in vivo Modeling and Simulation in Drug Research and
Development
Lehr T
1 Clinical Pharmacy, Saarland University, Campus Building C2 3, Room 2.33, 66123 Saarbrücken, Germany
The application and implementation of modeling and simulation in drug research and development has significantly
changed in the past decades. The enhancements were and are mainly triggered by improved computational
resources, the increased implementation of conceptual biological understanding, and the need to optimize the drug
research and development process. Nowadays, modeling and simulation techniques are frequently applied, whereas
the level of detail of these models can differentiate between empirical and mechanistic. However, the level of detail
and the model type applied is highly variable and depending on the stage of the drug research/development process,
the a priori knowledge available (i.e. in vitro or preclinical data) and the question that needs to be answered. Overall,
there is still an urgent need for a more efficient drug research and development process. The application of modeling
and simulation might be a cornerstone to meet this need.
The objective of this presentation is to provide a brief overview about the application of modeling and simulation in
the drug research and development process. The concept and impact of modeling and simulation shall be illustrated
by selected examples.
Sessionvorträge Moleküle 29

Estimating the In Vivo Release Behaviour of Extended- and Immediate-Release Products using the
Biorelevant Dissolution Stress Testers
Garbacz G 1,2 , Koziolek M 1 , Klein S 1 , Weitschies W 1
1 University of Greifswald, Department of Pharmaceutics and Biopharmaceutics, Felix-Hausdorf-Straße 3, 17489 Greifswald, Germany
2 Physiolution GmbH, Walther-Rathenau-Strasse 49a, 17489 Greifswald, Germany
Introduction
The estimation of the in vivo drug delivery behavior oral dosage forms by in vitro dissolution tests is a prerequisite for
successful development of immediate release (IR) and modified release (MR) formulations. Unfortunately, the
relevant aspects of the physiology of the gastro-intestinal (GI) tract cannot be simulated realistically using
compendial dissolution tests. Thus, the development of predictive in vitro test systems capable of biorelevant
simulation of GI transit conditions appears mandatory for rational dosage form development and characterization.
Materials and methods
In order to improve the predictive power of dissolution testing, we developed a novel dissolution test apparatuses
mimicking the physical conditions of the fasting gastrointestinal passage of IR and MR dosage forms which is
enabled by application of different dissolution vessels [1]. Parameters that can be depicted by our devices include GI
pressure exerted by gastrointestinal (GI) motility, shear stresses generated during phases of GI-transport,
temperature changes, media flow and emptying patterns and intermittent contact with intestinal fluids.
Results
The test results obtained for MR formulations indicate the susceptibility of modified release dosage forms towards
mechanical stresses of biorelevant intensity [2, 3]. Moreover, the relationship between the mechanical resistance of
MR tablets and the physico-chemical properties of the dissolution media was demonstrated. Our test results for IR
formulations clearly indicated that both the dosage form disintegration and the dissolution are impacted by
mechanical stress events of biorelevant intensity, temperature changes and different media flow conditions as they
may occur in the fasting stomach.
Conclusion
The study results confirmed the usability of this novel method and the corresponding test protocols for the estimation
of the in vivo drug delivery characteristics of solid oral dosage forms. Moreover, the study demonstrated the
applicability of the novel methods for the identification of undesired drug release behavior in vivo.
Acknowledgments: Federal Ministry of Education and Research (GASTROMAX, FKZ 13 N11368-370) is kindly acknowledged for the financial
support.
References:
1. Garbacz, G., et al., Eur. J. Pharm. Biopharm. 2008, 70(2): 421-482.
2. Garbacz, G., et al., Expert Opin. Drug. Deliv. 2010, 7(11): 1251-1261.
3. Garbacz, G., et al., J. Pharm. Pharmacol. 2012, 64(7): 944-968.
30 Sessionvorträge Moleküle

Improved input parameters for diffusion models of skin absorption
Hansen S1 ; Schaefer U2 1 Helmholtz Institute for Pharmaceutical Research Saarland, Saarland University, 66123 Saarbruecken, Germany
2 Biopharmaceutics and Pharmaceutical Technology, Saarland University, 66123 Saarbruecken, Germany
To use a diffusion model for predicting skin absorption requires accurate estimates of input parameters on model
geometry, affinity and transport characteristics [1]. We determined input parameters for diffusion models of skin
absorption focusing on partition and diffusion coefficients. These include experimental methods, extrapolation
approaches, and correlations that relate partition and diffusion coefficients to tabulated physico-chemical solute
properties.
Exhaustive databases on lipid-water and corneocyte protein-water partition coefficients were collected and analyzed.
Based on these improved equations were deduced which allow the prediction of solute partitioning into corneocytes
and lipids from the octanol water partition coefficient logKow and improve predictions of the macroscopic SC-water
partition coefficient.
With diffusion coefficients the situation is more complex. Due to the still ongoing discussion of lipophilic and polar
absorption routes across the SC the representation of these pathways varies greatly between individual models
which therefore require very different diffusion coefficients or mass transfer coefficients. A plethora of theories has
been used to obtain estimates. As a common principle all diffusion and mass transfer coefficients are inversely
related to a measure of molecular size. The strength of this size dependence may be different (less pronounced in an
aqueous environment than in the highly organized SC lipid bilayers, more pronounced in bilayer crossing than in
lateral diffusion).
In order to improve modeling of skin absorption from realistic (i.e. finite) doses it is essential to consider slow
equilibrating processes such as reversible protein binding [2, 3]. High keratin binding and slow desorption kinetics are
responsible (among other factors) for the formation of the stratum corneum reservoir reported especially in the
context of corticosteroid absorption. We can demonstrate that equilibrium binding and the rate of desorption are both
functions of solute lipophilicity. Our results prove that slow desorption from keratin may be a major contributor to the
stratum corneum reservoir. Also they prove that reservoir formation is relevant for lipophilic solutes independent of
drug class, thus allowing new options for topical pharmacotherapy.
From these studies we can conclude that in order to improve modeling of skin absorption in the future diffusion
models should include the vertical stratum corneum heterogeneity, slow equilibration processes, the absorption from
complex non-aqueous formulations, and an improved representation of dermal absorption processes. This will
require input parameters for which no suitable estimates are yet available.
References:
[1] S. Hansen, C.M. Lehr, U.F. Schaefer, Improved input parameters for diffusion models of skin absorption, Advanced Drug Delivery
Reviews, (2012) accepted.
[2] S. Hansen, D. Selzer, U.F. Schaefer, G.B. Kasting, An extended database of keratin binding, Journal of Pharmaceutical Sciences, 100
(2011) 1712-1726.
[3] S. Seif, S. Hansen, Measuring the stratum corneum reservoir: desorption kinetics from keratin, Journal of Pharmaceutical Sciences,
accepted (2012).
Sessionvorträge Moleküle 31

Interindividual gastrointestinal erosion and inter- as well as intraindividual gastrointestinal transit
time variability of hydrophilic matrix tablets
Jain A K1 ; Abrahmsson B3 ; Abrahmsén-Alami S3 ; Anschütz M2 ; Blume H2 ; Donath F2 ; Knopke C2 ;Schug B3 ;
Söderlind E3 ; Tajarobi F3 , Viridé A3 , Weitschies W1 ;Welinder A3 1 Department of Biopharmaceutics and Pharmaceutical Technology, University of Greifswald, Greifswald, Germany
2SocraTec R&D GmbH, Oberursel, Germany
3AstraZeneca R&D Mölndal, Sweden
Purpose
The aim of this study was to investigate the interindividual gastrointestinal erosion rate (%/hr.) as well as variability in
gastric emptying and colon arrival time of magnetically marked hydroxpropylmethylcellulose based matrix tablets.
Materials and methods
Table 1 Formulation composition of the hydroxypropylmethylcellulose matrix tablets
Formulation 1* Formulation 2* Formulation 3* Formulation 5*
Methocel K4M % (w/w) 23 10.1 - -
Methocel K100 % (w/w) 17 30 40 20
Dicalcium phosphate % (w/w) 57.6 57.5 57.6 77.6
* Additionally all formulations contain 1.4% (w/w) Iron oxide (E172) and 1 % (w/w) Sodium Stearyl Fumarate
Circular biconvex tablets of 350 mg with a diameter of 10 mm were prepared by direct compression at AstraZeneca
R&D Lund, Sweden. In vivo gastrointestinal performance of tablets was investigated by magnetic marker monitoring
(MMM) [1, 2] in a single centre, open label, cross over clinical study which was carried out in 5 healthy male
volunteers. Formulation 1 and Formulation 2 were administered in fasted state while Formulation 3 and Formulation
5 were administered under fasting as well as fed (30 min. after standard FDA breakfast) conditions. Mean, standard
deviation (SD) and percentage relative standard deviation (%RSD) for gastrointestinal erosion rate, gastric emptying
time and colon arrival time were calculated for each formulation as well as in each volunteer.
Results
Low variability in between-subject gastrointestinal erosion rate (9-29 %RSD) was observed between the subjects.
However, high between-subject variability was observed for gastric emptying time (56-110% RSD) while
interindividual variability was low for colon arrival time (0-39 %RSD). In only one volunteer, the Formulation 3, when
administered under fed condition reached the colon at 260 min. Formulation 5 when administered under fed condition
did not reach the colon in any volunteer. High intraindividual variability in gastric emptying time (59-110% RSD) was
observed for all the formulations when administered in fasted state. Moderate intraindividual variability in colon arrival
time (19-52 %RSD) was observed for all the formulations when administered in fasted state.
Conclusion
Erosion rate was found to be dependent on the formulation composition and exhibits low interindividual variability.
Gastric emptying time didn’t exhibit dependency on the formulation composition and/or on the volunteers and shows
high inter- as well as intra individual variability.
References:
1. Weitschies, W. et al.: J. Control Release. 2005, 108(2-3): 375–385
2. Weitschies, W. et al.: Adv. Drug Deliv. Rev. 2005, 57(8): 1210–1222
32 Sessionvorträge Moleküle

Session Moderne Trenntechniken
Continuous Improvement in pharmaceutical analytics: faster, more precise and more selective
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
2 Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, China
The increasing need to properly answer to pharmaceutical and bioanalytical challenges induced tremendous
progresses in electrophoresis and chromatography. Meanwhile Capillary Gel Electrophoresis (CGE), also known as
Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS), is established in the pharmaceutical industry replacing
SDS-PAGE gel electrophoresis, for example for the purity control of monoclonal antibodies. In order to quantify these
components with sufficient precision using the same quality control method, a relative standard deviation (RSD [%])
for the quantitative analysis under 2% is required. A reliable and highly precise CGE method could be obtained after
thorough optimisation. It was crucial to increase the sample concentration and the injection volume in order to
achieve sufficiently high signal-to-noise ratios (>70). The application of hydrodynamic injection is beneficial for the
precision of the method compared to the traditionally used electrokinetic one. Linearity was demonstrated and limits
of detection and quantitation were estimated. Both injection modes were compared in long series runs (n=48).
Furthermore, the use of an internal standard was investigated. Thus, the RSD [%] of the migration time was reduced
from 0.9% to 0.2% and the RSD [%] of peak areas was greatly improved. However, the normalisation to the total
area further reduced the influence of the injection error. RSDs [%] for the peak area ratios of typically between 1 and
2% were provided.
Affinity Capillary Electrophoresis (ACE) can nicely detect interactions of proteins by the mobility changes which occur
in the presence of various ligands [1]. ACE is able to explore changes in the conformation as well as charge changes
of proteins. Six repeats were done with a very good precision due to the use of a special rinsing procedure [2]. The
RSDs [%] of the migration times and the mobility ratios were typically below 2%, very often below 0.2%. The
influence of various charged species e.g. metal ions, such as Cu2+ , Mn2+ and Mg2+ , pharmaceutical cations as
ephedrine hydrochloride and pirenzepine dihydrochloride or anions such as glutamic acid and succinic acid was
tested on the migration behavior of BSA, ß-lactoglobulin and ovalbumin. Furthermore the influence of
phosphorylation on protein interactions was investigated at the activation site of the extracellular signal-regulated
kinase 1 (ERK1). Organic cations and metal ions showed clearly different interactions with the proteins. Together
with the advancements in mass spectrometry, NMR, and in other techniques such as THz spectrometry, deeper
understanding of the behaviour of biomolecules will become possible, which in turn will strongly support the
discovery of new pharmaceuticals.
Y. Xu greatly acknowledges the funding from the Alexander von Humboldt Foundation
References:
1. Busch, M. H. A. et al.: J. Chromatogr. A 1997, 777(2): 311-328.
2. El-Hady, D. et al.: J. Pharm. Biomed. Anal. 2010, 52(2): 232–241.
Sessionvorträge Moleküle 33

Untersuchung von Proteinen und Metaboliten - Was können moderne Massenspektrometer leisten?
Sinz A1 1 Abteilung für Pharmazeutische Chemie & Bioanalytik, Institut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-
Langenbeck-Str.4, D-06120 Halle/Saale
Die Massenspektrometrie hat sich in den vergangenen Jahren zu einer immer bedeutenderen Methode für die
Analyse klinischer Proben im Hochdurchsatz entwickelt. So findet man Massenspektrometer vor allem in den
Bereichen, in denen die klassischen Verfahren, wie Immunoassays, limitiert sind, wie beispielsweise in der
toxikologischen Analytik oder im Therapeutischen Drug Monitoring. Ebenso besitzt die Massenspektrometrie ein
großes Potential für die qualitative und quantitative Untersuchung des Proteoms und Metaboloms in
Körperflüssigkeiten, worauf in diesem Beitrag näher eingegangen wird.
Biotransformation of the NR2B-selective NMDA receptor antagonist ifenprodil
Falck E; Wünsch B
Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-Universität, Hittorfstr. 58-62, 48153 Münster, Germany
The NR2B-subunit of the NMDA receptor has a particular binding site which is named after ifenprodil (1) [1]. This
compound shows a high affinity to the NMDA receptor. Its antagonistic affect can be used for the therapy of
neurodegenerative diseases such as schizophrenia, epilepsy or parkinson’s disease.
1
The presentation will show the metabolism of ifenprodil, regarding the phase I and phase II metabolites resulting from
hydroxylation and conjugation reactions.
The metabolites were obtained by biotransformation with rat liver microsomes. Identification was possible using
HPLC-ESI-MSn . The structures of the resulting fragments were confirmed by recording the exact mass yielding from
time-of-flight detection.
References:
1. Williams, K.: Curr. Drug Targets 2001, 2: 285-298
34 Sessionvorträge Moleküle

Macroscopic Questions and Molecular Answers: Measuring Optical Rotation in Latest Chiral
Separation Techniques
Thiessen A; Bertram N
Anton Paar OptoTec GmbH, Albert-Einstein-Str. 5, 30926 Seelze-Letter, Germany, www.anton-paar.com
Optical instruments are used in bio analytics to determine physical properties such as optical rotation or refractive
index of chemical as well as biological compounds. The optical methods are among the oldest methods known, yet
do the macroscopic optical properties tell a lot about the behaviour on the nano and molecular level. Technical
innovations have dramatically improved the data quality of optical measurements in general and polarimeters
especially within the last years, making the determination of optical rotation of chiral substances as accurate and
traceable as never before.
Pharmaceutical drugs often come as chiral substances. Their enantiomers may differ in their pharmacodynamic and
pharmacokinetic interactions with receptors, enzymes and proteins in general. Therefore, one enantiomer – the
eutomer – may have the desired pharmaceutical activity, whereas the other enantiomer – the distomer – may be
inactive or even cause unwanted side effects. To ensure the desired efficacy and to avoid unwanted adverse effects
of a drug, chiral substances may reliably be separated by the use of chiral HPLC columns.
Polarimeters are used as chiral detectors in HPLC setups. But also after the separation process, a traceable and
accurate polarimetric analysis is mandatory to verify the qualitative and quantitative separation of those enantiomers.
Spectral polarimetry can be used to differentiate optically active substances by their rotational dispersion and may
shed light onto molecular structures.
We will present the possibilities of using polarimeters as rotation sensitive HPLC detectors and discuss principles
and instrumentation for spectral polarimetry. We will then introduce novel developments in polarimetry to minimise
the risk of measuring errors. This includes a live and recordable visualisation of the sample conditions inside the
sample cell, avoiding errors from bubbles or particles. Saving a picture of the filling condition greatly improves the
data traceability for pharmaceutical applications. The technology also indicates proper temperature distributions
within the sample, raising the bar for high quality data generation in polarimetric measurements like never before.
Another innovation automatically identifies the sample cell and checks its suitability for the selected method in order
to avoid human error.
We will briefly discuss how the enhanced traceability and data security improves compliance with pharmaceutical
industry standards.
Sessionvorträge Moleküle 35

Session Naturstoffe
Hepatic bioactivity of phenolic natural compounds
Kraus B 1 ; Decker M 1 ; Wolff H2 1 Lehrstuhl Pharmazeutische Biologie, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
2 Carl Zeiss Microscopy, Kistlerhofstrasse 75, 81379 München, Germany
The liver is the largest organ in the human body and manages a great number of functions. Its role as clearance
organ bears the risk that substances that should be degraded or eliminated are hepatotoxic and lead to tissue
damage [1]. The capacity of a substance to produce liver damage often results from the interaction of a series of
complex cellular processes, involving uptake, biotransformation and elimination of the substance [2].
There are a number of medicinal plants and natural compounds, e.g. silymarin/silibinin from milk thistle (Silybum
marianum (L.) GAERTN.), which posess hepatoprotective properties and are used for the therapy of liver diseases [3].
The mechanism of hepatoprotection by these compounds is generally by exerting multiple effects, including e.g.
antioxidant, immunomodulatory, and antiinflammatory activities. In recent years Xanthohumol (XAN), a prenylated
chalkone found in hop (Humulus lupulus L.) cones, has been reported to show hepatoprotective activity and to inhibit
hepatic inflammation and fibrosis [4].
We are interested in the hepatic bioactivity of prenylated and not prenylated chalcones from Humulus lupulus and
(the putative hepatotoxic) Piper methysticum G. FORST. as well as metabolites and structural derivatives thereof,
looking for structure activity-relationships. For this, we turned our attention to cellular bioavailability of XAN,
visualizing and tracing its uptake, intracellular accumulation and distribution [5].
In addition, we studied the hepatic bioactivity of the chalcones and metabolites. The influence of the substances on
cell viability and mitotoxicity was determined and antiinflammatory mechanisms were adressed.
In a further project, we analysed the potential of the flavonolignan silibinin to act hepatoprotective against tacrineinduced
liver cell damage in hepatic stellate cells. We determined the capability of silibinin to reduce tacrinemediated
cyto- and mito-toxicity applying either an equimolar mixture of silibinin and tacrine or a stable hybrid
structure with silibinin covalently connected to tacrine [6].
Acknowledgments: We like to thank Katharina Zenger and Magdalena Motyl for carrying out the cellular assays, Xinyu Chen for synthesis of
the silibinin-tacrin-hybrid, Petr Jirásek and Susanne Vogel for synthesis of chalcones and metabolites, and Jörg Heilmann for fruitful discussion
and support (all from Lehrstuhl Pharmazeutische Biologie, University Regensburg).
References:
1. Ramadori, G. et al.: J. Physiol. Pharmacol. 2008, 59 Suppl 1, 107-117.
2. Guillouzo, A.: Environ. Health. Perspect. 1998, 106 Suppl 2, 511-532.
3. Muriel, P., Rivera-Espinoza, Y.: J. Appl. Toxicol. 2008, 28, 93-103.
4. Dorn, C., et al.: Mol. Nutr. Food Res. 2009, 54 Suppl 2:S205-13.
5. Wolff, H., et al.: J. Agric. Food Chem. 2011, 59(24):12893–12901.
6. Chen, X., et al.: J. Med. Chem. 2012, DOI: 10.1021/jm300246n.
36 Sessionvorträge Moleküle

Mode of cell death induction by pharmacological V-ATPase inhibition
von Schwarzenberg K1 ; Wiedmann R M1 ; Oak P2 ; Schulz S3 ; Zischka H3 ; Wanner G4 ; Efferth T5 ; Trauner D6 ;
Vollmar A M1 1Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University, 81377 Munich, Germany
2Department of Pharmacy, Pharmaceutical Biology-Biotechnology, Ludwig-Maximilians-University, 81377 Munich, Germany
3Institute of Toxicology, Helmholtz Center Munich, German Research Center for Environmental Health, 85764 Neuherberg, Germany
4Department of Biology I, Ludwig-Maximilians-University, 82152 Martinsried, Germany
5Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, 55099 Mainz, Germany
6Department of Chemistry, Ludwig-Maximilians-University, 81377 Munich, Germany
Recently V-ATPase, a multisubunit proton pump, has come into focus as an attractive target in cancer cell invasion.
However little is known about the role of V-ATPase in cell death and especially the underlying mechanisms remain
mostly unknown. We used archazolid B, a macrolide of myxobacterial origin being a novel very potent inhibitor of the
V-ATPase as an experimental drug as well as a chemical tool to decipher V-ATPase related cell death signaling. We
found that archazolid induced apoptosis in highly invasive tumor cells at nanomolar concentrations while showing
almost no cytotoxicity in non tumorus cells. Apoptosis was caspase-dependant and executed by the mitochondrial
pathway shown by breakdown of mitochondrial potential, leakage of cytochrom c from the mitochondria and
activation of caspase-9. Prior to apoptosis induction archazolid lead to the activation of stress responses such as
autophagy and glycolysis. Autophagy was induced at concentrations that do not alkalize lysosomes and was shown
by degradation of p62 or fusion of autophagosomes with lysosomes. Autophagy was found to be a survival
mechanism at low concentrations but could not be accomplished at higher concentrations due to lysosome
alkalization by V-ATPase inhibition. Archazolid also lead to glycolysis induction which is mediated by the hypoxiainducible-factor-1
alpha and induced due to energy stress. This was shown by a decline of the ATP level and arrest
of energy consuming processes (AMPK, mTOR, eIF2alpha). As silencing HIF1alpha increases apoptosis, glycolysis
was suggested to be an adaptive mechanism. We conclude that archazolid leads to energy stress which activates
autophagy and glycolysis and finally leads to apoptosis. We propose V-ATPase as a promising drugable target in
cancer therapy caught up at the interplay of apoptosis, autophagy and cellular/metabolic stress.
Sessionvorträge Moleküle 37

Cyanobacteria in Anticancer Natural Products Research
Niedermeyer T H J1 ; Daily A2 ; Swiatecka-Hagenbruch M1 , Moscow J A2 1 Cyano Biotech GmbH, Magnusstr. 11, 12355 Berlin, Germany
2 Hematology-Oncology Department of Pediatrics of the University of Kentucky, 740 South Limestone, Lexington, Kentucky 40536, USA
Cyanobacteria are a prolific source of natural products. Many secondary metabolites isolated from cyanobacteria
show remarkable cytotoxicity. In the first part of this talk, cyanobacteria and some of the most interesting compounds
that have potential for the exploitation as lead compounds or have already been advanced into clinical studies (e.g.
the cryptophycins and dolastatins) are highlighted. In the second part, microcystins are presented as novel leads.
Microcystins, rather known as hepatotoxins produced by cyanobacteria, are currently also studied for their potential
as leads for anticancer drug development. They potently inhibit the eukaryotic protein phosphatase families PP1 and
PP2A [1]. Microcystins display their toxicity after transporter-mediated uptake by the cell. Three human proteins are
thought to be able to mediate this uptake, the organic anion transporting polypeptides OATP1B1, OATP1B3, and
OATP1A2. Liver cells express OATP1B1 and OATP1B3 transporters and are thus able to absorb microcystins from
the blood, resulting in a pronounced liver toxicity of microcystins. OATP1B3 is also expressed in hepatocellular
carcinoma cells (HCC) as well as gastrointestinal and lung tumors. Recent data offer support that the OATP1B3
protein is also expressed in a significant percent of breast and colon tumors [2, 3]. Interestingly, compared to
OATP1B1, OATP1B3 is found only in low abundance in liver cells. Thus selectivity that favors OATP1B3 over
OATP1B1 should lead to a decreased hepatic clearance / toxicity and an increased uptake in OATP1B3-expressing
tumors, creating a therapeutic window for the respective compound.
Since about 90 naturally occurring microcystins with variations in the seven constituting amino acids are known and
many more structures are not described yet, there is high potential for isolating variants with unique properties. In
fact, in our initial screening of 20 microcystins, we have found four microcystin structural variants with an IC50 in the
low nanomolecular range and with transporter selectivity that favors OATP1B3 over OATP1B1 by a factor of more
than ten. Some of these microcystin variants have not yet been described in the literature.
Testing of further microcystin variants, the elucidation of their structures and the deduction of structure-activity
relationships will enable us to find and create more selective and potent compounds.
OH
HN
References:
1. Watanabe, M. et al., Toxic Microcystis (CRC Press) 1995.
2. Muto, M. et al., Cancer Sci. 2007, 98(10): 1570–1576.
3. Lee, W. et al., Cancer Res. 2008, 68(24): 10315–10323.
O
NH 2
NH
N
NH
38 Sessionvorträge Moleküle
COOH
O
H
N
O
O
N
H
N
O
O
COOH
Structure of a typical microcystin (Microcystin LR)
NH
HN
O

Applied phytochemical research – an industry’s perspective
Stintzing F C
WALA Heilmittel GmbH, Dorfstrasse 1, 73087 Bad Boll/Eckwälden, Germany
An overview is given on research conducted on selected European plants used for cosmetics and complementary
medicine. A first key aspect will deal with the composition of a lipophilic extract from Cydonia oblonga and its
ramifications for cosmetic purposes [1]. The impact of location and harvest date on the phenolic composition at the
example of Salvia officinalis [2], Alchemilla mollis and A. vulgaris [3] will be a second focal point. A third topic will
cover the biotransformation of hydrophilic compounds from Betula pendula [4], Hamamelis virginiana [5,6], Nicotiana
tabacum [7] and Atropa belladonna [8] upon lactic acid fermentation. Fourth, the findings obtained by an in-depth
screening including lipophilic and hydrophilic compounds of Mercurialis perennis will be presented [9,10,11]. Finally,
most recent data on the stability evaluation of selected essential oils during exposure to light and elevated
temperatures will be shown also covering new approaches for quality control measures [12,13,14,15].
The data presented demonstrate that phytochemical research on the European flora is most rewarding and deserves
a more attentive dedication even on plants that are seemingly studied well. The impact of processing resulting in
specific compound patterns but also differing bioactivities should encourage both industry and academia to join
forces in discovering and making use of the plethora of phytochemicals in local plants.
Acknowledgments: The author is grateful to all co-authors and collaborators for their common interest and enthusiasm in conducting the
investigations presented.
References:
1. Lorenz, P. et al.: Anal. Bioanal. Chem. 2008, 391(2): 633-646.
2. Schnitzler, P. et al.: Phytomed 2008, 15(1/2): 62-70.
3. Duckstein, S.M. et al., Ztschr. Naturforsch C/ J. Biosci. 2012: submitted.
4. Millet, A., Stintzing, F., Merfort, I.: J. Pharm. Biomed. Anal. 2009, 49(5): 1166-1171.
5. Duckstein, S.M.., Stintzing, F.C.: Anal. Bioanal. Chem. 2011, 401(2): 677-688.
6. Duckstein, S.M. et al.: Phytochem. Anal. 2012: doi.10.1002/pca.2359.
7. Millet, A., Stintzing, F., Merfort, I.: J. Pharm. Biomed. Anal. 2010, 53(2): 137-144.
8. Schwarzenberger, M. et al.: Pharmazie 2012, 67(5): 460-466.
9. Lorenz, P. et al.:, Phytochem. Anal. 2009, 21(3): 234-245.
10. Lorenz, P. et al., Ztschr. Naturforsch C/J. Biosci. 2010, 65(3/4): 174-179.
11. Lorenz, P. et al., Phytochem. Anal. 2012, 23(1): 60-71.
12. Turek, C., Stintzing, F.C.: Anal. Bioanal. Chem. 2011, 400(9): 3109-3123.
13. Turek, C., Stintzing, F.C.: J. Food Sci. 2011, 76(9): C1365-C1375.
14. Turek, C., Stintzing, F.C.: Food Res. Int. 2012, 46(1): 341-353.
15. Turek, C., Kirschmann, N., Stintzing, F.C.: Ztschr. Arznei Gewürzpfla/J. Med. Spice Plants 2012, 17(2): 73-79.
Sessionvorträge Moleküle 39

Session Klinische Wirkstoffentwicklung
Single- and multiple-transfected cell lines as tools for the analysis of drug transport and of the
interplay of drug transport and drug metabolism
König J, Fahrmayr C, Fromm M F
Institute of Experimental and Clinical Pharmacology and Toxicology, Department of Clinical Pharmacology and Clinical Toxicology, Friedrich-
Alexander-Universität Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany
In liver drug disposition and effects are determined by the coordinated processes of drug uptake, drug metabolism
and drug export. Following transporter-mediated drug uptake (e.g. by the organic anion transporting polypeptide
OATP1B1, gene symbol SLCO1B1) and subsequent phase I (e.g. by the cytochrom P450 enzyme CYP3A4) and / or
phase II metabolism (e.g. by UDP-glucuronosyltransferase 1A1) parent compounds or their metabolites are
frequently exported by members of the ABC transporter superfamily (e.g. by the apically localized export pump
MRP2, gene symbol ABCC2) into bile. So far, mostly single-transfected cells recombinantly overexpressing the
protein of interest were used to investigate its role in drug disposition. This setup may be useful for determining the
substrate spectrum of transporters or metabolizing enzymes or for the analysis of transporter-mediated drug-drug
interactions but results in limited data regarding the interplay of drug transport and drug metabolism. Therefore,
double- and multiple-transfected cell lines have been established expressing an uptake transporter together with an
export pump [1, 2, 3] or expressing an uptake transporter simultaneously with the phase II enzyme UGT1A1 and the
export pump MRP2 [4]. The latest development was the establishment of a quadruple-transfected cell line
recombinantly overexpressing the uptake transporter OATP1B1, the phase I enzyme CYP3A4, the phase II enzyme
UGT1A1 and the apically localized export pump MRP2. This presentation summarizes data regarding the usage of
single- and multiple-transfected cell lines for the analysis of drug transport, transporter-mediated drug-drug
interactions and the interplay of drug transport and drug metabolism.
References:
1. Cui et al.: Mol Pharmacol 2001, 60: 934-943.
2. Letschert et al.: J Pharmacol Exp Ther 2005, 313: 549-556.
3. Nies et al.: Naunyn Schmiedebergs Arch Pharmacol 2007, 376: 449-461.
4. Fahrmayr et al.: Br J Pharmacol 2012, 165: 400-406.
40 Sessionvorträge Moleküle

Methods to investigate the clinical relevance of drug transporters
Oswald S
University Medicine of Greifswald, Center of Drug Absorption and Transport, Department of Clinical Pharmacology, Felix-Hausdorff-Str. 3,
17487 Greifswald, Germany
Transporter proteins are crucial determinates in the pharmacokinetics (absorption, distribution and excretion) of
many drugs. Many of these transporters are highly expressed in tissues that are of outstanding relevance in drug
disposition such as the intestine, the liver, the kidneys and at physiologically relevant blood-tissue barriers such as
the blood-brain-barrier or the placenta.
In general, one can distinguish between SLC (solute carrier) transporters which mostly facilitate the cellular uptake of
their substrates, and ABC (ATP-binding cassette) transporters such as P-glycoprotein (P-gp, ABCB1) which pump
their substrates out of the cell in an energy-dependent manner.
So far hundreds of these transporters have been described, whereas the clinical impact of only few candidates (e.g.
P-gp, OATP1B1) is well established. On the other side, there is an urgent need to know the distinct cellular transport
properties of a certain drug to avoid unwanted transporter-based drug-drug interactions (by induction or inhibition),
high inter-subject variability in drug disposition (e.g. by genetic variants) and safety issues.
Consequently, current guidelines from regulatory agencies (e.g. FDA, EMA) recommend comprehensive in vitro
characterization of new molecular entities with regards to their transporter affinity. After verification that a certain drug
may be a substrate for a certain transporter protein clinical studies in humans are recommended to conclude on the
potential clinical impact.
The presentation will provide a brief overview about clinical state-of-the-art approaches as well as experimental
studies in humans to conclude on the clinical relevance of the most important ABC and SLC transporters.
Sessionvorträge Moleküle 41

The functionality of uptake transporters in primary hepatocytes of different species can be verified
by estrone-3-sulfate
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
2Department of Clinical Pharmacology, University Medicine of Greifswald, Greifswald, 17487, Federal Republic of Germany
3Charité Virchow Klinik, Berlin, 13353, Federal Republic of Germany
4BG Trauma Hospital, Tübingen, 72076, Federal Republic of Germany
Introduction: Primary mammalian hepatocytes are used for several in vitro applications like testing of drug
metabolism, toxicity and transporter assays. However, little is known about species specific differences or similarities
in the activity of uptake and efflux transporter. Therefore, we started a species specific characterization and
compared the uptake of different substrates in hepatocytes from pharmacological relevant species which are
commonly used for drug testing.
Materials & Methods: Human, rat, dog and monkey hepatocytes were incubated in serum free media. 2-4 days after
cell isolation a time and concentration dependent uptake of [3H]- estrone-3-sulfate (E3S), -bromosulfophthaleine
(BSP), -digoxine (Dig) and -taurocholic acid (TA) at 4°C and 37°C was measured by liquid scintillation counting.
Competition assays were performed using E3S as substrate and rifampicin, BSP, Dig or TA as competitors.
Results: All hepatocytes showed a time-dependent and saturable increase in E3S and BSP uptake at 37°C
compared to the uptake at 4°C. By contrast, Dig and TA showed a time-dependent and saturable transport only in
hepatocytes of humanoids and rats, but not in dog. Moreover Dig uptake was about 10 fold higher in human than in
rat and monkey hepatocytes. E3S revealed a high affinity to human (Km = 12.9 ± 10.1 µmol/l; Vmax = 84.2 ± 30.3
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;
Vmax = 10.0 ± 2.1 pmol/mg × min). In rat hepatocytes E3S showed an increased uptake with increasing
concentrations but no kinetic data could be calculated. For BSP and Dig no pharmacokinetic data could be calculated
in any species and TA was transported only into hepatocytes of monkey (Km = 17.5 ± 12.5 µmol/l; Vmax = 5.8 ± 2.1
pmol/mg × min). Rifampicin clearly inhibited the E3S uptake in monkey (IC50 = 7.9 (2.4; 25.9) µmol/l) and dog
hepatocytes while uptake of E3S in rat hepatocytes was not influenced. E3S uptake was also inhibited by increasing
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
(177; 226) µmol/l) hepatocytes. Dig and TA did not significantly influence the E3S uptake in primary hepatocytes in
dog, rat and monkey hepatocytes.
Discussion: Our data suggest that among all substrates tested E3S is the most suitable substrate to verify the
functionality of uptake transporters in primary hepatocytes of humanoids, dogs, and rats. However, major differences
were observed in the uptake of drugs within the species tested here. These species specific differences in
hepatocellular uptake should receive attention when data obtained in drug transport, metabolism and toxicity assays
are transferred from animal experiments to humans.
This work was supported by the Ministry of Economy, Labour and Tourism of Mecklenburg-Vorpommern (Grant V630-F-108-2011/040 and
V630-VB-196-2011/038).
42 Sessionvorträge Moleküle

Solubilizing agents influence the pharmacokinetics of probe drugs by transporter interaction
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 Greifswald, Germany
2 Institute of Diabetes “Gerhardt Katsch” e.V., Greifswalder Str. 11e, 17495 Karlsburg, Germany
3 Institute of Pharmacy, Dpt. of Biopharmaceutics and Pharmaceutical Technology, Felix-Hausdorff-Str. 3, 17487 Greifswald, Germany
Background & Aims: Pharmaceutical excipients are supposed to be pharmacologically inactive by definition.
However, there is ample evidence that solubilizing agents (e.g. Cremophor ® EL) might significantly influence CYP450
dependent drug metabolism and efflux transport by ABCB1 and ABCC2 [1, 2, 3]. So far, little is known about the
influence of pharmaceutical excipients on uptake transporters, which were shown to be involved in the disposition of
many drugs. Therefore, we investigated the influence of polyethylene glycol 400 (PEG), hydroxypropyl-ß-cyclodextrin
(HPCD), Solutol ® HS15 (SOL) and Cremophor ® EL (CrEL) on the organic anion-transporting polypeptides (OATP)
1A2, 1B1, 1B3 and 2B1 and on the Na + /taurocholate cotransporting polypeptide (NTCP) in vitro. Furthermore, we
studied the impact of PEG, HPCD and SOL on the pharmacokinetics of the probe drugs acetaminophen (AAP),
talinolol (TAL) colchicine (COL) and cyclosporine A (CSA) in rats.
Materials & Methods: In vitro inhibitory effects of the excipients were studied in competition assays using HEK293
cells stably transfected with OATP1A2, 1B1, 1B3, 2B1 and NTCP, respectively, and in each case two established,
radio-labelled reference substrates. Intracellular accumulation of taurocholic acid (TA; OATP1A2, NTCP), estrone-3sulfate
(E3S; OATP1A2, 1B1, 2B1), bromosulfophthaleine (BSP; OATP1B1, 1B3, 2B1, NTCP) and estradiol-17ßglucuronide
(E17ßGln; OATP1B3) was measured by liquid scintillation counting after cell lysis. For pharmacokinetic
investigations, solutions of each probe drug with each excipient were administered intravenously as single or multiple
doses to male Wistar rats (each N=6). Drug in saline served as control. Blood samples, urine, feces and organs were
collected and drug concentrations determined by LC-MS/MS or HPLC-UV.
Results: In vitro, PEG selectively inhibited the uptake of TA and E3S by OATP1A2 (IC50 0.05% and 0.14%) but not by
1B1, 1B3, 2B1 and NTCP. HPCD strongly inhibited the TA, E3S and E17ßGln uptake by OATPs (IC50 0.001 –
0.24%). SOL and CrEL were potent inhibitors of all investigated transporters with the strongest effect on the TA, BSP
and E3S uptake by OATP1A2 and OATP2B1 (IC50 0.0003 – 0.01%). In vivo, all investigated excipients were able to
significantly modulate the pharmacokinetics of at least one probe drug. Pharmacokinetics of CSA were most
strikingly influenced by all three solubilizing agents, its single and multiple dose AUC was increased 2-5-fold and
volume of distribution (VD) was reduced to a similar degree while the elimination half-life (t1/2) remained unchanged.
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
times. HPCD increased VD of TAL, supported by a dramatic accumulation of TAL in kidneys and duodenum.
Furthermore t1/2 of TAL was doubled by HPCD. PEG only caused minor changes in the kinetics of TAL and COL but
significantly increased renal clearance of unchanged AAP and reduced renal clearance of AAP conjugates.
Conclusion: Frequently used solubilizing agents were shown to substantially influence intestinal and hepatic uptake
transporters in vitro and to significantly modulate the pharmacokinetics of drugs in the rat. This might lead to
unwanted drug interactions or adverse drug effects, which should be considered in drug development. However, the
clinical relevance should be evaluated in further investigations.
References:
[1] Bravo González, R.C. et al.: Biopharm. Drug Dispos. 2004, 25(1): 37–49.
[2] Hanke, U. et al.: Eur. J. Pharm. Biopharm. 2010, 76(2): 260–268.
[3] ten Tije, A. J. et al.: Clin. Pharmacokinet. 2003, 42(7): 665–685.
Sessionvorträge Moleküle 43

Sessionvorträge Targets
Session Proteomics und Metabolomics
OMICs approaches in the analysis of population-based cohorts – Potential avenues for biomarker
screening
Völker U, Ameling S1 , Endlich K2 , Felix S B3 , Gesell-Salazar M1 , Hammer E1 , Hoffmann W4 , Homuth G1 , Jehmlich N1 ,
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
2 Institute of Anatomy and Cell Biology
3 Clinic for Internal Medicine B
4 Institute for Community Medicine
5 Department of Restorative Dentistry, Periodontology and Endodontology
6 Clinic for Internal Medicine A
7 Institute for Clinical Chemistry and Laboratory Medicine, University Medicine Greifswald, D-17475 Greifswald, Germany
Different OMICs approaches are currently widely applied in the screening for diagnostic and prognostic biomarkers in
case-control settings or for the exploration of the pathophysiology of different diseases. On the other side populationbased
cohorts provide an extremely valuable resource particularly for the study of prevalent diseases. One such
study, the Study of Health in Pomerania (SHIP), has been established in the last 15 years at the University of
Greifswald and it is internationally acknowledged for its comprehensive high-quality phenotyping and quality control
measures (1). The first wave (SHIP-0) was a cross-sectional epidemiological survey in West Pomerania, in the
North-East of Germany, conducted from 1997-2001 and comprising 4310 participants. Two additional waves (SHIP-1
and 2) with expanded phenotyping were conducted between 2001-2006 and 2008-2012. Finally, a separate stratified
random sample of 8016 adults aged 20 to 79 years was drawn for SHIP-TREND for which examination started in
2009 and until October 2012 more than 3700 subjects participated.
Recently, we started to perform different OMICs analyzes with biomaterials of SHIP and SHIP-TREND, which started
with genome-wide genotyping and metabolome analysis of urine. Joining epidemiological, clinical and functional
genomics expertise we are now active partners of international consortia addressing the genetics of cardiovascular
diseases (2, 3).
The OMICs analyses are currently being expanded to include metabolomics measurements of plasma, expression
profiling of whole blood and proteomics approaches for the characterization of biofluids such as plasma, urine and
saliva. In the talk the progress in the analysis of the two population-based cohorts using OMICs technologies will be
presented.
The second part will be used to demonstrate, how such population-based cohorts can be used to provide reference
values in case-control settings particularly in the field of cardiovascular research. The application of these
technologies will be illustrated using dilated cardiomyopathy as an example (4).
References:
1. Völzke, H. et al.:Int J Epidemiol 2011, 40: 294-307.
2. Newton-Cheh, C. et al.: Nat Genet 2009, 41: 666-676.
3. Suhre, K. et al.: Nat Genet 2011, 43: 565-569.
4. Ameling, S. et al.: Eur Heart J 2012, in press.
44 Sessionvorträge Targets

Drug target fishing in silico and in vitro
Efferth T
Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128
Mainz, Germany
Mechanisms of action of drugs are frequently difficult to unravel in a comprehensive fashion. Elegant techniques
which appeared in recent years represent transcriptome-wide and proteome-wide expression profilings by
microarrays to connect gene and protein expression signatures to drug, genes and diseases.
Although clinical parameters are valuable for the determination of the prognosis of larger cohorts of patients, they are
frequently less helpful to predict the treatment success for each individual patient due to inter-individual variability
and heterogeneity. Therefore, personalized medicine aims to develop individualized treatment options adapted to
factors relevant for the prognosis of each single patient. Molecular biomarkers identified by “-omics” technologies are
expected to predict the likelihood of individual responsiveness (or toxicity) to treatment. This information can then be
used to optimize treatment schedules in a custom-made fashion.
However, microarray-based data do not allow conclusions on the protein targets of drugs. Considering that there is a
huge amount of potentially drugable proteins, but that only a few hundred drug targets have been exploited so far,
the development of drug target fishing techniques is urgently required. Identification of drug targets is mostly
performed by fishing proteins with a modified drug from a protein solution isolated from cells or tissues. Activitybased
probe profiling and compound-centric chemical proteomics are established methods for this purpose.
Pointing to individualized cancer therapy, a current concept is to identify mutations in “driver” genes of cancer
development and progression and to specifically develop small molecules or antibodies against patient-specific
mutated proteins in cancer tissues. The hope is not only to improve individual response rates of tumors, but also to
reduce side effects in normal tissues.
In this presentation, we give an overview of current bioinformatical and wet-lab technologies to identify and verify
drug targets and present selected examples.
References:
1. Volm, M.et al., Clin. Cancer Res. 2002, 8(6): 1843-8.
2. Gillet, J.P./Efferth, T. et al., Cancer Res. 2004, 64(24): 8987-93.
3. Efferth, T. et al., Mol. Cancer Ther. 2006, 5(8): 1986-94.
4. Efferth, T. et al., Mol. Cancer Ther. 2008, 7(1): 152-61.
5. Konkimalla, V.B., Efferth, T. Biochem. Pharmacol. 2010, 79(8): 1092-9.
6. Youns, M. et al., Planta Med. 2010, 76(17): 2019-25.
7. Sertel, S. et al., PLoS One 2012, 7(5): e35584.
8. Eichhorn, T. et al., Mol. Biosyst. 2012, 8(4): 1311-8.
Sessionvorträge Targets 45

From whole-organism to sub-cellular localization of metabolites by mass spectrometry imaging
Liebeke M
Department of Surgery and Cancer, Imperial College London, Sir Alexander Fleming Building, London, SW7 2AZ, UK
(m.liebeke@imperial.ac.uk)
Localization of metabolites in tissues or even within single cells is of emerging interest for comprehensive
understanding of biological systems. Advances in mass spectrometry imaging (MSI) allow the mapping of individual
molecules across a spatial distribution, but depending on the method used this distribution image is limited by the
technical possibilities of the desired device. We report here the use of matrix assisted laser desorption ionization
(MALDI) FTICR-MS to image tissue cross-sections (0.5cm 2 – 20µm resolution) and the application of secondary ion
mass spectrometry (SIMS) to further elucidate the cellular distribution of selected small molecules (0.5mm 2 – 100nm
resolution). Both MSI techniques gained comparable image results for the whole tissue cross-section but showed
distinct differences for high spatial resolution imaging. The advantages of TOF-SIMS and MALDI MSI approaches
will be pointed out, as well as problems and limitations of both techniques. Results of clinical interest (infected mouse
lung) and environmental biology (earthworm) will be presented. We show the power of the whole-organism to subcellular
metabolite localization, which may be able to reveal novel possible physiological implications in the analyzed
organism.
Figure 1 images from tissue sections generated by TOF-SIMS, observed ions indicated. Images for are presented in a gray scale from black to
white. Higher brightness corresponds to higher signal intensities. Total counts (TC) correspond to the total ion signal from the specified ion.
Maximum counts (MC) correspond to the maximal number of counts in a pixel. The field of view is 500 × 500 μm 2 .
46 Sessionvorträge Targets

Incorporation of unnatural amino acids to monitor conformational changes in peroxisome
proliferator-activated receptor alpha (PPARα)
Schwarz R, Tänzler D, Ihling C H, Kölbel K, Sinz A
Dept. Pharm.Chem./Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Halle/Saale, Germany
Chemical cross-linking combined with an enzymatic digestion and mass spectrometric analysis of the reaction
products has evolved into an alternative strategy to identify protein-protein and protein-ligand interactions. Here, we
present the investigation of conformational changes in PPARα upon ligand binding by photo-affinity labeling
combined with mass spectrometry. Peroxisome proliferator-activated receptors (PPARs) belong to the superfamily of
nuclear receptors. PPARα plays a crucial role in lipid metabolism and poses an important target for designing
antidyslipidemic drugs for the treatment of the metabolic syndrome.[1].
Expression and purification of PPARα variants were optimized in respect to obtain high protein yields. The
photoreactive amino acid para-benzoylphenylalanine (Bpa) was incorporated at position 258 of PPARα for
conducting photo-affinity labeling studies (Figure 1). Bpa incorporation was confirmed by peptide mass fingerprint
analyses. PPARα variant L258Bpa was employed for photo-cross-linking experiments in the absence and presence
of the PPARα ligand GW6471. After the photo-cross-linking reaction, reaction mixtures were enzymatically digested
and peptides were analyzed by high-resolution mass spectrometry (nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS).
The distance constraints imposed by the photochemical cross-links served to monitor conformational changes in
PPARα upon ligand binding.
Conformational changes in PPARα that had already been suggested in previous studies [2] were verified. In the
presence of the PPARα antagonist GW6471 a high number of cross-linked products were identified, which were
mainly localized in the region comprising amino acids 215 to 226. These cross-links can only be accomodated if the
flexible omega loop in PPARα, harboring the photo-amino acid Bpa258, exerts a huge conformational change and
thus closes the ligand binding pocket according to a “mouse trap” principle. Our strategy demonstrates that photoaffinity
labeling followed by a mass spectrometric analysis of the reaction products allows monitoring conformational
changes in target proteins in a highly efficient manner.
Figure 1: Three dimensional structure (pdb: 1KKQ) of GW6471-bound PPARα; Leu-258 that was replaced by Bpa is shown in green, Ω-loop
(yellow), activation function helix 2 (orange), and GW6471 (magenta).
References:
[1] Plutzky et al.: Am J Cardiol 2003.
[2] Müller et al.: J Med Chem 2009.
Sessionvorträge Targets 47

Session Transporter und Ionenkanäle
TRP channels as regulators of blood pressure and fertility
Freichel M
48 Sessionvorträge Targets

Role of Ca 2+ -activated K + Channels in Breast Cancer Proliferation
Lukowski R
Sessionvorträge Targets 49

Transporters in human platelets: physiological function and impact for pharmacotherapy
Jedlitschky G 1 , Rauch B 1 , Greinacher A 2 , Kroemer H K 1
1Department of Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald, Felix-Hausdorff-Str. 3,
17487 Greifswald, Germany
2Department of Immunology and Transfusion Medicine, University Medicine Greifswald, Sauerbruchstrasse, 17487 Greifswald, Germany
Platelets contain many biologically active molecules, such as factors triggering platelet aggregation, growth factors
and many other compounds, which are secreted upon platelet activation. Transporters mediate accumulation of
these molecules in platelet granules and –upon platelet activation- their translocation across the plasma membrane.
From a more pharmacologic point of view, platelets represent pharmacokinetic micro-compartments, in which the
combination of uptake and elimination transport, possibly together with intracellular metabolism determines
pharmacokinetics of drugs. As a result of transporter-mediated uptake, drugs can act on the target structures inside
the platelets and provoke therapeutically positive effects or negative side effects. On the other hand, elimination of
drugs from the platelet by export pumps can limit the effect of the drug and lead to drug resistance.
Several members of the two major transporter families, ATP-binding cassette (ABC) transporters and solute carriers
(SLCs), have been identified in platelets. 1 An example of an ABC transporter is MRP4 (ABCC4), which has been
shown to facilitate ADP accumulation in dense granules. 2 Several additional functions of MRP4 in platelets have
been proposed, since MRP4 is a very versatile transporter and its localization can be shifted from granules to the
plasma membrane upon activation of the platelets and under certain pathophysiological conditions. These functions
include the release of lipid mediators as well as a role in aspirin resistance. Several other ABC proteins have been
detected in platelets with functions in glutathione and lipid homeostasis including ABCC1 and ABCC3 as well as
several members of the ABCA subfamily. 3 The serotonin transporter (SERT, SLC6A4) in the platelet plasma
membrane represents a well characterized example of the SLC family. Furthermore, expression of the organic anion
transporter OATP2B1 (SLCO2B1), a high affinity transporter for certain statins, has been demonstrated in platelets.
Changes in transporter localization and expression can affect platelet function and drug sensitivity. Thus, due to their
various functions, platelet transporters can either serve as drug targets or as drug delivery systems and these
functions can be greatly altered in the setting of inherited or acquired platelet defects. Therefore, a detailed
understanding of the expression, function and regulation of transporters in platelets will add to a better understanding
of platelet biology and in many ways may result in improved therapeutic options.
References:
1. Jedlitschky, G. et al. Blood. 2012, 119(15):3394-3402.
2. Jedlitschky, G. et al. Am.J. Pathol. 2010, 176(3):1097-1103.
3. Niessen, J. et al. Pharmacogenet. Genomics. 2010, 20(6):396-400.
50 Sessionvorträge Targets

Expression analysis of drug transporter proteins in RPMI 2650 cell line and excised human nasal
mucosa
Reichl S
Institut für Pharmazeutische Technologie, Technische Universität Carolo-Wilhelmina zu Braunschweig, Mendelssohnstraße 1, 38106
Braunschweig, Germany
The nasal mucosa is an interesting site for application of drugs with systemic action, since it has a number of
advantages as a delivery route, including easy accessibility, extensive vascular supply and avoidance of
gastrointestinal degradation as well as first pass metabolism. Therefore more and more applications for nasal
systemic drug delivery have been developed during the last decades, and their number is still increasing [1].
In the development of drugs for nasal application, identification and characterization of the different uptake and efflux
systems are necessary. P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRP) are classified as
ATP binding cassette (ABC) transporters based on their sequences, organisation of the ATP-binding domains and
efflux function. ABC proteins represent a large family of integral membrane transporters that utilize the energy of ATP
hydrolysis to carry specific substrates across membranes [2]. The solute carrier gene (SLC) superfamily encodes
another large family of membrane-bound transporters, located in almost every cellular and organelle membranes.
Proteins of the SLC family include passive transporters, ion-coupled transporters and exchangers [3].
A comparison was performed between excised human nasal mucosa from turbinectomy surgeries and our in vitro
model based on immortalized human nasal epithelial cells (RPMI 2650) concerning the mRNA expression of different
efflux and uptake transporter proteins. First investigations to prove functionality of different ABC transporters in RPMI
2650 cells was carried out using permeation studies and uptake assays.
The mRNA expression of eight ABC transporters as well as 13 SLC transporters of excised human tissue and RPMI
2650 epithelial model were investigated and compared. The results showed promising similarity in the possible
presence or absence of these efflux and influx transporters in human tissue and the in vitro model. A similarity
between RPMI 2650 models and human nasal mucosa has already been shown for barrier properties to evaluate
passive drug permeation [4].
To verify the functionality of the first transporter protein, P-gp, bidirectional transport studies using rhodamine 123
with RPMI 2650 cells grown on polyethylene terephthalate filter were performed. Indirect immunofluorescence
staining for P-gp was also carried out. The findings confirmed the assumption that P-gp is not functionally expressed
in these epithelial cells.
An uptake assay using 5(6)-carboxy-2′,7′-dichlorofluorescein as a substrate for MRP1 and MRP5 was carried out.
The result indicated functional expression of these efflux transporters in RPMI 2650 cells. Identification and
localization of the responsible transporter proteins have to be proved by immunohistochemistry.
Further investigations will be focused on western blot, immunohistochemical analysis and permeation as well as
uptake studies using specific substrates for active transport to verify the protein expression and functionality of
further drug transporters in human nasal mucosa and in vitro model based on RPMI 2650 cell line.
Acknowledgments:
We are grateful to the German Federal Institute for Risk Assessment (BfR), which funded this work under grant no. 3-1329-469. Furthermore,
the authors thank Prof. Dr. Schroeder, Dr. Schmidt, Städtisches Klinikum Braunschweig and Dr. Reintjes, HNO-Praxis Schlosscarree
Braunschweig for the supply of human nasal mucosa specimens.
References:
1. Dimova, S. et al.: Toxicology In Vitro 2005, 19: 107–122.
2. Jones, P.M., George, A.M.: Cell. Mol. Life Sci. 2004, 61: 682–699.
3. He, L., Vasiliou, K., Nebert, D.W.: Human Genomics 2009, 3: 195–206.
4. Wengst, A., Reichl, S.: Eur. J. Pharm. Biopharm. 2010, 74: 290–297.
Sessionvorträge Targets 51

Session Nukleäre Rezeptoren und Transkriptionsfaktoren als Wirkstofftargets
Investigation on the estrogen receptor alpha selectivity of 2,3,5-triaryl-1H-pyrroles
Gust R
Institute of Pharmacy, University of Innsbruck, 6020 Innsbruck, Austria
The estrogen receptors ERα and ERβ are ligand-inducible transcription factors which belong to the nuclear hormone
receptor superfamily. ER activation or inhibition influences growth, differentiation and physiological function of
several organs. Selective ER modulators (SERM) like raloxifene or tamoxifen are used as partial ERα antagonists in
the therapy of osteoporosis and hormone dependent ERα-positive breast cancer, respectively. Despite their obvious
benefits, SERMs may cause some serious side effects, including thromboembolisms and endometrial cancer, which
urgently require the search for new derivatives.
In this study we wanted to design molecules that bind exclusively to ERα, which is over-expressed in hormonedependent
breast cancer cells. If the compounds displayed cytotoxic activity too, we would have an adequate drug
for the therapy of the ER-positive mammary carcinoma.
We started our investigations from the observation that triarylpyrazoles (e.g. PPT) but not related 2,4,5triarylimidazoles
showed high agonistic/antagonistic potency and ER subtype selectivity. This finding documents that
the position of the N-atoms of the heterocycle in relation to the aromatic rings determines the ER binding and
subtype selectivity.
In continuation of this study we synthesized 1H-pyrroles whose N-position in relation to the aryl rings at the
heterocycle was selected from the position in PPT resulting in Type A pyrroles or Type B pyrroles.
While Type A pyrroles showed strong degradation under physiological conditions, Type A pyrroles were stable and
activated the ER only in U2-OS/ERα cells. For all pyrroles no transactivation could be observed in ERß containing
U2-OS cells, which documents that they are selective ERα agonists in cellular systems. Additionally, the most
interesting drug 2,3,5-tris(2-fluoro-4-hydroxyphenyl)-1-propyl-1H-pyrrole inhibited the growth of hormone-dependent
tumor cells comparable to the established drug 5-fluorouracil and therefore fulfills the above mentioned
pharmacological profile.
52 Sessionvorträge Targets

Chemical epigenetics: histone modifying enzymes as targets to regulate transcription
Jung M
Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität, Albertstr. 25, 79104 Freiburg,
Background
Epigenetics is defined as inheritable phenotypic traits that determined by mechanisms beyond the DNA sequence.
Histone modifications are an important component of the epigenetic machinery and we are working on the drug
discovery of epigenetic inhibitors as well as the use of small molecule substrates to test for cellular enzyme activity.
Material and Methods
We have used focussed library and virtual screening in collaboration with Wolfgang Sippl (University of Halle) to
identify potential inhibitors of epigenetic targets. The candidates were then screened in in-vitro assays in our lab and
in collaborating labs (M. Lübbert, R. Schüle, University Freiburg Medical Centre) for phenotypic and functional
assays. We have used a fluorogenic substrate to screen cell culture, animal tissue samples and blood cells for
histone deacetylase activity and have validate this using western blotting of acetyl-histones.
Results
A combination of virtual and focussed library screening has identified new inhibitors of histone deacetylases as well
as the histone kinase PRK1. The PRK1 inhibitor lestaurtinib shows a suppression of androgen dependent gene
transcription in prostate cancer cells1 . A carbamate has been identified as a prodrug for hydroxamic acids with
increased intracellular activity2 and we currently work on testing different protecting groups and their effect on cellular
permeability.
We thank the Deutsche Forschungsgemeinschaft (Ju295/7-1 and Ju295/9-1, within SPP 1463) for funding.
References:
1. Köhler, J. et al.: PLoS ONE 2012, 7: e34973.
2. Schlimme, S. et al. ChemMedChem 2011, 6: 1193-8.
Sessionvorträge Targets 53

Helenalin derivatives and their impact on transcriptional control of mediators involved in rheumatic
diseases
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
Freiburg, Germany
2 Depart. Anesthesiology and Critical Care Medicine, University Hospital Freiburg, Freiburg, Germany
3 Inst. Pharmaceutical Scienecs, Pharmaceutical Bioinformatics, Albert-Ludwigs University, Hermann-Herderstr. 9, 79104 Freiburg, Germany
Helenalin derivatives are sesquiterpene lactones from the pseudoguaianolide type. They are considered as the
effective compounds in Arnica montana flowers which preparations have been proven to exert beneficial effects in
rheumatic complaints, such as osteoarthritis and rheumatoid arthritis (1). We here report studies on the molecular
mode of actions which contribute to a better understanding of these beneficial effects.
Both rheumatic diseases are characterized by chronic joint destruction. Whereas IL-1β and TNF-α are well-known
players in this process it has become more and more obvious that IL-17 is also involved (2). This cytokine stimulates
fibroblasts, macrophages and chondrocytes which for their part release various mediators with effects on
inflammation, cartilage damage and bone erosion. Interestingly, IL-17 does not only induce TNF-α and IL-1β, but
was shown to have also additive or even synergistic effects with TNF-α and IL-1β during the induction of cytokine
expression and joint damage in vitro and in vivo.
We could show with helenalinbutyrate that mRNA levels of IL-17 are reduced in primary human T-cells.
Consequently, pretreatment of human primary chondrocytes with helenalinisobutyrate or
dihydrohelenalinmethacrylate drastically reduced gene and protein expression of matrix metalloproteinase 1 (MMP1)
and 13 (MMP13) at low concentrations with helenalinbutyrate being mostly active. Additionally, this sesquiterpene
lactone lowered synergistically upregulated mRNA and protein of COX-2 by IL-17 and IL-1β. Transcription of MMPs
is tightly regulated by the transcription factors AP-1 and NF-κB which were demonstrated to be directly targeted by
the helenalin derivatives. The effect on NF-κB has already been studied intensively (3) in contrast that one on AP-1.
Impacts on these transcription factors were supported by molecular docking studies.
Acknowledgments: We are grateful to Bioforce company for financial support.
References:
1. Merfort, I. Curr. Drug Targets. 2011, 12: 1560-73.
2. Koenders, M.I., Joosten, L.A.B., van den Berg, W.B., Ann. Rheum. Dis. 2006, 65: 29-33.
3. Garcia-Pineres, A. et al., J. Biol. Chem. 2001, 276: 391113-20.
54 Sessionvorträge Targets

Rescuing mutant p53 using halogen-enriched fragment libraries (HEFLibs)
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
Pharmacy, Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany
2 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom
The destabilizing cancer mutation Y220C creates a druggable surface crevice in the DNA-binding core domain of the
transcription factor and tumor suppressor p53 [1,2]. We developed a strategy exploiting halogen bonding for lead
discovery to stabilize the mutant with small molecules. We designed halogen-enriched fragment libraries (HEFLibs)
as starting points to complement classical approaches [3]. From screening of HEFLibs and subsequent structureguided
design, we developed substituted 2-(aminomethyl)-4-ethynyl-6-iodophenols as p53-Y220C stabilizers. Crystal
structures of their complexes highlight two key features: (i) a central scaffold with a robust binding mode anchored by
halogen bonding of an iodine with a main chain carbonyl and (ii) an acetylene linker, enabling the targeting of an
additional subsite in the crevice. The best binders showed induction of apoptosis in a human cancer cell line with
homozygous Y220C mutation. Structure-activity relationships show correlations to QM-based interaction energies of
halogen-bonding. Our structural and biophysical data suggest a more widespread applicability of HEFLibs in lead
identification, yielding lead structures that feature binding modes hardly obtainable by other techniques.
References:
1. Joerger, A. C.; Ang, H. C.; Fersht, A. R.: Proc. Natl. Acad. Sci. U. S. A. 2006, 103(41): 15056-61.
2. Boeckler, F. M. et al.: Proc. Natl. Acad. Sci. U. S. A. 2008, 105(30): 10360-10365.
3. Wilcken, R. et al.: J. Am. Chem. Soc. 2012, 134(15): 6810–6818.
Sessionvorträge Targets 55

Novel leads for PPARs and FXR: a computer-aided drug design approach
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
Peroxisome proliferator-activated receptors (PPARs) and farnesoid X receptor are in the main focus of
pharmaceutical research for a wide range of metabolic disorders. Novel findings in this area indicate that although
these targets have been investigated intensively, there is still an unmet need for novel safe and efficient lead
structures. This presentation demonstrates the impact of computer-aided drug design techniques applied to
discovery and optimization of novel lead structures for PPARs and FXR. We present three different approaches to
identify novel lead structures: virtual screening, de novo design and in silico drug repurposing.
In our virtual screening study we were able to identify a potent and selective PPARa agonist using a combination of
pharmacophore- and shape-based screening [1]. The software retrieved a potent hit (EC50 = 44 nM), with >100fold
selectivity against PPARγ. In a second application, we designed a lead structure de novo, starting from the known
PPARα agonist GW590735 [2]. In the design process, two moieties were replaced by bioisosteric groups suggested
by SQUIRRELnovo, which are responsible for compound potency. The new lead structure yielded submicromolar
activity on PPARα (EC50 = 0.51 µM) in a cell-based reporter-gene assay [3].
Furthermore we were able to identify the molecular basis for the glucose-lowering effects of imatinib based on FXR
activation using a machine-learning approach [4]. We applied self-organizing maps to self-organizing map (SOM) is a
virtual screening method for correlation of molecular structure and potential biological activity [5]. We used a SOM
trained with known FXR ligands to screen the DrugBank database [6] for potential ligands for FXR. We were able to
identify six approved drugs out of the Drugbank as FXR modulators (ketoconazole, pentamidine, dobutamine,
imatinib, papaverine and montelukast) by using a SOM for screening of the DrugBank database. We show FXR
modulation by selected compounds in a full length FXR transactivation assay and modulation of a FXR target gene
by imatinib.
Acknowledgments: We gratefully acknowledge financial support from the Else-Kröner-Fresenius-Stiftung, Merz Pharmaceuticals, Lipid
Signaling Forschungszentrum Frankfurt (LiFF), Oncogenic Signaling Frankfurt (OSF) and Fonds der Chemischen Industrie for financial
support.
References:
1. Proschak, E., Zettl, H., Tanrikulu, Y., Weisel, M., Kriegl, J.M., Rau, O., Schubert-Zsilavecz, M., Schneider, G., ChemMedChem 2009, 4:
41–44.
2. Sierra ML, Beneton V, Boullay A-B, Boyer T, Brewster AG, Donche F, et al., J Med Chem 2007, 50: 685–95.
3. Proschak, E., Sander, K., Zettl, H., Tanrikulu, Y., Rau, O., Schneider, P., Schubert-Zsilavecz, M., Stark, H., Schneider, G., ChemMedChem
2009, 4: 45-48.
4. Steri, R., Achenbach, J., Steinhilber, D., Schubert-Zsilavecz, M., Proschak, E., Biochem Pharmacol 2012 accepted.
5. Schneider P, Tanrikulu Y, Schneider G., Curr Med Chem 2009, 16: 258–66.
6. Knox C, Law V, Jewison T, Liu P, Ly S, Frolkis A, et. Al., Nucleic Acids Res 2011, 39: D1035–1041.
56 Sessionvorträge Targets

Inhibitors of class I histone deacetylases induce 5-lipoxygenase expression
Steinhilber D
Goethe-University Frankfurt, Institute of Pharmaceutical Chemistry/ZAFES, Max-von-Laue Str. 9, D-60438 Frankfurt, Germany
5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes. We have previously shown that the histone
deacetylase (HDAC) inhibitor trichostatin A (TSA) activates 5-LO transcription via recruitment of Sp1, Sp3 and RNA
polymerase II to the proximal promoter [1, 2].
In order to identify the HDACs involved in the regulation of 5-LO promoter activity isoform-specific HDAC inhibitors
were applied. 5-LO promoter activity and mRNA expression was upregulated by the class I HDAC inhibitors apicidin
and MS-275 but not by class II inhibitors.
In order to analyze the chromatin modifications at the 5-LO promoter associated with HDAC inhibition, the time
course of 5-LO mRNA induction by trichostatin A was analyzed and the concomitant changes in histone
modifications at the 5-LO promoter in HL-60, U937 and Mono Mac6 cells was determined. ChIP analysis revealed
that trichostatin A increases acetylation of histones H3 and H4 at the 5-LO core promoter in HL-60 and U937 cells
whereas no significant changes were observed in Mono Mac6 cells. The appearance of H3 and H4 acetylation
preceded the 5-LO mRNA induction whereas in all three cell lines, induction of 5-LO mRNA expression correlated
with histone H3 lysine 4 trimethylation (H3K4me3), a marker for transcriptional activity of gene promoters.
Induction of 5-LO mRNA expression by class I HDAC inhibitors coincides with the upregulation of 5-LO promoter
activity as determined by reporter gene assays. The data suggest that in contrast to calcitriol and TGFβ [3], induction
of 5-LO mRNA expression by HDAC inhibition mainly occurs on the 5-LO promoter level.
Acknowledgments: We gratefully acknowledge financial support from the Else Kröner-Fresenius-Stiftung and DFG (GRK757).
References:
1. Schnur, N., Seuter, S., Katryniok, C., Rådmark, O. and Steinhilber, D., Biochim Biophys Acta 2007, 1771: 1271-1282.
2. Klan, N., Seuter, S., Schnur, N., Jung, M. and Steinhilber, D., Biol Chem 2003, 384: 777-785.
3. Stoffers, K.L., Sorg, B.L., Seuter, S., Rau, O., Radmark, O. and Steinhilber, D., J Mol Biol 2010, 395: 884-896.
Sessionvorträge Targets 57

Session Thrombozytenfunktion und Gerinnung
Novel oral anticoagulants – State of the art and aspects of their application
Alban S
Institute of Pharmacy, Christian-Albrechts-University of Kiel, Gutenbergstr. 76, 24118 Kiel, Germany
Since thromboembolic diseases, i.e. myocardial infarction, stroke and pulmonary embolism (PE), represent the major
cause of death in the Western world, anticoagulants are a class of life-saving drugs. Whereas there have been some
advances in short- and medium-term anticoagulation during the last two decades, the only option for long-term
anticoagulation were the vitamin K antagonists (VKA) so far.
This has changed in 2008 with the approval of two novel oral anticoagulants (NOA): the direct thrombin inhibitor
(DTI) dabigatran etexilate (Pradaxa ® ) und the first direct Factor Xa-Inhibitor (DXI) rivaroxaban (Xarelto ® ) for
prevention of venous thromboembolism (VTE) after hip and knee replacement operation. In 2011, apixaban
(Eliquis ® ), another oral DXI, was approved for this proof of concept indication and dabigatran etexilate and
rivaroxaban were licenced for stroke prevention in atrial fibrillation and rivaroxaban additionally for treatment of deep
vein thrombosis (DVT) and prevention of recurrent DVT and PE following acute DVT.
Therefore, it’s time for an update: How the anticoagulation in clinical practice changes with the NOA? What are the
differences between the NOA and the VKA as well as between the three NOA? Which patients are candidates for
switching from VKA to NOA? What is about practical aspects like monitoring anticoagulant efficacy, interruption for
surgical or invasive procedures and management of bleeding? Finally, are Marcumar ® and heparin injections about
to fold?
58 Sessionvorträge Targets

Novel platelet inhibitors – State of the art and aspects of their application
Greinacher A
Sessionvorträge Targets 59

Naturally occurring PF4/polyanion complexes prime for heparin-induced thrombocytopenia as a
misdirected host defense mechanism
Krauel K1,2 ; Weber C3 ; Jaax M1 ; Hackbarth C 1 ; Brandt S2 ; Block S2 ; Hammerschmidt S3 ; Greinacher A1 1 Institut für Immunologie und Transfusionsmedizin
2 Zentrum für Innovationskompetenz – Humorale Immunreaktionen bei kardiovaskulären Erkrankungen
3 Abteilung Genetik der Mikroorganismen, Interfakultäres Institut für Genetik und Funktionelle Genomforschung, Ernst-Moritz-Arndt-Universität
Greifswald, Germany
Heparin-induced thrombocytopenia (HIT) is caused by highly immunogenic complexes of negatively charged heparin
and positively charged platelet factor 4 (PF4). Some of the resulting antibodies against PF4/heparin complexes
activate platelets via the platelet FcγIIa receptor, leading to thrombin generation and a prothrombotic state. We have
recently proposed that HIT might be the result of a misdirected bacterial defense mechanism, as PF4 bound to
bacteria also exposes the epitopes to which PF4/heparin antibodies bind [1]. Consistently, we showed that
PF4/heparin antibodies are highly significantly associated with periodontitis, one of the most prevalent human
infections mostly induced by Gram-negative bacteria [2].
We now identified potential PF4 binding structures on the surface of Gram-negative bacteria using different bacterial
mutants with defined variations in their lipopolysaccharide (LPS) sequences [3]. Binding of biotinylated PF4 was
quantified by flow cytometry, and exposure of the antigenic epitope was assessed by adsorption and elution of
PF4/heparin antibodies. Bacterial mutants lacking the O-antigens and the core of LPS but still exposing lipid A
showed strongest PF4 binding, mediated by the phosphate groups of lipid A. As both the lipid A on the surface of
Gram-negative bacteria and the amino acids of PF4 contributing to polyanion binding are highly conserved, our
results further support the hypothesis that neoepitope formation on PF4 after binding to bacteria is an ancient host
defense mechanism. The distance of two phosphate groups on lipid A is rather similar to the distance of phosphate
groups on DNA or RNA. We therefore assessed the interaction of PF4 and nucleic acids. By enzyme-immunoassay
and functional studies, we found cross-reactivity of PF4/heparin antibodies with PF4/DNA complexes in vitro.
Systematic assessment of different RNA- and DNA-constructs revealed length and number of double-stranded
segments of nucleic acids as risk factors for complex formation with PF4. This has consequences for drug
development, as we accordingly found immunogenicity as a class effect of aptamers (DNA/RNA based therapeutics).
PF4/aptamer complexes bound PF4/heparin antibodies in vitro and induced PF4/aptamer antibodies in a mouse
model.
We next asked the question, which structural changes of PF4 may trigger B-cell activation using circular dichroism
(CD) spectroscopy. Pronounced structural changes of PF4, leading to expression of anti-parallel beta sheets,
occurred during the interaction with highly charged polyanions known to be immunogenic in vivo. Only minor or no
changes were found for weakly charged or neutral polymers. This allows to predict immunogenicity of drugs exposing
polyanions by CD spectroscopy.
Through desulfation of heparin, the new compound 2-O, 3-O desulfated heparin (ODSH, ParinGenix) has low
anticoagulant activity but the residual negative charge still allows interaction with PF4. We found that ODSH inhibits
platelet activation by PF4/heparin antibodies by disruption of PF4/heparin complexes [4]. In addition, ODSH does not
induce the conformational changes of PF4 analyzed by CD spectroscopy. Therefore a mixture of heparin and ODSH
might be an interesting option to reduce the risk of HIT which has to be addressed in clinical studies.
In summary, polyanions induce structural changes of PF4 leading to an adverse immune reaction, which is a
misdirected humoral host defense mechanism. CD spectroscopy can be applied to screen new drugs for their
potential to induce immunogenic structural changes of PF4 and partially desulfated heparin can be applied to
antagonize the adverse biological effects of PF4/polyanion complex antibodies.
References:
1. Krauel, K. et al.: Blood 2011, 117(4): 1370-1378.
2. Greinacher, A. et al.: Blood 2011, 118(5): 1395-1401.
3. Krauel, K. et al.: Blood 2012, epub.
4. Krauel, K. et al.: Blood 2012, 119(5): 1248-1255.
60 Sessionvorträge Targets

Session Neue Targets und Testsysteme
PfGSK-3: A target for new antimalarial drugs?
Kunick C1 , Fugel W1 , Ratin M2 , Kruggel S3 , Oberholzer A E4 , Dzikowski R5 , Pressburger N5 , Ferandin Y2 , Lemcke T3 ,
Meijer L2 1 Technische Universität Braunschweig, Institut für Medizinische und Pharmazeutische Chemie, Beethovenstraße 55, 38106 Braunschweig,
Germany
2 Protein Phosphorylation & Human Disease Group, CNRS, Station Biologique, Place Georges Teissier, B.P. 74, 29682 Roscoff, France
3 Universität Hamburg, Institut für Pharmazie, Bundesstraße 45, 20146 Hamburg, Germany
4 Structural Biology Community Laenggasse (sbcl), 3000 Bern, Switzerland
5 Department of Microbiology & Molecular Genetics, The Kuvin Center for the Study of Infectious and Tropical Diseases, IMRIC, The Hebrew
University-Hadassah Medical School, Jerusalem 91120, Israel
Malaria is one of the most threatening infectious diseases in tropical areas, causing hundreds of thousands fatal
cases each year. [1] Although efficacious medicines are available for treatment and prophylaxis, the emergence of
resistance is urging for new drugs with alternative pharmacological mechanisms. The inhibition of plasmodial protein
kinases has repeatedly been suggested as a strategy for the development of antimalarial drugs throughout the last
decade. [2-4] In spite of several efforts in this direction, protein kinase inhibitors have not yet reached clinical trials as
antimalarial drugs. PfGSK-3, the plasmodial homologue of the human glycogen synthase kinase-3 (HsGSK-3), was
recently identified from Plasmodium falciparum, the causative agent of malaria tropica. With a view to evaluate
PfGSK-3 as potential target for new antimalarial agents, several known GSK-3 inhibitors were tested on PfGSK-3. [5]
Unfortunately, these compounds were either none-selective or showed selective inhibition of the human enzyme.
Therefore, compound libraries were tested to identify new selective PfGSK-3 inhibitors. A hit compound from this
campaign was employed as structural template for the design of a congener series. Analysis of structure-activity
relationships and structure-guided design enabled the preparation of selective PfGSK-3 inhibitors with in vitro
antiplasmodial activity in the single-digit micromolar concentration range.
Acknowledgments: The work was funded in part by the Commission of the European Communities (Contracts LSHB-CT-2004-503467 and
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
R. D., N. P., and C. K). S. K. was supported by a PhD grant from the University of Hamburg, Germany.
References:
1. WHO: World Malaria Report 2010 (http://www.who.int/malaria/world_malaria_report_2010/worldmalariareport2010.pdf).
2. Doerig, C., Meijer, L.: Expert Opin Ther Targets 2007, 11(3): 279–290.
3. Zhang, V. M., Chavchich, M., Waters, N. C.: Curr Top Med Chem 2012 12(5): 456–472.
4. Jirage, D., Keenan, S. M., Waters, N. C.: Infect Disord Drug Targets 2010, 10(3): 134–146.
5. Droucheau, E. at al.: Biochim Biophys Acta 2004, 1697: 181–196.
Sessionvorträge Targets 61

Chemical Oncology – Converging Cancer Genetics, Structural Biology and Medicinal Chemistry
Rauh D
Technische Universität Dortmund, Fakultät Chemie, Chemische Biologie, Otto-Hahn-Str. 6, 44227 Dortmund
The 512 protein kinases encoded by the human genome are a prime example of nature's ability to create diversity by
introducing variations to a highly conserved theme. Layers of regulatory mechanisms involving different combinations
of post-translational modifications, intramolecular contacts, and intermolecular interactions control the activity of each
kinase domain. Ultimately, they all achieve their effect by favoring particular conformations that promote or prevent
the kinase domain from catalyzing protein phosphorylation. The central role of kinases in various diseases has
encouraged extensive investigations of their biological function and three-dimensional structures, yielding a more
detailed understanding of the mechanisms that regulate protein kinase activity by conformational changes.[1] I will
discuss these regulatory mechanisms and show how conformational changes can be exploited for the design of
specific inhibitors that lock protein kinases in inactive conformations.[2, 3] In addition, I highlight our recent
developments to monitor ligand-induced structural changes in protein kinases and for screening and identifying
inhibitors that stabilize enzymatically incompetent kinase conformations.[4-8]
References:
1. Rabiller, M., et al., Arch Pharm 2010, 343(4): 193-206.
2. Getlik, M., et al., J Medicinal Chem 2009, 52(13): 3915-26.
3. Getlik, M., et al., Eur J Med Chem 2012, 48: 1-15.
4. Simard, J.R., et al., Nature Chem Biol 2009, 5(6): 394-6.
5. Simard, J.R., et al., J Am Chem Soc 2009, 131(51): 18478-88.
6. Simard, J.R., et al., J Am Chem Soc 2010, 132(12): 4152-60.
7. Simard, J.R., et al., J Am Chem Soc 2009, 131(37): 13286-96.
8. Schneider, R., et al., J Am Chem Soc 2012, 134(22): 9138-41.
62 Sessionvorträge Targets

Development of lysine mimic quinazoline-based selective inhibitors of a Jmj-C histone demethylase
family
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
Square 5, 00185 Rome, Italy
2 Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA
The histone proteins are subjected to various post-translational modifications, such as methylation, acetylation,
phosphorylation, and ubiquitylation. These modifications, interfering with RNA polymerase activity and DNA
transcription, represent one of the main mechanisms of the epigenetic modulation of gene expression. Aberrant
expression of histone-modifying enzymes has been linked to various human diseases, such as neurological
disorders and cancer. While studies on histone acetylation are known from years, studies on histone
methylation/demethylation and relative modulators are at their infancy [1].
N
N
HN
HN
MeO
N
MeO
N
MeO N N
N
H2N O N NH
N
BIX-01294 MC3076
N
H2N MeO
O
HN
N
N Cl
H2N
MeO
O
N
N
N NH
N
MC3073 MC3074
BIX-01294, a diazepin-quinazoline-amine derivative able to inhibit G9a and G9a-like protein (GLP) lysine
methyltransferases and to reduce methylation levels of H3K9 at several G9a target genes [2], was our starting point
to design novel putative inhibitors of histone methyltransferases and/or demethylases acting on H3K9, (G9a/GLP and
KIAA1718, respectively). Indeed, G9a and KIAA catalyze opposite reactions on H3K9 (the first adding and the latter
removing the Me2 histone mark), and may recognize Lys in either un-methylated or methylated state, it being the
substrate or reaction product, alternatively. Thus, we replaced the C7-methoxy group of BIX with a Lys-mimic side
chain (5-aminopentyloxy chain), obtaining increased G9a/GLP inhibition (MC3076) respect to BIX [3]. Since MC3076
as well as BIX were found by us able to inhibit also the demethylase KIAA1718, on the basis of crystallographic
studies performed on MC3076 complexed with either GLP or KIAA, we prepared a new series of quinazoline analogs
in order to improve their potency and selectivity against KIAA demethylase.
We proceeded to the simplification of the MC3076 structure by insertion of small substituents at the C2 and/or C4
quinazoline ring positions, so obtaining new quinazolines (MC3073, MC3074, etc.) highly potent against KIAA1718
and devoid of any GLP inhibiting activity [4]. Some of these derivatives (MC3074) were also inactive against the
JARID family of Jmj-C histone demethylases so demonstrating a promising capability to discriminate between
different Jmj-C histone demethylase families [4].
References:
1. Varier, R.A.; Timmers, H.T.M. Biochim Biophys Acta 2011, 1815(1): 75-89.
2. Kubicek, S. et al.: Mol Cell 2007, 25(3): 473-481.
3. Chang, Y. et al.: J Mol Biol 2010, 400(1): 1-7.
4. Upadhyay, A.K. et al.: J Mol Biol 2012, 416(3): 319-327.
Sessionvorträge Targets 63

Profiling of epigenetic targets using peptide microarrays
Schutkowski M; Rauh D; Roessler C; Masch A
Martin-Luther-University Halle-Wittenberg, Institute of Biochemistry & Biotechnology, Department Enzymology, Kurt-Mothes-Strasse 3, 06120
Halle, Germany
Acetylation of lysine residues in proteins is one of the most frequently occurring post-translational modifications
playing a major role in protein-protein-interaction as well as in regulating DNA transcription and metabolic processes.
Using acetyl coenzyme A as donor for the acetyl group, lysine acetyltransferases are transferring the acetyl group
onto the ε-amino-group of lysine residues in proteins. The positive charge of the side chains is neutralized, thus
remodeling biological activity of enzymes and protein-protein-interaction. Bromodomains as protein interaction
modules are recognizing specifically the acetylated lysine side chains.
To understand substrate specificity of lysine acetyltransferases and binding specificity of bromodomains, we created
a high-density peptide microarray displaying 6800 peptides derived from all known acetylation sites in human
proteins in both unmodified and acetylated form resulting in more than 13.600 different features per microarray pair
(acetylome microarray). The acetylome microarrays were treated with lysine acetyltransferases in the presence of
acetyl-CoA followed by an optimized mixture of anti-acetyl-lysine-antibodies.
Additionally, treatment of the acetylome microarray with Sirtuins, NAD + -dependent protein lysine-deacetylases
involved in regulation of central physiological functions, such as energy metabolism, cell cycle progression, and
aging processes, resulted in decrease of signals for several of the acetylated peptides in comparism to the control
experiment in the absence of NAD + . We were able to identify subsite specificities and substrate sequences for all of
the seven human Sirtuin isoforms. Besides known substrates our results provide novel substrate candidates for the
different Sirtuin isoforms which could be confirmed in subsequent solution phase experiments.
Principally, each enzyme transfering residues to the side chains of lysines could be analysed with the acetylome
microarray using either radioisotopic labeling (lysine-methyltransferases and labelled AdoMet, poly-ADPribosyltransferases
and labelled NAD + ) or antibody-based readout (poly-ADP-ribosyltransferases and ethenoNAD +
followed by anti-ethenoNAD + -antibody, ubiquitin/SUMO transfering enzymes followed by anti-ubiquitin/SUMOantibodies).
64 Sessionvorträge Targets

Session Tumortherapie und Resistenzmechanismen
Anti-infectives research to combat drug resistant tumors?
Hartmann R W 1,2
1 Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, 66123 Saarbrücken, Germany
2 Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123 Saarbrücken, Germany
In the last decades there have been great efforts to develop new tumor therapeutics and in the last 25 years
approximately 100 new drugs (NCEs) have been admitted to the market. The clinical situation however has only
improved slightly which is due to the fact that the tumors develop resistance against the new drugs as well. The big
amounts of money that have been invested into basic research to elucidate resistance mechanisms have brought
new interesting findings but (so far) have not resulted in more efficient drugs. Also in antibiotics therapy the
emergence of resistant bacterial strains is a severe problem and research is focused on strategies to investigate how
the development of resistant bacterial strains can be avoided. In this context recent results from our group will be
presented aiming at a novel therapy for Pseudomonas aeruginosa infections by interference with its unique PQS
quorum sensing system. Finally the question will be raised whether insights from antiinfectives research could help to
discover novel strategies to combat drug resistant tumors.
A new class of glutathione peroxidase inhibitors is able to reverse resistance to chemotherapeutics
in human B-cell lymphoma cell lines
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, Ernst-Moritz-Arndt University Greifswald, 17489 Greifswald,
Germany
2 Clinic for Haematology and Oncology, Ernst-Moritz-Arndt University Greifswald, 17475 Greifswald, Germany
The development of chemoresistance in cancer cells poses a major risk for chemotherapy failure. A combination of
anticancer drugs and agents that suppresses adaptation in cancer cells is a potent method to increase the efficiency
of therapies. GUMBUS and DOGUM, two cell lines from patients with non-Hodgkin lymphomas, were used to
investigate the process of drug resistance. Cells were treated with the anticancer drugs methotrexate, etopiside,
cisplatin and bortezomib to induce resistance. Assays showed a significantly increased amount of glutathione
peroxidase 1 (GPx1) in treated cells compared to untreated. The approach now is to inhibit GPx1 to target the antioxidative
pathway in these cancer cells. A virtual screening of a self-composed compound library docked to the
active site of the X-ray crystal structure of bovine GPx1 yielded promising structures. In an enzyme assay a class of
acylhydrazone heterocyclic proved to be GPx inhibitors. Furthermore, lymphoma cells lost their resistance to anticancer
drugs when treated with these hydrazones. This concept could be refined to a new strategy in
chemotherapies.
Sessionvorträge Targets 65

Chemoresistance of ovarian cancers against platinum complexes
Kassack M U; Hamacher A
Heinrich-Heine-Universität Düsseldorf, Institut für Pharmazeutische und Medizinische Chemie, Pharmazeutische Biochemie,
Universitätsstrasse 1, 40225 Düsseldorf, Germany. matthias.kassack@uni-duesseldorf.de
Ovarian cancer is the most lethal gynaecological cancer. Surgery and a combination chemotherapy including
carboplatin (cisplatin) are the primary treatment options. Initially, most ovarian cancers are chemosensitive. However
rather soon, resistance mechanisms develop and lead to relapse and therapeutic failure. Many resistance
mechanisms against platinum compounds are known but this has not yet been translated into improved therapy. One
reason is a lack of systematic studies to overcome chemoresistance using resistance mechanisms as additional
therapeutic targets and second, relevant mechanisms driving the development of chemoresistance have only partly
been studied. Comprehensive analysis of cancer cells on a genomic, transcriptional and translational level show that
the development of chemoresistance is a complex biological process involving both, selection and adaptation
processes (1, 2).
cisplatin resistance factor
7
6
5
4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 12 16 20 24
No of treatment cycles [weeks]
Resistance development over 6 months in A2780 ovarian cancer cells
To study the development of chemoresistance, we
have undertaken a novel approach of generating
resistant cell lines. Ovarian cancer cell lines were
treated intermittently according to a clinically
relevant dosing scheme over a period of 6
months. By that procedure, we have obtained
pairs of sensitive and resistant cell lines that were
analysed using MTT assay, whole genome
expression microarrays, and (phospho) proteome
profiling of kinases. Bioinformatic analysis
revealed a number of potential novel targets to
overcome chemoresistance. Subsequent inhibitor
studies in combination with carbo/cisplatin could
demonstrate a reversal of chemoresistance
against platinum compounds.
In conclusion, bioinformatic analysis allows establishing drug resistance signatures with potential prognostic value.
Furthermore, novel therapeutic targets have emerged establishing a new approach to overcome chemoresistance
against carbo/cisplatin in ovarian cancer.
References:
1. Eckstein N, et al. Cancer Res. 2009; 69(7): 2996-3003.
2. Gosepath EM, et al. Int J Cancer. 2008; 123(9): 2013-9
66 Sessionvorträge Targets

Medicinal Chemistry with Organometallics – From Catalysts to Anticancer Drugs?
Ott I
Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, Beethovenstr. 55, 38106 Braunschweig, Germany
Despite the tremendous success of platinum based cancer chemotherapeutics, transition metal based drugs are rare
on the current drug market as well as in medicinal chemistry and drug design. However, metal complexes offer
interesting opportunities for drug development based on their structural and kinetic properties, which differ in many
ways from traditional “organic” compounds.[1]
Organometallics are metal complexes with metal-carbon bonds, which have been attracting increasing attention in
general chemistry over the last decades and enabled an enormous progress in the field of catalysis. Not surprisingly
this has led to a broader knowledge of the properties of available organometallic functional groups and thereby also
new approaches for inorganic medicinal chemistry have emerged.[2,3]
We have been working over the last years on several types of organometallics including metal complexes with Nheterocyclic
carbene (NHC) ligands or gold alkynyl complexes as new antitumor drugs (see the figure for some
examples).[4-6] For many of these compounds we could confirm distinct biological properties such as antiproliferative
effects in cultured tumor cells, selective inhibition of the enzyme thioredoxin reductase, triggering of reactive oxygen
species (ROS) formation or anti-angiogenic effects. Binding to albumin, cellular uptake and intracellular distribution
have been studied for selected examples by high resolution atomic absorption spectroscopy (HRCS-AAS) in order to
provide a more detailed picture of their behaviour on the cellular level.
In the presentation a short overview of the topic will be given and selected examples of our current projects will be
presented.
N
N
Au
Cl
O
Au P
Figure: examples for bioactive organometallics
Sessionvorträge Targets 67
N
N
Ru
Cl Cl
Acknowledgments: Financial support by the DFG (project FOR 630, “Biological Function of Organometallic Compounds”) is gratefully
acknowledged.
References:
1. Alessio, E: Bioinorganic Medicinal Chemistry (Wiley) 2011.
2. Gasser G, Ott I, Metzler-Nolte N: J Med Chem 2011, 54(1): 3–25.
3. Noffke, AL et al.: Chem Comm 2012, 48: 5219–5246.
4. Rubbiani, R et al.: J Med Chem 2011, 54(24): 8646–8657.
5. Oehninger, L et al.: ChemMedChem 2011, 6: 2142–2145.
6. Meyer, A et al.: Angew Chem 2012, accepted.

NHC-Silver(I) and NHC-gold(I) complexes as bioorganometallic anticancer and antibacterial drugs
Tacke M; Hackenberg F; Patil S; Lally G; Streciwilk W
UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
The synthesis of N-heterocyclic carbene (NHC) silver(I) acetate complexes with varying lipophilic benzyl-substituents
at the 1 and 3 positions of the (benz)imidazole ring was achieved by reaction of silver(I) acetate with the
corresponding (benz)imidazolium bromide or iodide salts [1-7]. These NHC-silver(I) acetate derivatives exhibit
interesting structural motifs in the solid state and proof to be soluble and stable in biological media. The preliminary
antibacterial activity of all the compounds was studied against Gram-negative bacteria Escherichia coli, and Grampositive
bacteria Staphylococcus aureus using the Kirby-Bauer disk-diffusion method. Almost all the NHC-silver(I)
acetate complexes have shown high antibacterial activity compared to the NHC-precursors. The antibiotic lead
compound SBC3, which is synthesised from 4,5-diphenyl-1,3-dibenzyl-imidazole, reaches an antibacterial activity
comparable to marketed modern antibiotics. In addition, the NHC-silver complexes had their cytotoxicity investigated
through MTT based preliminary in vitro testing on the human renal cancer cell line Caki-1 in order to determine their
IC50 values. Here, NHC-silver(I) acetate complexes were found to have IC50 values as low as 1.2 μM for (1-methyl-
3-(p-cyanobenzyl) benzimidazole-2-ylidene) silver(I) acetate, which has become the anticancer lead structure SBC1.
The best corresponding NHC-Au(I)Cl complex SBC2 reaches an IC50 value of 10 μM against the cancer cell line
CAKI-1 [8,9]. In addition, results of SBC1 against drug-resistant cancer cell lines, transport and target assays as well
as in vivo testing in zebrafish and mouse will be communicated.
The authors acknowledge IRCSET and UCD for funding.
References:
1. F. Hackenberg, A. Deally, G. Lally, S. Malenke, H. Müller-Bunz, F. Paradisi, S. Patil, D. Quaglia, M. Tacke, Int J Inorg Chem 2012,
DOI: 10.1155/2012/121540
2. S. Patil, A. Deally, B. Gleeson, F. Hackenberg, H. Müller-Bunz, F. Paradisi, M. Tacke., Z Allg Anorg Chem 2011, 637: 386–396.
3. S. Patil, A. Deally, B. Gleeson, H. Müller-Bunz, F. Paradisi, M. Tacke. Novel Benzyl-Substituted N-Heterocyclic Carbene–Silver Acetate
Complexes: Synthesis, Cytotoxicity and Antibacterial Studies, Metallomics, 2011, 3: 74–88.
4. S. Patil, K. Dietrich, A. Deally, B. Gleeson, H. Müller-Bunz, F. Paradisi, M. Tacke, Helv Chim Acta 2010, 93: 2347–2364.
5. S. Patil, A. Deally, B. Gleeson, H. Müller-Bunz, F. Paradisi, M. Tacke, Appl Organomet Chem 2010, 24: 781–793.
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
2010, 1020–1031.
7. S. Patil, M. Tacke., „NHC-Silver(I) Acetates as Bioorganometallic Anticancer and Antibacterial Drugs. Insights into Coordination,
Bioinorganic and Applied Inorganic Chemistry.“ Edited by M. Melník, P. Segľa, M. Tatarko., Press of Slovak University of Technology,
Bratislava, 2011: 555–566.
8. L. Kaps, B. Biersack, H. Müller-Bunz, K. Mahal, J. Münzner, M. Tacke, T. Mueller, R. Schobert., J Inorg Biochem 2012, 106: 52–58.
9. S. Patil, A. Deally, F. Hackenberg, L. Kaps, H. Müller-Bunz, R. Schobert, M. Tacke., Helv Chim Acta 2011, 94: 1551–1562.
68 Sessionvorträge Targets

Sessionvorträge Tabletten
Session Nanomedizin
Nanomedicines for targeted drug delivery to the inflamed intestinal mucosa
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
Research (HZI), Saarland University, Campus A4.1, 66123 Saarbruecken, Germany
2 Biopharmacy and Pharmaceutical Technology, Saarland University, 66123 Saarbruecken, Germany
3 Hebrew University of Jerusalem, Faculty of Medicine, School of Pharmacy, Institute for Drug Research, Jerusalem 91120, Israel
Inflammatory bowel diseases (IBD) such as Crohn’s disease or Ulcerative colitis are autoimmune, chronic, episodic,
inflammatory conditions of the gastrointestinal tract. Classical therapy of IBD with steroids or immunomodulators
delivered via pellets, capsules, or tablets is often inefficient mainly due to limited drug release time and enhanced
elimination of drug carriers as the result of diarrhoea. The emergence of biologicals has significantly increased the
treatment options for IBD in recent years. Still, these new therapeutics have to be applied systemically resulting in
sometimes severe adverse effects. Nanomedicine may enhance the efficacy of conventional IBD therapeutics and
open up new routes of application for next generation drugs: Passively targeting the inflamed intestinal areas,
nanoparticles of ~100 nm size have been shown to accumulate in the inflamed tissue, while bigger particles of 1 or
10 µm showed no specificity for diseased intestinal areas [1]. Moreover, polylactide-co-glycolide (PLGA)
nanoparticles loaded with rolipram showed higher and prolonged anti-inflammatory activity and reduced central
nervous adverse effects in a TNBS rat model [1].
Further investigating this EPR (enhanced permeability and retention) – like effect we developed a co-culture model of
the inflamed intestine based on Caco-2 enterocytes and blood derived macrophages and dendritic cells [2]. Via
addition of the cytokine interleukin-1β a reversible inflammation can be induced. In response to the stimulus a
decrease in barrier function, a re-organization of tight junctions, a release of inflammation markers and increased
mucus production can be observed mimicking pathophysiological phenomena observed in IBD in vivo.
Different nanoformulations of the glucocorticoid budesonide were applied in the in vitro system and evaluated for
their mechanism of accumulation and anti-inflammatory activity [3]. Both budesonide loaded PLGA nanoparticles and
the free drug solution were able to recover epithelial barrier function (quantified via TEER) and to reduce release of
inflammation marker IL-8. However, treatment with free budesonide solution was only effective for a short time as
after 48 h a rebound of IL-8 release was observed. In contrast PLGA budesonide treated cells retained low IL-8
levels. The prolonged activity of the nanoparticulate formulation can be ascribed to the formation of local drug
depots, as particles accumulate between the cells in the area of the tight junctions. A liposomal budesonide
formulation seemed to be preferentially taken up and processed by the immune cells in the co-culture model. The
resulting dose dump negatively affected the barrier function and resulted in increased IL-8 levels.
Aiming to further enhance the efficacy of nanocarrier accumulation, active targeting ligands for the inflamed intestinal
mucosa were investigated. In the inflamed colonic mucosa of colitis patients an increased expression of transferrin
receptor (TfR) has been reported both in the basolateral aspects of crypts and on the apical surface [4]. Indeed anti-
TfR immunoliposomes applied the luminal side showed greatly enhanced adherence in vivo compared to nonspecific
immunoliposomes.
As a first topical application of a biological in IBD therapy a nanoformulation of the potent, anti-inflammatory cytokine
IL-10 was developed. IL-10 is a promising IBD therapeutic, but failed in clinical trials upon subcutaneous injection,
due to its short serum half-life and severe adverse effects at higher doses. Drug loaded albumin nanoparticles were
prepared by spray drying and embedded in Eudragit S-100 microparticles to ensure stability during gastrointestinal
passage. The nanoparticle in microparticle oral system succeeded in protecting IL-10 integrity during the preparation
process and releasing bioactive cytokine at the desired pH of 7.4.
References:
1. Collnot, E.M., H. Ali, and C.M. Lehr, J Control Release 2012.
2. Leonard, F., E.M. Collnot, and C.M. Lehr, Mol Pharm 2010. 7(6): 2103–2119.
3. Leonard, F., et al., ALTEX 2012, 29(3): 275–285.
4. Harel, E., et al., PLoS ONE 2011, 6(9): e24202.
Sessionvorträge Tabletten 69

Selective gene vehicles for enhanced DNA and siRNA delivery
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
2Department of Cardiology, St. Georg Hospital Eisenach, Mühlhäuser Strasse 94/95, D-99817 Eisenach, Germany
Gene therapy is believed by many to be the therapy of the 21st century. Successful gene therapy or gene
knockdown depends on the delivery of nucleic acid molecules into cell. While viral vectors have shown high gene
transfer efficacies, their drawbacks include immunogenic/inflammatory responses, limited loading capacity and
issues with regards to large scale manufacturing and quality control. Consequently, numerous non-viral gene delivery
vectors including liposomes (lipoplexes), polycationic polymers (polyplexes) and organic or inorganic nanoparticles
(nanoplexes) have been developed and reported in recent years [1,2,4]. General issues in non-viral transfection
systems are the delivery of DNA for gene transfection into the nucleus or small interfering RNAs (siRNAs) for the
induction of RNA interference (RNAi). RNAi is a powerful method that has been applied successfully for the downregulation
of genes in both functional genetic analysis and gene therapy. It relies small interfering RNAs (siRNAs),
and since all other components of the RNAi machinery are provided by the target cell /target tissue, the efficient
siRNA delivery is of critical importance and has proven to be the bottleneck for successful RNAi applications.
Atherosclerosis is characterized by the buildup of lipid-rich plaques within the blood vessel walls of large arteries, and
underlies the clinical conditions of myocardial infarction, chronic stable angina, stroke and peripheral vascular
disease. The process of atherogenesis is induced by a low-grade inflammatory process in the vascular wall, leading
through various steps to the eventual formation of atheromatous plaque. These plaques may also become
progressively unstable due to local reaction to pro-inflammatory cytokines and proteinases (e.g. IFN-gamma,IL-
18,CD40–CD154, or IL 1 etc.), leading to plaque ulceration or rupture. Additionally activated endothelial cells express
adhesion molecules such as P-selectin and E-selectin, VCAM or ICAM leading to a pro-inflammatory environment
and a mediated activation of clotting cascade [3]. The gene overexpression of such target molecules make them
promising candidates for the treatment by siRNA formulations.
The aim of this research was to develop a gene-transfection system based on site specific lipoplexes and
lipopolyplexes which would possess a nano-scaled size below 200 nm. For the preliminary experiments a luciferase
reporter gene bearing plasmid DNA (pcmv beta) was used instead of siRNA for the therapeutic application (down
regulation of the targets TNF-alpha, VEGF). The pDNA was pre-condensed with cationic polymers [1] and/or
complexed [2,4] with different lipid mixtures (DPPC, DOPC, DSPC, Saint, Tetraetherlipids or Cholesterol etc.) to
improve the stability/efficiency and lowering the toxicity. The gene vehicles were targeted to E-selectin via surface
modification with E-Sel antibodies. The transfection efficacy and cell viability was investigated on the HUVEC and
CHO-E cells under shear flow conditions.
Acknowledgments: This study was supported by “Deutscher Akademischer Austauschdienst“ (DAAD) and by “Deutsche Forschungsgemeinschaft”
(Forschergruppe FG 495 and FG 629, UB). We are grateful to Prof. Dr. J. Aigner for the development of the polyplex vehicles
and the technical support.
References:
1. Schäfer J, Höbel S, Bakowsky U, Aigner A: Biomaterials 2010, 31(26): 6892–6900.
2. Kneuer C et al.: J Nanosci Nanotechnol 2006, 6(9-10): 2776–2782.
3. Kneuer C et al.: Drug Discov Today 2006, 11 (21-22): 1034–1040.
4. Tabatt K et al.: J Controlled Release 2004; 97: 321–332.
70 Sessionvorträge Tabletten

Modulating immune responses of human cells by adjuvant loaded particulate carriers
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
2Berlin-Brandenburg Centre for Regenerative Therapies, Berlin and Teltow, Germany
Building an efficient immunological memory to avoid infections remains a challenge
for various types of pathogens, particularly when purified, poorly immunogenic antigenic
structures should be used for safety reasons. Therefore, antigens are often
combined with cocktails of immunostimulating compounds, which in a rather uncontrolled
process induce a proinflammatory environment and frequently have led to
discussions about adjuvant induced severe vaccine reactions. For enabling tailored
immune responses including efficacy against intracellular pathogenes, carrier systems
are of high interest, as they may co-deliver the antigen(s) and a specific adjuvant
[1]. Particles from poly[(rac-lactide)-co-glycolide] (PLGA) have shown their
suitability as protein carriers as they are very efficiently taken up into cells by
Fig. 1: Intracellular localization of
MP, reproduced from [3] with
permission, Copyright 2006.
phagocytosis [2] (Fig. 1) and, moreover, facilitated phagolysosomal escape to the cytosol as relevant to induce
immunity against intracellular pathogens. The carrier size should be in the upper nanosize range or, preferentially,
microparticles (MP) < 10 µm, in order to efficiently enable phagocytosis as uptake mechanism and deliver sufficient
levels of payload.
Importantly, the MP carriers themselves were shown to be inert and did not induce activation of antigen presenting
cells (APC) such as human primary dendritic cells (DC) [3], which is required to tailoring the immune response by the
selectively added adjuvant. When poly[(riboinosinic acid)-co-(ribocytidylic acid)] as a Toll-like receptor 3 (TLR3)
agonist was bound to surface-functionalized MP [3] in a layer-by-layer approached, only ligand loaded but not blank
particles induced a full antigen-independent maturation of DC [4].
As the safety profile of TLR ligands in general is occasionally subject to
concerns and addressed presently in clinical studies, alternative defined
adjuvants may be required. Therefore, in this study, N-acetylmuramyl–
L-alanyl–D-isoglutamine (MDP) and γ-D-glutamyl-meso-diaminopimelic
acid (iE-DAP) as ligands of cytosolic receptors sensing nucleotide and
oligomerization domains (NOD) were evaluated for their capability to
induce DC maturation and cytokine release after encapsulation into
PLGA MP [5]. This concept was rationalized by the carriers’ capability
to support phagolysosomal escape, thus making available their payload
to the compartment where NOD receptors are expressed. Microencapsulated
NOD agonists but not blank MP or soluble NOD agonists
induced a dose-dependent maturation of DC (Fig. 2), illustrating the
efficiency of the particulate carrier in combination with phagocytosis as
transport mechanism. A proinflammatory DC phenotype with
Fig. 2: Dose dependent maturation of DC. remarkable high levels of released cytokines particularly for iE-DAP
were detected. Importantly, for selectively detecting effects of the studied adjuvant, a strategy is required to exclude
immunogenic impurities and prevent erroneous interpretations of cell activation in the case of impurity-burdened MP.
By different reporter cell lines not sensing MDP and iE-DAP but selectively expressing other pathogen associated
molecular pattern, the purity of the employed carriers and the specificity of results were proven. Based on this, further
exploration of these carriers is planned in vivo.
Acknowledgments: D. Lorenzen, Institute for Tumor Therapy, Duderstadt, Germany, for support in previous cell studies [Fig. 1; Ref. 2-4].
References:
1. Rappuoli, R., Aderem, A.: Nature 2011, 473: 463–469.
2. Wischke, C. et al.: Eur J Pharm Biopharm 2006, 62: 247–253.
3. Wischke, C. et al.: J Contr Rel 2006, 114: 359–368.
4. Wischke, C. et al.: Int J Pharm 2009, 365: 61–68.
5. Wischke, C. et al.: 2012 manuscript in revision.
6. Mathew, S. et al.: 2012 manuscript submitted.
Sessionvorträge Tabletten 71

Acid-Degradable Dextran Particles for the Delivery of Biotheropeutics
Wich P R1 ; Fréchet J M J2 1 Institut für Pharmazie und Biochemie, Johannes Gutenberg-Universität Mainz, Staudingerweg 5, 55128 Mainz, Germany
2 College of Chemistry, University of California Berkeley, CA 94720-1460, USA
Polymer-based drug, vaccine and gene delivery systems can be easily modified and are particularly attractive
because of their ability to perform several critical functions simultaneously. These include delivering therapeutic or
other bioactive agents to a specific site via a targeting mechanism, increasing the circulation times of drugs in the
body, protecting drugs from degradation, or increasing the bioavailability of poorly soluble drugs.
Recently, a new class of modular and acid-degradable, polymeric particles using acetal-modified dextran (Ac-DEX)
was developed.[1,2] This biocompatible material can be formulated into particles using a variety of common
emulsion-based techniques enabling control over both size and morphology. Due to their pH sensitivity, Ac-DEX
particles can selectively and rapidly release their encapsulated payload under mildly acidic conditions including those
found in sites of inflammation, tumor tissue, or endocytic vesicles. Upon hydrolytic degradation, Ac-DEX reverts back
to FDA-approved dextran without the generation of acidic byproducts that could damage the encapsulated payload or
lead to inflammation, as has been observed with some other commonly used polymer systems. In addition, Ac-DEX
particle degradation rate can be easily tuned within the time scale of relevant cellular processes. This release
tunability combined with their low-toxicity and payload versatility make Ac-DEX particles an ideal platform for a wide
range of biotherapeutic delivery applications including immunotherapy [2] and gene delivery.[3]
References:
1. Bachelder E.M. et al.: J Am Chem Soc 2008, 32: 10494–10495.
2. Broaders, K.E. et al.: Proc Natl Acad Sci USA, 2009, 106: 5497–5502.
3. Cohen, J. L. et al.: Bioconj Chem 2011, 13: 1902–1905.
72 Sessionvorträge Tabletten

Characterization of Bacterial Nanocellulose as Drug Delivery System for Low and High Molecular
Weight Drugs
Müller A1 ; Moritz S1 ; Wiegand C 2 ; Wesar F 3 ; Munteanu M 4 ; Hessler N 4 ; Kralisch D 4 ; Müller F A 3 ; Hipler U-C 2 ;
Fischer D1 1 Department of Pharmaceutical Technology, Friedrich-Schiller-University Jena, Otto-Schott-Str. 41, 07745 Jena, Germany
2 Department of Dermatology, Friedrich-Schiller-University Jena, Erfurter-Str. 35, 07743 Jena, Germany
3 Institute of Materials Science and Technology, Friedrich-Schiller-University Jena, Loebdergraben 32, 07743 Jena, Germany
4 Institute of Technical Chemistry and Environmental Chemistry, Friedrich-Schiller-University Jena, Lessingstr. 12, 07743 Jena, Germany
Synthesized by bacteria of the genus Gluconacetobacter the biopolymer bacterial nanocellulose (BNC) has already
been proven to be advantageous for various biomedical applications as tissue engineering, implant materials, skin
substitutes and wound care [1, 2]. The use of this biomaterial as drug delivery system offers the opportunity to
combine its beneficial material properties (e.g. high purity, mechanical stability and biocompatibility) with the positive
effects of incorporated drugs and opens up new promising fields of application.
Comparative loading and release studies with high molecular weight protein drugs (bovine serum albumin: BSA,
luciferase) as well as different low molecular weight antiseptics (polihexanide: PHMB, PVP-iodine, octenidine) were
performed using planar BNC fleeces produced in a static cultivation process. Therefore BNC samples were loaded
by immersion in aqueous drug solutions and released after different incubation times. Influence of drug
concentration, water content of the BNC samples, incubation time as well as temperature were investigated.
Quantification of the different drugs was performed by UV spectrophotometry (BSA, 278 nm; PHMB, 235 nm; PVPiodine,
352; octenidine, 281 nm), BCA assay and luminescence measurements (luciferase assay system). Drug
distribution in the samples was analyzed by staining of BSA loaded samples with BCA assay reagent and the use of
red brown coloured PVP-iodine. Sample morphology was studied by SEM analysis. Furthermore, drug release data
were examined according to Ritger-Peppas equation. Polyacrylamide gel electrophoresis of released BSA as well as
measurement of biological activity of released luciferase was conducted to study protein stability. Antibacterial
efficacy of antiseptic BNC extracts prepared according to DIN EN ISO 10993-12 against Staphylococcus aureus was
tested by microplate laser nephelometry. Furthermore, a direct challenge test (JIS L 1902:2002) was performed for
measuring the antibacterial efficacy of PVP-iodine loaded samples. Cell proliferation of human HaCaT keratinocytes
incubated with extracts of BNC loaded with antiseptics was quantified using a luminometric ATP-luciferase assay for
cytotoxicity studies. The proteins as well as the low molecular antiseptic drugs showed the same release pattern,
characterized by a burst in the beginning followed by a slower release rate until 48 hours. An overlay of diffusion- and
swelling-controlled drug transport mechanisms was found by mathematical analysis (Ritger-Peppas equation) for
both types of drugs. Optimized loading techniques allowed a homogenous drug distribution in the loaded samples.
Protein stability could be maintained during loading and release as shown by gel electrophoresis as well as biological
enzyme activity. Extracts of BNC hydrogels loaded with PHMB and octenidine showed a strong inhibition of
Staphylococcus aureus growth. Antiseptic efficacy of PVP-iodine loaded BNC could be confirmed with the direct
challenge test. Cell proliferation of HaCaT keratinocytes in cytotoxicity assays was found to be dependent on
antiseptic drug type and extraction ratio. Whereas PVP-iodine extracts had no influence on cell proliferation, PHMB
and octenidine extracts exhibited a decrease of cell proliferation at different extraction ratios (PHMB: 0.1 g:50 mL,
octenidine: 0.001 g:50 mL), which has to be considered for determination of active loading concentration in wound
dressing development.
In conclusion, obtained results characterize drug loading to and release from the BNC biomaterial and confirm the
suitability of BNC for drug delivery applications for high molecular as well as low molecular weight drugs.
Acknowledgments: This study was funded by the Thuringian Ministry of Education, Science and Culture (B714-10032) as well as the European
Fund for Regional Development. Additionally, the authors would like to thank Ramona Brabetz as well as Elena Pfaff for the excellent technical
assistance.
References:
1. Kralisch, D. et al.: Biotechnol Bioeng 2010, 105(4): 740–747.
2. Klemm, D. et al.: Angew Chem Int Ed 2011, 50(24): 5438–5466.
Sessionvorträge Tabletten 73

Biomedical applications of magnetite in the respiratory system – Toxicological considerations and
mechanistic studies in A549 lung cells
Könczöl M1,2 , Gminski R1 , Gieré R3 , Merfort I2 , Mersch-Sundermann V1 1 Institut für Umweltmedizin und Krankenhaushygiene, Universitätsklinikum Freiburg
2 Institut für Pharmazeutische Biologie und Biotechnologie, Universität Freiburg
3 Institut für Geowissenschaften, Universität Freiburg
Magnetite, a ferromagnetic iron oxide, represents a promising material within the broad field of nanomaterials for
biomedical applications [1]. Beside its use in magnetic resonance imaging or hyperthermia, research has been
focused on its potential to serve as an inhalative carrier system for drugs or vaccines. The lack of consistent data on
pulmonary toxicity requires more systematic investigation of magnetite nano- and micrometer particles. In a former
study, magnetite was thoroughly investigated with regard to its potential to induce toxic effects and to influence
signaling pathways. It was clearly demonstrated that reactive oxygen species (ROS)-formation led to mitochondrial
damage and genotoxic effects in A549 cells [2]. Based on these findings, we here report studies which elucidate the
origin of magnetite-mediated ROS-formation and their influence on cell cycle of A549 human lung epithelial cells.
Additionally, effects on p53 and p21 activation were evaluated by western blots.
After exposure of A549 to magnetite particles, an increased superoxide production, measured by electron
paramagnetic resonance (EPR), was detected, while the glutathion (GSH) level decreased significantly. Moreover,
the particles were able to induce ROS-formation in an acellular environment. Mitochondria or soluble iron did not
contribute considerably to the ROS production. We could show that incubation of A549 cells with inhibitors of
NADPH-oxidase (NOX) prior to particle treatment led to decreased ROS formation measured by EPR and DCFH-DA
assay. Analysis of cell cycle distribution yielded a pronounced sub-G1-peak, which cannot be linked to increased cell
death. Western blot analysis showed no activation of p53 but upregulation of p21 in A549. Surprisingly, exposure to
magnetite leads to p21-mediated G1-like arrest, which has only reported before for low concentrations of microtubule
stabilisation drugs, but so far never after particle exposure. Importantly, the arrested sub-G1 cells were viable and
showed no caspase activation. In summary, magnetite induced a variety of cellular responses in A549 cells in vitro.
Since it cannot be excluded that these toxic effects may be also of relevance in vivo, a fundamental safety profile has
to be compiled before considering nanomaterials for biomedical use in human.
References:
1. Kim, J. et al., Arch Toxicol 2012, 86(5): 685–700.
2. Könczöl, M. et al., Chem Res Toxicol 2011, 24(9): 1460–1475.
74 Sessionvorträge Tabletten

Session Pharmaverfahrenstechnik
Formulation screening with colloidal drug carrier systems
Bunjes H
Institute of Pharmaceutical Technology, TU Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany
As a large fraction of newly identified drug candidates is poorly water soluble the use of enabling formulation
technologies becomes increasingly important. In principle, this applies to all potentially relevant routes of
administration but dosage forms for systemic administration, in particular via the parenteral (especially intravenous)
and oral route are usually of special interest. Usually, the development of viable dosage forms for poorly soluble
substances is a comparatively time-consuming process. This creates a kind of dilemma in regard to the rapid
availability of a rather large number of potential drug candidates by modern drug discovery methods. The situation is
further complicated by the usually very limited amount of drug substance available for formulation studies in early
development. In order to rapidly provide suitable formulations for the required tests with poorly water soluble drug
candidates the availability of formulation screening methods for the fast development of formulations with a minimum
amount of drug candidate substance would thus be highly desirable. Ideally, the formulations identified this way
would also bear the potential to be developed into marketable products.
Colloidal carrier systems like drug nanosuspensions or different types of lipid dispersions (such as liposomes, mixed
micellar systems or several types of lipid nanoparticles) are a prominent formulation option for poorly soluble drug
candidates. Properly designed colloidal formulations are, in principle, applicable via all potential routes of
administration and may also be developed into drug products. Compared with more conventional dosage forms (e.g.,
drug solutions) most of them are, however, also comparatively demanding, in particular with regard to their way of
preparation and their physicochemical stability. Only a few of them can be prepared by simple mixing (e.g. mixed
micellar solutions) that would allow for easy screening. For others (like liposomes), small scale dispersion methods
are well established but the whole process of formulation is still quite time-consuming.
The preparation of colloidal carrier systems with micro- and nanostructured devices like by high-pressure
microchannel or premix membrane emulsification [1, 2] or in miniaturized milling devices [3] offer novel approaches
for small scale formulation preparation. Beyond formulation screening with a drug candidate of interest, they are also
very useful for the optimization of formulations and in basic formulation research. The work with such preparation
methods may, however, still require several experiments to determine the loading capacity of a lipid carrier system
for a specific drug. In order to solve this task in a more efficient way, loading of pre-formed colloidal carrier systems is
an interesting option [4]. This presentation will introduce and discuss different approaches for formulation screening
with a special focus on the development of colloidal lipid carrier systems for poorly water soluble substances.
References:
1. Finke, J. et al.: Chem Eng J 2012, accepted for publication.
2. Joseph, S., Bunjes, H.: J Pharm Sci 2012, 101(7): 2479–2489.
3. Juhnke, M., Berghausen, J., Timpe, C.: Chem Eng Technol 2010, 33(9): 1412–1418.
4. Kupetz, E. et al.: 8 th PBP World Meeting 2012, Istanbul, Turkey.
Sessionvorträge Tabletten 75

Pharmaceutical Extrusion Technology – Recent developments
Thommes M
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitaetsstrasse 1, 40225 Duesseldorf, Germany
Pharmaceutical extrusion technology has become an important processing technology within the last few decades.
For this reason, academia, as well as industry, is focusing research efforts on this area. It is likely that extrusion will
become one of the most important technologies for many pharmaceutical applications.
In the last few years the co-rotating twin screw extruder has become the common extruder type due to its versatility.
Its modular setup allows for broad manufacturing process customization to meet different product requirements.
Such flexibility allows multiple unit operations to be performed in a single machine, thereby increasing efficiency.
Based on the plastification method, extrusion processes can be differentiated as wet, hot melt, and cold extrusion.
These have been investigated and used during the past 30 years. Each of these methods has its own advantages
leading to specific applications, such as pelletization, taste masking, and solubility enhancement.
Recent investigations deal with the process understanding (QbD) and downstream processing such as film extrusion
and injection moulding. A lot of research efforts also deal with down-scaling of extrusion equipment to make it
suitable for small-scale production and early development.
Pharmaceutical extrusion technology is an innovative technology that continues to become more relevant, due to its
many assets as compared with other pharmaceutical technologies.
76 Sessionvorträge Tabletten

Hot melt extruded dosage forms
Rein H
Pharmazeutische Technologie, Universität Bonn, Gerhard-Domagk-Straße 3, D-53121 Bonn-Endenich, Germany
1. Introduction
Hot melt extrusion is gaining more and more interest in pharmaceutical industry. One of the main advantages, apart
from improving the bioavailability of an API, is the possibility to move away from batch processing towards
continuous production. Mostly the extrusion process itself is already well adjusted. One of the major problems right
now is giving shape to the final products.The aim of our work was to develop continuous processes for the
production of pellets and extruded tablets (extrudets).
2. Pellets [1]
Spherical starch pellets were directly and continuously produced using hot-melt extrusion and die-face pelletisation.
In contrast to conventional pelletisation procedures, a discontinuous spheronisation step is not necessary. Pellets
were produced based on different starches and several other polysaccarids, four different active ingredients
(ibuprofen, paracetamol, phenazon and tramadol-HCl) and various additives. The resulting pellets exhibit a large
mechanical stability, low porosity, small surface area and a a very narrow particle size distribution even in the micron
scale. The drug is either dispersed or dissolved in the polysaccharid melt. Drug loadings of up to 80% are
achievable. The dissolution rate depends on pellet size, pellet porosity, the velocity with which the penetrant diffuses
into the pellet-core, the water solubility of the API, and the molecular structure, composition and the swelling behavior
of the used polysaccarid. The fractional drug release from non eroding starch pellets can be described with the
Noyes-Whitney equation. [2]
3. Tablets/extrudets
Biplan tablets were produced by using a co-rotating twin-screw extruder (ZSE 27 HP-PH, Leistritz AG, D-Nürnberg).
For the final shaping of the tablets, the still elastic strands were cut with a rotary fly-knife cutting machine (Dynamat,
Metzner, D-Neu Ulm), which is generally used in plastics industry for cutting of wires, cables or seals. [3] Another
important aspect of our work concerns data-acquisition during the cutting process. Monitoring the energy of each cut
gives an interesting insight into the process. This might be applied as a useful in-process tool.
Tablets were produced based on several starches and various different polysaccharides, which were well known in
the food industry, e.g. gelatine, gummi arabicum, pectine or locust bean gum and several additives. Depending on
the used polysaccharide the production of melting tablets, lozenges or dosage forms with fast or prolonged API
release is possible.
Acknowledgments:
1, Roquette, F-Lestrem, donation of starches.
2. Leistritz Extrusionstechnik, D-Nürnberg, allocation of a twin-screw extruders (ZSE 18 HP-PH and ZSE 27 HP-PH).
3. Metzner Maschinenbau, D-Neu-Ulm, allocation of a rotary fly-knife cutting machine (Dynamat 20/40).
References:
1. Bialleck S, Rein H,: Eur J Pharm Biopharm 2011, 79: 440–448.
2. Bialleck S, Rein H,: Stärke 2012, 64: 408–419.
3. Kudernatsch, H., 2011, Pharm Ind 2011, 73: 2050–2060.
Sessionvorträge Tabletten 77

Nanogrinding of naproxen: influence of process parameters on product quality
Bitterlich A1 , Komoß C2 , Dengler M, Bunjes H 2 , Kwade A1 1Institute for Particle Technology, TU Braunschweig, Volkmaroder Str. 5, 38104 Braunschweig, Germany
2Institute of Pharmaceutical Technology, TU Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany
Nanosized active pharmaceutical ingredients (APIs) increasingly attract interest in pharmaceutical industries. Up to
now, five drug-containing nanoparticulate formulations are on the market [1]. In micronized form, all corresponding
APIs exhibit a poor water solubility and, consequently, a low bioavailability after oral administration. By reducing the
particle size, the dissolution rate can be strongly increased which leads to an improved biopharmaceutical profile.
Wet media milling is often used to produce API nanoparticle suspensions. In this study the influence of the process
parameters on product quality during the nanogrindnig process was investigated. A planetary ball mill (PM400,
Retsch GmbH) was modified according to [2] to enable parallel small scale screening with 12 samples of 25 mg API
each. Inside the grinding chambers, the anti-inflammatory API naproxen (kindly provided by Novartis Pharma AG) in
aqueous suspension experienced high shear and compression forces between grinding media of different size,
material and shape which were brought into relative motion by centrifugal forces.
In a first step screening experiments were conducted, using various polymers and surfactants as stabilizers to
identify an optimum formulation against agglomeration phenomena. With this formulation (10 % naproxen, 2.5 %
Kollidon ® 30) the influence of process parameters on product quality was investigated. Within a certain range, the
product quality (regarding particle size and distribution width) increased by using small grinding beads, spherical
grinding media, high centrifugal forces and a high density of the grinding media.
In addition, some interesting stabilizer and process parameter dependent phenomena were observed during the
grinding process: Different product shapes were obtained by varying the stabilizer. When using PVP (Kollidon ® 30) or
PVA (Mowiol ® 3-85) grades, the product particles exhibited a round morphology after the grinding process. By using
Tween ® 80 or HPMC (Pharmacoat ® 603) as stabilizer, however, the product particle shape was more needle-like
(figure 1). Furthermore, the product particle shape could be influenced by adjusting the process parameters: By
applying grinding media made from yttrium stabilized zirconia with a diameter of 100 µm at high centrifugal forces of
the mill, even the morphology of the product particles stabilized with Kollidon ® 30 changed from “round” over “needlelike”
to finally “disc-like” over a grinding time of 960 min in the modified planetary ball mill. Apparently, the high
(mechanic) energy transferred to the product led to a growth of the API crystals under these process conditions
rather than to a real comminution process.
Figure 1: TEM micrograph of a Mowiol ® 3-85 (left) and Tween ® 80 (right) stabilized
naproxen suspension after 4 h grinding time
References:
1. Merisko-Liversidge, E., Liversidge, G.G.: Adv Drug Deliv Rev 2011, 63: 427–440.
2. Juhnke, M., Berghausen, J., Timpe, C.: Chem Eng Technol 2010, 33: 1412–1418.
78 Sessionvorträge Tabletten

Session Bioanalytik: Testsysteme und Datenqualität
Image Processing in medicinal microscopy
Matz M1 , Schumacher K2 , Rustenbeck I2 , Baumann K1 1Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany.
2Institute of Pharmacology, Toxicology and Clinical Pharmacy, TU Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany.
In the last decade, total internal reflection fluorescence microscopy (TIRF-M) was established as a standard method
for observing molecular phenomena close to the cell membrane. It is used here to study the behavior of insulin
granules. The obtained TIRF images and video sequences are usually evaluated with commercial software
packages, which allow counting of the labeled cell components and tracking of single labeled particles (i.e. insulin
granules).
Here we present a new and sophisticated program for the exploration of TIRF video sequences, enabling the user to
simultaneously count and track all visible labeled objects. Moreover, the tracked objects can be characterized with
respect to velocity, size, kinetic behavior, and exocytosis. The program starts with background subtraction. Next, a
3D convolution is carried out for noise reduction. Afterwards all fluorescing objects are tracked down and are being
followed. A specifically tailored clustering routine in combination with an edge filter, which detects jumps, is used
exocytosis detection.
While methods for object identification and single particle tracking are well established in the literature, this talk
focuses on new methods for the characterization of granule tracks. The goal is to estimate whether a particular object
moves due to diffusion or some kind of directed movement.
A detailed analysis of the detection of exocytitic events will also be presented. Simply using an edge filter on the
tracks yields too many false positive and false negative events. The clustering routine employed here, is able to
identify even vague increases in fluorescence intensity, which is highly related to membrane fusion and the release
of vesicle content.
Sessionvorträge Tabletten 79

The use of microscale thermophoresis (MST) for protein characterization and formulation
Besheer A1 , Abstiens K1 , Winter G1 , Baaske P2 , Duhr S2 1 Pharmaceutical Technology and Biopharmaceutics, Department of Pharmacy, Ludwig-Maximilians-Universität, Butenandtstrasse 5-13, D-
81377 Munich, Germany.
2 NanoTemper Technologies GmbH, Flößergasse 4, D-81369 Munich, Germany.
Microscale thermophoresis (MST) is the drift of dissolved molecules along a micron-sized temperature gradient [1]
(Figure 1). The strength and direction of this molecular thermal diffusion depend mainly on the molecular surface
area, the strength of ionic shielding and on the molecule’s hydration shell [2]. Changes in these properties lead to
alterations in the thermophoretic motion, which make MST a very sensitive tool to analyse structural changes in
proteins. Accordingly, MST was successfully applied for the measurement of macromolecular interactions and
binding affinities [3]. By Applying MST to investigate protein unfolding, we found out that the changes in protein
hydration accompanying unfolding lead to significant changes in thermophoresis, allowing the determination of the
protein’s melting point (Tm). For instance, it was possible to determine Tm for recombinant human granulocyte colony
stimulating factor (rh-GCSF) at different pHs and sucrose concentrations, and validate the results by fluorescence
and microcalorimetric measurements. In this respect, MST offers a number of advantages as it is a label-free
technique, requiring very small sample volumes (< 3 µl) and is able to measure protein thermophoresis over a wide
concentration range (nM to mM). Furthermore, it is an entirely optical method, which is contact-free and therefore
minimizes contamination of the sample. In comparison to other methods, it is not dependant on the surroundings of
tryptophan and does not require the use of external fluorescent dyes like in fluorescence-based techniques, nor is it
limited by low concentration ranges like microcalorimetry and circular dichroism, or the need for relatively
concentrated solutions as in ATR-FTIR. The potential of MST to deliver information about protein unfolding can thus
be exploited over a wide range of applications in protein science.
Figure 1. A) Experimental setup
for label-free MST involves the
use of UV-LED fluorescence for
concentration measurement
through the intrinsic tryptophan
fluorescence. An IR-Laser
produces a temperature
gradient on the order of
0.1 K/µm. Capillaries containing
< 3 µl samples are heated to
the desired temperature.
Samples can show B) a
thermophobic behaviour, where
molecules diffuse away from the
IR-Laser or C) a thermophilic
behaviour moving towards it.
References
1. Iacopini, S., Rusconi, R., & Piazza, R., Eur Phys J E Soft Matter 2006 19: 59–67.
2. Duhr, S. & Braun, D., Proc Natl Acad Sci USA 2006 103: 19678–19682.
3. Wienken, C .J., Baaske, P., Rothbauer, U., Braun, D., & Duhr, S., Nat Commun 2010 (1): 100.
80 Sessionvorträge Tabletten

High-Throughput Protein Melting Temperature Analysis by Differential Scanning Fluorimetry
Menzen T, Frieß W
Ludwig-Maximilians-Universität München, Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics; Butenandtstraße 5, D-
81377 Munich, Germany
Unfolding and aggregation of therapeutic proteins are severe problems in drug formulation, because they are linked
to immunogenic reactions and reduced efficiency. Thus, the investigation of stabilizing conditions is essential during
development. The protein melting temperature (Tm) resembling the temperature at which half of the protein is in a
denatured state, is commonly used to assess overall thermal stability. Stabilizing conditions will shift Tm to higher
values whereas destabilizing effects reduce Tm. Due to the wealth of formulation parameters, such as buffers, pH,
excipients, and combinations thereof a high-throughput Tm assay would be beneficial. Differential Scanning
Calorimetry (DSC) as the gold standard for Tm analysis even in an automatized capillary system is limited to a few
samples per day of several hundred microliters of volume. Differential Scanning Fluorimetry (DSF) was introduced in
2001 by Pantoliano and coworkers and allows the determination of Tm by tracking the thermal unfolding of the
protein by means of an environmental sensitive fluorescent dye [1]. The application of DSF for high-throughput Tm
analysis of therapeutic proteins with low consumption of both time and material has been described [2-4]. But this
method to determine Tm is limited in presence of surfactants which are frequently used as stabilizers in protein
formulation. SYPRO® Orange is commonly utilised because it shows bright fluorescence after binding to the
exposed hydrophobic structures of the unfolded protein. However, the unfolding transition of the protein is covered by
a high background fluorescence of the dye trapped inside the hydrophobic core of the micelles formed by the
surfactants present. In this study we demonstrate how to overcome this limitation to determine Tm of a therapeutic
monoclonal antibody (MAb) with DSF in presence of surfactants typically used as stabilizing agent polysorbate 20,
polysorbate 80, and poloxamer 188 in concentrations above the CMC. The molecular rotor probe DCVJ (4-
(dicyanovinyl)julolidine) is sensitive to changes in protein structure and shows less interactions with micelles [5]. By
careful correction of the background fluorescence of corresponding placebo samples, Tm values of surfactant
containing protein formulations can be determined, whereas it is not possible solely based on the original sample
traces. The resulting Tm values correlate very well with Tm values obtained by DSC and in surfactant free
formulations. Tm values could be determined in a concentration range between 4 and 40 mg/ml MAb with DCVJ and
SYPRO® Orange in presence of surfactants. Despite the limited sensitivity of DCVJ compared to SYPRO® Orange,
DCVJ allowed Tm determination in various cases in which SYPRO® Orange failed. Additionally, we observed that
the appearance of turbidity marking the formation of aggregates of this MAb is linked to the second unfolding
transition (Tm2). This indicates that unfolding of specific domains is decisive for aggregation. We conclude that DSF
is a very promising method for high-throughput Tm analysis during therapeutic protein formulation development and
can also be used in presence of surfactants, a previously critical limitation.
References:
1. Pantoliano, M. W. et al.: J Biomol Screen 2001, 6(6): 429–440.
2. He, F. et al.: J Pharm Sci 2010, 99(4): 1707–1720.
3. Goldberg, D. S. et al.: J Pharm Sci 2011, 100(4): 1306–1315.
4. Li, Y. et al.: J Pharm Sci 2011, 100(6): 2120–2135.
5. Hawe, A. et al.: Pharm Res 2010, 27(2): 314–326.
Sessionvorträge Tabletten 81

NMR spectroscopy of high molecular weight protein complexes
Nagaraj M1 ; Schubeis T1 ; Ahmed M1 ; Zimmer A1,2 ; Ritter C1 1 Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany
2 current address: Karolinska Institutet, SE-171 77 Stockholm, Sweden
High-resolution structural information of protein targets is of crucial importance for rational drug design approaches.
X-ray crystallography and solution Nuclear Magnetic Resonance (NMR) are standard techniques to determine the
structures of soluble proteins. However, large protein complexes are still extremely challenging to study, as they are
difficult to crystallize or inherently non-crystalline, as is the case e.g. for fibrillar proteins. Recent advances in NMR
spectroscopy, isotope labelling schemes and protein sample preparation strategies have enabled new approaches to
obtain atomic-resolution structural details, and even high-resolution structures, of ordered, high molecular weight
protein complexes. Examples include fibrillar aggregates, microcrystalline samples, oligomeric assemblies and
membrane proteins.
Here, we present solution- and solid state NMR based approaches [1-3] and specific isotope labelling strategies to
study the structural details of the amyloid-like bacterial virulence factor curli. Amyloid fibrils are ordered aggregates
that have a high content of specific eta-sheet secondary structure. Long associated with fatal human diseases such
as Alzheimer’s, Parkinson’s and prion diseases, the amyloid fold is now known to be formed natively by a diverse set
of proteins without toxic side-effects. Amyloid fibrils appear to be particularly abundant as adhesive surface
structures on bacteria forming multi-cellular communities called biofilms. Understanding the structural properties of
these amyloid fibrils, as well as the details of their biogenesis, is therefore of great interest for infection research. In
addition, they allow the straight-forward correlation of structure and function within the native organism. They are
thus promising targets to study general aspects of amlyoid formation.
T. Schubeis greatly acknowledges a scholarship from the HZI Graduate School. M. Ahmed greatly acknowledges the funding from the German
Research Foundation.
References:
1. Ritter, C. et al.: Nature 2005, 435: 844–848.
2. Siemer, A.B. et al.: Angew Chem Int Ed 2005, 44: 2441–2444.
3. Wasmer, C. et al.: J Mol Biol 2010, 402(2): 311–325.
82 Sessionvorträge Tabletten

Session Spezielle Darreichungsformen
Orodispersible dosage forms – novel concepts, innovative products, new therapeutic options
Breitkreutz J
Heinrich Heine University Düsseldorf, Institute of Pharmaceutics and Biopharmaceutics, 40225 Düsseldorf, Germany
Orodispersible dosage forms are pharmaceutical drug formulations which rapidly disintegrate into multiple particles in
the oral cavity. Whereas oral lyophilisates 1 and orodispersible tablets (ODTs) 2 have been used for years in various
indications throughout the world, orodispersible films (ODFs) 3 and orodispersible minitablets (ODMTs) 4 have been
licensed most recently for the European and the national German market. Orodispersible films have been recently
included in the Ph.Eur. for the first time. Some further production methodologies such as electrospinning are
currently under investigation, but have not reached industrial production scale yet.
The gained popularity of orodispersible drug formulations is due to the fact that these rapidly disintegrating dosage
forms combine beneficial properties of both liquid and solid dosage forms. They enable improved drug stability over
the shelf-life and usually do not contain any potential harmful ingredients like preservatives or antioxidants, but offer
precise dosing, ease of administration and swallowing. Hence, they are ideal dosage forms for patients with diseaserelated
swallowing issues 5 , in particular for the paediatric and geriatric population 6 .
Some recent advances in material science and improved engineering processes seem to overcome the previously
used energy and time consuming freeze-drying technology for preparing orodispersibles. Ready-to-use tabletting
excipients such as Ludiflash ® , Pharmaburst ® , Pearlitol Flash ® or Parteck ® ODT promise easy and safe production by
simple mixing and direct compression. However, the resulting disintegration times are usually much higher as for the
oral lyophilisates. More recently the ODMT concept was introduced by our group using orodispersible minitablets of
only 2 mm in diameter or even less made from ready-to-use excipients or self-prepared mixtures which exhibit short
disintegration times below 5 s which is very similar to the marketed oral lyophilisates. As the total minitablet mass is
restricted to about 7 mg, the maximum drug load is also limited to approximately 2.5 mg. Complying with the Ph.Eur.
specifications regarding uniformity of dosage units is a major issue for low-dosed drug substances. A subsequent
granulation step may improve the content uniformity. Orodispersible films can be produced by various methods, such
as solvent-casting, melt extrusion or drug printing. Disintegration times are much higher than for lyophilisates and
ODMTs, but often the film is not sensed at the application site and therefore, the disintegration times might be of
minor importance for patients’ acceptance and therapy adherence. The drug dose is defined by the film area and
thickness, usually containing up to 15-20 mg drug substance. Inclusion of taste masking agents such as ionexchange
resins or cyclodextrins may further lower the maximum drug load.
Although the recent developments and products are quite promising and offer new therapeutic options in various
conditions, the specifications in the pharmacopoeias and the requirements of the regulatory bodies are still not
harmonized and mostly even useless. Especially mechanical properties of the orodispersible dosage forms,
disintegration and dissolution testing need new validated equipment and analytical methods to ensure the quality of
the advanced drug dosage forms.
References:
Seager H., J Pharm Pharmacol 1998, 50: 375–382.
Brown D., Drug Deliv Technol 2003, 3: 58–61.
Hoffmann E.M., Breitenbach A., Breitkreutz J., Exp Opin Drug Deliv 2011, 8: 299–316.
Stoltenberg I., Breitkreutz J., Eur J Pharm Biopharm 2011, 78: 462–469.
Stegemann S., Gogol M., Breitkreutz J., Int J Pharm 2012, 430: 197–206.
Breitkreutz J., Boos J., Exp Opin Drug Deliv 2007, 4: 37–45.
Sessionvorträge Tabletten 83

Challenges in Developing Gastroretentive Dosage Forms
Neumann M; Schneider F, Weitschies W
Institute of Biopharmaceutics and Pharmaceutical Technology, Department of Pharmacy, University of Greifswald, Center of Drug Absorption
and Transport, F.-Hausdorff-Str. 3, 17487 Greifswald, Germany
Similarly to food, solid oral dosage forms show highly variable gastric emptying, depending e.g. on their size, volume
and viscosity of gastric contents, intragastric flows, pH-profiles in the gastrointestinal (GI) tract or caloric content of
coadministered food [1]. Furthermore, due to the interdigestive migrating motor complex (IMMC), especially the
housekeeping waves, both particles and monolithic dosage forms are subject to antral forces in the stomach [2].
Once they are emptied through the pylorus, absorption of drugs from the human intestine is a complex and also
highly variable process. Hence, consideration of human physiology is the major prerequisite in the design of dosage
forms for drugs that may benefit from the targeted delivery in the stomach.
Since years, it is an ongoing challenge to overcome physiological processes of gastric emptying, and thereby
achieving prolonged gastric residence times of oral dosage forms in a controlled and reproducible manner.
In general, so called gastroretentive dosage forms are supposed to remain in the stomach over a long period of time,
and thus enable controlled drug delivery by delayed gastric emptying. Furthermore, gastroretention is suitable for
drugs that exhibit e.g. regio specific absorption in the upper small intestine (absorption windows), low solubility or
stability at high pH-values or short plasma half-lives [3]. By means of prolonging gastric residence time the
bioavailability of drugs can be increased.
During the last decades different gastroretentive dosage forms have been designed, which can be classified into four
major categories: (i.) floating systems that are able to swim on top of gastric contents; (ii.) mucoadhesive systems
that attach to the stomach walls; (iii.) high density systems that deposit in the antrum; and (iv.) unfolding or
expanding systems that enlarge in size, and thus cannot pass the pylorus [4].
Our aim is to develop and evaluate new gastroretentive dosage forms, which remain in the stomach in a controlled
manner. On one side, we focus on the design of fast expandable hydrogels of different shapes and the formulation of
a swelling gastroretentive dosage form for pH independent release of acyclovir. This antiviral drug is widely used in
therapy and prophylaxis of herpes infections and exhibits low bioavailability of only 10-30 % primarily due to its
narrow absorption window in the upper small intestine [5].
On the other side, another challenge is the development of in vitro and in vivo test methods to evaluate potential
gastroretentive dosage forms. Critical physiological conditions cannot be reflected by the application of rather simple
in vitro tests as they were used in the past. However, data from tracking of dosage forms in the human GI tract via
commonly applied techniques like gamma scintigraphy, X ray, MRI and magnetic marker monitoring (MMM) raised
the need for more biorelevant in vitro test methods [6]. Therefore, our dosage forms are tested by use of a novel,
mechanical stomach model developed in our laboratory, which simulates antral pressures and housekeeping waves.
Financial support from the German Federal Ministry of Education and Research is gratefully acknowledge (FKZ 13N11370).
References:
1. Streubel, A., Siepmann, J., Bodmeier, R.: Curr Opin Pharmacol 2006, 6: 501–508.
2. Wilson, C.G., Crowley, P.J. (eds): Controlled Release in Oral Drug Delivery (Controlled Release Society) 2011.
3. Klausner, E.A. et al.: J Control Release 2003, 90: 143–162.
4. Bardonnet, P.L. et al.: J Control Release 2006, 111: 1–18.
5. Arnal, J. et al.: J Pharm Sci 2008, 97(12): 5061–5073.
6. Kagan, L., Hoffman, A.: Expert Opin Drug Deliv 2008, 5(6): 681–692.
84 Sessionvorträge Tabletten

Innovative nanoparticular dosage forms – advantages in pharmacokinetics and administration
Rischer M
Losan Pharma GmbH, Department of Drug Delivery & Innovation Projects, Otto-Hahn-Strasse 13, 79395 Neuenburg am Rhein, Germany
Oral and parenteral dosage forms containing nanoparticles in the pharmaceutical development have been proposed
and used over more than 10 years. However, very often results have been published for the application of
nanosuspensions only not considering the difficulties (e.g. agglomeration, loading, scale-up, manufacturing costs)
which may appear if the downprocessing to the final dosage form is performed. In addition sometimes surfactants
or polymers as stabilizers have been used which could not be regarded as safe and toxicological assessments or
studies need to be performed. Basically, these reasons should be mainly responsible that only a few dosage forms
containing nanoparticles have entered the final commercial market yet. Losan Pharma has now completed several
studies with up-to-date Active Pharmaceutical Ingredients (BCS II compounds) and also projects with New
Chemical Entities from Research collaborations and has implemented a process of a cost-effective production of
nanoparticles in oral dosage forms (e.g. tablets). Key figures of this innovative process will be presented as well as
case studies proving the advantages of nanoparticulate dosage forms. Also a comparison of in-vitro with in-vivo
data will be made for certain cases. Finally, new convenient water-free “on the go” applications for such
formulations will be presented. Although, many research companies nowadays try to avoid insoluble new
compounds, there is still a majority of API´s present which do need new and innovative formulations and
applications. Nanoparticulate formulations do also help to reduce environmental contamination by reducing the
dosage for application an aspect which is be more and more considered (e.g. by AMNOG) for future developments.
Sessionvorträge Tabletten 85

Development and pharmaceutical technological applications of Diels-Alder hydrogels
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
2 Universitaet Regensburg, Department of Pharmaceutical Technology, 93040 Regensburg
Since the pioneering work of Otto Wichterle in the 1950s, polymeric hydrogels received much interest due to their
chemical versatility and favorable properties. Hydrogels essentially consist of water-insoluble polymers with infinite
molecular weight that bind large amounts of water. Their rheological behavior is between those of liquids and solids,
which makes them ideal candidates for injectable soft tissue implants. In fact, hydrogels have been proposed as
wound dressings, as carrier systems for the controlled release of drugs, as three-dimensional scaffolds for cell
transplantation, and for many other biomedical or technical applications. However, despite many years of research,
finding suitable cross-linking reactions is still an issue. On the one hand, the chosen reaction has to be highly
efficient in order to obtain high cross-linking densities. But on the other hand, the cross-linking reaction must occur
under mild conditions to be compatible with peptides, proteins, or living cells.
O
O
O
O
O
O
N
O
O
O
N
During our research on hydrogels, the Diels-Alder reaction came into our focus. The Diels-Alder reaction is a [4+2]
cycloaddition reaction between a conjugated diene and a substituted alkene which occurs in water without any
catalysts or initiators. The high efficiency and selectivity makes this reaction an ideal, biocompatible cross-linking
reaction. In our group, star-shaped poly(ethylene glycol) is functionalized with furyl and maleimide groups yielding
two complementary macromonomers. Upon mixing the two macromonomers, highly elastic hydrogels with low
swelling ratio are formed within minutes. Currently, we are investigating the suitability of these hydrogels as carrier
systems for the controlled release of pharmacologically active proteins. Special emphasis is put on the delivery of
antibodies, e.g. to treat common eye diseases such as age-related macular degeneration. Other potential
applications are the treatment of postoperative wounds or in the field of tissue engineering.
Acknowledgements: Financial support from Deutsche Forschungsgemeinschaft (grant number GO 565/16-1) is gratefully acknowledged.
O
O
86 Sessionvorträge Tabletten
O
O
N
H
O
N
H
O
O
O
O

Session Optimierte Patientenversorgung
Medication safety research as challenge for pharmaceutical scientists
Jaehde U
Institute of Pharmacy, Clinical Pharmacy, University of Bonn
Various studies have shown that drug therapy is a high-risk process demanding particular attention by all health care
professionals. Many adverse drug events are caused by medication errors, such as overdosing in patients with organ
dysfunctions or inadequate consideration of drug-drug interactions. Whereas there are strict regulatory requirements
to assure the safety of drugs before and after approval (product safety), there are only few systematic approaches to
enhance the safety of the entire medication (patient safety). The Federal Ministry of Health (BMG) has therefore
published an action plan for "Improving medication safety in Germany” which defines specific measures to be taken
by professional institutions and organizations dealing with drug therapy.
As part of this action plan the BMG supports various individual projects and encourages scientists to initiate research
aiming at developing new concepts and models for the improvement of medication safety. At the University of Bonn
various research projects have been conducted in collaboration with pharmacy practitioners as well as physicians
from various medical disciplines. Based on two examples, the challenges and opportunities of medication safety
research for pharmaceutical scientists will be discussed.
Clinical pharmacists to optimise patient safety in drug therapy
Bertsche T
Department of Clinical Pharmacy, University of Leipzig, Germany
Objectives
Drug-related problems (DRP) are frequent in hospitals. Particularly, they occur in prescription and administration and
lead to death, prolonged hospital stay, and severe adverse drug reactions (ADRs). Different reasons, such as
knowledge deficits, account for DRPs. In a setting of limited resources, however, intervention strategies to improve
patient safety have to be prioritised to the most frequent and severe DRPs and have to be tailored to the causes.
Participants and methods
At a university hospital, we identified DRPs in routine care by instructed monitors (clinical pharmacists) and assessed
knowledge deficits by a questionnaire survey. DRPs were classified according to their prevalence, potential risk, and
reasons for their occurrence using a decision-matrix model. Tailored interventions such as teaching sessions, clinical
pharmacist interventions, and newly developed clinical decision support systems were implemented in hospital
settings including e.g. intensive care units, paediatric wards, or cancer patients.
Results
In drug administration, we identified physico-chemical incompatibilities and drug administration by gastric tube as
most prevalent DRPs (n = 1376 processes). DRP prevalence decreased by 59% (p = 0.003, incompatibilities, adult
ICU patients, n = 1108 drug pairs) and by 94% (p < 0.001, gastric tube administration in children, n = 1164
processes). In a follow-up analysis these effects were sustainable. In drug prescription, guideline adherence in pain
treatment for cancer patients increased by 81% (p < 0.001, n = 100 patients), ADRs caused by drug interactions
decreased by 43% (p = 0.001, n = 265 patients) and excessive dosing in patients with renal insufficiency by 51%
(p < 0.001, n = 68).
Conclusions
Tailored interventions in routine care by clinical pharmacists based on teaching sessions and newly developed
electronic systems decreased rates of DRPs and DRP-related ADRs.
Sessionvorträge Tabletten 87

Safety of pharmacotherapy in nursing homes
Thürmann P A
Clinical Pharmacology, Faculty of Health (Department of Human Medicine), HELIOS Clinical Centre, 42283 Wuppertal, Germany
Nursing home residents are seen as a vulnerable population, due to their old age, multimorbidity, high frequency of
dementia and subsequent polypharmacy. Moreover, nursing home residents receive the highest number of
psychotropics. Many of these psychotropic drugs are so-called potentially inappropriate medication for the elderly
(PIM) as defined by Beers (1) and adopted to the German market by Holt et al. (2). Moreover, nursing home
residents are prone to experience drug-related problems (DRPs) and adverse drug-related events (AEs) (3). So far,
no data were available on the frequency and severity of AEs and DRPs in German nursing homes. We thus aimed to
investigate DRPs and AEs in German nursing homes and to develop a concept for an inter-disciplinary intervention in
order to improve safety of drug therapy in nursing homes.
Methods: Drug- related problems and drug-related adverse events were detected by trained clinical pharmacists in
nursing homes willing to participate over a period of four weeks. The study protocol was approved by the responsible
ethics committee, written informed consent was obtained from the residents or their responsible caregivers. Medical
documentation and nurses notes were screened by trained pharmacists, medication was documented and potential
DRPs and AEs were documented in an ACCESS database. Potential AEs were discussed in a multi-disciplinary
team, were causality assessment was performed. Frequency of ongoing and new AEs during the 30-days period was
calculated as the 30-day-prevalence of AEs per 100 nursing home residents months (NHRMs), whereas the 30-daysincidence
included only those AEs which started during the 30-days period and were also related to 100 NHRMs.
Results of the cross-sectional survey were presented in a multi-disciplinary expert workshop and served to develop a
concept to improve safe prescribing and monitoring of pharmacotherapy in nursing homes. This intervention was
implemented in 4 nursing homes, where the prescribing general practitioners, nurses and pharmacists received
special training in geriatric pharmacotherapy. In nursing homes Drug Safety Teams (DSTs) were built consisting of
trained nurses and pharmacists, who followed a pre-specified protocol of medication checks, residents monitoring
and communication with physicians.
Results: 778 nursing home participants in 11 nursing homes participated in the first survey. In total 102 AEs were
detected in 80 seniors (10.3 %). 37 of these AEs (59.7 %) were judged as preventable by the experts and 4 AEs (6.5
%) were ameliorable. The 30-days-prevalence of AEs was estimated with 12.94 AEs/100 NHRMs, the incidence
came to 7.87 AEs/100 NHRMs. 1.493 DRPs were detected, where almost 50 % concerned the documentation, 35 %
the storage and 15 % dispension of drugs. Three months after the implementation intervention the 30-daysprevalence
came to 15.93 AEs per 100 NHRMs and the incidence to 7.62 AEs/100 NHRMs. In contrast to the first
cross-sectional survey a much higher number of adverse events concerning the central nervous system was
observed, probably due to the increased awareness of the nurses. The intervention proved to be feasible, most
nurses would recommend it for other nursing homes.
Discussion: Data on frequency of AEs in nursing homes obtained in this study are in accordance with data published
in the literature (3). A prospective, cluster-randomized trial will be performed in two different regions of Germany to
demonstrate the effectiveness of the intervention with reduction of AEs as primary outcome variable.
The study was funded by the German Ministry of Health, grant no.: 1501/54401.
References:
1. Beers MH, Ouslander JG, Rollingher I, Reuben DB, Brooks J, Beck JC., Arch Intern Med 1991, 151: 1825–32.
2. Holt S, Schmiedl S, Thürmann PA., Dtsch Arztebl In. 2010, 107: 543–51.
3. Gurwitz JH, Field TS, Judge J et al., Am J Med 2005, 118: 251–258.
88 Sessionvorträge Tabletten

Identification of predictors for drug related problems in the context of home medication review
Fiß T1 ; Hoffmann W2 1 German Center for neurodegenerative diseases (DZNE), site Rostock/Greifswald; Ellernholzstreet 1/2; 17487 Greifswald, Germany
2 University Greifswald, Institute for Community Medicine; Ellernholzstreet 1/2 , 17487 Greifswald
Introduction: Drug related problems (DRP) are affecting success of pharmacotherapy, cause drug-induced
hospitalisations high costs, and limit health related quality of life. Identification of DRP and their predictors is needed
for focussed intervention and implementation of innovative health supply concepts. Several data sources are
available for identification of predictors for DRP. Especially routine data from the statutory health insurances have a
very strong role in this field. However, these data are limited due to incomplete data. Routine data only reflect health
care services which are paid by the statutory health insurance. Clinical tests as plasma level of several vital
parameter or cognitive tests are not available. Hence there is a need for drug utilisation studies in the context of
health services research.
Methods: We used datasets from two studies (AGnES: Arzt-entlastende, Gemeinde-nahe, E-Health-gestützte,
systemische Intervention; IDemUck: inegriertes Demenznetzwerk Uckermark) to evaluate determinants for drug
related problems and estimated needs for a structured medication management. Patients received a structured
medication review1 in their households and subsequent intervention. The pharmaceutical care was delegated in the
AGnES-studies to the local pharmacy whereas the patient’s GP conducted intervention in IdemUck.
Results: We found a high proportion of drug related problems. Especially the intake of potentially inappropriate
medication as well as intake of anticholinergig drugs was found as problematic. Out of the AGnES-patients aged > 65
years a total of 18% (n= 134) of the patients received 163 inappropriate drugs according to the Beers’ criteria. The
intake of PIM was slightly associated with self-reported falls (Phi: 0.1074; P= 0.0244). Multivariate logistic regression
showed significant results for the number of taken substances (OR = 1.176; 95% CI 1.121-1.234, P< 0.001).
Additionally we found the intake of anticholinergics (according to the ACBS list) as determinant for impaired cognition
(OR 10; 95-CI 1.2-85.3). As a 2nd problem we identified the occurrence of potentially clinically relevant drug-drug
interaction. In n=454 patients (58.3%) we found at least one drug-drug interaction with serious or moderate
relevance. Adjusted for age and gender, multiple binary logistic regression showed significant results for the number
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
diagnose of the muscular-skeletal disease (OR 1.74; 95%-CI 1.2-2.5).
Due to the pharmacist’s intervention in the AGnES-studies we found significantly improved DRP (adherence, drugdrug
interaction). In contrast to these results we found an increased prevalence of potentially clinically relevant drugdrug
interaction from 42.1% to 47.4% in the IDemUck-study.
Discussion: In the context of home medication reviews we identified a large array of DRP. Due to several additional
tests we could find some associations to clinical outcomes as falls and impaired cognition. Overall the number of
taken drugs was the main risk factor for the occurrence of DRP. Subsequent intervention by a pharmacist is
necessary to improve DRP.
However data are limited due to small number of patients and short follow-up period. We could only find first clues for
determinants which must be evidenced in further analysis. Hence linkage with routine data could help for long term
follow-up. Clinical outcomes as hospitalisation and mortality could be measured with these data.
As one model of evaluate efficacy of tailored intervention including long term effects we have included medication
management into the study DelpHi-MV (dementia: life and person centred help in Mecklenburg Western Pomerania,
clinicaltrials.gov identifier NCT01401582). Results of the analysis of determinants for the occurrence of DRP were
integrated into the study algorithms.
References:
1. Fiss T, Ritter CA, van den Berg N et al. Pa Wo Sci 2010, 32(5):566-74.
Sessionvorträge Tabletten 89

Session Arzneimittelüberwachung/Regulatory Affairs
Analyzing the use of (Q)SAR models for the evaluation of potential genotoxic impurities: A
cookbook in support of ICH M7
Sutter A
Bayer AG
• Assessing the (Q)SAR systems used
• Defining what exactly is expert knowledge
• Evaluating current practice in pharma companies: PhRMA and EFPIA surveys
• Discussing (Q)SAR reporting
Control strategy for an active coating process based on systematic process development (DoE) and
in-line Raman spectroscopy (PAT)
Funke A
Bayer Pharma AG, Global Pharmaceutical Development, Müllerstraße 178, 13353 Berlin, Germany
Introducing active ingredients into pharmaceutical dosage forms via film coating onto tablets (active coating) is
unusual and challenging due to high quality requirements with regard to both intra-tablet coating uniformity and
accuracy of average coating amount per tablet. The variability has been reduced by systematic process development
using design of Experiments (DoE). A process control strategy has been elaborated using in-line Raman
spectroscopy as an IPC for the endpoint determination of the coating process (PAT). As a result, the active coating
process reproducibly leads to a drug product fulfilling the predefined quality requirements.
90 Sessionvorträge Tabletten

Human Tissues and Cells – Advanced Therapies Medicinal Products
Astner I1, 2
1 GAA, Petzvalstr. 18, 38104 Braunschweig, Germany.
2 ZLG – EFG 04, Sebastianstr. 189, 53115 Bonn, Germany.
Tissues and Cells for transplantation offer a wide and expanding field of medicinal products for treating a variety of
diseases in humans. Alike the better known organ transplants tissues and cells are exchanged worldwide to
overcome transplant shortages or to ensure better donor–recipient matching.
Furthermore human or xenogenous tissues and cells are used as starting material for manufacturing (personalized)
advanced therapies medicinal products (ATMPs) or Medical Devices. Gene therapy, somatic cell therapy and tissue
engineering medicinal products offer new treatment opportunities for human diseases or dysfunctions like
regeneration, repairing or replacement of human tissues.
For tissues, cells and ATMPs it is essential to establish high standards of quality and safety for donors and patients,
on the one hand and to provide exchange of these products within the (sometimes short) expiry date of the cell
products, on the other hand.
As human and xenogenous materials are heterogeneous similar to biologicals the quality management and the
validated manufacturing process is an essential part of the product.
References:
1. DIRECTIVE 2004/23/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 31 March 2004 on setting standards of quality
and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells: OJ L 102,
2004: 48
2. COMMISSION DIRECTIVE 2006/17/EC of 8 February 2006 implementing Directive 2004/23/EC of the European Parliament and of the
Council as regards certain technical requirements for the donation, procurement and testing of human tissues and cells, OJ L 38, 2006: 38
3. REGULATION (EC) No 1394/2007 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 13 November 2007 on advanced
therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004, OJ L324, 2007: 121
Sessionvorträge Tabletten 91

Analysis of illegal medicinal products using NMR
Schramek N
Bavarian Health and Food Safety Authority, Veterinärstr. 2, Oberschleißheim, 85764, Germany
The number of adulterated food supplements and illegal medicinal products to be analyzed at the Official Medicinal
Control Laboratories raises steadily. More and more often unknown substances can be found as illicit additives in
food supplements [1]. Fast and feasible analytical methods are required to deal with this challenge. Despite its lack of
sensitivity Nuclear Magnetic Resonance (NMR) spectroscopy can be used as a fast screening method. Moreover
structural information can be obtained from NMR experiments required for the identification of unknown substances
[2, 3].
This presentation gives a short overview on how 1- and 2-dimensional NMR spectroscopy can be useful to official
medicinal control laboratories. A novel sildenafil-analogue, found as adulterant in a food supplement is presented [4].
References:
1. Venhuis B. J., de Kaste D.: J Pharm Biomed Anal 2012, DOI:10.1016/j.jpba.2012.02.014.
2. Trefi S., Gilard V., Balayssac S., Malet-Martino M., Martino R.: Magn Reson Chem 2009, 47: 163–173.
3. Holzgrabe U., Malet-Martino M.: J Pharm Biomed Anal 2011, 55: 679–687.
4. Wollein U., Eisenreich W., Schramek N.: J Pharm Biochem Anal 2011, 56: 705–712.
92 Sessionvorträge Tabletten

Poster

001
An in vitro test system to examine release in the vitreous humor
Loch C1; Nagel S1; Guthoff R²; Seidlitz A1; Weitschies W1 1 University of Greifswald; Center of Drug Absorption and Transport (C_DAT), Institute of Pharmacy, Felix-
Hausdorff-Strasse 3, Greifswald, 17487; Germany
2 University of Rostock; Department of Ophthalmology, Doberaner Strasse 140, Rostock, 18055; Germany
Introduction
The vitreous body has become an interesting location for the administration of injections or
implants in modern ophthalmology [1]. Such intravitreal dosage forms are loaded with
active agents, which are released over a prolonged period of time. A test method simulating
the situation in vivo is needed to observe the release behaviour of these new dosage forms.
To simulate this situation the Vitreous Model (VM) was developed.
Methods
The corpus of the VM consists of glass. Size and shape are assimilated to the in vivo
situation. Gel insertion and sampling can be performed via a vent at the top. To simulate
the movement of the eye the VM was placed on an altered orbital shaker. The modified
polyacrylamide gel (PAA-gel), which is used as vitreous substitute in this study, consists of
a 30% solution of acryl amide and standard Ringer buffer solution. The viscose solution
was filled in the corpus and the acryl amide monomers were cross-linked. The water
content, pH, relative density, refractive index and viscosity of the PAA-gel were examined
and compared experimentally to porcine and to human vitreous humor by literature
analysis. Furthermore, during a period of 3 hours the distribution behaviour of fluorescein
sodium solution (1.25 mM) injected into the PAA-gel and in the porcine vitreous humor was
investigated by using the VM.
Results
The water content (99%) and pH (7.4) of the PAA-gel were equal to the values obtained for
porcine and human vitreous humor. Also, the results for the relative density (1.0013) and
refractive index (1.3385) showed good accordances (deviation < 1%). Slight deviations
were found for the viscosity. The shape and volume of the VM correspond to the natural
vitreous body.
In the distribution study further agreements were achieved. For analysis the porcine
vitreous body and the substitute were divided in two halves. 82% of the detected amount of
fluorescein sodium was found at the side at which the injection occurred for the PAA-gel
and 75% for the natural vitreous humor.
Conclusion
The results of the distribution study and the tested physical and chemical properties of the
PAA-gel were in good accordance with the properties of porcine and human vitreous
humor. Along with the related shape of the VM, the new model seems suitable to perform in
vitro experiments, which are capable of simulating in vivo conditions. Through the
possibility of insertion of intravitreal injections and implants in the VM, biorelevant
distribution and release studies may become feasible.
Acknowledgments: The authors would like to thank to the staff members of the glazier’s workshop of the
Faculty of Mathematics and Natural Sciences for their excellent technical assistance. This project was
funded by the Federal Ministry of Education and Research (BMBF) within REMEDIS “Höhere Lebensqualität
durch neuartige Mikroimplantate” (FKZ: 03IS2081).
References:
1. Baino, F. et al.: Acta Biomaterialia 2011, 7(3): 921-935.
002
Diversified influences of arginine and guanidine on proteins
Redweik S; Wätzig H
Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstr. 55, 381106
Braunschweig, Germany
Although arginine and guanidine exhibit the same partial structure their influence on
proteins is very diversified [1]. In this study these influences on the proteins BSA and ßlactoglobulin
were analyzed by Affinity Capillary Electrophoresis (ACE). These influences
on the proteins were also tested in the presence of urea in the Tris-buffer system because
there is a concentration dependent inhibition of arginine to the urea induced protein
unfolding of BSA [1].
ACE is a powerful tool to detect interactions between proteins and various ligands due to
mobility shifts of the protein [2]. Therefore the difference of the protein electrophoretic
mobility is detected without any ligand in the buffer and with the addition of a defined ligand
concentration. In order to compensate for changes on the migration time, which are not
due to ligand binding, the mobility ratio of an EOF-marker and the protein is used [3]. Six
repeats were done with a very good precision due to the use of a special rinsing procedure
[4]. The differences of the mobility ratios Rf (without addition of ligand) and Ri (in presence
of a defined ligand concentration) were normalized and the confidence intervals were
estimated to demonstrate the significance of these interactions. The achieved small
confidence intervals showed clearly different influences of the tested ions on BSA and ßlactoglobulin
with and without urea in the buffer.
References:
1. Gosh, R. et al.: Biochemistry 2009, 48(5): 1135-1143.
2. Busch, M. H. A. et al.: J Chromatogr A 1997, 777(2): 311-328.
3. Bose, S. et al.: J Chromatogr B 1997, 697(1-2): 77-88.
4. El-Hady, D. et al.: J Pharm Biomed Anal 2010, 52(2): 232–241.
003
The new cardioactive compounds from Leonurus cardiaca L. (Ph.Eur.)
Savtschenko A1 2, Dhein S1, Rauwald H W2 1 Clinic for Cardiac Surgery, University of Leipzig, Strümpellstraße 39, D-04289 Leipzig, Germany
2 Department of Pharmaceutical Biology, University of Leipzig, Johannisallee 21–23, D-04103 Leipzig,
Germany.
Just recently, aqueous preparations of Leonurus cardiaca – such as a newly developed
refined extract – were reported to exert a series of effects on cardiac electrophysiology,
such as ICa.L blockade, a reduction of the repolarising current IK.r, as well as the prolongation
of both AP-duration and the activation time constant of the If current, while INa was strikingly
unaffected [1]. This synergistic action on multiple electrophysiological targets may limit
potential adverse effects, especially the risk of proarrhythmia, and is thus in accordance
with the traditional use of this medicinal plant as an antianginal in European herbalism [2].
Therefore, several extract samples were examined for substances potentially contributing
to these pharmacological activities. In this screening procedure, ferulic acid (4-hydroxy-3methoxy-trans-cinnamic-acid)
and stachydrine were detected as cardioactive compounds of
the refined extract. Although a variety of pharmacological actions on the cardiovascular
system have been reported for this caffeic acid derivative [3], specific molecular
pharmacological actions on cardiac ion channels – as observed for the L. cardiaca refined
extract [1] – have not been reported for the isolated compound up to now. In subsequent
voltage clamp experiments carried out according to a protocol previously developed in our
group [4], ferulic acid at concentration of 3 µM influences neonatal rat ventricular
cardiomyocytes by ICa.L blockade and AP-duration shortening while INa was not affected.
Epicardial mapping experiments on isolated rabbit hearts perfused according to the
Langendorff technique showed an increase of basic cycle length and relative coronary flow,
as well as a reduction of left ventricular pressure. Thus mimicking parts of the effect of the
previously described L. cardiaca refined extract. In voltage clamp experiments stachydrine
at concentration of 1 mM and depending on cell type increases the ICa.L current and
prolongs the AP-duration of neonatal rat ventricular cardiomyocytes, while INa was
unaffected. Thus, ferulic acid and stachydrine contribute to the cardiac effects of L.
cardiaca; however, other compounds from L. cardiaca may also contribute according to a
‘multi-component / multi-target’ active principle [1].
References:
1. Ritter, M. et al.: Planta Med. 2010, 76: 572-82.
2. Fuchs, L.: New Kreutterbuch (Basel edit on) 1543.
3. Wang, BH., Ou-Yang JP.: Cardiovascular Drug Reviews 2005, 23(2): 161-172.
4. Scheffler, A. et al.: J. Ethnopharmacol. 2008, 120: 233-240.
004
Quantification of tumor-promoting and skin irritating diterpene esters in
homeopathic mother tinctures
Kummer, J., Melzig, M. F.
Institute of Pharmacy - Pharmaceutical Biology, Freie Universität Berlin, Königin-Luise-Str. 2+4,
14195 Berlin, Germany
Some plants from the family Euphorbiaceae were used in the past as traditional remedies.
With ingenol melbutate there is now a highly active therapeutic for the topical field therapy
of actinic keratosis [1]. The active ingredients are diterpene esters, which are known to be
tumor-promoting and skin irritating since Hecker et al. discovered TPA (12-Otetradecanoylphorbol-13-acetate)
in the oil of Croton tiglium L. seeds [2].
There are two basic chemical structures such as the alcohols phorbol and ingenol. These
two alcohols form a great variety of different esters with different activities. Available
preparations of these esters are homeopathic mother tinctures. For standardization and the
monographic description in pharmacopoeias it is necessary to establish an easy method to
quantify these esters from herbal preparations, e.g. homeopathic mother tinctures.
To avoid the difficult analysis of the different esters and to quantify all the toxic esters it´s
necessary to hydrolyse them and to determine the resulting alcohols by HPLC. The alkaline
hydrolysis is not suitable for all structures because of a bad recovery [3].
We will discuss the different approaches for gentle methods to hydrolyze the esters. The
enzymatic hydrolysis with different enzymes such as pectinase from Aspergillus niger and
esterase from pig liver and Bacillus subtilis didn t result in complete hydrolysis. Other
esterases like enzymes from rabbit or rat liver seem to be more selective for diterpene
esters [4].
Ingenol Phorbol
References:
1. Lebwohl, M. et al., N Engl J Med 2012, 366: 1010-1019.
2. Hecker, E., Schmidt, R., Prog Chem Org Nat Prod 1974, 31: 377-467.
3. Gläser, S., Untersuchungen zu einem möglichen Gesundheits- und Krebsrisiko durch pflanzliche
Arzneimittel sowie industriell genutze Rohstoffe aus Euphorbiaceen – Quantitative Bestimmung von
irritierenden und tumorpromovierenden Diterpenestern und Evaluierung durch biochemische und
biologische Tests (Thesis, Universität Heidelberg) 1991.
4. Saito, M., Egawa, K., J Biol Chem 1984, 259: 5821-5826.
005
Various Modes of High Performance Liquid Chromatography for Analysis of
Monoclonal Antibodies
Grotefend S1; Wätzig H1 1 Technische Universität Braunschweig, Institute of Medicinal and Pharmaceutical Chemistry, Beethovenstr.
55, 38106 Braunschweig, Germany
Protein quantitation as well as the detection of protein aggregates becomes more and more
important since protein based pharmaceuticals (biologicals), especially monoclonal
antibodies (mAb), were established as potent drugs in anticancer therapy or for treatment
of autoimmune based diseases. Accurate and precise methods have to be found for this
purpose, due to the high efficiency of these drugs and its behaviour to form aggregates,
which may lead to adverse effects like anaphylaxis or injection side reactions after drug
application even in small amounts [1]. Quality control purposes could not been reached by
classic protein quantification methods such as the Bradford assay or SDS-PAGE, yet. In
contrast different HPLC separation modes possess great selectivity as well as high
precision in monoclonal antibody analysis. An IgG1-type antibody could be precisely
analyzed (RSD % Monomer = 2.54 % day-to-day precision, n=60) by using High
Performance Size Exclusion Chromatography (HP-SEC), which also enables the complete
separation from monomer, aggregates and a low molecular weight fragment [2]. Weak
Cation Exchange Chromatography (WCX), which is also already successfully applied in
protein, especially antibody analysis, will be analyzed for its suitability in precise and
selective monoclonal antibodies analysis. The high selectivity of this mode allows
separating antibodies with different C-terminal lysine residues (proved by carboxypeptidase
B addition) [3]. The separation modes will be compared in terms of precision, selectivity,
analysis time as well as effort during sample and mobile phase preparation.
94 Poster

References:
1. Carpenter, J.F. et al, J. Pharm. Sci. 2010, 99 (5): 2200-2208.
2. Grotefend, S. et al, Oral presentation on HPLC 2011 Budapest, Hungary.
3. Weitzhandler, M. et al, Proteomics 2001, 1: 179-185.
006
Synthesis of novel Janus Kinase Inhibitors via Copper Catalyzed Azide-Alkyne
Cycloaddition
Gehringer, M. and Laufer, S.
University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany
Janus Kinases (JAKs) are non-receptor protein tyrosine kinases mediating signalling
through the JAK-STAT (Signal Transducer and Activator of Transcription) pathway. The
Janus Kinase family has four members: JAK1,2,3 and TYK2. Being crucial signal
transducers for a variety of cytokines, growth factors, and interferons, JAKs are involved in
numerous pathologies including malignancies, myeloproliferative disorders and
autoimmune diseases.
In contrast to the ubiquitous expression of the other JAK family members, JAK3 is
predominantly expressed in hematopoietic cells. In mammals, the lack of functional JAK3
causes immunodeficiencies while not disrupting the function of non-immune cells.
Therefore, targeting JAK3 is a promising strategy to generate a novel class of
immunosupressant drugs with limited side effects. [1]
Recently, Ruxolitinib a small-molecule JAK1/2 inhibitor was approved by the US Food and
Drug Administration for the treatment of patients with intermediate or high-risk
myelofibrosis. [2]
In search for novel JAK3 inhibitors we replaced the Ruxolitinib pyrazole ring by a 1,4disubstituted
1,2,3-triazole accessible thought copper catalyzed azide-alkyne
cycloaddition. [3] Compared to the laborious synthesis of the corresponding pyrazoles, click
chemistry offers a rapid and efficient access to triazols with various substitution patterns
facilitating their optimization towards JAK3 inhibition.
References:
[1] Cornejo , M. G. et al., Int. J. Biochem. Cell. Biol. 2009, 41, 2376-2379.
[2] Mesa, R. A. et al., Nature Rev. Drug Discov. 2012, 11, 103-104.
[3] Sharpless, K.B. et al., Angew. Chem. Int. Ed., 2002, 41, 2596–2259.
007
Synthesis of water soluble aziridine-2,3-dicarboxylate-based cysteine protease
inhibitors with leishmanicidal activity
Fey, P. 1; Schad, C. 2; Schurigt, U. 3; Moll, H. 3; Schirmeister, T. 1
1Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, 55099 Mainz,
Germany
2Institute of Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, 97074
Wuerzburg, Germany
3Institute for Molecular Infection Biology, Infection Immunology Unit, University of Wuerzburg,
Josef-Schneider-Str. 2, 97080 Wuerzburg, Germany
Cysteine proteases of Leishmania are proven targets for disease control of leishmaniasis,
because they play a key role in parasite growth, differentiation, pathogenicity and
virulence.[1]
Starting from two selective protease inhibitors RV122C (Boc-(S)-Leu-(R)-Pro-(S,S)-
Azi(OBn)2) and RV212C (Boc-(R)-Leu-(S)-Pro-(S,S)-Azi(OBn)2) of cathepsin L and CL-like
proteases, a series of structural isomers, stereoisomers and derivatives was synthesized
and tested for antileishmanial activity against L. major. [2] Out of this series CS128 (Boc-
(S)-Leu-(R)-Pro-(R,R)-Azi(OBn)2) is the most promising candidate. The improvement of the
water solubility of this compound is the main goal of the present work.
According to docking results with RV122C in the active site of human cathepsin L[3] the
Boc protecting group as well as one of the benzyl esters are not necessary for productive
interactions with the enzyme. CS128, only differing in the configuration at the aziridine ring
carbons, is proposed to have a comparable binding mode and therefore will be modified at
the identified positions.
We first focussed on the synthesis of aziridin-2,3-dicarboxylates with different ester
moieties. One of these is used to yield a free acid-group after hydrogenolysis, which will
increase water solubility. The other is designed to be stable against hydrogenolysis, but
enabling the introduction of different liphophilic esters increasing affinity by deeply
extending into the hydrophobic S2-pocket of the enzyme.
Additionally, substitution of the protecting group with hydrophilic groups like glycols was
chosen as alternative possibility to increase water solubility.
O
O
HN
(S)
N
O
(R)
N
HO
O
R
O O
(S S) + (R R)
References
1. Ponte-Sucre, A. et al., Antimicrob. Agents Chemother. 2006, 50, 2439-2447.
2. Schurigt U. et al., Antimicrob. Agents Chemother. 2010, 54, 5028-5041.
3. Vicik R. et al., ChemMedChem 2006, 1, 1126-1141.
008
Synthesis, characterization and screening of Dengue Virus Type 2 NS2B-NS3
protease inhibitors
Holloway, S. 1; Wu, H. 1; Bodem, J. 2; Snitko, M. 2; Welker, A. 3; Sotriffer, C. 3; Klein, C. 4;
Schirmeister, T. 1
1 Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55099 Mainz,
Germany
2 Institute of Virology and Immunology, University of Würzburg, Versbacher Strasse 7, D-97078
Würzburg, Germany
3 Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074
Würzburg, Germany
4 Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer
Feld 364, D-69120 Heidelberg, Germany
Dengue, a mosquito-borne viral infection of humans transmitted by Aedes aegypti and
Aedes albopticus mosquitoes, has become a continuing global threat, since 2.5 billion
people living in tropical and subtropical regions of the world (predominantly in Asia, Africa
and the Americas) are at risk of infection. [1,2] It is caused by a flavivirus with a single
positive-stranded RNA genome [3,4] and four distinct serotypes. [5] The dengue virus
infection can result in classic dengue fever, dengue hemorrhagic fever (DHF) or dengue
shock syndrome (DSS). [6,7] The dengue virus genome encodes a serine protease with a
classical catalytic triad (His51, Asp75 and Ser135) [8,9] which is responsible for the posttranslational
proteolytic processing of the polyprotein precursor and essential for the viral
replication [10,11], making it an important and attractive therapeutic target. [12]
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
influence on enzyme inhibition due to a variation of the amine residue on the one hand and
the aromatic cinnamic acid residue on the other hand, and b) diaryl thioethers. The possible
binding modes are analyzed by docking studies. The synthesized compounds are screened
in fluorometric enzyme assays and HPLC-based assays using a fluorogenic AMC-derived
substrate. Since a good specifity for the DENV-2 NS2B-NS3pro should be achieved, other
serine proteases, such as trypsin, chymotrypsin and thrombin are included in our screening
procedures.
References
1. Guzman, M. G. et al.: Nat. Rev. Microbiol. 2010, 8: S7–S16.
2. Halstead, S. B.: The Lancet 2007, 370: 1644–1652.
3. Li, H. et al.: J. Virol. 1998, 73(4): 3108–3116.
4. Chambers, T. J. et al.: Annu. Rev. Microbiol. 1990, 44: 649–688.
5. Morita, K., Tanaka, M., Igarashi, A.: J. Clin. Microbiol. 1991, 29: 2107–2110.
6. Gubler, D. J.: Clin. Microbiol. Rev. 1998, 11(3): 480–496.
7. Rigau-Pérez, J. G. et al.: The Lancet 1998, 352: 971–977.
8. Bazan, J. F., Fletterick, R. J.: Virology 1989, 171: 637–639.
9. Melino, S., Paci, M.: FEBS Journal 2007, 274: 2986–3002.
10. Falgout, B. et al.: J. Virol. 1991, 65: 2467–2475.
11. Zhang, L., Mohan, P. M., Padmanabhan, R.: J. Virol. 1992, 66: 7549–7554.
12. Zheng, Y. et al: Bioorg. Med. Chem. Lett. 2006, 16: 36–39.
009
Substituted vinyl sulfones as covalent and reversible cysteine protease
inhibitors
Kesselring, J. 1; Schneider, T. 1; Engels, B. 2; Sotriffer, C.; 3 Schirmeister, T. 1
1 Institute of Pharmacy and Biochemistry, University of Mainz, GERMANY
2 Institute of Physical and Theoretical Chemistry, Universiy of Würzburg, GERMANY
3 Institute of Pharmacy and Food Chemistry, University of Würzburg, GERMANY
Cysteine proteases play vital roles for the life cycles, nutrition and pathogenesis of a variety
of parasites causing infectious tropical diseases [1,2]. Therefore a promising strategy for
the treatment of these diseases is the inhibition of these proteases.
At present a large number of potent and irreversible cysteine protease inhibitors of different
classes, including peptidyl vinyl sulfones, have been reported [3]. The inhibition mechanism
of such vinyl sulfones results from the addition of the enzyme’s active site cysteine residue
in a Michael type reaction [4].
QM and QM/MM calculations have proposed substituted vinylsulfones which should be able
to form a covalent, but reversible bond with the cysteine sulfur of the protease’s active site.
Such a reversible reaction should be possible by choosing suitable substituents and leaving
groups attached to the α- and β-position of the vinyl sulfone moiety, e.g. nitrile groups, and
thiolates, respectively. This leads to a thermoneutral or slightly endergonic vinylic
substitution or addition reaction. To confirm these calculations a series of highly
functionalized vinyl sulfones with varying peptidic residues were synthesized and reacted
with phenylethanethiol as an analogue of the active site’s cysteine residue. The progression
of the reaction was monitored by NMR- and quantitative IR spectroscopy, which allows the
determination of rate constants, equilibrium constants, and reaction energies. Furthermore
the inhibitory potencies of the synthesized compounds were evaluated by enzyme assays
with various cysteine proteases.
References:
[1] R.P. Hanzlik, J. Med. Chem. 1984, 27(6), 711.
[2] P.J. Rosenthal and coworkers, Ant microb. Ag. Chemother. 2003, 47(1), 154.
[3] J.C. Powers, J.L. Asgian, O.D. Ekici, K.E. James Chem. Rev. 2002, 12, 4639.
[4] J.T. Palmer, D. Rasnick, J.L. Klaus, D. Bromme J. Med. Chem. 1995, 38 (17), 3193.
010
Genome-inspired drug discovery in the predatory bacterium Cupriavidus
necator
Nett, M. 1; Kreutzer, M. F. 1; Kage, H. 1
1 Junior Research Group “Secondary Metabolism of Predatory Bacteria”, Leibniz Institute for
Natural Product Research and Infection Biology / Hans-Knöll-Institute, Beutenbergstr. 11a, 07745
Jena, Germany
Whole genome sequencing has illuminated the secondary metabolic proficiency of
Poster 95

microbes – underappreciated for years based on conventional isolation programs – and has
helped set the foundation for a new natural product discovery paradigm based on genome
mining [1]. Furthermore, it is becoming increasingly evident that the potential for the
biosynthesis of bioactive compounds is more abundant than previously anticipated. In
recent years, many neglected microorganisms have emerged as alternative resources of
chemical diversity [2].
Bacteria that prey on other organisms as a food source are particularly appealing in this
context. We assume that a large fraction among these micropredators evolved or acquired
antibiotic biosynthesis genes to promote their feeding strategy. As a proof-of-concept, we
analyzed the genome of the predatory bacterium Cupriavidus necator for the presence of
such genes. C. necator had previously been reported to secrete a peptidic molecule, which
sequesters copper from the environment and is important for the killing of competing
predacious species [3], but the structure of the involved metabolite was never resolved. We
now identified a promising gene cluster that could account for the biosynthesis of this
copper chelating natural product. The candidate locus includes genes for the nonribosomal
assembly of a peptide as well as for the incorporation of bidentate ligand groups. To
retrieve the associated natural product, we disrupted one of the biosynthesis genes by
insertional mutagenesis and subsequently compared the metabolite profiles of mutant and
wild-type strain. This approach led to the discovery of a family of linear lipopeptides,
exemplified by the main metabolite cupriachelin [4]. When tested in an agar diffusion assay,
cupriachelin and its congeners exhibited strong effects against numerous bacteria,
including all identified prey organisms of C. necator.
Acknowledgments: We gratefully acknowledge the Bundesministerium für Bildung und Forschung for
supporting our research involving genomics and genome-inspired drug discovery (BMBF GenoMik-Transfer
programme; grant no. 0315591A).
References:
1. Nett, M.; Ikeda, H.; Moore, B. S.: Nat. Prod. Rep. 2009, 26(11): 1362-1384.
2. Winter, J. M.; Behnken, S.; Hertweck, C.: Curr. Opin. Chem. Biol. 2011, 15(1): 22-31.
3. Casida, L. E.: Appl. Environ. Microbiol. 1987, 53(7): 1515-1518.
4. Kreutzer, M. F.; Kage, H.; Nett, M.: J. Am. Chem. Soc. 2012, 134(11): 5415-5422.
011
Aziridine-based inhibitors of HIV-1 protease
Grün, J. 1; Rieger, M. 2; Welker, A. 3; Bodem, J. 4; Schneider, T. 1; Engels, B. 2 ; Sotriffer, C. 3;
Kiefer, W. 1; Schirmeister, T. 1;
1 Institute of Pharmacy and Biochemistry, University of Mainz, GERMANY
2 Institute of Physical and Theoritical Chemistry, Universiy of Würzburg, GERMANY
3 Institute of Pharmacy and Food Chemistry, University of Würzburg, GERMANY
4 Institute of Virology and Immunology, University of Würzburg, GERMANY
The HIV-1 protease represents an aspartate protease essential for proper virion assembly
and maturation. [1-3] Many competitive inhibitors of this protease are available with FDA
approval. [4,5] But there is a rapid rise of strains that encode mutant proteases resistant to
these reversible protease inhibitors. [6,7] We explored the ability of QM/MM models to
accurately describe the inhibition reaction of the HIV-1 protease by epoxide- and aziridinebased
inhibitors. In contrast to their epoxide counterparts, the mechanisms and binding
modes of aziridine-based inhibitors have much more rarely been the subject of
investigations, e.g. no X-ray data for complexes with HIV-1 PR or SIV PR exist.
Computations [8-11] predict their inhibition mechanism to be similar to that of epoxides, but
differences result from the stronger basicity of the aziridine. Accordingly, aziridine-based
inhibitors should be ideally suitable for aspartate proteases which act in more acidic
environments. [12] This was indeed shown by recent work. Employing docking approaches
the HIV-1 PR structure is used to predict possible substitution patterns of such new
inhibitors with improved binding affinities. We present synthetic approaches for these new
optimized aziridine-based inhibitors.
References:
1. Frankel, A. D.; Young, J. A. T. Annu Rev B ochem 1998, 67, 1.
2. Seelmeier, S.; Schmidt, H.; Turk, V.; Vonderhelm, K. P Natl Acad Sci USA 1988, 85, 6612.
3. Brik, A.; Wong, C. H. Org Biomol Chem 2003, 1, 5.
4. Rose, J. R.; Craik, C. S. Am J Resp Crit Care 1994, 150, 176.
5. Wlodawer, A.; Erickson, J. W. Annu Rev Biochem 1993, 62, 543.
6. Metzner, K. J.; Rauch, P.; von Wyl, V.; Leemann, C.; Grube, C.; Kuster, H.; Boni, J.; Weber, R.;
Gunthard, H. F. J Infect Dis 2010, 201, 1063.
7. Bihani, S. C.; Das, A.; Prashar, V.; Ferrer, J. L.; Hosur, M. V. Biochem Bioph Res Co 2009, 389, 295.
8. Helten, H.; Schirmeister, T.; Engels, B. J Phys Chem A 2004, 108, 7691.
9. Helten, H.; Schirmeister, T.; Engels, B. J Org Chem 2005, 70, 233.
10. Vicik, R.; Helten, H.; Schirmeister, T.; Engels, B. Chemmedchem 2006, 1, 1021.
11. Vicik, R.; Busemann, M.; Baumann, K.; Schirmeister, T. Curr Top Med Chem 2006, 6, 331.
12. Dunn, B. M. Chem Rev 2002, 102, 4431.
012
Bicyclic acetals – potential inhibitors of Golgi α-mannosidase II
Juchum, M. 1; Kiefer, W. 1; Schneider, T. 1; Seibel, J. 2; Sotriffer, C. 2; Engels, B. 3; Schirmeister,
T. 1;
1 Institute of Pharmacy and Biochemistry, University of Mainz, GERMANY
2 Institute of Pharmacy and Food Chemistry, University of Würzburg, GERMANY
3 Institute of Physical and Theoretical Chemistry, University of Würzburg, GERMANY
Cancer – predominantly trachea, bronchus and lung cancer – causes 5.9 % (3rd place in the
top ten after ischemic heart disease and stroke) of deaths in high-income countries and 2.4
% (7 h place) of deaths in the world [1]. Unregulated spreading of cells, invasion of healthy
tissue and especially metastasis characterize the pathology of these diseases [2].
Selectins, carbohydrate recognizing proteins, play a crucial role in binding metastasizing
cancer cells [3]. Ligands for selectins are often modified glycosylation patterns on the
surface of the cancer cells [4]. Studies have shown an overexpression of different sugar-
hydrolyzing enzymes in these cells, making such enzymes promising targets for new anticancer
drugs [5]. Especially inhibition of the Golgi alpha-mannosidase II (GM II) has shown
tumor repression [6].
GM II, a glycosyl hydrolase, is a 125 kDa type II transmembrane protein [7] that plays an
essential role in the N-glycosylation pathway of asparagine side chains. The high specific
cleavage of two mannose units (α-(1,3) and α-(1,3)) of the intermediate
GlcNAcMan5(GlcNAc)2 takes place in the active site of the enzyme, two aspartate residues
and a zinc cation involved. GM II is a retaining glycosidase and cleaves the sugars in a twostep-SN2-mechanism
that preserve the configuration of the anomeric C-atom [8].
Already available inhibitors, mostly derivatives of swainsonine, have side effects, due to low
selectivity [9]. The goal of the presented project is the synthesis of selective, covalentreversible
inhibitors with a long resting time in the catalytic site of the enzyme. QM
calculations and docking simulations have shown that bicyclic acetals are promising
candidates, in terms of both, high affinity to the target enzyme and reaction kinetics. Based
on L-gulose, we synthesized 1-2 and 1-6 bridged species. New strategies like cycloaddition
and especially olefin ring-closing metathesis (RCM) were also evaluated as new
synthetic pathways to yield the desired acetals.
References:
1. World Health Organization. Media Centre: The top ten causes of death (Updated June 2011).
http://www.who.int/mediacentre/factsheets/fs310/en/index.html (accessed March 19, 2012).
2. Kohn, E. C., Liotta, L. A.: Cancer Res 1995, 55: 1856-1862.
3. Kim, Y. J. et al.: PNAS 1998, 95: 9325-9330.
4. Kim, Y. J., Varki, A.: Glycoconjugate J. 1997, 5: 569-576.
5. Rambaruth, N. D., Dwek, M. V.: Acta Histochem. 2011, 113: 591-600.
6. Goss, P. E. et al.: Clin. Cancer Res, 1997, 3: 1077-1086.
7. Moremen, K. W., Touster, O.: J. Biol. Chem. 1986, 261: 10945-10951.
8. Petersen, L. et al.: J. Am. Chem. Soc. 2010, 132: 8291-8300.
9. Kuntz, D. A. et al.: ChemBioChem 2010, 11: 673-680.
013
Tacrine Derivatives and its dimers as Potential Antiprotozoic Agents
Schmidt I1; Schurigt U2; Pradel G2; Holzgrabe U1 1Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074
Würzburg, Germany
2Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Str. 2, 97080
Würzburg, Germany
Three of the most important tropical infectious diseases are Malaria with an estimated 655
000 deaths in 20101, Leishmaniasis with an estimated incidence of 2 million new cases per
year2 and Human African Trypanosomiasis with 7139 new cases as reported in 20103. The
development of new and cheap agents for the treatment is urgently needed. In our previous
investigations antiprotozoal activity of tacrine (e.g. IC50 = 12.5 µM for Plasmodium
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
(e. g. IC50 = 0.095 µM for Plasmodium falciparum). This dimer and derivatives with
carbonyl- and hydroxyfunctions at the saturated ringsysteme (R1) exhibit antiprotozoal
activity partially in nanomolar range of concentration, but associated with moderate
cytotoxicity. Hence our first task was to identify the optimal length of the linking alkyl chain.
Therefore we synthesized the above mentioned compounds with various chain length from
C6 to C12. The antiprotozoal activities of this dimers against Plasmodium falciparum,
Leishmania major, Trypanosoma brucei and cytotoxicity on macrophages J774.1 were
determined in vitro and showed that the length of 7 to 9 methylene units is most suitable.
Furthermore our results demonstrated that the introduction of substituents like nitro or
chlorine in the aromatic moiety leads to a similar antiprotozoal effect and reduced
cytotoxicity. Therefore our future goal will be the design and synthesis of active compounds
with decreased cytotoxicity.
1
Acknowledgments: We acknowledge the Deutsche Forschungsgemeinschaft (SFB 630) for support.
References:
1. WHO; www.who.int; World Malar a report 2011.
2. WHO; www.who.int; Technical Report Series 949.
3. WHO; www.who.int; Fact Sheet N° 259.
014
Development of MIP-Inhibitors
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
Würzburg, Germany
2 Defence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, UK
3 Research Center for Enzymology of Protein Folding, Max-Planck Institute Halle, Weinbergweg 22,
06120 Halle, Germany
4 Department of Biopolymers, University of Bayreuth, Universitätsstrasse 30, 95447 Bayreuth,
Germany
Major virulence factors known as macrophage infectivity potentiators (MIPs) facilitate the
infection of cells with different pathogenic bacteria like Legionella pneumophila or
Burkholderia pseudomallei. Both pathogens manifest in different diseases, L. pneumophila
is the causative agent of Legionnaires‘ disease, whereas B. pseudomallei induces
melioidosis which emerges in Southeast Asia and Northern Australia. An infection with
B. pseudomallei may occur by inoculation through skin lesions or occasionally by
inhalation. Human infection with Legionellosis transpires as well by inhalation of
contaminated aerosols, typically being air-conditioning systems, whirlpools or even
showers, i.e. systems with stagnant, warm water. The MIP of L. pneumophilia assists the
Legionella to transmigrate epithelial cells and extracellular matrix (ECM) of lung tissue and
to intrude alveolar macrophages. Both MIPs of L. pneumophila and B. pseudomallei show
peptidyl propyl cis/trans isomerase (PPIase) activity [1,2]. These surface proteins belonging
to the family of the FKBP binding protein form a stable complex with immunosuppressive
96 Poster

drugs FK506 or rapamycin which inhibit the enzymatic function of the mentioned MIPs [3].
Due to the immunosuppressive effects Legionnaires‘ disease and melioidosis can not be
treated with these drugs, thus novel inhibitors had to be applied. With the aid of
computational analysis, subsequent syntheses and biological screening molecules with a
pipecolin acid moiety (A, B) were established. These compounds showed an activity
against L. pneumophila with an IC50 of 9.0 µM for A and 32 µM for B [1]. Also, a satisfying
activity against B. pseudomallei was detected for both substances. In HSQC-NMR binding
studies an interaction of synthesized compounds A, B and the MIP of L. pneumophila could
be verified. Furthermore, an interaction was found with substance B and the MIP of
B. pseudomallei. To continue studies NMR binding experiments with compound A and the
MIP of B. pseudomallei will be carried out.
Acknowledgments: Financial support DFG (SFB 630)
References:
1. Juli, C. et al. J. Med Chem. 2011, 54(1): 277–283.
2. Norville, I.H. et al. Infection and Immunity 2011, 79(11): 4299–4307.
3. Fischer, G. et al. Mol. Microbiol. 1992, 6(10): 1375–1383.
015
Discovery of novel protease inhibitors from the Caribbean sponge Plakortis
halichondrioides.
Oli S, 1 Tabares P, 2 Hentschel-Humeida U, 2 Schirmeister T3 1 Institut für Pharmazie und LMC, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
2 Julius-von-Sachs Institut, Universität Würzburg, Julius-von Sachs Platz 3, 97082 Würzburg,
Germany
3 Institut für Pharmazie und Biochemie, Johannes Gutenberg-Universität Mainz, Staudinger Weg 5,
55128 Mainz, Germany
Marine sponges of the family Plakinadae are known to be rich sources of structurally
unique and biologically active metabolites.[1] In this study, bioactive compounds with
protease inhibiting properties were discovered from the sponge Plakortis halichondrioides
following bioactivity-guided fractionation. The structures of the endoperoxides plakortide E
and plakortin were identified by means of MS and NMR spectroscopy. Plakortide E showed
inhibitory activities against papain-like proteases such as cathepsins B and L, as well as
against SARS-CoV PLpro and rhodesain. The ester plakortin was identified due to its activity
against the parasite protease rhodesain from Trypanosoma brucei rhodesiense.
Procedures of isolation, spectroscopic data, enzyme inhibition data as well as in vitro
results are presented.
Plakortide E
Plakortin
References:
[1] Noriko Sata. et al.: J. Nat. Prod. 2005, 68, 1400-1403.
016
New therapy against HIV: Syntheses of inhibitors of the vif - elongin-C
interaction
Menrath, C. 1; Matz, F. 2; Welker A. 2; Nowotny, B. 3; Bodem, J. 3; Rethwilm, A. 3, Sotriffer, C. 2,
Schirmeister, T. 1
1 Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55099 Mainz,
Germany
2 Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074
Würzburg, Germany
3 Institute of Virology and Immunology, University of Würzburg, Versbacher Strasse 7, D-97078
Würzburg, Germany
AIDS (acquired immunodeficiency syndrome) is a disease caused by HIV-1 and HIV-2
(human immunodeficiency virus). In the end of 2010, about 33 millions of people were
infected with HIV. 1
The today’s most successful therapy is HAART (highly active anti-retroviral therapy) using
combinations of drugs inhibiting essential viral enzymes thus blocking virus replication.
In the human immune system, the proteins of the APOBEC3 family play essential roles in
the defence against retroviral infections, in case of HIV especially APOBEC3G and 3F are
involved. These proteins possess cytidine deaminase acivity thus inducing various
mutations in the viral nucleic acid. Such hypermutation ultimately destroys the coding and
replicative capacity of the virus, resulting in nonviable virions. However, the viral protein vif
(viral infectivity factor) interacts with APOBEC3G and triggers the ubiquitination and
degradation of APOBEC3G via the proteasomal pathway, thus overcoming its native
antiviral activity. APOBEC3G and vif complex formation also involves binding of the human
protein elongin C.
On the basis of the crystal structure of elongin C, we identified new inhibitors of the vif-eloC
interaction by means of virtual screening and docking studies. We present syntheses and
test results of these compounds exhibiting a new mechanism of action against retroviral
infections. 2 3 4 5 6
References:
1. Epidemie Report Update (UNAIDS 2010).
2. K. N. Bishop,R. K. Holmes, A. M. Sheehy, M. H. Malim Science 2004, 305 (5684), 645.
3. S. L. Sawyer, M. Emerman, H. S. Malik, PLoS. Biol. 2004, Sep; 2(9), E275.
4. A. M. Sheehy, N. C. Gaddis, J. D. Choi, M. H. Malim, Nature 2002, 418 (6898), 646 – 50.
5. R. S. Harris, M. T. Liddament, Nat. Rev. Immunol. 2004, 4, 868 – 877.
6. M. J. Wichroski, K. Ichiyama, T. M. Rana, J. Biol. Chem. 2005, 280 (9), 8387 – 8396.
017
Synthesis of 1-benzoxepines as NR2B selective NMDA receptor antagonists
Wendling, F. 1; Wünsch, B. 1
1 Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms Universität
Münster, Hittorfstraße 58-62, 48149 Münster, Germany
While the NMDA receptor is responsible for a number of physiological effects, including
neuronal development, overactivation of NMDA receptors has been associated with
pathological conditions such as Alzheimer’s Disease, Parkinson’s Disease and depression.
[1]
Functional NMDA receptors are composed of four subunits containing at least one NR1(ah)
subunit assembled with NR2 (A-D) and/ or NR3 (A, B). [2] The NR2 subunits vary in their
distribution throughout the CNS. As the NR2B subunit is less distributed in the cerebellum
[3], addressing it selectively has been reported to result in decreased cognitive and motoric
side effects. [4]
Therefore design, synthesis and structure-affinity relationships of NR2B selective
antagonists are of great interest as these compounds represent possible therapeutic agents
for various neurological conditions.
Using Ro-25,6981 1 as a lead compound, which binds with high affinity (Ki = 6 nM) at the
NR2B subunit [5], we develop structurally related molecules with various substituents and
stereochemistry.
On this poster we present a Dieckmann cyclisation as key step for the synthesis of
benzoxepines 2 (X=O), which we developed as conformationally restricted Ro-25,6981
analogues.
1 2
References:
1. Dingledine, R., et al.: Pharmacol. Rev. 1999, 51(1): 7-62.
2. Chatterton, J.E., et al.: Nature 2002, 415(6873): 793-798.
3. Gogas, K.: Curr. Opin. Pharmacol. 2006, 6 (1):68-74.
4.Nikam, S.S., Meltzer, N.T.: Curr. Pharm. Design 2002, 8(10): 845-855.
5. Pinard, E., et al.: Bioorgan. Med. Chem. Lett. 2001, 11(16):2173-2176.
018
Identification of the bioactive principles underlying the vascular barrierprotecting
action of the Crataegus extract WS ® 1442
Fuchs, S. 1; Willer, E. A. 1; Hartung, E. 1; Vollmar, A. M. 1; Potterat, O. 2; Hamburger M. 2; Fürst,
R. 1
1 Department of Pharmacy, Center for Drug Research, Pharmaceutical Biology, University of
Munich, Butenandtstr. 5-13, 81377 Munich, Germany
2 Department of Pharmaceutical Sciences, Pharmaceutical Biology, University of Basel,
Klingelbergstr. 50, 4056 Basel, Switzerland
We have recently discovered that the hawthorn (Crataegus spp.) special extract WS ® 1442,
widely used for the treatment of mild forms of heart failure, effectively prevents
inflammation-induced endothelial hyperpermeability in vitro and in vivo [1]. WS ® 1442
affects key systems of endothelial barrier regulation, such as the F-actin cytoskeleton,
adherens junctions (VE-cadherin), the contractile machinery (myosin light chain), and the
intracellular calcium concentration [1]. The extract targets two major endothelial signaling
cascades: it blocks the barrier-destabilizing Ca2+/PKC/RhoA pathway and activates the
barrier-protecting cAMP/Rap1/Rac1 signaling [1]. In the search for the constituents
responsible for these effects, 4 fractions (A-D) of WS ® 1442 were prepared (Sephadex LH-
20 column chromatography, kindly provided by Dr. Willmar Schwabe GmbH & Co. KG,
Karlsruhe, Germany). We could show that the two signaling cascades affected by the
extract are separately targeted by two different fractions: only fraction B, containing small
phenolic compounds, flavonoids, and lipophilic constituents, blocked the calcium signaling
and only fraction C, consisting of oligomeric proanthocyanidins (OPCs), activated the cAMP
pathway in endothelial cells.
Intention of the present study was to identify the bioactive compounds by bioguided
fractionation. Regarding fraction B, 9 subfractions (preparative HPLC) were analyzed for
their influence on a thrombin-evoked rise of the intracellular calcium concentration in
primary human endothelial cells (ratiometric, microscopic analysis using Fura-2). Only one
subfraction blocked the thrombin effect and was thus judged as active. A preliminary HPLC-
DAD-MS study revealed that this fraction contains lipophilic, UV inactive substances,
suggesting that phenolic compounds and flavonoids are not responsible for the observed
action on calcium levels. A detailed phytochemical analysis of this subfraction (isolation,
structure elucidation) is in progress.
Fraction C was divided into 6 subfractions (preparative HPLC). The microscopical analysis
of endothelial cortactin phosphorylation, a downstream event of the cAMP-triggered
signaling evoked by WS ® 1442, served as read-out parameter for bioactivity. 2 fractions
were highly active, 3 exhibited a slight activity, and one was completely inactive. Further
separation (Sephadex LH-20 column chromatography) of the two highly active fractions
resulted in 10 subfractions, of which 8 were active. Due to this very broad activity
distribution and the well-known difficulties in precisely separating OPCs, we decided to test
isolated OPCs of WS ® 1442. Procyanidin B2 (an OPC dimer) and procyanidin C1 (an OPC
trimer) were confirmed to trigger endothelial cAMP signaling (cAMP ELISA, phosphocortactin
Western blots and microscopical analysis).
In summary, we could provide first insights into the bioactive principles that underlie the
polypharmacological action of WS ® 1442. The inhibition of calcium signaling seems to be
evoked by lipophilic, non-phenolic compounds, which we currently aim to identify.
Moreover, we could show the OPCs procyanidin B2 and C1 to be responsible for the
activation of endothelial cAMP signaling by WS ® 1442.
Poster 97

Acknowledgments: This work is supported by the German Research Foundation (DFG, FU691/7-1). WS ®
1442 was kindly provided by Dr. Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany.
References:
1. Bubik, M. F. et al.: J. Mol. Cell. Cardiol. 2011, 52(1): 196-205.
019
HPTLC of purified hyaluronan oligosaccharides: reagent-free derivatization
and densitometric quantification on amino-modified silica, TLC–ESI–Q-TOF-MS
coupling on normal phase
Rothenhöfer, M. 1; Scherübl, R. 2; Bernhardt, G. 1; Heilmann, J. 2; Buschauer, A. 1
1 Lehrstuhl für Pharmazeutische/Medizinische Chemie II, Universität Regensburg, 93040
Regensburg, Germany
2 Lehrstuhl für Pharmazeutische Biologie, Universität Regensburg, 93040 Regensburg, Germany
By the development of powerful high performance thin layer chromatography (HPTLC)
techniques, planar chromatography has become a cost-effective and convenient alternative
to high performance liquid chromatography (HPLC). It allows parallel analysis of multiple
samples and, owing to a multitude of derivatization possibilities, attractive options for the
detection of unfavorable analytes. For instance, oligosaccharides derived from enzymatic
cleavage of hyaluronan by hyaluronidases pose a particular analytical challenge.
Nevertheless, there is a strong need for purified hyaluronan fragments as pharmacological
tools to shed light on the physiological and pathophysiological role of hyaluronidases,
hyaluronan, and its degradation products.
Therefore, we developed and validated an efficient HPTLC method for qualitative and
quantitative analysis of small saturated hyaluronan oligosaccharides consisting of 2 to 4
hyalobiuronic acid moieties. Uniform and reagent-free in situ derivatization is enabled by
the use of amino-modified silica as stationary phase. Subsequent quantification by
densitometric scans in reflectance mode revealed very low limits of detection of 7 to 19
pmol per band, depending on the oligosaccharide.
In view of the applicability of HPTLC for the quality control of hyaluronan oligosaccharides,
proof of principle was provided for unambiguous identification of the substances by direct
ESI–Q-TOF-MS coupling. For this purpose standard silica TLC plates were used and high
resolution mass spectra were obtained for all three analytes.
020
Effects of STW 5 and STW 6 on rat ileal and colonic preparations: A
comparative study
Nieber, K1, Voß, U. 1, Michael, S. 2, Kelber, O. 3, H. Abdel-Abziz3 Weiser, D. 3
1Institut für Pharmazie, Universität Leipzig, 04013 Leipzig,
2Löwen-Apotheke-Waldheim, 04736 Waldheim,
3Wissenschaftliche Abteilung, Steigerwald Arzneimittelwerk GmbH, 64295 Darmstadt
STW 5 (Iberogast ) is a fixed combination of nine plant extracts with Iberis amara (STW 6)
as one of its components. It is successfully used for treatment of functional dyspepsia and
irritable bowel syndrome (IBS). Because clinical data suggest inflammatory involvement in
the etiology of IBS the influence of STW 5 and STW 6 on tone and acetylcholine (ACh)induced
contractions of intact and inflamed intestinal preparations was examined. We used
1 - 1.5 cm long ileum and colon preparations of male Wistar rats to analyze region specific
differences. The inflammation was induced by intraluminal instillation of 2,4,6trinitrobenzene
sulfonic acid (TNBS, 10 mM, 30 min). Incubation with STW 5 (64 – 512
µg/ml, 30 min) concentration dependently reduced the tone and decreased ACh-induced
contractions of untreated ileal and colonic preparations. STW 6 in equivalent concentrations
(3 – 24.1 µg/ml) neither affected the tone nor the contractility. TNBS-induced inflammation
was accompanied by a significant reduction of ACh-induced contractions and
morphological disturbances. Co-incubation of TNBS with STW 5 (512 µg/ml) or STW 6
(24.1 µg/ml) partially normalized the TNBS-induced attenuation of ACh-induced
contractions and the morphological damage in ileum preparations, whereas in inflamed
colon segments only the co-incubation of TNBS with STW 6 in a high concentration (24.1
µg/ml) revealed similar effects.
In conclusion, STW 5 influenced ACh-induced contractions and tone in untreated ileal and
colonic preparations, whereas STW 6 did not contribute to these effects. In TNBS-inflamed
ileum preparations STW 5 as well as STW 6 normalized morphological and contractile
disturbances, while in colon preparations STW 6 but not STW 5 was effective. Our study
confirms region specific effects of STW 5 and its fresh plant component STW 6.
021
The PI3/Akt signaling pathway is involved in endothelium-dependent relaxation
by Salix spp. in porcine coronary arteries
Kaufeld, A. M.; Pertz, H. H.; Kolodziej, H.
Institut für Pharmazie, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin
Numerous lines of evidence have shown cardioprotective and cardiotonic effects of
polyphenols [1]. Preliminary studies demonstrated that 2,3-cis procyanidins of Nelia meyeri
Schwant. induced an endothelium-dependent relaxation of isolated porcine coronary
arteries [2]. The aim of the present study was to gain insight into the molecular mechanism
by which 2,3-trans procyanidins of willow bark elicit blood vessel relaxation.
The willow bark procyanidin sample comprising a mixture of dimeric to hexameric 2,3-trans
flavan-3-ols [3] induced a concentration-dependent relaxation in pre-contracted
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
transport chain). Furthermore, the extract-induced relaxation was abolished by mechanical
removal of the endothelium, addition of wortmannin (inhibitor of phosphoinositide 3-kinase;
PI3K), as well as by manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; cell
permeable mimetic of superoxide dismutase). Similarly, the response was completely
inhibited by the combination of charybdotoxin, apamin (Ca2+-activated K + channel blockers)
and L-NAME. This showed that the endothelium-derived hyperpolarizing factor (EDHF) and
NO (EDRF) were involved in the relaxant response to Salix spp.. In contrast, the addition of
ICI 182,780 (estrogen receptor antagonist) or pertussis toxin (inhibitor of Gi proteins) did not
affect the relaxation. These findings indicated that willow bark procyanidins triggered the
activation of the PI3/Akt pathway. Western Blot analysis using human umbilical vein
endothelial cells (HUVEC) demonstrated that Salix procyanidins concentration-dependently
induced Akt and eNOS phosphorylation. The addition of wortmannin, MnTMPyP or
rotenone significantly inhibited key phosphorylation sites.
In conclusion, the present data confirm procyanidin-induced production of potent
vasoprotective factors including NO and EDHF. The results support the view that
polyphenols have beneficial effect on endothelial cell functions of blood vessels.
References:
1. Rasmussen, S.E. et al.: Mol. Nutr. Food Res. 2005, 49: 159–174.
2. Kaufeld, A.M., Pertz, H.H., Kolodziej, H.: Planta Med. 2011, 77: 1403.
3. Kolodziej, H.: Phytochemistry 1989, 29: 955–960.
022
Development of NR2B-selective NMDA receptor antagonists
Lütnant I. 1; Wünsch B. 1
1 Institut für Pharmazeutische und Medizinsche Chemie der Westfälischen Wilhelms-Universität
Münster, Hittorfstraße 58-62, D-48149 Münster, Germany
The N-Methyl-D-Aspartate (NMDA) receptor is an excitatoric amino acid receptor belonging
to the class of ionotropic glutamate receptors. The receptor is a ligand-gated ion channel
highly permeable for Ca2+-ions consisting of four subunits. Seven subunits named NR1,
NR2 A-D and NR3 A, B have been identified. A functional NMDA receptor contains at least
one NR1-subunit and one NR2-subunit. The receptor is distributed all over the brain, but its
composition differs depending on the region in the brain in which it is expressed. [1, 2]
The NMDA receptor shows unique properties such as a co-agonism of (S)-glutamate and
glycine, permeability for Na +-, K +-, Ca2+-ions and a voltage-dependent Mg2+-block. When the
channel is opened Ca2+-ions enter the cell and cause activation of several pathways
leading to different effects amongst others neuronal development. Overstimulation of the
NMDA receptor causes cell death and therefore “neurodegeneration”. [3, 4]
Ifenprodil (1) is a non-competitive NMDA receptor antagonist with a high affinity towards the
ifenprodil binding site on the NR2B subunit. Due to the fact that ifenprodil also binds to 5-
HT1A, 5-HT2 and σ- receptor, it causes many adverse effects.
To reduce the adverse side effects on the one hand and to improve the neuroprotective
effects on the other hand, compounds with increased selectivity have been developed
derived from the lead structure ifenprodil (1). [4]
NR2B-selective NMDA receptor antagonists were found to be of value because the NR2B
subunit is not expressed in the cerebellum. [5] Therefore NR2B-selective antagonists lead
to reduced motoric side effects. They seem to be useful compounds considering the
therapy of Parkinson´s and Alzheimer´s disease because they are capable to inhibit the
effects of an overstimulation of the NMDA receptor.
We develop a new group of potential NR2B-selective NMDA receptor antagonists with a
benzimidazolone bioisoster (2) of the phenol of ifenprodil (1).
References:
1. Dingledine, R. et al.: Pharmacol. Reviews 1999, 51(1).
2. Goebel, D.J., Poosch, M.S.: Mol. Brain Res. 1999 69: 164-170.
3. Stark, H, Graßmann, S., Reichert, U.: Pharm. In unsrere Zeit 2000, 29(3): 159-166.
4. Stark, H, Graßmann, S., Reichert, U.: Pharm. In unsrere Zeit 2000, 29(4): 228-236.
5. Monyer, H. et al.: Neuron. 1994, 12: 529-540.
023
Chain modified oxa-analogs of primaquine and some of its derivatives
Leven, M. 1; Held, J. 2; Tschan, S. 2; Delves, M. 3; Plouffe, D. 4; Mordmüller, B. 2; Kurz, T. 1
1 Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine Universität Düsseldorf,
Universitätsstr. 1, 40225 Düsseldorf, Germany
2 Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstr. 27, 72074 Tübingen,
Germany
3 Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7
2AZ, United Kingdom
4 Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San
Diego, CA 92121, United States of America
The 8-aminoquinoline antimalarial primaquine 1 affects multiple life cycle stages of
Plasmodium species. It exhibits activity against developing and dormant liver stages of P.
vivax and P. ovale. Thus it is used for the treatment of symptomatic patients with P. vivax or
P. ovale malaria and for the presumptive anti-relapse therapy in persons heavily exposed to
these plasmodia. As 1 also influences developing liver stages of P. falciparum, it is used as
a primary prophylactic agent. Due to its activity against gametocytes of all species of
human malaria, 1 is recommended as a transmission-blocking agent in endemic regions. Its
weak inhibitory activity against asexual blood stages of all Plasmodium species calls for a
combination therapy with an effective blood schizontocidal antimalarial agent. Serious
adverse effects including hemolysis in persons with glucose-6-phosphate dehydrogenase
(G6PD) deficiency and methemoglobinemia limit the therapeutic potential of 1. Its
application requires a previous test for G6PD activity. 1 is contraindicated in pregnancy and
in children under the age of four years [1,2].
In contrast to 1, clinical phase III drug candidate tafenoquin 2 demonstrates potent
inhibitory activity against blood stages of P. falciparum and P. vivax. Preliminary data
suggest that 2 is less toxic than 1, but hemolysis in persons with G6PD deficiency has also
been reported.
The (-)-enantiomer NPC1161B 3, currently in preclinical trials, affects effectively blood
stages of P. berghei in mice and is less hematotoxic in beagle dogs than 1. In addition, 3
inhibits effectively sporogony in vitro [3,4].
98 Poster

In order to improve activities and to reduce side effects numerous derivatives of 1 have
been prepared and biologically evaluated. An established concept for the modification of
lead structures is the bioisosteric replacement of methylene groups in alkyl chains by
bivalent atoms like oxygen and sulfur. In an attempt to optimize structural features of 1 we
now report on the synthesis and biological evaluation of side chain modified oxa-analogs of
1 and some of its derivatives.
References:
1. Hill, D. R. et al.: Am. J. Trop. Med. Hyg. 2006, 75(3): 402–415.
2. Vale, N., Moreira, R., Gomes, P.: Eur. J. Med. Chem. 2009, 44(12): 937-953.
3. Nannayakara, N. P. D. et al.: Antimicrob. Agents Chemother. 2008, 52(6): 2130-2137.
4. Delves, M. et al.: PLoS Med. 2012, 9(2): e1001169. doi:10.1371/journal.pmed.1001169.
024
Enantioselective Synthesis of Spirocyclic σ1 Ligands
Holl, K. 1; Wünsch, B. 1
1 Institut für Pharmazeutische und Medizinische Chemie, Westf. Wilhelms-Universität Münster,
Hittorfstr. 58-62, D-48149 Münster, Germany
σ receptors represent a unique class of receptors, consisting of two subtypes, σ1 and σ2
receptors. They are widely distributed in the human body, including the central nervous
system, as well as in many tissues of the periphery, e.g. heart, lung, liver, kidney [1]. The σ1
receptor has been associated with various neuronal diseases, such as schizophrenia,
depression and Alzheimer’s disease. In addition to that, an overexpression of σ1 receptors
in certain tumor cells was reported [2]. Therefore, high-affinity σ1 ligands have potential for
the therapy and diagnosis of CNS diseases and cancer. Fluorinated ligands can be used as
PET tracers [3].
A variety of spirocyclic ligands with high affinity for the σ1 receptor has been synthesized,
but so far, only racemic mixtures have been characterized. We report on the first
enantioselective synthesis of this kind of ligands, in order to detect relationships between
the absolute configuration and the σ1 affinity.
Starting from 2-bromostyrene (1), a series of spirocyclic σ1 ligands with a benzopyrane
structure was synthesized, using Sharpless asymmetric dihydroxylation as key step.
Furthermore, several variations in position 4 were carried out, including fluoro derivatives
Br
OH
N
R
OH
OH
1 2 3
Acknowledgments: We thank Deutsche Forschungsgesellschaft for financial support.
References:
1. Matsumoto, R. R. et al.: Eur. J. Pharmacol. 2003, 469(1-3): 1-12.
2. Maurice, T. Su, T.: Pharmacol. Ther. 2009, 124(2): 195-206.
3. van Waarde, A. et al.: Curr. Pharm. Des. 2010, 16(31): 3519-3537.
025
Comparative effect of proanthocyanidin enriched extracts on the in vitro
inhibition of bacterial and viral neuraminidase
Quosdorf, S. J.; Kolodziej, H.
Institut für Pharmazie, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin
Influenza infections are still a major threat to human health and emergence of resistance to
anti-influenza drugs is a cause for concern. Expanding the range of antiviral drugs that
effectively inhibit influenza viruses is therefore a matter of urgency. Neuraminidase (NA), a
key enzyme in the influenza life cycle and pathogenesis, has been established as a primary
drug target. Accordingly, NA inhibition assays represent a promising strategy for finding
new antiviral compounds. However, the substrates used in such functional assays are
recognized by both bacterial and viral NAs [1]. Due to limited comparative data, IC50 values
obtained from either setup must be regarded with caution. The current study was carried
out to examine the validity and significance of the origin of NAs in a functional NA inhibition
test system.
Using an established fluorescence based in vitro assay, the inhibitory potential of a series
of polyphenolic samples (Pelargonium sidoides DC, Salix spp, Nelia meyeri Schwant. and
Diospyros kaki L.) was measured for bacterial (Vibrio cholerae) and viral NA (Baculovirus).
Zanamivir (Relenza ®) and oseltamivir carboxylic acid served as positive controls. Selection
of the test substances is based on preliminary studies regarding prominent in vitro inhibition
of bacterial NA and differences in their proanthocyanidin patterns [2]. In this assay the
substrate 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (MUNANA) is cleaved by NA
to produce the fluorescent compound 4-methylumbelliferone (MU) [3]. The quantity of MU is
inversely proportional to the inhibitory activity of the tested sample.
The results demonstrate that the inhibitory potential of the proanthocyanidin enriched
extracts differ significantly and depend strongly on the origin of NA under the same
experimental conditions. All extracts were more effective against Vibrio cholerae NA (IC50
ca. 1 µg/mL – ca. 19 µg/mL) compared to Baculovirus NA (IC50 ca. 19 µg/mL – ca. 58
µg/mL). Interestingly and in contrast, zanamivir and oseltamivir carboxylic acid exhibited
much stronger inhibitory activities against the viral NA (IC50 ca. 4 µg/mL and ca. 1 µg/mL,
respectively) than against the bacterial NA (IC50 ca. 16 µg/mL and ca. 40 µg/mL,
respectively). Therefore it appears reasonable to select preferably viral NAs when testing
samples for anti-influenza potentials or searching for promising antiviral compounds. On the
OR
N
Bn
O
other hand, the test system should be adapted to a given pathogenic condition that may
well include bacterial NAs. The observed difference in susceptibility of the NAs is an
important parameter affecting generated results in the applied test system.
References:
1. Grienke, U., et al.: Nat. Prod. Rep. 2012, 29(1): 11–36.
2. Janecki, A., Kiderlen, A.F., Kolodziej, H.: Planta Med. 2009, 75(9): 989.
3. Potier, M., et al.: Analytical Biochemistry. 1979, 94(2): 287–296.
026
Spirocyclic isochromane-piperidines as new lead structures against
atherosclerosis
Strunz, A. K. 1; Wünsch, B. 1
1Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-Universität
Münster, Hittorfstraße 58-62, D-48149 Münster, Germany
The chemokine receptor 2 (CCR2) is a G-protein coupled receptor (GPCR) and part of the
chemokine system, a complex network consisting of soluble proteins and their receptors.
Chemokines and their receptors mediate effects on the cellular signalling pathway. The
CCR2 receptor is expressed on various immune cells and regulates cellular movement and
activation.
The interaction of the endogenous ligand MCP-1(monocyte chemoattractant protein 1) with
the CCR2 receptor plays a crucial role in several inflammatory processes [1]. In the chronic
inflammatory disease of atherosclerosis, this interaction leads to the mobilization of
activated monocytes into the subendothelial tissue [2].
On one hand CCR2 antagonists can stop the growth and progression of atherosclerotic
plaques by inhibition of early key steps, on the other hand [ 18F]-labeled CCR2 antagonists
can be applied in PET (positron emission tomography) studies to analyze atherosclerotic
plaques.
The synthesis of new CCR2 antagonists with a spirocyclic substructure will be presented on
this poster. The main building block is a spirocyclic system which consists of an
isochromane (A) with a piperidine ring (B), prepared by Oxa-Pictet-Spengler reaction [3].
The cyclization is performed with various phenylethanol derivatives. Different benzylamines
(D) lead to different test compounds. The introduction of an aromatic substituent R5 might
lead to compounds with increased CCR2 affinity [4]. The four variable building blocks (A-D)
can be combined in different ways.
References:
1. Gerard, C., Rollins, B. J.: Nat. Immunol. 2001, 2 (2): 108-115.
2. Galkina, E., Ley, K.: Annu. Rev. Immunol. 2009, 27: 165-197.
3. Larghi, E. L., Kaufman, T. S.: European Journal of Organic Chemistry 2011, 2011 (27): 5195-5231.
4. Butora, G. et al.: Bioorg. Med. Chem. Lett. 2006, 16 (18): 4715-4722.
027
Reverse α-aryl substituted β-thia-analogs of fosmidomycin as 1-deoxy-Dxylulose
5-phosphate reductoisomerase (Dxr) inhibitors
Lienau, C. 1, Kunfermann, A. 2, Hähn, S. 1, Konzuch, S. 1, Behrendt, C. T. 1, Illarionov, B. 3,
Held, J. 4, Mordmüller, B. 4, Fischer, M. 3, Groll, M. 2, Kurz, T. 1
1 Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine Universität Düsseldorf,
Universitätsstr. 1, 40225 Düsseldorf, Germany
2 Center for Integrated Protein Science Munich, Lehrstuhl für Biochemie, Technische Universität
München, Lichtenbergstr. 4, 85747 Garching, Germany
3 Hamburg School of Food Science, Universität Hamburg, Grindelallee 117, 20146 Hamburg,
Germany
4 Institut für Tropenmedizin, Eberhard Karls Universität Tübingen, Wilhelmstr. 27, 72074 Tübingen,
Germany
Inhibition of enzymes of the non-mevalonate pathway (MEP pathway) is a promising
concept for the development of novel anti-infective drugs [1,2]. The non-mevalonate
pathway of isoprenoid biosynthesis is the only source of the isoprenoid precursors
isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAP) in several pathogenic
bacteria and apicomplexan protozoa including the causative agents of malaria, Plasmodium
spp.. The MEP pathway is absent in mammalians but essential for various pathogenic
bacteria and Plasmodium spp.. A validated target for the development of novel
antimalarials within the MEP pathway is the 1-deoxy-D-xylulose 5-phosphate
reductoisomerase (Dxr) [3,4]. Reverse α-aryl substituted carba- and oxa-analogs (1,2) of
the natural product fosmidomycin are highly active Dxr inhibitors and show potent
antiplasmodial activity [3,4]. Further structural modifications of analogs (1,2) led to
previously unreported reverse α-aryl substituted β-thia-isosters of fosmidomycin.
References:
1. Jomaa, H. et al.: Science 1999, 285: 1573-1576.
2. Rohmer, M. et al.: Curr. Opin. Investig. Drugs 2004, 5: 154-162.
3. Behrendt, C. T. et al.: J. Med. Chem. 2011, 54: 6796-6802.
Poster 99

4. Behrendt, C. T. et al.: ChemMedChem 2010, 5(10):1673-1676.
028
Influence of the indirubin derivative 6BIO on proliferation and metastasis of
breast cancer cells
Braig S. 1, Kressirer C. 1, Bischoff F. 1, Meijer L. 2, Zahler S. 1, Vollmar A.M. 1
1Department of Pharmacy, Center for Drug Research, Butenandtstr.5-13, 81377 Munich, Germany
2CNRS, Station Biologique, Roscoff, France
Initially, indirubins were discovered as the active component of a well known traditional
Chinese medicine. Since then, it was shown that natural indirubins and also its synthetic,
cell permeable derivative 6BIO (6-bromo-indirubin-3-oxime) display a remarkable potential
to inhibit glycogen synthase kinase 3 (GSK-3) and cyclin-dependent kinases (CDKs) by
interacting with the ATP-binding pocket of these kinases.
By an inverse virtual screening approach phosphoinositide-dependent kinase 1 (PDK1) was
proposed by us as a yet unknown target of 6BIO. Western blot analysis and confocal
microscopy revealed that treatment of breast cancer cells with 6BIO resulted in a reduced
phosphorylation of AKT, an important downstream target of PDK1. Since about 70% of all
breast cancer carcinomas express moderate to high levels of activated PDK1, we
investigated the impact of 6BIO treatment on different breast cancer cell lines by analyzing
the anti-proliferative and cytotoxic effects as well as the influence on reduction of metastatic
capabilities of these cells.
As determined by crystal violet staining and xCELLigence experiments, incubation of
SkBr3, MDA-MB-231, MCF7 and AKT overexpressing SkBr3 breast cancer cell lines with
increasing concentrations of 6BIO led to a dose-dependent inhibition of proliferation. In
addition, 6BIO treated breast cancer cells showed a strongly diminished long-term
clonogenic survival rate.
Furthermore, Nicoletti assays and Hoechst staining indicated that stimulation of breast
cancer cell lines with high doses of 6BIO led to an induction of apoptosis.
Cells treated with sub-toxic doses of 6BIO displayed a strongly reduced attachment
capacity, as shown by adhesion assays on fibronectin and xCELLigence experiments.
Scratch assays as well as chemotaxis assays revealed a significantly diminished migratory
potential of 6BIO treated breast cancer cells compared to control cells. Furthermore, 6BIO
significantly disrupted the invasion capacity of the cells. The impact of 6BIO treatment on
the migration and invasion inhibition might be due to altered localization of the well-known
migration associated molecules FAK and Rac1 within the cells, as shown by confocal
microscopy.
In summary, we were able to show that 6BIO inhibits PDK1 signal transduction in different
breast cancer cell lines and reduces proliferation, clonogenic survival, attachment,
migration and invasion of the cells. Thus, the indirubin derivative 6BIO might serve as a
highly promising potential therapeutic agent for metastatic breast cancer cells.
029
Discovery of the first antagonists of PqsR to interrupt Pseudomonas
aeruginosa cell-to-cell communication
Steinbach, A. 1; Lu C1; Kirsch, B. 1; Zimmer, C. 1; de Jong, J.C. 1; Henn, C. 1; Maurer, C.K. 1;
Müsken, M. 2; Häussler, S. 2 3; Hartmann, R.W. 1
1 Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123 Saarbrücken,
Germany
2 Twincore, Department of Pathophysiology of Bacterial Biofilms, Feodor-Lynen-Str. 7, 30625
Hannover, Germany
3 Helmholtz Centre for Infection Research, Chronic Pseudomonas Infection Research Group,
Inhoffenstr. 7, 38124 Braunschweig, Germany
P. aeruginosa coordinates group behaviors such as virulence factor expression and biofilm
formation via a cell-to-cell communication system known as quorum sensing (QS). The
transcription of target genes are regulated via a pqs QS system that functions via the signal
molecules PQS and its precursor HHQ that interact with their receptor PqsR. Targeting
PqsR to develop quorum sensing inhibitors is considered as novel strategy to attenuate P.
aeruginosa pathogenicity [1]. In order to discover PqsR antagonists, a ligand-based drug
design approach was followed to synthesize a set of HHQ and PQS derived compounds.
HHQ (R = H) R 1 = ED or EW groups
PQS (R = OH) R 2 = alkyl or alkylether
Agonistic or antagonistic properties were determined in a heterologous β-galactosidase
reporter gene assay in E. coli to examine effects of side chain modifications and
substitutions at the benzene moiety. An n-heptyl chain in 2-position was found to be
optimal. Most importantly, the introduction of strong electron withdrawing functions like CN,
NO2 or CF3 in 6-position of HHQ led to the discovery of the first antagonists (IC50 values of
259 nM, 51 nM and 54 nM), while HHQ analogues with the same substituents in 7- or 8position
or other substituents and all PQS derivatives were moderate to weak agonists.
Direct evidence for the binding of a selected antagonist (6-CN HHQ) to PqsR was provided
by surface plasmon resonance (SPR) biosensor experiments (KD=57 nM). Moreover, 6-CF3
HHQ reduced pyocyanin production by 74 % (3 µM) in the target organism P. aeruginosa
[2]. The aqueous solubility of the antagonists could be improved by the introduction of O
into the alkyl side chain or CONH2 into 3-position.
The first antagonists of PqsR are highly valuable scientific tools for in-depth study of
protein-ligand interactions and provide a promising starting point for the development of a
new anti-infective strategy.
References:
Deziel E et al., Mol. Microbiol. 2005, 55: 998–1014.
Lu, C. et al.; Chem. Biol. 2012, 19: 381–390.
030
Diarylpyrrolizines as Inhibitors of Microsomal Prostaglandin E2 Synthase-1
(mPGES-1) or as Dual Inhibitors of mPGES-1 and 5-Lipoxygenase (5-LO)
Keck, P. R. W. E. F. 1; Liedtke, A. J. 2; Koeberle, A. 3; Werz, O. 3; Schollmeyer, D. 4; Laufer, S.
A. 1
1 Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, University of
Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany
2 Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4,
53121 Bonn, Germany
3 Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, University of
Jena, Philosophenweg 14, 07743 Jena, Germany
4 Institute Organic Chemistry, University Mainz, Duesbergweg 10-14, 55099 Mainz, Germany
Prostaglandin E2 (PGE2) is one of the most abundant and powerful prostanoids with diverse
biological activities. As a key cyclic lipid mediator derived by the cyclooxygenase (COX)
from arachidonic acid (AA), it is involved in the development and perpetuation of
inflammation seen in diseases such as rheumatoid arthritis (RA). PGE2 has been implicated
in the development of peripheral and central sensitization during nociceptive processing
(e.g., hyperalgesia, allodynia) and in tumorigenesis[1]. Nonsteroidal anti-inflammatory
drugs (NSAIDs), including selective COX-2 inhibitors, have analgesic, antipyretic and antiinflammatory
properties. The main mechanism of action of NSAIDs is the inhibition of the
COX isoenzymes. However, inhibition of the COX pathway by NSAIDs also reduces gastro
protective PG synthesis, resulting in the well-known gastrointestinal side effects. In
addition, the resulting increase in leukotriene (LT) biosynthesis due to shunting of AA to the
5-lipoxygenase (5LO) pathway activated by the 5-lipoxygenase activating protein (FLAP)
causes additional gastrointestinal injury[2,3].
A dual COX/FLAP inhibition by Licofelone (ML3000) is an approach to minimize the side
effects[2]. Licofelone also shows an inhibitory activity on the mPGES-1[4]. Going
downstream, we synthesized and evaluated inhibitors for the mPGES-1, based on the
diarylpyrrolizine scaffold of Licofelone while conserving the activity on the FLAP respective
5-LO[1].
Additionally, two crystal structure have been obtained to determine the angle of the aryl
substituents[5,6].
References:
1. Liedtke, A. J. et al. J. Med. Chem. 2009, 52(15): 4968-4972.
2. Laufer, S. A. et al. J. Med. Chem. 1994, 37(12): 1894-1897.
3. Laufer, S. A.; Gay, S.; Brune, K.: Inflammation and Rheumatic Diseases (Thieme) 2003.
4. Koeberle, A. et al. J. Pharmacol. Exp. Ther. 2008. 326(3): 975-982.
5. Keck, P. R. W. E. F.; Schollmeyer, D.; Laufer, S. Acta. Cryst. E 2011, E67(9): 2292.
6. Keck, P. R. W. E. F.; Schollmeyer, D.; Laufer, S. Acta. Cryst. E 2011, E67(9): 2417.
031
The actin cytoskeleton as potential antitumor target: Chondramide and its
effects on breast cancer cells
Förster F1; von Schwarzenberg K; Müller R2; Vollmar A
1 Department of Pharmacy - Center for Drug Research, Pharmaceutical Biology, LMU Munich,
Butenandtstaße 5-13, 81377, München
2 Institut für Pharmazeutische Biotechnologie, Universität des Saarlandes, Postfach 15 11 50,
66041, Saarbrücken”
The actin cytoskeleton is a crucial component to maintain cellular homeostasis. It is
important in processes like muscle contraction, trafficking of cellular compartments, mitosis,
invasion and migration. Thus, actin-binding compounds like Chondramide A (Chon A), a
cyclic depsipeptide of myxobacterial origin could be developed as potential anticancer
therapeutics. Aim of this study was to characterize antitumoral effects of Chon A in
mammary cancer cells in vitro.
In fact we could show that Chon A inhibits proliferation of two breast tumor cell lines MCF-7
and MDA-MB-231 in a low nanomolar range. The long term survival of these cell lines was
impaired by Chon A as the clonogenic survival was also decreased by about 60 % (MDA-
MB-231) and 80 % (MCF-7), respectively. To proof if the observed effects are due to actin
overpolymerization, the Triton-X 100 soluble fraction was analyzed for its actin content and
showed a time (24h – 72h) and dose dependent decrease, which suggested that Chon A
overpolymerized actin to its filamentous form that is not soluble in the Triton-X-100 fraction.
Clarifying the mechanisms underlying the antiproliferative effects of Chon A, we examined
whether Chon A induces apoptosis and/or premature cellular senescence. Annexin V/PI
costaining revealed a concentration dependent apoptotic cell death induced by 500 nM
Chon A in both cell lines. Cleavage of PARP, a known substrate of caspase-3, detected by
western blotting, supported the induction of apoptosis by Chon A, too. The induction of
cellular senescence was cell line specific, as MCF-7 showed an increased β-galactosidase
activity, compared to MDA-MB-231 which did not display this hallmark of senescence. The
cdk-inhibitor p21, which is important for senescence induction, was also increased with
Chon A 500 nM treatment in MCF-7 cells. These results suggest, that the inhibition of
growth in MDA-MB-231 is only due to apoptosis induction, whereas in MCF-7 cells both
processes apoptosis and senescence are involved. Furthermore, phosphorylation of
histone γH2A.X, an established sign for DNA damage, treated with Chon A was observed
in both cell lines. To this end, Chon A induces pronounced cell cycle arrest in G2-phase (40
% versus 20 % in untreated cells).
100 Poster

Our investigations reveal that Chon A is a potent inhibitor of breast cancer cell proliferation
via induction of apoptosis and premature cellular senescence. Induction of senescence has
not yet been reported for actin – binding drugs. Thus, further experiments will focus on the
mechanism underlying senescence induction. One major tool will be PCR and micro-array
technologies to possibly find new and valuable players and targets in senescence and
growth arrest signaling.
Acknowledgments: Supported by the DFG: FOR 1406 Vo 376/15-1
032
Phenylamino-substituted 5,11-dihydrodibenzo[a,d]cyclohepten-10-ones and
11H-dibenzo[b,f]-oxepin-10-ones as novel p38 MAP kinase inhibitors
Dorn, A. 1; Martz, K. E. 1; Laufer, S. A. 1
1 Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-Universität, Auf der
Morgenstelle 8, 72076 Tübingen
The p38 MAP kinase is a key player in signalling pathways regulating the biosynthesis of
pro-inflammatory cytokines like TNF-α and IL-1β. Small molecule p38 inhibitors suppress
the production of these cytokines making p38 a promising drug target for novel antiinflammatory
drugs.
We recently reported a novel series of dibenzepinone inhibitors belonging to the class of so
called linear binders [1]. Upon binding of the dibenzepinone inhibitor, the p38 MAP kinase
undergoes a Gly110 flip. The glycine flip is a small conformational rearrangement in the
hinge region of the p38 MAP kinase induced by the inhibitor and it provides selectivity for
p38 over other kinases with less flexible, non-glycine residues at this position. Hence the
carbonyl functionality of the dibenzepinone inhibitor is essential for the inhibitory activity
and selectivity as it forms two hydrogen bonds towards Met109 and Gly110 in the hinge
region.
In this study, we report the design, synthesis, and SAR of novel N-substituted 11Hdibenzo[b,f]oxepin-10-ones
and 5,11-dihydro-dibenzo[a,d]cyclohepten-10-ones as p38α
inhibitors. The main aim was to conserve the key interaction: the bidentate hydrogen bond
of the carbonyl oxygen of the inhibitor to the backbone NH of Met109 and the backbone NH
of Gly110. Docking studies predicted alternative positions for the carbonyl oxygen within
the scaffold of the inhibitor. Within these inhibitor structures, the carbonyl functionality was
moved on the bridge side and the linker atoms X and Y were varied to obtain distinctive
molecular geometries and additional interaction opportunities.
Initial investigations of the inhibitory activities and structure-activity-relationships of
phenylamino-substituted dibenzo[b,f]oxepin-10(11H)-one, 5,11-dihydro-(10H)-dibenzo[a,d]cyclohepten-10-one
and dibenzo[b,f][1,4] oxazepin-11(10H)-one inhibitors for p38 MAP
kinase were accomplished. The promising structural variations suggested by our docking
experiments did not result in any novel compounds as active as the lead compound. While
our initial hypothesis that substitution of a tricyclic scaffold would result in a favorable
position for the carbonyl functionality was not supported by our data, we did identify some
structural determinants that may be useful for the development of future p38 MAPK
inhibitors.
References:
1. Laufer, S. A. et al.: J. Med. Chem. 2006, 49: 7912-7915.
033
Long-Range Intramolecular S → N Acyl Migration: A Study of the Formation of
Native Peptide Analogues via 8-, 11-, 13-, 14-, 15-, and 16-Membered Cyclic
Transition States
Hansen, F. K. 1 2; Ha, K. 2; Chahar, M. 2; Monbaliu, J.-C. M. 2 3; Oliferenko, A. A. 2; Katritzky, A.
R. 2
1 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University of Düsseldorf,
Universitätsstr. 1, 40225 Düsseldorf, Germany. Email: finn.hansen@uni-duesseldorf.de
2 Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville,
FL 32611-7200, USA.
3 Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience
Engineering, Ghent University, B-9000 Ghent, Belgium.
Native chemical ligation (NCL) is the most widely used chemoselective ligation technique
based on a rearrangement concept nowadays. NCL involves the covalent condensation of
a peptide-thioester with N-terminal cystein peptide, followed by S to N acyl shift to form a
native amide linkage.[1] In recent years, various chemical ligation methodologies such as
sugar-assisted ligation, nucleotide-based ligation and chemical ligation using click
chemistry have been developed. The classical NCL method is limited to peptides
possessing an N-terminal cysteine residue. Unfortunately, cysteine is a relatively rare
amino acid (1.3% average content) and is not always available in a terminal position.
Moreover, some amino acid-cysteine bonds such as Pro-Cys, Asp-Cys, Glu-Cys and Lys-
Cys can be difficult to access by chemical ligation.[2]
To overcome this requirement of a specifically placed cysteine residue one approach is the
use of thiol ligation auxiliaries, but removable cysteine mimics can sterically hinder ligation
and difficulties can arise at the stage of auxiliary removal.[3]
A long-range S to N acyl migration methodology would avoid the requirement of a Nterminal
cysteine residue. We have now studied the feasibility of chemical ligation of Sacylated
cysteine peptides via 8-, 11-, 13-, 14-, 15-, and 16-membered cyclic transition
states.[4,5] Our microwave-assisted methodology allows the synthesis of native peptides
from non-terminal cysteine residues without the need of ligation auxiliaries.[4,5]
Computational studies provide mechanistic evidence for the ligation process.[5] The impact
of pH on the long-range S to N acyl migration is exemplified using a S-acyl tetrapeptide.[5]
Acknowledgments: The authors gratefully acknowledge the Kenan foundation for financial support.
References:
1. Dawson, P. E. et al.: Science 1994, 266(5186): 776-779.
2. Haase, C. and Seitz, O.: Eur. J. Org. Chem., 2009, (13): 2096-2101.
3. Marinzi, C. et al.: Bioorg. Med. Chem. 2001, 9(9): 2323-2328.
4. Hansen, F. K. et al.: Org. Biomol. Chem. 2011, 9(20): 7162-7167.
5. Ha, K. et al.: J. Org. Chem., 2012, 77(6): 2637-2648.
034
Synthesis and pharmacological evaluation of thiazole-featured pirinixic acid
derivatives acting as dual 5-lipoxygenase and microsomal prostaglandin E2
synthase-1 inhibitors
Hanke, T. 1; Popella, S.-D. 2; Liening, S. 2; Dehm, F. 2; Lämmerhofer, M. 3; Werz, O. 2;
Schubert-Zsilavecz, M. 1
1Goethe University of Frankfurt am Main, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany
2Friedrich Schiller University of Jena, Philosophenweg 14, 07743 Jena, Germany
3Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany
Imbalances in the lipid signaling network contribute to the pathogenesis of a large number
of human diseases. The metabolic fate of arachidonic acid (AA) plays a crucial role within
this network and is associated with pathophysiological conditions such as inflammation,
analgesia, asthma and cancer.
The metabolic pathway of AA can be divided into two different ways: The formation of
prostaglandins (PGs) catalyzed by cyclooxygenases (COXs) and the biosynthesis of
leukotrienes (LTs) initialized by 5-lipoxygenase (5-LO). Non steroidal anti-inflammatory
drugs (NSAIDs) and COX-2 selective inhibitors (coxibs) are the most wide-spread drugs in
the therapy of inflammation. However, especially long-term use of these drugs is closely
related to severe side effects such as gastrointestinal and renal complications (NSAIDs) or
an increased cardiovascular risk (coxibs) due to the suppression of physiological relevant
prostaglandins [1]. Consequently, new pharmacological strategies for anti-inflammatory
therapy are urgently needed. One promising approach is the selective inhibition of
downstream-acting enzymes such as the microsomal prostaglandin E2 synthase-1
(mPGES-1), which catalyzes the formation of PGE2 from PGH2. PGE2 is the most
prominent mediator in inflammatory pain. On the other hand, LTs produced by 5-LO are
important inflammatory mediators which act as bronchoconstrictors and increase vascular
permeability. The dual inhibition of 5-LO and mPGES-1 is considered as a novel strategy to
avoid COX-related side effects such as the analgesic asthma syndrome and to maintain the
physiological prostaglandin levels.
The structural basis of the presented compounds is pirinixic acid, which is inactive on both,
mPGES-1 and 5-LO. Especially the introduction of n-alkyl chains in α-position led to potent
dual 5-LO / mPGES-1 inhibitors. Furthermore, a broad modification of the lipophilic
backbone is tolerated with an equal or increased activity [2]. Herein we present a novel class
of pirinixic acid derivatives featuring a thiazole-scaffold at the lipophilic backbone. The
resulting thiazole-substituted derivatives show balanced dual inhibition of mPGES-1 and 5-
LO with IC50 values mainly in the submicromolar range.
References:
1. Koeberle, A. et al.: Curr. Med. Chem. 2009, 16: 4274–4296.
2. Koeberle, A. et al.: J. Med. Chem. 2008, 51: 8068–8076.
035
Optimization of transport conditions for the shipping of 3D human cornea
constructs used for in vitro drug absorption studies
Beißner N1; Reichl S1 1 Institut für Pharmazeutische Technologie, Technische Universität Carolo-Wilhelmina zu
Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig
In vitro data of transcorneal absorption behaviour of ophthalmic drugs may be obtained by
permeation studies using either excised cornea taken from experimental animals or corneal
cell culture models. In recent years we developed serum-free cultivated 3D cornea
constructs (HC) based on immortalized human corneal cells exhibiting similar barrier
properties as excised porcine and rabbit cornea [1]. In a prevalidation study we generated
SOPs for HC cultivation and permeation procedures and transferred SOPs in two other cell
Poster 101

culture labs. Furthermore, in this study using seven ophthalmic drugs we demonstrated that
our cornea model exhibits lower intra- and inter-laboratory variances than excised animal
cornea [2]. The wide use of validated cell culture models normally requires valid cultivation
in few labs and standardized shipment without loss of their functionality. In the present
study we evaluated optimal transport conditions for the shipping of our cornea construct.
Human cornea constructs were cultivated under serum-free conditions on permeable
polycarbonate filters (Transwells ®) using SV40 immortalized human keratocytes (HCK-Ca)
and human corneal epithelial cells (HCE-T) as described before [1]. After finishing of
cultivation HC were kept in medium under submerged conditions and stored in different
transport containers for various time periods changing shipment parameters as temperature
and CO2 content. Cell viability after simulated transport was evaluated via MTT testing.
Barrier properties of HC expressed as epithelial integrity was estimated by transepithelial
electrical resistance (TEER) measurements.
Both storage temperature and CO2 content had great impact on viability and TEER after 24
and 48 h of transport simulation. Storage at 4 °C and 20 °C without additional CO2 support
resulted in decreased TEER values. In addition, the viability was diminished at 4 °C. In
contrast, keeping cornea constructs at lowered temperature in 5% CO2 atmosphere yielded
viable HC exhibiting constant TEER values. However, lowering CO2 content resulted in
strongly decreased TEER values, which couldn’t be recovered after cultivation for additional
24 h in incubator (37 °C, 5% CO2, humidified atmosphere). Sealing of storage container led
to TEER values similar to those obtained for HC stored under incubator conditions at same
temperature. The combination of sealed transport containers and lowered temperature (22
– 24 °C) caused only marginal reduction of TEER values after 24 h storage. Various
preparatory treatments of cell culture medium as pre-warming to 37 °C or 24 h equilibration
in incubator atmosphere (5% CO2) before use seemed to have no significant influence on
the resulting corneal barrier properties after shipment simulation. Additionally, the container
material (polystyrene, polyamide) didn’t show significant impact.
Our results suggest CO2 atmosphere and medium pH, respectively, as critical parameters
during transport of HC. Thus, an optimized transport system should ensure constant CO2
atmosphere during shipment. In the next step appropriate transport container will be
developed to ensure these requirements. Furthermore, real time transport experiments
have to prove the usefulness of our developed shipment SOP.
Acknowledgments: We are grateful to the German Federal Institute for Risk Assessment (BfR), which
funded this work under grant no. 3-1328-488.1.
References:
1. Hahne, M., Reichl, S., International Journal of Pharmaceutics, 2011, 416, 268-279
2. Hahne, M. et al., Journal of Pharmaceutical Science (accepted and in press,) 2012, Prevalidation of a
human construct as an alternative to animal corneas for vitro drug absorption studies
036
Type-I-allergic diseases and a possible way to treat them: new clozapinederived
Histamine H1-/H4-receptor-ligands and their pharmacological
characterization
Gobleder S. 1, Wittmann H.-J. 2, Elz S. 1 Straßer A. 3
1 Department of Pharm./Med. Chemistry I, Universitätsstr. 31, 93040 Regensburg
2 Faculty of Chemistry and Pharmacy, Universitätsstr. 31, 93040 Regensburg
3 Department of Pharm./Med. Chemistry II, Universitätsstr. 31, 93040 Regensburg
The number of patients that suffer from allergic rhinitis and conjunctivitis (the so called
“hay-fever”) increases every year, a trend that indicates the need of good pharmaceutical
treatment. A very common and well-tried therapy is the combined p.o. and local application
of “antihistamines”, drugs with antagonistic effects at H1R, such as cetirizine and azelastine
to name only a few. Recent research results pointed out, that not only the H1R but also the
H4R is involved in type-I-allergic diseases. [1] Good therapeutic response could be
achieved in the acute murine asthma model with a combined application of mepyramine
(H1-antagonist) and JNJ7777120 (H4-antagonist). [2] These results suggest that addressing
both, H1R and H4R, could be a new therapy-option of hay-fever and other type-I-allergic
diseases like histamine induced itch or inflammatory asthma.
Screening compounds with good affinity to H1R and H4R, we choose the antipsychotic drug
clozapine (1) which has high antagonistic affinity to H1R and moderate agonistic affinity to
H4R. [3] Soft structure modifications, e.g. exchanging the 5-nitrogen by a 5-oxygen and
shifting the 8-chlorine to position 7 lead to an optimized clozapine-derivative (2) with
increased agonistic affinity at H4R and constant high antagonistic affinity to H1R. [4] The
aim of the project was to get a closer insight into structure-activity relationships at H1R and
H4R and, according to our results, to convert the agonism at H4R into an antagonism or
inverse agonism by modifying the clozapine-molecule in different ways.
In a first part of the project we reduced the 5H-dibenzo[b,e][1,4]diazepine-structure of
clozapine to an “open” (E)-N-((phenyl)methylene)aniline structure and varied the kind and
position of substituents on both aromatic rings. Examples of the synthesized compounds
are shown in (3), (4) and (5). Since our compounds showed, compared to clozapine, a
decreased affinity to hH1R and hH4R, we continued synthesizing compounds with
dibenzo[b,f][1,4]oxazepine-structure and added new non-halogenide substituents to this
optimized clozapine derivatives (6), (7) and (8). All compounds were tested at the guinea
pig ileum in presence of histamine and in a competition binding assay at hH1R and hH4R.
For selected compounds ((6), (7) and (8)) we performed a GTPγS assay for hH2R, hH3R
and hH4R to get information about receptor-selectivity and potency. First data revealed
selectivity towards hH1R and/or hH4R for all tested compounds. To explain structure-activity
relationships on a molecular level, molecular modelling studies at appropriate models of
hH1R and hH4R were performed.
R 1
R 2
N N
X
N
R1 Cl,R2 H,X NH 1
R 1 H,R 2 Cl,X O 2
R 1
R 2
N N
N
R1 R2 H,R3
R3
Cl 3
R1 Cl,R2 R3 H 4
R1 H,R2 Cl,R3 NH2 5
R 1
R 2
pA2 pKi pKi
gpH1R hH1R hH4R
1 n.d. 8,6 5,9
2 7,6 7,8 7,0
N N
O
N
NH2
R1 Cl,R2 H 6
R 1 H,R 2 Cl 7
R1 R2 H 8
3 6,6 6,1 4,7
4 6,9 6,0 4,9
5 5,0 4,1 6,6
6 8,9 8,2 6,1
7 7,9 7,7 6,5
8 n.d. 8,0 5,1
References:
1. Thurmond, R.L. et al.: Nature Reviews Drug Discovery 2008, 7 : 41-53.
2. Deml, K.-F. et al.: Mol. Pharmacol. 2009, 76 : 1019-1030.
3. Appl, H. et al.: Nauyn-Schmiedeberg’s Arch. Pharmacol. 2012, 385(2) : 145-170.
4. Smits, R. A. et al.: J. Med. Chem. 2006, 49 : 4512-4516.
037
Optimization of capillary electrophoresis-based analysis of 5-lipoxygenase
metabolites.
Abromeit H; Schaible A M; Werz O, Scriba G K E
Department of Pharmaceutical/Medical Chemistry, Friedrich-Schiller-Universität, Philosophenweg
14, 07743 Jena, Germany
A new sensitive method using α-cyclodextrin-modified micellar electrokinetic
chromatography has been developed to separate and quantify arachidonic acid metabolites
of the lipoxygenase pathways in human polymorphonuclear leukocytes, i.e. leukotriene B4,
6-trans-leukotriene B4, 6-trans-12-epi-leukotriene B4, 5(S)-hydroxy-6-trans-8,11,14-ciseicosatetraenoic
acid, 12(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, and 15(S)hydroxy-6-trans-8,11,14-cis-eicosatetraenoic
acid. The electrophoresis system was
optimized with regard to the pH, boric acid, SDS and α-cyclodextrin concentration as well
as separation voltage and temperature using a three level resolution IV fractional factorial
design and a five level circumscribed central composite design. The optimized conditions
included 80 mM sodium borate buffer, pH 10.07, containing 16.6 mM sodium dodecyl
sulfate and 15 mM α-cyclodextrin, using a separation voltage of 12.5 kV at 23 °C.
Sensitivity was enhanced employing head-column field amplified sample stacking which
resulted in limits of quantification between 30 and 50 ng/mL and limits of detection between
10 and 17 ng/mL after solid phase extraction of the lipoxygenase products. The method
was validated according to the recommendations of the International Conference on
Harmonization and applied to the determination of the lipoxygenase metabolites in
polymorphonuclear leukocytes upon stimulation with Ca2+-ionophore A23187 and
arachidonic acid. Robustness was confirmed using a three level resolution IV fractional
factorial design. In conclusion, the novel method is suitable for the analysis of various
arachidonic acid metabolites produced by living cells and may be used for evaluation of
lipoxygenase inhibitors.
038
Carnosol and Carnosic acids from Salvia officinalis inhibit microsomal
Prostaglandin E2 Synthase-1
Bauer, J1., Kuehnl, S. 2, Rollinger, J. M. 2, Scherer, O. 4, Northoff, H. 3, Stuppner, H. 2, Werz,
O. 4, Koeberle, A. 4
1 Department for Pharmaceutical Analytics, Pharmaceutical Institute, University of Tuebingen,
Tuebingen, Germany);
2 Institute of Pharmacy / Pharmacognosy and Center for Molecular Biosciences Innsbruck,
University of Innsbruck, Innsbruck, Austria
3 Institute for Clinical and Experimental Transfusion Medicine, University Medical Center,
Tuebingen, Germany
4 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University of Jena, Jena,
Germany
Prostaglandin E2 (PGE2), the most relevant eicosanoid promoting inflammation and
tumorigenesis, is formed by cyclooxygenases (COXs) and PGE2 synthases from free
arachidonic acid. Preparations of the leaves of Salvia officinalis are commonly used in folk
medicine as an effective antiseptic and anti-inflammatory remedy and possess anticancer
activity. Here, we demonstrate that a standard ethyl acetate extract of S. officinalis
efficiently suppresses the formation of PGE2 in a cell-free assay by direct interference with
microsomal PGE2 synthase (mPGES)-1. Bioactivityguided fractionation of the extract
yielded closely related fractions that potently suppressed mPGES-1 with IC50 values
between 1.9 and 3.5 µg/ml. Component analysis of these fractions revealed the diterpenes
carnosol and carnosic acid as potential bioactive principles inhibiting mPGES-1 activity with
IC50 values of 5.0 µM. Using a human whole-blood assay as a robust cell-based model,
carnosic acid, but not carnosol, blocked PGE2 generation upon stimulation with
lipopolysaccharide (IC50 9.3 µM). Carnosic acid neither inhibited the concomitant
biosynthesis of other prostanoids [6-keto PGF1a, 12(S)-hydroxy-5-cis-8,10-transheptadecatrienoic
acid, and thromboxane B2] in human whole blood nor affected the
activities of COX-1/2 in a cell-free assay. Together, S. officinalis extracts and its ingredients
carnosol and carnosic acid inhibit PGE2 formation by selectively targeting mPGES-1. We
conclude that the inhibitory effect of carnosic acid on PGE2 formation, observed in the
physiologically relevant whole-blood model, may critically contribute to the antiinflammatory
and anticarcinogenic properties of S. officinalis.
039
Investigation of the degradation of the model tetrapeptide Gly-Phe-Asp-GlyOH
at alkaline pH by capillary electrophoresis including identification of the
degradation products by CE-ESI-MS
Brückner, C. 1; Scriba, G. K. E. 1
1 Friedrich Schiller University, Department of Pharmaceutical and Medicinal Chemistry, School of
Pharmacy, Philosophenweg 14, 07743 Jena, Germany
Asparagine and aspartic acid are prone to chemical degradation such as deamidation,
isomerization, and enantiomerization. [1-3] Enantiomerization and isomerization proceed
via the formation of an aminosuccinimidyl intermediate (Asu). Hydrolysis of the Asu peptide
yields native Asp and β-Asp (iso-Asp) peptides favouring the β-Asp peptide over the Asp
peptide by a ratio of 3-5:1. [1,2] Differentiation of the Asp isomers by mass spectrometry
can be achieved by the ratio of the complementary b- and y-ions generated during peptide
fragmentation. [4] In contrast, diastereomeric Asp peptides, i.e. peptides containing an L-
Asp or D-Asp moiety could not be distinguished based on their MS spectra.
102 Poster

The degradation of the model tetrapeptide Gly-L-Phe-α-L-Asp-GlyOH was investigated at
80°C and 25°C in sodium borate buffer pH 10. Separation and quantification of the
degradation products was achieved using a validated capillary electrophoresis assay. Peak
identification was obtained by CE-ESI-MS experiments and by co-injection of synthetic
reference compounds. Due to the differences in the MS spectra the degradation products
could be clearly assigned to the α-Asp or β-Asp linkage. While for α-Asp peptides b2-ion
formation competes with y2-ion formation, in case of β-Asp peptides the intensity of the y2ion
considerably exceeded that of the b2-ion. At 80°C a rapid decline of the starting peptide
was observed yielding a mixture of α-Asp and β-Asp as well as L-Asp and D-Asp containing
peptides. The β-aspartyl peptides exceeded the α-Asp linkage after 288 h by a ratio of
4.0:1. Furthermore, the peptides containing the D-Asp moiety were favored over the L-Asp
residues. At 25°C the tetrapeptide Gly-L-Phe-α-L-Asp-GlyOH proved to be relatively stable
compared to 80°C. After an incubation interval of 2350 h 88 % of the starting peptide was
still present in the incubation solution. Nevertheless, formation of the enantiomerization and
isomerization products could be demonstrated yielding β-Asp residues in higher amounts
as the respective α-D-Asp peptide.
References:
1. Aswad DW, CRC Press. 1995, 279.
2. Geiger T, Clarke S, J. Biol. Chem. 1987, 262(2):785-794.
3. Manning MC, Patel K, Borchardt RT, Pharm. Res. 1989, 6(11):903-918.
4. Lehmann, WD. et al., Protein Sci. 2000, 9(11):2260-2268.
040
Evaluation of the cytoprotective effects of individual and combined
constituents of the combination drug STW 5
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
2 Steigerwald Arzneimittelwerk GmbH, Scientific Department, Havelstr. 5, 64295 Darmstadt,
Germany
STW 5 (Iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of
functional dyspepsia and irritable bowel syndrome (IBS). We investigated the cytoprotective
action of individual herbal extracts of STW 5 in CaCo-2 cells. The CaCo-2 cells were
incubated with the herbal extracts and stimulated with LPS (10 ng/ml) for 2 hours. Possible
synergistic effects were determined by isobologram analysis of LPS-induced LDH release
as a measure for cell toxicity using a commercially available kit.
STW 5 (500.5 µg/ml) significantly inhibited LPS-induced LDH release by 74 %.
Comparable effects were found with lemon balm, whereas in equivalent concentrations to
STW 5 the effects of Iberis amara, peppermint, chamomile, angelica and milk thistle were
less potent. Caraway and celandine had stimulatory effects. Isobolgram analysis indicated
that a 2 h exposure to peppermint and Iberis amara (ratio 0.5/0.5) resulted in a synergistic
effect of 28 %. Less pronounced synergy was observed using the component ratio
0.75/0.25. An additive effect was present in the ratio 0.25/0.75. Application of peppermint
together with milk thistle (ratio 0.5/0.5) showed ratio-independent synergistic effects of 13-
16 % whereas the combined application of chamomile and Iberis amara had ratioindependent
additive effects. The combination of chamomile and angelica (ratio 0.5/0.5)
exerted an additive effect, the ratio 0.75/0.25 showed an antagonistic effect which
increased using the ratio 0.25/0.75.
Our observations confirm that the components of STW 5 contribute to different extents to its
cytoprotective effect in CaCo-2 cells and clearly indicate that the individual herbal extracts
can contribute synergistically, additive and antagonistically to the overall effects of STW 5.
The type of effect was determined in some cases by the ratio of the constituents.
041
Novel synthetic analogues of toxiferine I and their pharmacological
characterization at α7 nAChRs, muscle-type nAChRs, and the allosteric
binding site of muscarinic M2 receptors
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
Cairo, Egypt
2 Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawai'i at Hilo, Hilo,
HI 96720, USA
3 Pharmacology and Toxicology Section, Institute of Pharmacy, Rheinische Friedrich-Wilhelms-
University, D-53121 Bonn, Germany
4Institut für Pharmazie and Lebensmittelchemie, Universität Würzburg, Am Hubland, 97074
Würzburg, Germany
5 Department of Medicinal Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100
Copenhagen, Denmark
Toxiferine I is a major bisquaternary alkaloid of calabash-curare and Strychnos species. [1]
Its semi-synthetic diallyl analogue alcuronium (Alloferin ®) is in clinical use as muscle
relaxant. Both compounds display high binding affinities for the muscle-type nAChRs from
the electric organ of Torpedo californica. [2] In addition to their neuromuscular-blocking
activity, toxiferine I and alcuronium have been reported to be moderately potent antagonists
at α7-nAChRs [3], and to possess moderate to high affinity for the allosteric binding site of
M2-mAChRs, respectively. [4] Interestingly, the structurally related caracurine V analogues
show a different pharmacological profile displaying considerably lower affinity for the
muscle-type nAChRs, [2] higher antagonistic activity at α7-nAChRs, [3] and similar
allosteric potency at M2-mAChRs, [4] when compared to the correspondingly substituted
bisnortoxiferine analogues. Here, we describe the synthesis and pharmacological
evaluation of novel bisquaternary toxiferine I analogues with structurally modified side
chains. In particular, a series of non-symmetrical derivatives lacking only one alcohol group
(1a-c), and of symmetrical compounds lacking both alcohol functions (2a-c) have been
examined. The findings help delineate the structural requirements for the action at different
AChRs and design ligands selective for one of the subtypes.
Poster 103
OH
H
N
R
N H
H
H
H N
R
N
H
toxiferine I, R Me
alcuronium, R allyl
OH
2Cl -
H
N H H
H
O
O
H
H
H N
H
N
R +
+ R
N
caracurinium V analogues
R Me, allyl, pNO 2 -benzyl
2X -
1
R
H
2 N
R
N H
H
H
H N
2
R
N
H
CH 3
2X -
1a c R 1 -CH 2 OH, R 2 Me, allyl, pNO 2 -benzyl
2a c R 1 -CH 3 , R 2 Me, allyl, pNO 2 -benzyl
Acknowledgments: Bonn University, Mechthild Kepe, Würzburg University, Elfriede Ruckdeschel, Matthias
Grüne
References:
1. Battersby, A.R.; Hodson, H.F. Q. Rev. Chem. Soc. 1960, 14, 77-103.
2. Zlotos, D.P. et al.: Bioorg. Med. Chem., 2004, 12, 6277-6285.
3. Jensen, A.A.; Zlotos, D.P.; Liljefors, T. J. Med. Chem. 2007, 50, 4616-4629.
4. Zlotos, D. P. et al.: J. Med. Chem. 2004, 47, 3561-3571.
042
Ligand based approaches to reveal the active conformation of highly flexible
azecine derivative
Enzensperger C1, Robaa D2, Schulze M3, Lehmann J1 1 Institute for Pharmacy, Department of Medicinal Chemistry, Philosophenweg 14, D-07743 Jena,
Germany
2 Institute for Pharmacy, Department of Medicinal Chemistry, Wolfgang-Langenbeck Str. 4 D-06120
Halle-Wittenberg, Germany
3 Temporarily at ‘Institute de Genomique Fonctionnelle-CNRS’, Montpellier cedex 05, France
Annulated azecine derivatives are highly flexible compounds and some of them exert very
high affinity for dopamine and serotonin receptors. In the crystal structure there are
obviously two enantiomers present, which give rise to the question about the eutomer,
dystomer and the active conformation (in figure 1 LE300 (1) is shown). We succeeded to
synthesize, screen and X-ray an enantiopure chiral benzindolo azecine (2).[1]
Figure 1: Both enantiomers of LE300 (1) present 1:1 in the crystal (left). The absolute
configuration and conformation of the chiral eutomer (2) (right)
This crystal structure is the only conformation that allows a rigid alignment with
conformationally constricted and stereochemically defined templates like asenapine (3) and
ecopipam (4). In that case, the indole moiety can only be superimposed with the aromatic
ring not containing a chloro subtitutent.
O
Cl
1
H
N
H
N CH3
3
H
N CH3
S
CH3
Cl
HO
N
H
N CH3
Figure 2: Rigid alignment of the X-ray structure of the chiral eutomer (2) with asenapine (3)
and ecopipam (4)
This hypothesis is also supported by observed SARs in the class of benzazepine- and
benzindolo- azecine derivatives. The most important structural features of these
compounds seem to be the shape and the site of protonation of the basic nitrogen. We are
now also able to explain, why azecine derivatives with a different annulation pattern are
inactive.[2]
References:
1. Robaa, D.et al., J Med Chem 2011, 54: 7422-6.
2. Schulze, M. et al., Bioorg Med Chem 2009, 17: 6898-907.
2
1
N CH3
CH3
S
043
Design, synthesis, biological testing and SAR of 5-Pyridinyl-2-thioimidazole
derivatives as selective JNK3-inhibitors over p38α
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-
University, Auf der Morgenstelle 8, 72076 Tuebingen, Germany
Many inflammatory conditions are driven by both p38α and JNK3 MAPK.[1] To differentiate
the contribution of each kinase, selective inhibitors are necessary. For p38α this might be
the case[2], but as of today only few selective JNK3 inhibitors with good ADME (Absorption,
Distribution, Metabolism, Excretion) properties are available.
JNK-kinases are c-jun NH2-terminal serine/threonine mitogen activated kinases which are
Cl
HO
4
N CH3
Cl
H
O
2
H
N CH3

mainly activated by cytokines and environmental influences.[3][4] JNK3-kinases are
believed to play a central role in the pathology of neurologic diseases such as
cerebrovascular accidents, Parkinson's and Alzheimer's disease.[5] Therefore it has
become an attractive and valid drug target.
5-Pyridinyl-2-thioimidazole derivatives are known as p38α-inhibitores.[6] Due to the
sequential and steric similarity of p38α- and JNK3-kinase, we assumed to gain active and
selective JNK3-inhibitores by introducing different substitution patterns for R1, R2, and R3.
At the edge of the hydrophobic region II in JNK3-kinase, there are Asn152 and Gln155,
whereas Asp112 and Asn152 are shown in p38α-kinase.[3] In order to create repulsion
between the Asp112 and p38α, we introduced anionic substituents at the aminopyridine
scaffold.
Furthermore, we tried to hit the Asp112[3] by introducing carboxylic moieties at the
imidazole nitrogen. In order to target the conserved but steric diverse Arg107 and
Asn194[3], we synthesised carboxylic substituents linked by a sulfide at R1 resulting in 50
nM inhibition of JNK3 with about 10 fold selectivity against p38α.
References:
1. Kamenecka T.,: J. Biol. Chem. 2009, 284(19): 12852-61.
2. Koebler S. C., et al., Nat. Chem. Biol. 2012, 8(2): 141-3.
3. Scapin G. L., et al., Chem. Biol. 2003, 10: 705-712.
4. Johnson G. L., et al., Science 2002, 298: 1911-1912.
5. Bogoyevitch M. A., Trends Mol. Med. 2005, 11: 232-239.
6. Laufer S. A., et al., J. Med. Chem. 2008, 51(14), 4122-4149.
044
8-Substitued Indolo[3,2-f][3]Benzazecines Receptor Antagonists: Synthesis
and Activity of Racemic and Enantiopure Derivatives
Robaa, D. 1; Enzensperger, C. 2; AbulAzm, S.; 3 Lehmann, J. 2
1Institute for Pharmacy, Department of Medicinal Chemistry, Wolfgang-Langenbeck Str. 4, 06120
Halle
2Institute for Pharmacy, Department of Medicinal Chemistry, Philosophenweg 14, 07743 Jena,
Germany
3Department of Medicinal Chemistry, Faculty of Pharmacy, El Khartoum Square 1, 21521
Alexandria, Egypt
Partially hydrogenated indolo[3,2-f][3]benzazecines represent a structurally novel class of
dopamine receptor antagonists, distinguished by nanomolar affinities as well as their
preference for the receptors of the D1 family [1,2].
Derivatives of the lead indolo[3,2-f][3]benzazecine derivative LE 300 (1), substituted at
position 8 with three different residues, namely methyl, hydroxymethyl and carboxylic acid,
were prepared. The 8-methyl and 8-hydroxymethyl derivatives were obtained as the
separated R- and S- enantiomer as well as the racemic mixture, while the amino acid
derivative could only be obtained in a racemized form. The separate enantiomers showed
significantly different affinities; the (8S)-methyl and (8R)-hyroxymethyl derivatives where the
substituents point below the reference plane of the indolo[3,2-f][3]benzazecine scaffold
were markedly more active than their enantiomeric counterparts. The racemic 8-carboxy
derivative was highly interesting since it showed a pronounced selectivity for the D5receptor,
even against D1.
11
10
12
13 N
H
1
1
(LE300)
9
8
CH3 N 6
7
15 5
1
2
3
N
H
R
CH3 N
Derivatives with substituents
pointing below the projection
place
References:
1. Hoefgen, B. et al.: J. Med. Chem. 2006, 49, 760–769.
2. Witt, T.; Hock, F. J.; Lehmann, J. J. Med. Chem. 2000, 43, 2079–2081.
N
H
R
CH3 N
Derivatives with substituents
pointing above the projection
place
045
Influence of API and Surfactant Selection on Nanosuspensions Properties
Comparing Two Different Top Down Methods
Lestari, Maria L.A.D1; Müller, R.H1; Möschwitzer, J.P 1 2 *
1 Freie Universität Berlin, Institute of Pharmacy, Biopharmaceutics & NutriCosmetics, Kelchstrasse
31, 12169 Berlin, Germany
2 Pharmaceutical Development, Abbott GmbH &Co.KG, Ludwigshafen, Germany
* Corresponding author: Telephone: 030-838-50703, email: info@janmoeschwitzer.de
High Pressure Homogenization (HPH) and wet ball milling (WBM) are two well known top
down methods for producing nanosuspension. Both methods are based on different
diminution principles. These different principles may affect the stabilization of the
nanosuspension as well as process efficiency. Besides these two factors also the active
pharmaceutical ingredients (API) and surfactants are playing an important role for the
particle size reduction process. Amphotericine B and resveratrol were used as model drugs
in this study. Various surfactants (anionic, cationic, nonionic, polymeric and combination)
were tested at specified concentration level. Nanosuspensions were produced with low
energy wet ball milling using yttrium stabilized milling beads (diameter 0.5 mm) with electric
stirring system at 900 rpm for 72h. HPH was done by two pre-milling cycles at 150 and 500
bar and 20 cycles at high pressure of 1500 bar. The nanosuspensions were characterized
using photon correlation spectroscopy (PCS), laser diffractometry (LD), light polarized
microscopy and laser doppler anemometry (zeta potential). Results showed that LE-WBM
of resveratrol produced nanosuspensions with mean particle sizes below 500 nm and a
narrow size distribution. In case of WBM only TPGS led to a mean particle size larger than
1000nm. In contrast, HPH of resveratrol resulted in general in larger particle sizes above
1000 nm. Only nanosuspensions stabilized with sodium cholate had particle sizes below
1000 nm. For amphotericine B, some nanosuspensions had similar particle sizes
irrespectively of the employed technique. Sodium cholate and a combination of sodium
cholate-poloxamer were identified as most suitable stabilizers for both techniques. In
conclusion, both models behaved differently regarding the optimal stabilizer applied. When
LE-WBM technique was used, stabilizer selection was less important since most of the
nanosuspension produced had small mean particle sizes. For the HPH method, the
selection of the right stabilizer system in combination with the API was more important.
046
inSARa: SAR interpretation by network navigation
Wollenhaupt, S. 1; Baumann, K. 1
1 Institut für Medizinische und Pharmazeutische Chemie, Technische Universität Braunschweig,
Beethovenstr. 55, D-38106 Braunschweig, Germany
Structure-Activity-Relationship (SAR) analysis of small molecules is a fundamental and
challenging task in drug discovery. The knowledge of this relationship between structural
determinants and bioactivity is of high value for the medicinal chemist e.g. in the leadoptimization
process or de-novo-design. For the elucidation of SARs the recognition of
molecular similarities is a crucial step due to the assumption that molecules with similar
properties or features exhibit similar biological activities [1].
The question arising from this is how to define molecular similarity ideally. Fingerprintbased
similarity is an established and widely used concept [2]. Fingerprints represent
molecules in the form of binary vectors which show the presence or absence of
substructures or other structural features. Although fingerprints are fast to compute and well
suited for processing large data sets, the interpretation of the results is difficult and the
concept appears less intuitive to a medicinal chemist. A promising and more intuitive
alternative is the maximum common substructure (MCS), the largest common substructure
of a pair of molecules. One drawback is the high computational complexity which prevents
large-scale applications such as the analysis of high-throughput-screening data. Moreover,
exact atom matching is a limiting factor which decreases the scaffold hopping potential and
hinders the identification of structurally less similar bioisosteric substructures.
To tackle these limitations our in-house approach inSARa (intuitive networks for Structure-
Activity-Relationships analysis) combines MCS similarity with the reduced graph (RG)
concept [3]. Reduced graphs promise a higher degree of abstraction as they provide a
conceptual representation of physicochemical properties and pharmacophoric features of a
molecule by reducing groups of atoms to single pseudoatoms while retaining the topology
between these functional units.
inSARa networks are derived by combining RG-MCS-calculations and iterative super- and
substructure searches. By linking the molecules with bioactivity data the networks can be
used for SAR interpretation. This opens up the possibility to display molecules sharing
common features as a function of bioactivity. Furthermore, inSARa networks are of great
value for the detection of so called activity cliffs [4], where minor structural changes lead to
a major change in bioactivity. Even though no bioactivity information is used to generate the
inSARa networks, they can also be used for compound classification and bioactivity
prediction.
References:
1. Johnson, M. A., Maggiora, G. M.: Concepts and applications of molecular similarity (Wiley) 1990.
2. Wawer, M. et al.: J. Med. Chem. 2008, 51(19): 6075-6084.
3. Gardiner, E. J. et al.: J. Chem. Inf. Model. 2007, 47(2): 354-366.
4. Stumpfe, D., Bajorath, J.: J. Med. Chem. 2012, 55(7): 2932-2942.
047
Dibenzosuberones as highly potent p38α MAP kinase inhibitors: Efficient use
of a hydrophobic pocket to improve activity
Fischer S1, Wentsch H1 , Karcher S1, Dorn A1, Laufer S1 1 Institute of Pharmacy, Department of Pharmaceutical Chemistry, University of Tuebingen, Auf der
Morgenstelle 8, 72076, Germany
The dysregulation of inflammatory cellular signal transduction cascades is known to be
involved in the development of many diseases including chronic inflammatory and
autoimmune diseases like rheumatoid arthritis (RA) and inflammatory bowel disease (IBD).
p38α MAP kinase is a key enzyme regarding the regulation of the pro-inflammatory
cytokines TNF-α and IL-1β and is therefore a promising target for the treatment of many
inflammatory diseases. 1 2
By now, successful development of p38α MAP kinase inhibitors as drugs has been
compromised by poor selectivity and / or high toxicity. 3 The ATP-binding pocket of kinases
is highly conserved, offering almost no possibilities to achieve selectivity. Therefore our
efforts concentrated on exploiting unique features of the enzyme. One of them is the ability
to perform the so called “glycine-flip”, which is already enforced by the suberone template.
Another one is the “selectivity-pocket”. This region is located adjacent to the ATP-bindingpocket.
Figure 1: Proposed binding mode of dibenzosuberones and essential interaction sites
(Hinge Region, Selectivity Pocket, Deep Pocket)
In contrast to other kinases, the “selectivity-pocket” is accessible because of the small
gatekeeper residue Thr106. As it is mostly surrounded by lipophilic residues, we had to
104 Poster

design moieties fitting exactly in this location. By extending these moieties towards the
deep pocket, we were able to achieve high potency in the p38α MAP kinase enzyme assay
down to an IC50 of 1nM.
References:
1. Badger et al., J. Pharmacol. Exp. Ther. 1996, 279, 1453-1461.
2. Boehm et al., J. Med. Chem. 1996, 39, 3929-3937.
3. Dominguez et al., Curr. Opin. Drug Discov. Devel. 2005, 8, 421-430.
048
Different column classification systems applied to calixarene- and
resorcinarene-bonded stationary phases
Chamseddin, C.; Jira, T.
Institute of Pharmacy, Pharmaceutical/Medicinal Chemistry, Ernst-Moritz-Arndt-University of
Greifswald, Friedrich-Ludwig-Jahn-Strasse 17, Greifswald D-17487, Germany.
The recently dramatic increase in the number of reversed phase columns could be an
advantage of this liquid chromatographic technique. However, due to the insufficiency of
information that is available from manufacturers in terms of the exact functionality of their
phases, it is problematic to determine which could be the best column for a given situation.
There is not a “super column” that will work best for all applications. As a result, there have
been several attempts to develop testing strategies to characterize column chemistries.
These column classification systems are typically based on one or more test mixtures to
identify the so called “column parameters”. Three different systems, originally developed for
reversed phases, were applied to evaluate the chromatographic properties of the
calixarene- and resorcinarene-bonded stationary phases. These are: Tanaka, USP, and
Snyder/Dolan approaches [1,2,3]. Although all column classification systems aim to
evaluate the “more or less” same characteristics, each system uses different test mixtures
in different chromatographic conditions. All of that make the resulted “column parameters”
using these systems deviate from one to another. Calixarene- and resorcinarene-bonded
stationary phases are reversed phases; however they show, depending on the analytes,
some additional interactions, since there steric, polar and ionic properties are different
compared to those of conventional alkyl-bonded phases.
The following table summarizes the parameters used by each system.
System
Tanaka
USP Snyder/Dolan
Parameter
Hydrophobicity
Hydrophobicity
Hydrophobicity
KPB
KEB
H
Hydrophobic
interactions
Hydrophobic selectivity
αCH2 = KPB/ KBB
Hydrophobic selectivity
αCH2 = KEB/ KT
Steric
Shape selectivity
___ Steric resistance
interactions αT/O = KT/ KO
S*
Hydrogen bonding Activity toward bases Hydrogen-bond
Hydrogen
capacity
KAm and TAm
acidity
bonding
αC/P = KC/ KP
A
and
ionic interactions
Acidic ion-exchange
αB/P = KB/ KP (pH 2.7)
Hydrogen-bond
basicity
Chelation
B
Total ion-exchange
TQ
Cation-exchange
αB/P = KB/ KP (pH 7.6)
activity
C
Am: Amitriptyline; B: Benzylamine; BB: n-butylbenzene; C: Caffeine; EB: Ethylbenzene; O: oterphenyl;
P: Phenol; PB: n-pentylbenzene; Q: Quinizarin (1,4-dihydroxyanthraquinone); T:
Triphenylene.
Acknowledgments: Chamseddin, C. has a PhD scholarship from the DAAD and the Syrian ministry of high
education.
References:
1. Kimata, K. et al.: J. Chromatogr. Sci.1989, 27: 721-728.
2. Bidlingmeyer, B. et al.: Pharmacopeial Forum. 2005, 31(2): 637-645.
3. Snyder, L.R. et al.: J. Chromatogr. A. 2004, 1057: 49-57.
049
Structure-based discovery of novel allosteric modulators addressing the
chemokine receptor CXCR3
Tschammer, N. 1; Bernat, V. 1; Schmidt, D. 2; Kolb, P. 2
1 Friedrich Alexander-University Erlangen/Nürnberg, Department of Chemistry and Pharmacy,
Chair of Medicinal Chemistry, Schuhstr. 19, 91052 Erlangen, Germany
2 Philipps University Marburg, Pharmaceutical Chemistry, Wilhelm-Roser-Str. 2, 35032 Marburg,
Germany
Small soluble proteins chemokines and their G protein-coupled receptors are the key
mediators of immune system function in health and disease. Irregularities in the regulation
and response of the chemokine receptor CXCR3 are associated with numerous pathologies
including autoimmune diseases (e.g. multiple sclerosis, rheumatoid arthritis, and psoriasis),
cancer, vascular disease and transplant rejection. Allosteric modulators that inhibit this
attractive pharmacological target could be thus used to treat or prevent a range of
pathologies.
We began the search for novel allosteric modulators addressing the chemokine receptor
CXCR3 with a structure-based virtual screen [1]. Recently resolved X-ray structure of
CXCR4 [2] served us as a template on which a homology model of CXCR3 was build. The
homology model of CXCR3 was further refined using a large library of known CXCR3
ligands in the combination with a set of non-binders. The homology model of CXCR3
characterized by the best docking score for recent clinical candidate AMG487 was chosen
as a basis for virtual screening. Nearly one million of “lead-like” compounds from ZINK, a
free database of commercially available compound for virtual screening, were docked.
From the 500 best ranked after visual inspection, a set of eight compounds was subjected
to biological testing measuring [ 35S]GTPγS incorporation. To facilitate the identification of
novel CXCR3 ligands we synthesized also a tritium labelled allosteric modulator of CXCR3
RAMX3, which was based on an 8-azaquinazolinone core of AMG487 [3]. The study of
allosteric modulators namely needs to differentiate between the ligand affinity toward the
receptor from the cooperativity exhibited towards the endogenous agonist like chemokine,
as the two properties are not correlated [4]. We discovered six novel chemical scaffolds
modulating the function of CXCR3. Interestingly, although we refined the homology model
with negative allosteric modulators of CXCR3, we identified also positive (PAM) and silent
(SAM) besides negative (NAM) allosteric modulators, indicating the delicate balance
between a molecules' function as SAM, NAM or PAM.
Our investigations demonstrate that a carefully refined homology model can provide a
valuable template for the discovery of novel pharmacophores for allosteric modulators even
for such challenging targets as chemokine receptors.
Acknowledgments: N.T. was supported by the German Research Foundation (DFG, TS 287/2-1).
References:
1. Kolb, P. et al.: P. Natl. Acad. Sci. USA 2009, 106(16): 6843-6848.
2. Wu, B. Et al.: Science 2010, 330(6007): 1066-1071.
3. Bernat, V. et al.: ChemMedChem 2012, Early View, doi: 10.1002/cmdc.201200184.
4. Keov, P. et al.: Neuropharmacology 2011, 60 (1) : 24-35.
050
Structural variations on the thiazolone derivative C06 as potent 5-lipoxygenase
inhibitor
Lill A1, Barzen S1, Rödl C B1, Steinhilber D1, Hofmann B1, Stark H1 1Goethe University, Institute of Pharmaceutical Chemistry, Frankfurt am Main, Germany
(h.stark@zafes.de)
Arachidonic acid released from cytoplasmic membrane upon external stimuli is the source
of one important group of inflammatory mediators: the leukotrienes (LTs). The key enzyme
of their biosynthesis is the iron-containing, heme-free 5-lipoxygenase (5-LO). LTs play a
pivotal role in inflammation, allergic disorders, asthma, cardiovascular diseases and cancer.
Up to now, only two LT-interfering drugs are marketed with limited success: the LT-receptor
antagonist Montelukast (Singulair®) and the iron chelating 5-LO inhibitor Zileuton (Zyflo®).
The need to discover new active ligands for anti-leukotriene therapy is still urgent [1,2].
Based on early virtual screening [3] and medium-throughput screening we previously
identified 5-(4-methoxybenzylidene)-2-p-tolylthiazol-4(5H)-one, C06, as a promising new
direct inhibitor of 5-LO [4,5].
S
N
O
C06 IC = 300nM
50
Poster 105
H3C O
CH 3
R
S
N
O
Our previous studies pointed out that 5-benzylidene-2-phenyl-thiazolinones are a new class
of 5-LO inhibitors. By variation of the substitution pattern we designed and synthesized
novel derivatives of C06 leading to a promising new inhibitors.
The compounds were either prepared by one-pot domino reaction using 2-sulfanylacetic
acid and the corresponding benzaldehyd and benzonitrile or in a two-step synthetic
procedure. All compounds were tested for their 5-LO inhibitory efficiency using a cell-free
and whole-cell (PMNL cells) based assay systems [4,5].
Kindly supported by LOEWE Schwerpunkte LIFF, OSF, NeFF, Anwendungsorientierte
Arzneimittelforschung (Fraunhofer IME) and EU COST Actions CM1103, BM1107, BM0806.
References:
1 M. Peters-Golden and W. Henderson, N. Engl. J. Med. 2007, 357: 1841-1854
2 O. Werz, O and D. Steinhilber, Pharmacol. Ther. 2006, 112: 701-718
3 G. Schneider et al., ChemMedChem 2008, 3: 1535-1538
4 B. Hofmann et al., J. Med. Chem. 2011, 54: 1943-1947
5 S. Barzen et al., Bioorg. Med. Chem. 2012, 20: 3575-3583
051
Myrrhinil-Intest � chamomile component affects contraction and morphology of
rat ileum/jejunum ex-vivo preparations in a concentration dependent manner
Vissiennon, C. 1; Raszek, M. 1; Goos, K.-H. 2; Nieber, K. 1
1 University of Leipzig, Institute of Pharmacy, Talstraße 33, 04103 Leipzig, Germany
2 REPHA GmbH Biologische Arzneimittel, Alt-Godshorn 87, 30855 Langenhagen, Germany
Myrrhinil-Intest � is a prescribed phytotherapy for the treatment of inflammatory intestinal
diseases. However, how its functional effects are exerted has not yet been fully elucidated.
One of its active ingredients, lyophilised extract of chamomile flowers, was tested on the
acute inflammation of rat ileum/jejunum preparations, experimentally induced by 2,4,6trinitrobenzene
sulfonic acid (TNBS, 10 mM, 30 min). At low concentrations tested (8.75-70
µg/mL) chamomile exhibited a basal tone enhancement of both the untreated controls and
TNBS-treated preparations. At higher concentrations this trend subsided at first with the
inflamed segments only (140 µg/mL), with subsequent inhibition of basal tone for both
untreated and TNBS-treated preparations (210-280 µg/mL).
Histological analysis revealed that the concentration-dependent effect correlated with a
protective function of chamomile from TNBS-induced morphological damage (100 mM, 30
min). Thus whilst pre-treatment with 70 µg/mL exhibited protective function, higher levels of
chamomile (280 µg/mL) did not. 10 mM TNBS also appeared to have 1.6-fold reduction
effect on acetylcholine (ACh)-induced contraction (EC50 0.9 µM vs. 0.58 µM for untreated
controls). This could be reversed with chamomile pre-treatment. However, this observation
was not statistically significant.
The importance of a chamomile treatment concentration-dependence is further
underscored by the fact that TNBS-inflamed segments also exhibit increased sensitivity to
chamomile inhibition of ACh-induced contractions (IC50 106 µg/mL vs. 175 µg/mL for
untreated controls), particularly in the higher concentration range.
052
Four novel antifungal compounds from the Baltic Sea cyanobacterium
Anabaena sp.
Bui, T. H. 1; Wray, V. 2; Nimtz, M. 2; Fossen, T. 3; Schröder, G. 4; Wende, K. 1; Mundt, S. 1
1Institute of Pharmacy, Department of Pharmaceutical Biology, Ernst-Moritz-Arndt-University,
Friedrich-Ludwig-Jahnstr. 17, D-17487 Greifswald
2Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38214 Braunschweig, Germany
3Department of Chemistry and Centre for Pharmacy, University of Bergen, N-5007 Bergen, Norway
R

4 Friedrich Loeffler Institute of Medical Microbiology, University Medicine Greifswald, Martin-Luther-
Str. 6, D-17475 Greifswald
18 Vietnamese and 4 German cyanobacterial strains were screened with the agar diffusion
assay for their antifungal activity to human pathogenous fungi. It was found that Anabaena
sp. strain Bio 33 collected from The Baltic Sea (Rügen Island, Germany) exhibited an
excellent and specific antifungal activity with inhibition zones from 21 to 32 mm against
Candida albicans, Candida krusei, Candida maltosa, Aspergillus fumigatus, Microsporum
gypseum, Trichophyton rubrum and Mucor sp. No cytotoxic activity against the human
bladder carcinoma cell line 5637 (IC50 > 500 μg/mL) and no antibacterial activity against
Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa
were observed. Bioassay-guided fractionation of the 50% methanolic water extract by silica
gel column chromatography followed by repeated reversed phase HPLC (column
LiChrospher RP-18, gradient of MeOH in H2O with 0.05% TFA) resulted in four white
amorphous solids. The structures were elucidated by 1D ( 1H) and 2D spectra (COSY,
TOCSY, NOESY, HSQC, HSQC edited TOCSY and gHMBC), HR-ESI-MS and confirmed
by amino acid analysis and sugar analysis. Spectroscopic data analysis afforded an
unambiguous sequence of R.CHO(S1).CHOH.CONH-Thr(1)-Thr(2)-Thr(3)-HOTyr(4)-
DeThr(5)-Gln(6)-Gly(7)-ThrNMe(8)(S2)-GlnCOOH(9) in which S1 is arabinose-(3-1)galacturonic
acid, S2 is a mannose and R is a aliphatic residue. The four compounds
distinguish from their cyclic/linear core and the presence of chlorine in the aliphatic part.
Culture optimization to enhance the yield of these antifungal peptides is currently
developed.
053
Synthesis of inhibitors of glutathione peroxidases for reversal of cancer drug
resistance
Wilde F1; Lemmerhirt H1; Emmrich T1; Schulz R1; Bednarski P J1, Link A1 1 Ernst-Moritz-Arndt University of Greifswald, Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald,
Germany
Glutathione peroxidases (GPx) contribute substantially to cell protection by reducing
reactive oxygen species (ROS) such as H2O2 and organic hydroperoxides and hence
regulating the redox status of cells to prevent cell death [1]. While H2O2 plays a vital role in
the induction of apoptosis [2], over-expression of GPx has been observed in cancer cells
that have acquired resistance to anticancer agents.
Following a combinatorial chemistry approach, a small library of test candidates was built
starting from a lead compound identified by a molecular modeling project [3].
deletion
of
substituent
replacement
of
basic
heterocycle
To determine the activity of the potential inhibitors, a recently established microtiter method
based on the inhibition of bovine erythrocyte GPx was applied, which allowed for a rapid
screening of compounds.
Here we present the general synthesis of the lead compound and the library members.
Characterization of this class of compounds is hampered by the formation of isomers
leading to complex NMR spectra. A rational for this finding is discussed.
References:
1. Takebe, G. et al.: J. Biol. Chem. 2002, 277(43): 41254-8.
2. Lopez-Lazaro, M.: Cancer Lett. 2007, 252(1): 1-8.
3. Schulz, R.: Diploma thesis, 2008.
adjustment
of
spacer length
modification
of
substitution
pattern
variation
of
aromatic system
054
Structural Insights into Retinal Guanylylcyclase / GCAP-2 Interaction
Determined by Cross-Linking and Mass Spectrometry
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
University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, 06120 Halle-Saale, Germany
2 Department of Technical Biochemistry, Institute of Biochemistry and Biotechnology, Martin-Luther
University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle-Saale, Germany
Two isoforms of retinal guanylylcylase (ROS-GC 1 and 2), localized in photoreceptor cells,
are responsible for the synthesis of cGMP, the second messenger in phototransduction [1].
ROS-GC 1 and 2 are regulated in a Ca2+-dependent manner from the intracellular site by
guanylylcyclase-activating proteins (GCAPs) [2]. GCAPs are hydrophobic N-terminally
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-
GC genes lead to different retinopathies [4]. Therefore, a detailed knowledge of the
complex formation between GCAPs and ROS-GC is crucial for the development of novel
drugs.
Interactions between a ROS-GC 1 peptide (amino acids 965-981 from the catalytic domain
[5]) and GCAP-2 were investigated applying two complementary types of cross-linking
chemistry. For mapping larger distances, the homobifunctional amine-reactive cross-linker
bis(sulfosuccinimidyl)glutatrate (BS2G) was used. To determine spatially close interactions,
the unnatural photoreactive amino acid photo-leucine that is activated by UV-A radiation,
was incorporated into ROS-GC peptides. After the cross-linking reaction the cross-linked
GCAP-2/GC peptide complexes were enzymatically digested and analysed by highresolution
mass spectrometry.
In the presence of Ca2+, several intramolecular cross-links were identified within N-
terminally myristoylated GCAP-2, all of which were in agreement with the published NMRstructure
(pdb 1JBA) of non-myristoylated GCAP-2. The C-terminal part of GCAP-2, which
is not resolved in the NMR-structure, seems to be highly flexible as indicated by the
numerous intramolecular cross-links within GCAP-2. Analysis of cross-linking samples
without Ca 2+ yielded a limited number of intramolecular cross-linking products of GCAP-2
itself. Several cross-links between the N-terminus of GC peptide and different lysines of
GCAP-2 were identified both in the presence and absence of Ca 2+. After photo-crosslinking,
a limited number of cross-links were obtained pointing to a structurally well-defined
interaction between GCAP-2 and the GC peptide in the presence of Ca 2+. In the absence of
Ca 2+ the structure of GCAP-2 appeared to be much more flexible as indicated by a large
number of diverse photo cross-linking products between GCAP-2 and GC peptide. Based
on the distance contraints imposed by the cross-links it was possible to create a model of
the complex between myristoylated GCAP-2 and the GC peptide that is formed in the
presence of Ca 2+ (Figure 1).
Figure 1: Structural model of the GCAP-2/GC peptide complex. The backbone of the most
representative GC peptide is colored red along with GCAP-2, which is shown as a light blue
cartoon. Cross-linking sites in GCAP-2 obtained from photoaffinity labeling, which were
used for modeling, are shown as light green spheres. Cross-linking sites obtained with
BS2G, which were used for modeling, are shown as light orange spheres. The reactive
sites in the GC peptide are shown as dark green (photo-Leu at positions 5 and 13) and
brown (Tyr-1) spheres.
References:
1. Yang R.B. et al.: Proceedings of the National Academy of Science of the United States of America 1995,
92(2): 602-606.
2. Sharma R.K.: Mol.Cell. Biochem. 2010, 334(1-2): 3–36.
3. Olshevskaya E.V. et al.: J. Biol. Chem. 1997, 272(22): 14327-14333.
4. Sato M. et al.: Graefes Arch. Clin. Exp. Ophthalm. 2005, 243(3): 235-242.
5. Duda T. et al.: Biochemistry 2005, 44(19): 7336-7345.
055
Triterpenes from the fungus Piptoporus betulinus
Alresly, Z. 1; Lindequist, U. 1; Lalk, M. 1; Porzel, A. 2; Arnold, N. 2 ; Wessjohann, L. A. 2
1Institute of Pharmacy, Ernst-Moritz-Arndt-University, Friedrich-Ludwig-Jahn-Straße 17, D-17489
Greifswald, Germany
2Leibniz – Institute of Plant Biochemistry, Department of Bioorganic Chemistry (NWC), Weinberg 3,
06120 Halle (Saale , Germany
The mushroom Piptoporus betulinus (Bull. : Fr.) P. Karst. (Polyporaceae), birch polypore,
growth as trunk parasit and saprophyt on Betula pendula Roth. and B. pubescens Ehrh.
(Betulaceae). Fruit bodies of this fungus are used in the European ethnomedicine for the
treatment of cancer and stomach diseases. The mushroom is also known as fungus of the
“iceman” from the Copper Age found in 1991, who carried P. betulinus fruiting bodies
attached to his clothing on his journey in the Alps [1].
The phytochemical knowledge about this mushroom and the justification of its
ethnomedicinal use are limited. Aim of our studies was the phytochemical examination of
extracts prepared from the fruiting bodies of collected mushrooms and the investigation of
isolated compounds for antimicrobial activity.
Fractionation of the ethyl acetate extract led to the isolation of a new bioactive triterpene
with a lanostane basic structure. By the help of spectroscopic methods it could be identified
as 3β-acetoxy-16 hydroxy-24- oxo-5a- lanosta- 8(9) diene -30- oic acid (Fig. 1). In addition,
seven known triterpenes, polyporenic acid C, betulinic acid, betulin, ergosterol peroxide,
9,11dehydroxyergosterol peroxide, 24-methylene-3-oxo-lanosta-8-en-21-oic acid, and
fomefficinic acid, could be isolated and identified by comparison with data from the
literature. All isolated compounds were tested for antimicrobial activity against some Grampositive
and Gram-negative bacteria as well as against some microscopic fungi. The new
triterpene, polyporenic acid C, and fomefficinic acid showed antimicrobial activity against
Staphylococcus aureus.
106 Poster
CH 3
CH3 OH
CH3 CH3 CH3 O O
H3C CH3 Fig. 1: 3β-acetoxy-16α hydroxyl-24-oxo-5α-lanosta-8(9) diene-30-oic acid
References:
1. Lindequist, U.; Niedermeyer, T.H.J.; Jülich, W.D.: eCAM 2005, 2(3): 285–299.
056
A1-barrigenol and rare 17,22-seco oleanolic acid glycosides from the leaves of
Pittosporum angustifolium.
Bäcker, C. 1; Jenett-Siems, K. 2; Siems, K. 3; Wurster, M. 1; Bodtke, A. 4; Chamseddin, C. 4;
Crüsemann, M. 1a; Lindequist, U. 1
1 Department of Pharmaceutical Biology, Institute of Pharmacy, Ernst Moritz Arndt University
Greifswald, Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany
2 Department of Pharmaceutical Biology, Institute of Pharmacy, Free University of Berlin, Königin-
O
OH
O
CH 3

Luise-Str. 2+4, 14195 Berlin, Germany
3 AnalytiCon Discovery GmbH, Hermannswerder Haus 17, 14473 Potsdam, Germany
4 Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Ernst Moritz Arndt
University Greifswald, Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany
1a Present address: Kekulé Institute of Organic Chemistry and Biochemistry, Rheinische Friedrich
Wilhelms University Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany
Pittosporum angustifolium LODD. (Pittosporaceae), a small tree known colloquially as
"gumby gumby", occurs in inland areas of most states of Australia. Especially the leaves
are used by Aboriginals for various medical applications [1]. Recently beneficial effects of
“gumby gumby” as a supportive agent for cancer treatment have been observed [2].
Phylogenetic studies described P. angustifolium as a distinct species, which previously was
considered as a variety of P. phillyreoides (syn. P. phillyraeoides; var. microcarpa) [1].
Phytochemical investigation of the leaves collected in Queensland, Australia, resulted in the
isolation of triterpene saponins known as eryngioside I [3] and new compounds named
pittangretosides A-I. Their structures were elucidated and characterized by NMR,
HRESIMS, hydrolysis and CD measurement. Eight saponins were identified as
A1-barrigenol derivatives whereas two compounds exhibit an unusual 17,22-seco backbone
of oleanolic acid.
Acknowledgments: We sincerely thank Dr. Rudolf Kunze, Berlin, Germany, for the stimulation of this work
and his continuous support as well as Dr. Cornelia Krasser, Yepoon, Australia and Mr. Klaus von
Gliszczynski for the collection and provision of the plant material. We also thank all of the colleagues at the
Institute who were involved in supporting this work.
References:
[1] Cayzer, L.W., Crisp, M.D., Telford, R.H., Aust. Syst. Bot. 2000, 13: 845-902.
[2] Patent, 2009. WO 2009/037225 A2; DE 10 2007 044 094 A1.
[3] Zhang, Z. et al., Phytochemistry 2008, 69: 2070-2080.
057
Homology Modeling of the Kinase Domain of DLK
Hasenpusch, D. 1; Oetjen, E. 2; Lemcke, T. 1
1 Institut für Pharmazie, Universität Hamburg, Bundesstr. 45, 201146 Hamburg, Germany
2 Institut für Klinische Pharmakologie und Toxikologie, Universitätsklinikum Eppendorf, Martinistr.
52, 20246 Hamburg, Germany
An interesting subgroup of protein kinases are the mixed-lineage kinases (MLKs). They
belong to the tyrosine kinase-like kinases (TKL) and show phylogenetic similarity to both
typical serine/threonine kinases and tyrosine kinases. Functional they are a member of the
serine/threonine kinases.
Two interesting members of this group are the DLK (dual leucine zipper kinase) and LZK
(leucine zipper kinase), which form homodimers mediated via hydrophobic interactions of
their leucine zippers. Acting as a MAP3K DLK was shown to result in the activation of the
downstream kinases p38 and JNK (jun-N-terminal kinase). In the insulin producing β-cells
within the pancreatic islets DLK was shown to mediate apoptosis induced by
immunosuppressants and by the cytokine TNFα and to inhibit insulin gen transcription [1–
4]. Given the importance of maintaining β-cell function and mass to prevent the
pathogenesis of diabetes mellitus, the inhibition of DLK represents an interesting and novel
target for developing potential drugs for the therapy of this chronic disease.
So far no crystal structure data of the DLK is available. As a starting point for a structure
based project to identify potential selective DLK inhibitors, we constructed threedimensional
homology models of the kinase domain of DLK. Suitable templates, which
represented different conformations of the kinases, namely the open and closed
conformation of the activation loop, were found in the phylogenetic neighborhood of DLK.
The actual model building was accomplished with MODELLER [5] using standard
procedures and subsequent loop refinement by the loop model routine. Models were
afterwards evaluated using a workflow that combines individual scorings of different
evaluation algorithms (e.g. PROCHECK, ProSA, ERRAT, MODELLER) in a rank-by-rank
ConsensusScoring [6]. Top ranking models were submitted to molecular dynamic
simulations of at least 2 ns, to test for general stability and to allow assuming their final
three-dimensional shape. These models will be used in a future virtual screening
experiment for the search of DLK inhibitors.
References:
[1] Plaumann, S. et al., Mol. Pharmacol. 2008, 73, 652–659.
[2] Klimpel, C., Boerchers, S., Oetjen, E., Arch Pharmacol. 2009, 379, 37-37.
[3] Oehmen, M.-J. et al., Arch. Pharmacol. 2010, 381, 36-36.
[4] Phu do, T. et al. Cell Signal. 2011, 23, 344-353.
[5] Sali, A., Blundel, T. L., J. Mol. Biol. 1993, 234, 779–815.
[6] Kruggel, S., Lemcke, T., Arch. Pharm. (Weinheim) 2009, 342, 327–332.
058
ChemicalToolBoX in action!
Grüning, B. A. 1; Lucas, X. 1; Chbeib, M. 1, Patel, H. 1, Günther, S. 1
1 Institute of Pharmaceutical Sciences, Hermann-Herder-Strasse 9, 79104, Germany
ChemicalToolBox is a set of tools integrated into a workflow-management system to enable
researchers easy-to-use, reproducible, and transparent access to cheminformatic libraries
and drug discovery tools. It includes standard applications for similarity and substructure
searches, clustering of compounds, prediction of properties and descriptors, filtering, and
many other tools that range from drug-likeliness classification to fragmentation and
fragment-merging.
ChemicalToolBox is based on open-source software, web-accessible, freely available, and
easily expandable. It can be downloaded and easily deployed locally or on a cluster.
Here we present some use-cases for the ChemicalToolBox that we have developed in our
lab to demonstrate its capabilities.
As a first application we have created ChemicalBoX, a comprehensive library of about 40
million unique small compounds collected from freely-accessible repositories. The
compounds included in ChemicalBoX have been filtered by physico-chemical properties
and clustered by similarity to allow fast navigation throughout millions of compounds
belonging to many different chemical classes.
In a similar project, called PurchasableBoX, a comprehensive library consisting of several
millions of purchasable small compounds was assembled. Both libraries are unique and, to
the best of our knowledge, are the largest freely-available libraries.
With the fragmentation tool included in ChemicalToolBox, we can build specialized
fragment libraries using databases of compounds as input. Those collections provide the
foundation for calculating focused Mass Spectrometry libraries and for enabling fast and
efficient screening of large amounts of MS data. Furthermore, these fragments can be the
input of a workflow we have created for fragment-based drug design: it merges the
fragments according to predefined synthetic route rules to form new promising compounds
that enlarge the known chemical space.
The use cases presented here provide an insight into the capabilities of ChemicalToolBoX.
By combinations of the various tools many more powerful applications can be designed.
059
The influence of saponins on the cell membrane-cholesterol
Böttger, S. 1, Melzig, M. F. 1
1 Institute of Pharmacy – Pharmaceutical Biology, Freie Universität Berlin, Königin-Luise-Str. 2+4,
14195 Berlin, GERMANY
As secondary plant compounds with amphiphilic properties saponins possess surface
activity. They can interact with physical surfaces such as those of aqueous solutions and
lower the surface tension [1]. They also interfere with biologic surfaces such as cell
membranes. The latter case may result in haemolysis and cytotoxicity as a consequence of
perturbation of the membrane integrity at higher saponin concentrations.
The mechanism behind this saponin characteristic is not yet completely understood, but
there are different indications that the membrane-cholesterol is decisively involved in this
process. Saponins have been described to bind/to complex cholesterol [1, 2, 3]. As
cholesterol is known to carry out a cell membrane stabilizing function [4], a loss of the
cholesterol itself or the loss of the ability to carry out this function will lead to cell membrane
instabilities and/or membranolysis.
We developed a cell culture model using ECV-304 urinary bladder carcinoma cells
involving 3H-marked cholesterol and investigated the influence of different types of
saponins on the cell membrane-cholesterol.
References:
1. Böttger, S., Hofmann, K., Melzig, M. F.: Bioorg. Med. Chem. 2012, 20(9): 2822-2828.
2. Glauert, A. M., Dingle, J. T., Lucy, J. A.: Nature 1962, 196(4858): 953-955.
3. Schlösser, E.: Can. J. Physiol. Pharmacol. 1969, 47(5), 487-490.
4. Meyer, T. et al.: Biospektrum, 2012, 18(2), 142-145.
060
Synthesis and testing of novel 2,4-thiazolidinedione derivatives as inhibitors of
the enoyl-ACP-reductase InhA of Mycobacterium tuberculosis
Vogel, S. 1; Wagner, S., Waltenberger, C., Sotriffer, C.A., Schirmeister, T. 2
1 Institute of Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, 97074
Würzburg , Germany
2 Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55128 Mainz,
Germany
InhA is part of the fatty acid synthase complex type II (FAS II) and catalyzes the reduction
of enoyl-acyl-[acyl-carrier-protein] derivatives of the chain length up to 56 carbons using the
essential cofactor NADH.[1] When inhibited, unsaturated fatty acids accumulate, and the
formation of the cell wall is disturbed.
Computational methods have been applied in a step-wise fashion in order to search for new
InhA inhibitors. Using ligand- and target-based approaches three pharmacophore models
were defined. For screening purposes the ‘drug-like’ subset of the ZINC database and the
vendor database distributed by CCG were used. Hits of the pharmacophore screening were
subdued to hierarchical filtering and visually inspected. The most suitable candidates were
selected for docking procedures with the programs MOE Dock and Autodock 3.0. For
validation purposes, known active compounds were included in the docking simulations.
The most promising hits were chosen for further studies and experimental testing of their
inhibitory activity.
For activity testing two enzyme assays based on the decrease of the absorption or
fluorescence intensity upon reduction of the cofactor NADH were established and validated
using triclosan as positive control. The enzyme InhA was overexpressed in E. coli and
purified.[2] The substrate 2-trans-octenoyl-coenzyme A was synthesized via the mixed
anhydride method.[3]
Based on the results of the screening of a large library of compounds and subsequent
docking experiments the most promising hits with 2,4-thiazolidinedione structure were
chosen for further optimization.
References:
1. X.Hong et al.: Biomacromolecules 2004, 5: 1052-1065.
2. Parikh et al.: Biochemistry 1999, 38: 13623-13634.
3. Goldman/Vagelos: J.Biol.Chem. 1961, 10: 2620-2623.
061
3-Hydroxypyrroles or Pyrrolin-3-ones as novel p38 MAP kinase inhibitors
Freitag, A. 1; Laufer, S. A. 1
1 Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-
Universität, Auf der Morgenstelle 8, 72076 Tübingen, Germany
p38 MAP kinase controls a plethora of cellular responses to extracellular stimuli such as
cellular stress. The kinase is part of a signalling pathway resulting in the biosynthesis of
pro-inflammatory cytokines like IL-1β and TNF-α. Since their levels are elevated in
chronical inflammatory diseases inhibition of p38 is a promising therapeutic target.
The small molecule inhibitor SB-203580 is one of the first known selective inhibitors of p38.
Poster 107

Due to X-ray crystal strucure analysis, the interactions of the pyridinylimidazole to the
binding site of the kinase are established [1].
Figure 1: left side: binding mode of SB-203580; right side: postulated binding mode of
pyrrolin-3-ones
Being iron-ligands, imidazoles lead to CYP-450 inhibition and probably hepatotoxicity. Our
strategy is to replace the core-heterocycle in order to reduce hepatic toxicity and to improve
the interaction with Lys53. These considerations led us to pyrrolin-3-ones.
Since established synthetic approaches to build pyrroles don’t suit this substitution pattern
a new route has been developed based on reference [2].
One major challenge is tautomerism of the target compound. Depending on substituents
and solvent 3-hydroxypyrrole and pyrrolin-3-one exist at equilibrium.
In position 2 unsubstituted pyrrolinones are extremely tending to polymerize, but the 3hydroxypyrrole
with an ethyl carboxylate substituent in position 2 could successfully be
crystallized and tested in a p38α MAP kinase assay (IC50 0,139 µM).
Acknowledgments: Katharina Bauer and Marcia Goettert for biological testing, Verena Schattel for molecular
modelling.
References:
1. Gallagher, T. F. et al.: Bioorg. & Med. Chem. 1997, 5(1): 49–64.
2. Bauer, H.: Justus Liebigs Annalen der Chemie 1970, 736:1-15.
062
On the Importance of Water Molecules on Ligand Binding in the ATPase of
GyrB
Münsterberg, M.; Lemcke, T.
Institut für Pharmazie, Universität Hamburg, Bundesstr. 45, 20146 Hamburg, Germany
Gyrase is a well-known ATP-dependent topoisomerase II, essential for prokaryotic cells. It
is the only known topoisomerase, which catalyses the negative supercoiling of DNA and
consists of two subunits GyrA and GyrB. While GyrA mainly processes the strand passage,
the ATP-hydrolysis is done by the N-terminus of GyrB [1]. Because of its significance in
prokaryotes, gyrase has become a widely used target in antibiotic therapy. Nevertheless,
only fluoroquinolones, inhibitors of the DNA-GyrA-complex, are available for therapy.
Although there are known inhibitors of the ATPase in GyrB, e. g. the aminocoumarines like
novobiocin, there are no drugs on the market which use this target [2]. Meanwhile, the
resistance of bacteria against all major antibiotics forces the development of new antibiotics
with superior effects [3].
Several x-ray crystal structures of GyrB complexes from the protein data bank [4], with
chemically different ligands, contain conserved water molecules. It is well known that water
molecules play an important role for the stability of protein-ligand complexes, because the
presence of water in the ligand binding site can increase or weaken the binding affinity of a
ligand [5,6]. The release of buried water molecules from hydrophobic binding site
environments very often has a beneficial effect on the free energy of binding of an inhibitor.
For a structure based virtual screening it is necessary to know, which water molecule is
important for the complex stability and ligand binding, and which water molecule might be
replaced during docking experiments.
Through the investigation of the GyrB ATPase protein-ligand complexes with different
methods and tools (MD simulations, SuperStar [7] and Rank and HINT [8]) we have
assessed the water molecules in the ATP-binding site. Even though there are huge
conformational differences within the structures, particular water molecules are necessary
for ligand binding in all complexes and thus for successful docking. Other, also conserved,
water molecules in hydrophobic environment might be replaced by a suitable ligand to
perform a successful docking.
Our results will be the foundation for the setup of a virtual high throughput screening in a
structure based design campaign to identify new and selective GyrB ATPase inhibitors.
References:
1. Champoux J. J.: Annu. Rev. Biochem. 2001, 70(1): 369–413.
2. Collin F., Karkare S., Maxwell A.: Appl. Microbiol. Biotechnol. 2011, 92(3): 479–497.
3. Boucher H. W. et al.: Clin. Infect. Dis. 2009, 48(1): 1–12.
4. Berman H. M. et al.: Nucleic Acids Res. 2000, 28(1): 235–242.
5. Lam P. Y. et al.: Science 1994, 263(5145): 380–384.
6. Mikol V., Papageorgiou C., Borer X.: J. Med. Chem. 1995, 38(17): 3361–3367.
7. Verdonk M. L., Cole J. C., Taylor R.: J. Mol. Biol. 1999, 289(4): 1093–1108.
8. Amadasi A. et al.: J. Med. Chem. 2008, 51(4): 1063–1067.
063
Natural surfactant of polyglycerol fatty acid esters type as stabilizers for lipid
nanoparticles
Keck CM1 2; Kovacevic A3; Müller RH1 1Institute of Pharmacy, Department of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Freie
Universität Berlin, Kelchstrasse 31, 12169 Berlin, Germany
2Applied Pharmacy Division, University of Applied Sciences Kaiserslautern, Campus Pirmasens,
Carl-Schurz-Strasse 10-16, 66953 Pirmasens, Germany
3Faculty of Pharmacy, Department of Pharmaceutical Technology and Cosmetology, University of
Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia
Polyglycerol fatty acid esters (PGE) are non-ionic surfactants with various applications in
pharmaceutical and cosmetic industries. They have been used for many years as food
additives and therefore designed as „orally safe” surfactants. The aim of the study
presented here was to evaluate the performance of PGE as potential stabilizer for SLN and
NLC. PGE may be formed by a polymerization of glycerol followed by an esterification with
fatty acids. The obtained mixture might vary in polymerization degree, kind and position of
esterified fatty acids. The surfactants investigated were differed in the kind and position of
esterified fatty acid (Fig. 1).
SLN composed of 10% Cutina ® CP as solid lipid and NLC containing 6% Cutina ® CP and
4% Miglyol ® 812 as a liquid lipid were produced by hot high pressure homogenization [1].
All samples were stabilized with 1% PGE. Characterization of the particles was performed
by photon correlation spectroscopy (PCS) and laser diffraction (LD). To investigate the
surface properties of the SLN and NLC zeta potential (ZP) was measured in distilled water
adjusted to conductivity of 50 μScm −1. Crystallinity of the SLN and NLC and respective solid
lipid i.e. solid lipid/liquid lipid blend used as a particle matrix was investigated by differential
scanning calorimetry (DSC).
The obtained results indicated that PGE showed a good efficiency in the stabilization of the
SLN and NLC. All samples have revealed size (z-ave) below 200 nm, with narrow size
distribution (polydispersity index < 0.15). The respective z-ave were in good agreement
with LD diameters indicating narrow and unimodal size distribution. PGE based SLN and
NLC possessed the same particle composition, but different crystallinity indices. Particles
stabilized with PI and PDI possessed lower crystallinity than PS based samples. The
differences in DSC parameters are obviously created by the different types of the
surfactants i.e. different interaction with the particle matrix and different surface tension
generated near to the surface of the particles. The differences in the internal structure of
the particles were also reflected in ZP values. With higher melting enthalpy and higher
crystallinity index higher ZP values were obtained.
Conclusions: PGE are suitable stabilizers for SLN and NLC. However, the tendency of the
lipid (used as a particle matrix) to create more crystalline matrix of the lipid nanoparticles
was favored by polyglycerol-6 ester type of the surfactant (PD) whereas the influence of the
polyglycerol-6 isoesters (PI, PDI) was not significant.
A B
C
Chemical structures of the PGE-surfactants used: A- polyglycerol 6-distearate (PD), B-
polyglycerol 6-diisostearate (PDI) and C- polyglycerol 6-monoisostearate (PI)
References:
1. Müller RH, Lucks JS. Arzneistoffträger aus festen Lipidteilchen - Feste Lipid Nanosphären (SLN). 1991,
German patent application P 41 5 62.6.
064
Lectine Mimetics Based on Multivalent Boronic Acids
Claes D1; Maison W1 1 Pharmaceutical and Medicinal Chemistry, Bundesstraße 45, 20146 Hamburg, Germany
Carbohydrates are playing an essential role in biological systems. They are used for energy
storage, as structural substances and for cellular recognition [1]. The so called glycocalix
covers cells and its molecular structure is dependent on the cell type, its level of
differentiation and the activity of carbohydrate modifying enzymes. The recognition of these
differences is a key component of pathogen detection by the native immune system and
makes carbohydrates promising targets for pharmaceuticals [2].
Natural carbohydrate binders are lectins, the first line of defense in our immune system [3].
Lectins show weak affinities to monosaccharides and are therefore binding carbohydrates
multivalently to strengthen these interactions. The geometry, composition and density of
binding epitopes has a profound influence on these binding processes [4]. Notably, many
lectins recognize their carbohydrate ligands in C3-symmetric complexes.
108 Poster
HO
B
O
peptide
O
NH
B O
HO
scaffold
HN O
peptide
O
N
H
boronolectin s
peptide
O
B OH
We propose to mimic lectins with trivalent assemblies of boronic acids. For this purpose we
have synthesized suitable trivalent scaffolds and benzboroxoles, which are known to bind
to non reducing sugars with significant affinities [5].

References:
1. Roseman S. J. Biol. Chem. 2001, 276(45): 41527–42542.
2. Dube D. H., Bertozzi C. R. Nat. Rev. Drug. Dis. 2005, 4(6): 477–488.
3. Lis H., Sharon N. Chem. Rev. 1998, 98(2): 637–674.
4. Kiessling L. L., Gestwicki J. E., Strong L. E. Curr. Opin. Chem. Biol. 2000, 4(6): 696–703.
5. Berube M., Dowlut M., Hall D. G. J. Org. Chem. 2008, 73(17): 6471–6479.
065
Heterologous expression of (putative) polyketide synthases and nonribosomal
peptide synthetases in Bacillus subtilis
Kumpfmüller, J. 1; Kabisch, J. 1; Wenzel, S. 2; Müller, R. 2; Pfeifer, B. 3; Nagel, S. 1; Hiddemann,
L. 1; Lalk, M. 1 Schweder, T. 1
1Institut für Pharmazie, Universität Greifswald, F.-L.-Jahnstr.17, 17489 Greifswald, Germany
2Department of Chemical and Biological Engineering, Science and Technology Center, Tufts
University, 4 Colby Street, Medford, MA 02155, USA
3Institut für Pharmazeutische Biotechnologie, Universität des Saarlandes, Universitätscampus,
Gebäude C2 3, 66123 Saarbrücken, Germany
Polyketides and non-ribosomal peptides constitute an important class of complex natural
products with wide-ranging therapeutic value. They are produced by multifunctional megaenzymes,
the polyketide synthases (PKSs) and the non-ribosomal peptide synthetase
(NRPS), respectively. Looking for new drugs, especially from unculturable microorganisms,
as many marine bacteria are, heterologous expression of putative PKS and NRPS clusters
becomes more and more important. Hence, there is a need for new hosts that can serve as
alternatives to existing expression systems.
In this study, we investigated Bacillus subtilis’s ability to express heterologous
PKSs/NRPSs. First three putative PKSs/NRPSs (RB386, RB6500 and RB11975) from the
marine planctomycete Rhodopirellula baltica were successfully integrated into the
chromosome of B. subtilis. Although all of the recombinant strains showed positive mRNA
results, overexpression of the heterologous protein could be detected and verified via MS
only for RB11975. HPLC-MS-analyses of cell and medium extracts are being used to
identify specifically produced metabolites.
Beside this, we chose the PKS deoxyerythronolide B synthase (DEBS) as a model gene
cluster since it was already successfully expressed by the heterologous hosts Escherichia
coli and Streptomyces coelicolor. The product, 6-deoxyerythronolide B (6dEB), is the
polyketide core of the antibiotic erythromycin. Due to the complexity of the 30kb large
DEBS-cluster it was necessary to develop a new method for its stepwise chromosomal
integration.
To optimize our expression host for the production of polyketides and non-ribosomal
peptides we performed a knockout of two large clusters: the NRPS-operon srfA (26kb,
coding for surfactin synthetase) and the PKS-cluster pksX (78kb, coding for bacillaene
synthase). Hence, we hope to provide more substrates for the heterologous PKS/NRPS
and to reduce the background for an enhanced detection of produced metabolites.
Metabolite analyses of the mutant strains are being used to elucidate the effect of these
mutants on the physiology of B. subtilis.
066
Fed Stomach Model – a novel test device for biorelevant simulation of
intragastric drug dissolution after food intake
Koziolek, M. 1; Görke, K. 1; Garbacz, G. 1, Weitschies, W. 1
1 Ernst-Moritz-Arndt-Universität Greifswald, Institut für Pharmazie, Biopharmazie &
Pharmazeutische Technologie C_DAT Center of Drug Absorption and Transport, Felix-Hausdorff-
Straße 3, D-17487 Greifswald
Food ingestion is known to alter physicochemical as well as mechanical conditions in the
upper gastrointestinal (GI) tract. Consequently, drug release from solid oral dosage forms
can be affected, which may have an impact on drug plasma levels by changing one or more
pharmacokinetic parameters. To predict food effects on drug release in the human
stomach, we designed an in vitro dissolution device for biorelevant simulation of fed gastric
conditions, the Fed Stomach Model (FSM). This study aimed at the examination of the
technical functionality of the FSM and the evaluation of the individual effects of the factors
applied.
The construction of the FSM is based on USP paddle apparatus. It is equipped with six
specially designed dissolution cells. Media circulation between dissolution cells and the
vessels of USP paddle apparatus is provoked by a peristaltic pump. By the use of stepping
motor and valve system, both controlled via custom-made computer software, the FSM
enables the simultaneous simulation of predefined movement profiles, flow and pressures.
These parameters are exerted by a central axis driven by the stepping motor and
connected to a pressure regulation module. In the present study, the drug release from
diclofenac extended release (ER) tablets in USP phosphate buffer pH 6.8 was investigated
to examine the individual relevance of the factors applied. In this study, the FSM was
operated in closed-loop mode and the amount of drug dissolved was measured on-line by
fiber optics.
It could be demonstrated with the aid of magnetically labeled tablets that the FSM was able
to simulate intragastric transport velocities equivalent to in vivo data generated by our
group [1]. Furthermore, a valve system connected to a pressure regulation module allowed
the application of biorelevant pressures by air inflation or deflation of a balloon. In
preliminary dissolution experiments, we demonstrated that drug release from diclofenac ER
tablets depended on dosage form movement as well as on pressure. It is thus feasible to
apply test algorithms specific for different gastric regions after food intake.
The Fed Stomach Model can be a valuable tool for biorelevant dissolution testing due to its
potential for precise and reproducible simulation of mechanical parameters present under
fed gastric conditions. Media flow including secretion and emptying, movement patterns
and pressures were integrated into test algorithms representing specific conditions in
fundus and antrum. Thus, the intragastric location of solid dosage forms can be considered
in the FSM. This enables a more realistic in vitro investigation of intragastric drug release.
Further experiments are needed to provide detailed information on mixing behavior and
hydrodynamics. In addition, the dynamics of gastric emptying will be considered in certain
test programs.
Acknowledgments: Federal Ministry of Education and Research (GASTROMAX, FKZ 13 N11368-370)
References:
1. Weitschies, W. et al.: J. Contr. Rel. 2005 108(2-3): 375-385.
067
Molecular Dynamics Simulations and MM/GBSA-Calculations of Docking
Poses to predict the native Binding Mode of thieno[2,3-b]pyridines in PfGSK-3
Poll, B. 1; Kruggel, S. 1; Kunick, C. 2; Brandt, W. 2; Meijer, L. 3, Lemcke, T. 1
1 Universität Hamburg, Institut für Pharmazie, Bundesstraße 45, 20146 Hamburg, Germany
2 Technische Universität Braunschweig, Institut für Medizinische und Pharmazeutische Chemie,
Beethovenstraße 55, 38106 Braunschweig, Germany
3 Protein Phosphorylation & Human Disease Group, CNRS, Station Biologique, Place Georges
Teissier, B.P. 74, 29682 Roscoff, France
In addition to HIV and Tuberculosis, Malaria is one of the most prevalent infectious
diseases in developing countries, causing over 200 mio. infections every year [1]. Malaria
tropica, the most serious form of malaria, is caused by Plasmodium falciparum. There are
potent malaria therapeutics on the market, however rapid emergence of resistance and
high costs necessitate the development of new drugs [2].
In cooperation with the University of Braunschweig and the CNRS in Roscoff (France) a
thieno[2,3-b]pyridine derivative has been identified as a potential lead structure to inhibit the
plasmodial Glycogen Synthase Kinase 3 (PfGSK-3) [3,4].
One of the crucial aspects of lead structure optimization is the identification of the correct
binding geometry of putative inhibitors. Homology models of PfGSK-3 have been
developed [5] and were used for docking- and 3D-QSAR-studies [6] as well as guide for
structural variations [4]. Different binding modes have been identified and investigated [6].
We used molecular dynamics simulations and MM/GBSA-calculations of the respective
protein-ligand-complexes considering pre-optimized calculation parameters [7] to further
evaluate the binding poses of a series of thieno[2,3-b]pyridine derivatives 1.
Poster 109
R1
1
The GBSA energies were correlated to the biologically measured IC50 values of the
individual compounds for complexes with the proposed binding mode and give further
evidence of the native binding mode [6]. The results of these investigations will be
presented.
References:
1. WHO, World Malaria Report 2010.
2. Noedl, H. et al.: N. Engl. J. Med. 2008, 359: 2619-2620.
3. Droucheau, E. et al.: Biochim. Biophys. Acta. 2004, 1697: 181-196.
4. Brandt, W.: Dissertation, Braunschweig 2009.
5. Kruggel, S., Lemcke, T.: Arch. Pharm. Chem. Life. Sci. 2009, 342: 327-332.
6. Kruggel, S.: Dissertation, Hamburg 2011.
7. Poll, B. et al.: MM-PBSA and MM-GBSA Calculation for Estimating the Free Energy of Binding of
Glycogen Synthase Kinase-3 Inhibitors 2011. Joint Meeting of the German and Austrian pharmaceutical
societies. Innsbruck. Austria.
068
Pharmacometrics – methodologies, establishment and opportunities of a
promising new discipline in Germany
Minichmayr I1 2; Kloft C1 1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin,
Kelchstr. 31, 12169 Berlin, Germany
2 and Graduate Research Training program PharMetrX, Germany
Background: The emerging science of pharmacometrics represents a multidisciplinary
research field bridging clinical pharmacy, pharmacology, medicine, statistics, engineering
and computational methods [mod. 1]. During the past years, pharmacometrics has been
gaining pivotal importance in drug development, regulatory approval, study design and
therapeutic decision making [2]. The present work presents an overview over different
concepts and methodologies within pharmacometrics, outlines possible applications and
gives examples of past pharmacometric accomplishments. Additionally, the current
establishment of pharmacometrics and educational resources in Germany are presented.
Methods: Seminal papers on pharmacometrics were reviewed. Prevalence of
pharmacometrics research at German Institutes of Pharmacy was assessed by systematic
literature search (PubMed, university homepages).
Results: Pharmacometrics applies computational models of biology, anatomy, physiology,
pharmacology, disease and systems biology to describe and quantify interactions between
drugs and patients [mod. 1]. Ultimately, the qualitative, quantitative and temporal
relationships between drug exposure (pharmacokinetics, PK), drug effects
(pharmacodynamics, PD) and favourable or unfavourable responses in connection with
disease and recovery progression are explored and predicted on a subcellular, cellular,
tissue, organ and organism level.
Various approaches exist to characterise drug-patient interactions, ranging from classical
empirical to more mechanistic pharmacometric models. The most prominent concepts –
often separately used in research – are:
1) Classical drug data analysis comprises single individual analysis and population basedmethodologies,
which allow for e.g. the assessment of both inter- and intra-individual and
unexplained variability. Within the latter, the standard two stage approach requires data rich
situations, whereas non-linear mixed effect (NLME) modelling is also apt for modelling
sparse data, typically obtained in patient care, due to simultaneous analysis of several
individuals.
2) Physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) models do not
start with study data but anatomical and physicochemical drug data, enabling a more
mechanistic simulation of drug profiles in individual tissues and blood. By means of lumping
techniques, a direct link to classical compartment models can be established [3].
3) Systems biology models consider even more detailed knowledge of underlying complex
biologic processes, e.g. cell signal transduction pathways and their subsequent gene
regulatory network.
Despite the widespread potential of pharmacometrics, educational resources (e.g. scientific
societies, textbooks, journal articles) in this field are scarce. At German Institutes of
R2

Pharmacy, pharmacometric modelling skills and competences are basically gained by
postgraduate training within the scope of a PhD (Universities of Berlin, Bonn, Duesseldorf,
Muenster, planned: Saarbruecken) or a structured graduate research training program
(PharMetrx - Pharmacometrics and Computational Disease Modelling at the Freie
Universitaet Berlin and the University of Potsdam, jointly with five industry partners).
Conclusion: Ideally, all three concepts of pharmacometrics should be linked and
combinedly integrated in future research. Given the tremendous progress and value of the
various applications of pharmacometrics in drug development (“model-based drug
development”), regulatory and therapeutic decisions (“model-based patient care”), the
demand for pharmacometricians is substantially growing. To meet the need in Germany,
several universities already provide postgraduate training. Nonetheless, further expansion
of pharmacometric expertise, particularly in undergraduate pharmacy curricula, is desirable.
References:
1. Barrett JS. et al.: J. Clin. Pharmacol. 2008, 48(5): 632-49.
2. Gobburu JV.: J. Clin. Pharmacol. 2010, 50(9 Suppl): 151-157.
3. Pilari S., Huisinga, W.: J. Pharmacokinet. Pharmacodyn. 2010, 37(4): 365-405.
069
Comparison of the ATP-Binding Sites of IR and IGF-1R Kinase Crystal
Structures and their corresponding TrK Homology Model Counterparts
Löhr, J. H. 1; Lemcke, T. 1
1 Universität Hamburg, Institut für Pharmazie, Bundesstraße 45, 20146 Hamburg, Germany
Receptor tyrosine kinases (RTKs) have been pursued as promising targets in cancer
therapy. Transforming extracellular signals into cellular responses, they take part in the
regulation of cell growth and survival. Deregulation of certain RTKs has been linked to
various human tumor conditions and already various small molecule inhibitors have been
approved for therapeutic use.[1]
The insulin-like growth-factor receptor 1 (IGF-1R) and the neurotrophic tyrosine kinase
receptor (TrK) are examples of cancer associated RTKs, where inhibition may prove to be
an effective therapeutic intervention.[2, 3]
With plenty of crystal structures available, structure based design of small molecule
inhibitors of the ATP-Binding site of the IGF-1R kinase is a feasible strategy. However, offtarget
activity on the highly homologous insulin receptor kinase (IR) poses a challenge. In
absence of published three-dimensional models of the TrK, construction and evaluation of
homology models has to precede any structure-based efforts.
Previously, homology models of both active and inactive states of the TrK-1 and TrK-2
kinase domains were built with suitable IR and IGF-1R X-ray structures serving as
templates. Using molecular interaction fields (MIFs) and the GRID/GOLPE approach, the
MIFs were investigated for statistical differences among the different crystal structures of
IR/IGF-1R and the corresponding TrK-1/2 homology models.[4-6] Based on these findings,
geometric and volumetric measurements were evaluated in order to pinpoint structural
differences that emerge from the set of over 30 crystal structures and 10 homology models.
A recently published partly solved crystal structure showing the TrK-3 kinase domain for the
very first time is also taken into account both to assess the homology models and to
highlight structural differences.[7]
References:
1. Arora, A., Scholar, E.M.: J. Pharmacol. Exp. Ther. 2005, 315(3): 971–979.
2. Li, R., Pourpak, A., Morris, S.W.: J. Med. Chem. 2009, 52(16): 4981–5004.
3. Nakagawara, A.: Cancer Lett. 2001, 169(2): 107–114.
4. Goodford, P.J.: J. Med. Chem. 1985, 28(7): 849–857.
5. Kastenholz, M.A. et al.: J. Med. Chem. 2000, 43(16): 3033–3044.
6. Baroni, M. et al.: Quant. Struct.-Act. Relat. 1993, 12(1): 9–20.
7. Albaugh, P. et al.: ACS Med. Chem. Lett. 2012, 3(2): 140–145.
070
Two-dimensional capillary gel electrophoresis (2-D-CGE) as powerful
instrument for quantitation and characterisation of biomolecules
Hahne, T.; Cianciulli, C.; Wätzig, H.
Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig,
Beethovenstr. 55, 38106 Braunschweig, Germany
Capillary gel electrophoresis (CGE) is a highly selective separation technique, used more
and more in pharmaceutical industry for analysis of biomolecules. Thus, CGE is replacing
conventional gel electrophoresis stepwise, because of some advantages such as ease of
handling and time-saving. Even if CGE is a powerful and selective tool for bioanalysis the
selectivity was increased by adding a second dimension. According to our approach the
molecules are separated due to their isoelectric point in the first dimension and then due to
their molecular weights in the following second dimension, as known from classical CGE. In
this study a protein mixture consisting of bovine serum albumin (BSA), ovalbumin, ßlactoglobulin,
myoglobin and a monoclonal antibody was used to investigate the
performance of this innovative separation technique. Reproducibilities, resolutions, signalto-noise
ratios and limits of quantitation with different concentrations of the biomolecules
were investigated.
071
A comparison: Quantification of therapeutically used organic nitrates via LC-
UV and LC-ESI-MS in biological samples after solvent and solid-phase
extraction respectively
Puerstinger, J. 1; Seeling, A. 1
1 Institute of Pharmaceutical/Medical Chemistry, Friedrich-Schiller University, Philosophenweg 12,
07747 Jena, Germany
Pentaerithrityl tetranitrate (PETN), isosorbide dinitrate (ISDN) and glyceryl trinitrate (GTN)
are organonitrate coronary vasodilators and, consequently, used in the treatment of
coronary insufficiency and angina pectoris. Sustained therapeutical effects of PETN and
ISDN are useful in the prophylaxis of attacks of angina pectoris but lead to a slower onset
in comparison to GTN which plays an important role in the management of cardiac
infarction. Furthermore, recent investigation points to a remarkable benefit of organic
nitrates in the therapeutic fields of diabetes mellitus [1] and pulmonary hypertension [2].
Bioactivation via several enzymatic reduction pathways [3] results in less nitrated polyols
which contribute to the vasodilator potency [4].
In order to quantify the therapeutically used organic nitrates and to monitor their
denitrification processes, we developed miscellaneous chromatographic separation
methods to determine PETN, ISDN and GTN as well as their less nitrated metabolites in
biological liquids such as human plasma and whole blood samples using solvent or solidphase
extraction for the segregation of the analytes and, subsequently, UV or ESI-MS for
their detection.
Acknowledgments: Measurements on LC-ESI-MS instruments could be realized in the research group of
Prof Christian G. Huber, Department of Chemistry and Bioanalytics, University of Salzburg, Austria.
References:
1. Wenzel P. et al.: Arterioscler. Thromb. Vasv. Biol. 2007, 27(8): 1729-1735.
2. Daiber A. et al.: Mol. Pharmacol. 2004, 66: 1372-1382.
3. Munzel T., Daiber A., Mulsch A.: Circ. Res. 2005, 97(7): 618-628.
4. Wenzel P. et al.: Br. J. Pharmacol. 2007, 150(4): 526-533.
072
Pulmonary deposition of three market dry powder inhalers using an idealized
pediatric and adult mouth-throat model
Below, A. 1; Bickmann, D. 2; Breitkreutz, J. 1
1 Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, 40225 Düsseldorf,
Germany
2 Boehringer Ingelheim Pharma GmbH & Co. KG, 55216 Ingelheim am Rhein, Germany
The efficiency of aerosol delivery to the patient´s lung depends on three main parameters:
inhalation device, formulation and patient. Marketed inhalation products are in most cases
developed according to the needs of adult patients and less for children. Changes in upper
airway geometry and breathing pattern due to maturation from childhood to adolescent are
usually not considered. Because of respiratory diseases like childhood asthma and cystic
fibrosis even young children have to use inhaled medicines. The efficiency of inhaled
products is especially in preschool children unknown. The complex interaction between
inhaled product and patient is the scope of this study. In a first step the influence of the
upper airway geometry in dependence of age on the performance of three market products
[1-2] was investigated. Afterwards, also the impact of different flow rates was considered.
For the in vitro assessment of particle deposition via next generation impactor (NGI), three
commercially available multidose dry powder inhalers (Salbu Novolizer ® [1], SalbuHexal ®
Easyhaler ® [2], Asmanex ® Twisthaler ®) were connected to two different inlets: idealized
pediatric and adult upper airway model. The pediatric model represents 4-5-year-old
children. In dependence of the device different steady flow rates through the model were
achieved. The inhalation volume was adjusted to 4L (adult model) and 1L (adult and
pediatric model). Deposited mass in the model (extrathoracic deposition) and at the model
outlet (pulmonary deposition) was collected, determined by HPLC and related to label
claim.
An influence of inhalation volume on pulmonary deposition (PD) was observed for the
Novolizer (15% vs. 22% at 75 L/min (4 kPa)). Easyhaler and Twisthaler PD were not
influenced by inhalation volume accordingly. The comparison of adult and pediatric models
has shown higher pulmonary doses using the adult mouth-throat model for all three
products, e.g. Twisthaler 21% vs. 27% (46 L/min (4 kPa)). Different flow rates have an
influence on product performance especially on Twisthaler performance. Using the pediatric
mouth-throat model PD increased from 15% (30 L/min (2 kPa) to 21% (46 L/min (4 kPa)).
Novolizer and Easyhaler performance is very robust; PD is almost the same for all
investigated flow rates.
Physical upper airway models as impactor inlets simulate the human upper airways more
correctly than the USP/Ph.Eur. inlet. Using the adult mouth-throat model higher pulmonary
doses were observed than for the pediatric model. Because there is still a lack of in vivo
data of preschool children using dry powder inhalers in vitro in vivo correlation is not
feasible. If in vivo deposition data will be available in future, the developed model may be
validated. Based on different product performance we believe that for pediatric product
developments child-appropriate models should be used.
References:
1. Below, A. et al.: 18th ISAM conference 2011
2. Below, A.: 8th PBP World Meeting 2012.
073
Biosynthesis of structurally diverse paracylophanes by Vietnamese
cyanobacteria
Preisitsch, M. 1, Neidhardt, I. 1, Pham, T. L. H. 1, Bodtke, A. 2, Niedermeyer, T. H. J. 3, Mundt,
S. 1
1 Institute of Pharmacy, Department of Pharmaceutical Biology, Ernst Moritz Arndt University
Greifswald, Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany
2 Institute of Pharmacy, Department of Pharmaceutical/Medicinal Chemistry, Ernst Moritz Arndt
University Greifswald, Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany
3 Cyano Biotech GmbH, Magnusstraße 11, 12489 Berlin, Germany
Cyanobacteria have been identified as a promising source of new, highly diverse, bioactive
natural products in the research of novel pharmaceutical leads. Especially cyclic peptides
and lipopeptides, alkaloids, lactones, amides, esters, derivatives of fatty and amino acids,
biosynthesized by marine cyanobacteria, represent the largest part of recently isolated
compounds with mainly antibiotic, antifungal, cytotoxic or enzyme-inhibiting activities [1]. In
contrast to these metabolites, naturally occurring paracyclophanes have been isolated less
frequently from cyanobacteria [2].
Bioassay-guided separation of the methanol extract from the Vietnamese freshwater
cyanobacterium Nostoc sp. CAVN 10 resulted in five [7.7]paracyclophanes,
carbamidocyclophanes A-E, including carbamido moieties and a varying substitution
pattern of the chlorinated butyl side chains. Carbamidocyclophanes A-C exhibited cytotoxic
activity against MCF-7 (breast cancer cell line) and Fl cells (human amniotic epithelial cell
line) and moderate antibacterial activity against Staphylococcus aureus (ATCC 6538)[3].
Further screenings for carbamidocyclophanes in various cyanobacteria, in particular in
Nostoc strains, revealed a second carbamidocyclophane producing strain, Nostoc sp.
CAVN 2. HPLC-DAD/IT-TOF-HRMS analysis of separated CAVN 2 cyclophanes resulted in
an identification of already published [2-4] and also previously unknown cyclophanes.
Feeding experiments with Nostoc sp. CAVN 2, using different halogen salts, yielded in the
first-time detection of brominated carbamidocyclophanes.
An optimized, highly rapid one-step extraction and separation procedure for the detection of
110 Poster

carbamidocyclophanes as well as closely related structural analogues and first insights into
the phylogenetic relationship of axenic carbamidocyclophane biosynthesizing
cyanobacteria are presented.
Acknowledgments: The authors thank Dr. M. Meixner for 16S rDNA amplification and sequencing of
investigated strains as well as Mr. S. Heiden for the assistance in the construction of the phylogenetic tree.
References:
1. Singh, R. K. et al.: J. Antibiot. 2011, 64(6):401-412.
2. Chlipala, G. E. et al.: J. Nat. Prod. 2010, 73(9): 1529-1537.
3. Bui, H. T. N. et al.: J. Nat. Prod. 2007, 70(4): 499-503.
4. Kang, H.-S. et al.: Phytochem. 2012, 79(0): 109-115.
074
Cannabinoids stimulate mesenchymal stem cell migration via a mitogenactivated
protein kinase pathway
Schmuhl, E. 1; Ramer, R. 1; Peters, K. 2; Hinz, B. 1
1 Institute of Toxicology and Pharmacology, University of Rostock, Schillingallee 70, D-18057
Rostock, Germany
2 Department of Cell Biology, University of Rostock, Schillingallee 69, D-18057 Rostock, Germany
Mesenchymal stem cells (MSC) are known to be involved in various regenerative
processes such as cardiac, ocular, skin and bone tissue healing. However, little is known
about the pharmacotherapeutical options aiming at tissue healing steps such as the
mobilization and homing of MSC. Here, we show that cannabidiol (CBD), a nonpsychoactive
cannabinoid, stimulates the migration of human adipose-derived MSC in both
Boyden chamber and in vitro scratch wound assays. In Boyden chambers CBD (0.01 – 3
µM) was shown to promote cell migration in a time- and concentration dependent manner.
This promigratory action was inhibited by AM-630 (CB2 receptor antagonist) and by O-1602
(G protein-coupled receptor [GPR] 55 agonist). Moreover, CBD activated the mitogenactivated
protein kinase (MAPK) pathway as evidenced by increased phosphorylation of
extracellular signal-regulated kinase (ERK) 1/2. Blockade of ERK activation by PD98059
prevented CBD-stimulated MSC migration, whereas inhibition of p38 MAPK by SB203580
was inactive in this respect. Furthermore, AM-630 and O-1602 were found to attenuate
CBD-induced ERK activation. An ERK-dependent promigratory action was likewise
demonstrated for the phytocannabinoid Δ9-tetrahydrocannabinol, the GPR55 antagonist O-
1918 and the selective CB2 receptor agonist JWH-133. We conclude that cannabinoids
promote MSC migration via receptor-dependent ERK activation, possibly contributing to
tissue healing.
075
Accuracy temperature control as a meaningful tool for microwave assisted
method development and optimization
Härter A
Anton Paar GmbH, Helmuth-Hirth-Str. 6, 73760 Ostfildern
During the last decade microwave-assisted synthesis has become a powerful tool for
pharmaceutical method development and optimization. Controlled microwave heating under
sealed vessel conditions has - shown to dramatically reduce reaction times, increase
product yields, and enhance product purities by reducing unwanted side reactions
compared to conventional synthetic methods. Modern microwave reactors control and
regulate important parameters such as temperature, pressure and power. Due to this
plurality of control parameters, the essential of accurate temperature for reaction
optimization is often unacknowledged.
We will introduce the basics of optimal temperature measurement, including tips for heating
- microwave transparent solvents, effects due to external cooling, the importance of reliable
stirring and the influence of reactor stability. The concept of accurate temperature control is
outlined, which enables performing optimized protocols in any applicable scale in any
instrument of our modular equipment family without reoptimization. This increases the
efficiency of microwave-mediated synthesis even further, speeding up the entire process of
academic and industrial research.
076
Using sedimentation velocity centrifugation to study the aggregation process
of proteins
Wolff, M. 1 2; Nagel-Steger L. 1 2; Willbold, D. 1 2
1 Heinrich-Heine-Universität, Institut für Physikalische Biologie, 40225 Düsseldorf, Germany
2 Forschungszentrum Jülich, ICS-6, 52425 Jülich, Germany
The pathogenic aggregation of proteins plays an important role in the development of a
number of neurodegenerative diseases. For example in Alzheimer’s or Parkinson’s Disease
the self-assembly of misfolded cellular proteins causes the formation of different oligomeric
structures which finally leads to amyloid fibril formation. In order to identify different
assemblies in the aggregation process we employ the method of sedimentation velocity
(SV) analysis. Using SV analysis gives us the opportunity to study protein size and shape
distributions in solution under defined conditions. The advantages of SV analysis are that 1)
no interaction with liquid or solid phases influences the measurement as known for other
separation techniques like e.g. size exclusion chromatography, and 2) that due to the
fractionation power of the method large aggregates do not impair the results as in light
scattering methods.
One of the most important targets in Alzheimer’s therapy is the 39 to 43 amino acids long
proteolytic fragment of the amyloid precursor protein, called amyloid β peptide for its
property to form amyloid fibrils. Beginning with the characterization of the hydrodynamic
properties of the monomeric Aβ peptide preparations, we will analyze early steps in
aggregate formation in order to identify small Aβ assemblies of defined size, which are
significantly populated in freshly prepared Aβ solutions. With this approach we want to gain
insight into mechanistic details of the aggregation process. Moreover this approach is
aimed at the identification of novel drug targets for Alzheimer’s disease therapy.
077
New Furanoid and seco-Labdanoid Diterpenes isolated from Leonurus
cardiaca L.
Brückner, A. 1; Hennig, L. 2; Rauwald, H. J. 1
1University of Leipzig, Department of Pharmaceutical Biology, Johannisallee 21-23, D-04103
Leipzig, Germany
2University of Leipzig, Department of Chemistry, Institute for Organic Chemistry, Johannisallee 39,
D-04103 Leipzig, Germany
Continuing our search for possible cardioactive single constituents of primary and refined
antiarrhythmic extracts of Leonurus cardiaca L. [1] we report herewith the isolation of three
new diterpenes besides known substances, like leosibiricin, sitosterol and stigmasterol [2].
The structures of isoleosibirin, leopersin F and 7-epi-leopersin F were determined by 1d/2d
1H/ 13C NMR spectroscopical experiments. These labdane type diterpenes have been found
only once in other plants of the genus leonurus, namely in the related Lamiaceae Leonurus
sibiricus and Leonurus persicus [3, 4]. Thus these compounds may prove useful as
analytical marker substances as well as possible pharmacologically active compounds, as
for example shown for the vasorelaxant activity of similar diterpenes isolated of Marrubium
vulgare [5].
References:
[1] Ritter, M et al.: Planta Med. 2010, 76: 572-582.
[2] Knöss, W.: Plant Cell Reports 1995, 14: 790-793.
[3] Tasdemir, D.; Wright, A.D.; Sticher, O.: J. Nat. Prod. 1996, 59: 131-134.
[4] Savona, G.; Piozzi, F.; Rodriguez, B.: Phytochemistry 1982, 21 (11): 2699-2701.
[5] El Bardai, S.; Morel, N.; Wibo, M.: Planta Med. 2003, 69: 75-77.
078
Characterization of the pharmacokinetic properties of visnagin and Ammi
visnaga water extract following oral administration in rats
Haug, K. G. 1; Weber, B. 1; Hochhaus, G. 1; Butterweck, V. 2
1 Department of Pharmaceutics, College of Pharmacy, University of Florida, 1600 SW Archer
Road, Gainesville, FL,32610, USA
2 Institute for Pharma Technology, School of Life Sciences, University of Applied Sciences
Northwestern Switzerland, Gruendenstrasse 40, 4132 Muttenz, Switzerland
The furanochromone visnagin is one of the main compounds of Ammi visnaga L. (syn.
Khella, Apiaceae) with potential effects on kidney stone prevention. After determination of
the pharmacokinetic (PK) properties of visnagin after intravenous bolus administration in
rats [1], the aim of the present study was the evaluation of the PK properties of visnagin
and an aqueous Ammi visnaga extract (AVE) after oral administration in rats. In two
separate experiments three doses of visnagin (2.5, 5, and 10 mg/kg) solubilized in 25%
Captisol® and three doses of AVE (standardized on visnagin and containing equivalent
amounts of visnagin) were administered by oral gavage to male Sprague-Dawley rats,
respectively. Plasma samples were extracted and subsequently analyzed using a validated
LC-MS/MS method [1]. Plasma concentration-time profiles (Figure 1) were explored by
non-compartmental analysis. Visnagin plasma exposure (median AUClast and AUCinf) was
significantly increased for all three doses (up to 10-fold for the low dose) when administered
as extract compared to the pure agent (Figure 2). For both the AVE and the pure
compound, AUClast and AUCinf increased disproportionately with an increase in dose (Figure
1 & 2). Visnagin resided significantly longer in the body when given in form of AVE with up
to three times longer median MRTlast and MRTinf for the low dose. Cmax values after AVE
administration were elevated and occurred at later time points in comparison to equivalent
doses of pure visnagin. The terminal half-life increased with dose for both AVE and pure
visnagin, reaching a maximum value of 1.94 h for the 10 mg/kg pure compound group.
In conclusion, exposure of visnagin is enhanced after extract administration and could
result in a superior efficacy of AVE compared to an equivalent dose of visnagin.
Figure 1: Visnagin plasma concentration (geometric mean � SEM) vs. time profiles after
administration of the pure compound (a) and AVE (b), sorted by dosing group (Low: 2.5
mg/kg, Medium: 5 mg/kg, High: 10 mg/kg).
Figure 2: AUCinf (a) and AUClast (b) (median + SD) of visnagin after administration of pure
compound vs. AVE, sorted by dosing group (Low: 2.5 mg/kg, Medium: 5 mg/kg, High: 10
mg/kg), numbers represent Bonferroni adjusted p-values, AUCinf values that are based
upon extrapolated AUC > 50% were excluded.
Reference:
Haug, K.G. et al.: Eur J Pharm Sci 2012, 45 (1-2): 79-89
079
Synthesis and biological studies on novel water-soluble gold(I)alkynyl
complexes as antitumor agents and thioredoxin reductase inhibitors.
Andermark, V. 1; Meyer, A. 1; Ott, I. 1
1 Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig,
Beethovenstraße 55, 38106 Braunschweig, Germany
Poster 111

Besides the use of gold(I) complexes, i.e. auranofin, as disease modifying antirheumatic
drugs (DMARDs)they are also known for their potential as antitumor drugs and highly
efficient inhibition of thioredoxin reductase (TrxR)[1], an enzymewhich is overexpressed in
tumor cells.TrxR contains a selenocystein in the active site, making it an excellent target for
soft lewis acids like gold(I)-complexes[2]. Inhibition of TrxR leads to increased reactive
oxygen species (ROS) levels, cell cycle arrest and can induce apoptosis [3].
This work bases on recent results, which show that phosphinegold(I)alkynyl complexes can
inhibit TrxR in the nanomolar concentration range and have anti-angiogenic properties in
zebrafish embryos[4].To improve the water-solubility of the phosphinegold(I)alkynyl
complexes the bulky, hydrophobic triphenylphosphine-ligand of the most active complexes
will be replaced by more polar phosphine ligands, while the most promising alkynyl ligands
will be kept.
The tri-(2-furyl)phosphinegold(I)chloride (3)complex is prepared by the reaction of
equimolar amounts of dimethylsulfidegold(I)chloride (1) and tri-(2-furyl)phosphine (2).
Afterwards 3 is reacted with an equimolar amount ofan alkyne (4) in basic conditions,
yielding aphosphinegold(I)alkynyl(5) complex (scheme 1). Synthesized compounds are
confirmed by 1H, 13C, 31P NMRand their purity is evaluated by elemental analysis.
The cytotoxicity of the synthesised complexes, as well asthe metal free alkynes and
phosphinesis evaluated using HT-29 colon carcinoma cells and MCF-7 breast cancer cells
in the colorimetric crystal violet assay. Additionallycurrent results onTrxR inhibition,
glutathione reductase inhibition and cellular uptake into tumor-cells will be presented.
Scheme 1.Synthesis of the compounds 3 and 5 as an example for the general synthetic
route.
Acknowledgments: Financial support bythe Deutsche Forschungsgemeinschaft (DFG, project FOR630)is
gratefully acknowledged.
References:
1. Ott, I.: Coord. Chem Rev.2009, 253: 1670-1681.
2. Gromer, S. et al.: J. Biol. Chem. 1998,273(32): 20096-20101
3. Urig, S., Becker, K.: Semin.Cancer Biol. 2006,16: 452-465
4. Meyer A. et al.:Angew. Chemie2012accepted
080
Influence of opioid receptor activation and inhibition on synaptic transmission
under hypoxia
Bloßfeld, M. 1; Merkenschlager, A. 2; Nieber, K. 1
1 University Leipzig, Institute of Pharmacy, Pharmacology for Natural Sciences, Talstraße 33,
04103 Leipzig, Germany
2 Universitätsklinikum Leipzig, Klinik und Poliklinik für Kinder und Jugendliche, Liebigstraße 20a,
04103 Leipzig
Activation of delta opioid receptors by [D-Ala2, D-Leu5] enkephalin (DADLE) has been
reported to have neuroprotective effects on rat embryonic cortical neurons and PC12 cells
under hypoxia. A neurotoxic impact has also been shown for micromolar concentrations
[1,2]. There are limited information about effects of naloxon, a mu receptor antagonist,
during hypoxia. The aim of the present study was to examine the influence of DADLE and
naloxone on the amplitude of electrically evoked postsynaptic potentials (PSPs) under
normoxic (95% O2, 5% CO2) and hypoxic (1% O2, 5% CO2, 94% N2) conditions. The
experiments were done using intracellular recordings on cortical pyramidal cells in brain
slices of adult rats. DADLE (1 µM) decreased the amplitude of PSPs. Naloxone (100 µM)
inhibited synaptic transmission in neurons from 10 week old rats whereas in neurons of
brain slices of rats older than 10 weeks no influence could be found. Naloxone (100 µM)
inhibited completely the effect of DADLE (1µM). Both components had no influence on
input resistance and membrane potential. Exposure of the slices to hypoxia (5 min) resulted
in a decreased amplitude of PSPs. DADLE (1 µM), superfused 5 min before hypoxia, did
not alter the hypoxia-decreased PSPs. Contrary, naloxone (100 µM) antagonised the PSP
amplitude reduction. This effect was age-independent. We conclude that during hypoxia
naloxon (100µM) but not DADLE (1µM) normalizes synaptic transmission as an indicativ of
a neuroprotective effect.
References:
1. Su, D.S. et al.: Neurosci. Lett. 2007, 423, 113-117
2. Sun K., Su D.S. and Wang X.: Brain Res. 2009, 1292, 100-106
081
Glutathione transferase isoenzymes in corneal tissues, human cornea
construct and corneal cell lines
Kölln C1; Reichl S1 1 Institut für Pharmazeutische Technologie, Technische Universität Carolo-Wilhelmina zu
Braunschweig, Mendelssohnstr. 1, 38106 Braunschweig, Germany
The human cornea represents the main barrier for active drug substances from ophthalmic
formulations. Therefore, excised cornea and corneal cell culture models are used for in vitro
drug absorption studies. However, little is known about drug metabolism during
transcorneal passage. The purpose of this study was to determine expression and activity
of glutathione transferases (GST) in human corneal cell lines and human cornea construct
in comparison to excised animal corneas. Isoenzymes GSTO1 and GSTP1 are supposed
to be considered in more detail.
Presence of GST isoenzymes in corneal cell lines, cornea construct and excised corneal
tissues was determined by western blot and immunohistochemical staining using specific
antibodies against isoenzymes GSTO1 and GSTP1. In this study corneal cell lines HCE-T
(epithelial), HCK-Ca (stromal) and HENC (endothelial) were investigated as well as tissue
engineered human cornea construct [1, 2]. Porcine and rabbit corneas were studied in
comparison. The activity was measured by spectrophotometrically methods. Total GST
activity was profiled using 1-Chloro-2,4-dinitrobenzene (CDNB) [3], specific GSTO1 activity
by using S-(4-Nitrophenacyl)-glutathione [4] and specific GSTP1 activity was investigated
using ethacrynic acid [5].
The presence of glutathione transferases in human cornea construct and all corneal cell
lines was verified on protein level by western blot and immunohistochemical staining,
respectively. In case of corneal cell lines, activity of investigated isoenzymes seems to be
comparable in HCE-T and HCK-Ca, whereas total GST activity was found to be highest in
HCE-T. In comparison, HENC showed lowest activity in all studies. Excised rabbit and
porcine corneas showed higher specific GSTP1 and GSTO1 activity as well as total GST
activity compared to human cornea construct.
Known to be present in human cornea, glutathione transferase activity is detectable in all
investigated human corneal cell lines, as well as in human cornea construct and excised
animal corneas.
Acknowledgments: We are grateful to the German Federal Institute for Risk Assessment (BfR), which
funded this work under grant no. 3-1328-30652054369.
References:
1. Hahne M, Reichl S: Int J Pharm. 2011, 416(1): 268-279.
2. Hahne M et al.: J Pharm Sci. 2012, accepted
3. Habig WH, Pabst MJ, Jakoby WB: J Biol Chem. 1974, 249(22): 7130-7139.
4. Board PG et al.: Anal Biochem. 2008, 374(1): 25-30.
5. Habig WH, Jakoby WB: Methods Enzymol. 1981, 77: 398-405.
082
Novel inhibitors of the arginine methyltransferase PRMT6
Wagner, T. 1; Jung, M. 1
1 Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79110
Freiburg, Germany
Background
Arginine protein methyltransferases (PRMTs) represent a SAM dependent class of protein
modifying enzymes that are able to mono- and dimethylate the guanidino group of specific
arginine residues within certain protein substrates e.g. histone proteins, p53, FOXO
transcription factor and other protein modifying enzymes where PRMTs usually methylate
glycine-alanine-arginine patches (GAR motifs). The human family of PRMTs consists of
eleven members (PRMT1-11) which are devided into two classes. Type I
methyltransferases are able to methylate arginine residues in an asymmetrical manner and
type II methyltransferases catalyse the formation of symmetrically substituted arginine
residues. PRMTs 1–4, 6 and 8 are type I enzymes and PRMTs 5, 7 and 9 are PRMTs of
type II. [1] PRMTs are involved in the regulation of various physiological processes like
apoptosis, cell differentiation, metabolism, DNA recombination and HIV tat transactivation.
Thus, PRMT inhibitors are interesting potential compounds for drug discovery. [2]
Inhibitor search is highly dependent on high throughput screening (HTS) which relies on
suitable assay systems. Here we used a new homogeneous bead based in vitro assay
system for screening new PRMT6 inhibitors. Therefore we surveyed some known PRMT1
inhibitors that have been identified in our group previously.
Material and Methods
A method for the expression and purification of PRMT1 was set up in order to be able to
screen larger amounts of compounds as potential inhibitors of this enzyme. Compounds
have been screened and PRMT1 inhibitors have been identified by the use of an ELISA
based, heterogeneous assay system (Delfia) in our group previously. In order to test the
selectivity of the PRMT1 inhibitors towards other arginine methyltransferases we used an
alphaLISA based method for exploring the enzyme PRMT6. Therefore a biotinylated
peptide fragment of the histone protein H3 (amino acids 1-21) is used as substrate for the
enzyme PRMT6. This peptide is able to bind to a streptavidine coated donor bead which
contains phthalocyanine as a photosensitizer which converts ambient oxygen to singlet
oxygen upon excitation with light at a wavelength of 680 nm. In the case of the peptide
being methylated by PRMT6 at the arginine residue at position 2 of the peptide after
addition of the cofactor S-adenosylmethionine (SAM) an antibody directed against this
modification and coated to acceptor beads is able to bind to the modified substrate but not
to the unmodified state. Thereby donor and acceptor beads are brought into close proximity
so that the singlet oxygen emitted by the donorbeads is able to convert a thioxene
derivative on the acceptor beads. The addition product degrades with chemoluminsecence
which finally leads to the excitation of the lanthanide europium which is also present in the
acceptor beads. The latter can be detected by time resolved fluorescence readout in an
EnVision plate reader (commercial detection system by PerkinElmer) at a wave length of
615nm.
Results
We were able to profile PRMT inhibitors against PRMT6 by using a new alphaLISA based
assay system. Most compounds inhibit both PRMT1 and PRMT6 in low micromolar
concentrations. But we could further show selectivity for PRMT1 towards PRMT6 for some
compounds. We also identified one compound that seems to inhibit PRMT6 selectively
compared to PRMT1. The presented assay protocols are also suitable for HTS of larger
compound libraries.
References:
1. Bissinger, E.-M. et al.: Med. Chem. Commun. 2010, 1(2): 105–172.
2. Bissinger, E.-M. et al.: Bioorg. Med. Chem. 2011, 19(12): 3717–3731.
083
The estrogen receptor as a target for anabolic effects of phytoecdysteroids in
mammals
Haupt, O1); Tchoukouegno Ngueu, S1); Diel, P1); Parr, MK1 2)
1 Center for Preventive Doping Research, German Sport University, Cologne, Germany
2 Institute of Pharmacy, Freie Universität Berlin, Germany
Phytoecdysteroids, such as ecdysterone (chemical structure in figure 1), are structurally
similar or identical to insect-moulting hormones and have been detected in various plant
species. In mammals ecdysteroids are reported to produce a range of effects including the
stimulation of protein synthesis and physical performance. Therefore, ecdysterone is
promoted as active component in several over-the-counter supplements and may be
misused by athletes to increase muscle mass.
The anabolic-androgenic potency of ecdysterone was studied in-vivo. In a Hershberger
assay analogue design male Wistar rats were treated for 21 days either with 5 mg/kg
ecdysterone s.c. or placebo. After necropsy, the weights of the prostate, seminal vesicle
and levator ani muscle were measured. The muscle fiber size of the soleus muscle and the
gastrocnemius muscle was determined. The anabolic effect of ecdysterone resulted in an
112 Poster

increase of the fiber size in both muscles. An androgenic effect could not be observed.
In order to elucidate the molecular mechanism involved in the anabolic activity of
ecdysterone, cell culture experiments were conducted using the mouse skeletal muscle cell
line C2C12. Differentiation of C2C12 cells towards myotubes was induced. The resulting
myotubes were incubated in the presence of ecdysterone, dihydrotestosterone (DHT) and
antiandrogenic or antiestrogenic substances, such as flutamide (an antiandrogen) and ZK
191703 (an antiestrogenic substance). Additionally co-incubation of with ecdysterone and
the antagonists was performed. Cells were fixed and myotube diameters were determined
by glutaraldehyde-induced autofluorescence and morphometry.
The treatment of the cells with ecdysterone resulted in an increased myotube-diameter.
This stimulation could be antagonized by an antiestrogen, which suggests an estrogen
receptor mediated mechanism. Flutamide, an anti-androgen, could not antagonize this
effect, which supports our hypothesis that the anabolic effect of ecdysterone is mediated by
an estrogen receptor mediated pathway.
In summary these observations are relevant for the development of new strategies for the
treatment of skeletal muscle injuries such as sarcopenia and cachectic diseases but also to
provide scientific data that allow for the classification of ecdysterone in preparations.
Currently in Germany a categorization either as novel food or as pharmaceutical is
discussed.
Figure 1: Chemical structure of ecdysterone
084
Regulation of 5-lipoxygenase gene expression by AF4 and MLL as
transcriptional regulators
Ahmad, K. 1; Marschalek, R. 2; Steinhilber, D. 1
1 Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main,
Germany
2 Institute of Pharmaceutical Biology, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
Besides its function in inflammation, recent findings support a role of 5-lipoxygenase (5-LO)
as critical regulator of leukemia, creating a further link between inflammation and cancer
[1]. The mechanism of this interaction still remains unclear.
5-LO which is encoded by the ALOX5 gene is an enzyme that catalyses the first two steps
in the biosynthesis of the pro-inflammatory leukotrienes derived from arachidonic acid [2]. It
is known that ALOX5 gene expression is controlled by 1,25-dihydroxyvitamin D3 (calcitriol)
and transforming growth factor β (TGFβ) [3]. The resulting upregulation of the 5-LO gene
expression is mediated by the binding of TGFβ effector proteins SMADs and the vitamin D
receptor (VDR) to response elements within the 5-LO gene and is due to transcript
elongation [4,5].
Concerning the transcript elongation it is described that the AF4 (ALL-1 fused gene on
chromosome 4) protein is involved in the regulatory process of the transcript elongation and
exhibits chromatin remodelling activities [6]. Furthermore the chromosomal translocation of
the AF4 gene and the MLL (mixed lineage leukemia) gene leads to the production of der4
(AF4-MLL) and der11 (MLL-AF4) fusion proteins. These fusion proteins are also associated
with the pathomechanism of leukemia [7].
To elucidate the involvement of AF4, MLL and their fusion proteins on the 5-LO gene
expression, reporter gene assay experiments were performed. Thus different 5-LO reporter
gene constructs were cotransfected with AF4, MLL, der4 or der11.
The results revealed that both AF4 and MLL are involved in the control of 5-LO gene
expression as transcriptional regulators. Furthermore the presented data suggest that der4
and der11 are able to deregulate the 5-LO gene expression either in the step of transcript
initiation or at the step of transcript elongation. Thus, MLL and its fusion proteins provide a
potential link between 5-LO and leukemia development.
Acknowledgments: Hans Kröner - Graduiertenkolleg
References:
1. Chen, Y. et al.: Nat Genet. 2009, 41(7): 783-92.
2. Rådmark, O. et al.: Trends Biochem Sci. 2007, 32(7): 332-41
3. Brungs, M. et al.: Proc. Natl. Acad. Sci. USA 1995, 92(1): 107-11
4. Seuter, S., Sorg, BL., Steinhilber, D.: Biochem Biophys Res Commun. 2006, 348(4): 1403-10
5. Stoffers, KL. et al.: J Mol Biol. 2010, 395(4): 884-96
6. Bitoun, E., Oliver PL., Davies, KE.: Hum Mol Genet. 2007, 16(1): 92-106
7. Gaussmann, A. et al.: Oncogene. 2007, 26(23): 3352-63
085
In vitro study of colonic microbial metabolization and absorption kinetics of
olsalazine in the TIM-2 model simulating the conditions in the proximal large
intestine
Glöckl G1;Venema K2;Wilson CG3; Weitschies W1 1Institute of Pharmacy, Ernst Moritz Arndt University, Greifswald, Germany
2TNO Healthy Living, Zeist, The Netherlands
3Institute of Pharmacy and Biomedical Sciences, Strathclyde University, Glasgow, UK
It is well-known that the colonic epithelium expresses a lot of transporters, either for uptake
or efflux, as well as metabolizing enzymes. The epithelial metabolism is superimposed by
the metabolic action of the microbiota colonizing the human colon. The complex interplay
between these processes is the origin of the limited understanding of the colonic absorption
process. The latter is of special importance when administering standard oral extended
release formulations.The microbial metabolic activity is difficult to investigate in vivo
(location) as well as in vitro (anaerobic environment). Therefore, TNO’s in vitro model of the
large intestine (TIM-2) wasdesignedto mimic the conditions in the proximal part ofthe
human colon [1].
Some prodrugs, e.g. for the medication of chronic inflammatory bowel diseases, are
specifically cleaved in the colon by means of microbial reducing enzymes. The aim of the
presented study is to investigate in vitrohow the azo-prodrugolsalazineis cleaved by
faecalmicrobiota and further absorbed using the TIM-2 model.
The computer controlled TIM-2 system consists of glass tubes and flexible walls allowing
the content to be mixed in a peristaltic movement. The device is continuously flushed with
nitrogen generating an anaerobic environment and kept at body temperature. The pH is
adjusted to 5.8 by adding NaOH. The system is further equipped with a set of hollow fiber
membranes allowing the fermentation products (predominately short chain fatty acids) to be
eliminated from the system. Therefore, a dialysis liquid containing electrolytes and vitamins
in a phosphate buffer pH 5.8 is pumped through the system. The volume of the luminal
content is kept at 120 mL using an integrated level sensor.For a continuous measurement
of the luminal redox potential an electrode (InLab ® Redox, Mettler) was integrated in a
specially designed glass compartment.
The system is inoculated with standard pooled faecalmicrobiota obtained from healthy
volunteers. For feeding, a standard ileal efflux medium mainly consisting of peptides and
polysaccharides is continuously delivered to the system. Each day 25 mL of luminal
contentare withdrawn and replaced by dialysis liquid to simulate passage of stool.
Olsalazine was utilized as model drug to evaluate the metabolic function of the microbiota.
The prodrugolsalazine is cleaved by microbial azoreductases to form two molecules of the
active mesalazine. Both compounds can easily be quantified with UV/Vis spectrometry in
the lumen as well as in the dialysate.
After addition of 500 mg of olsalazineto the TIM-2 system via the sampling port we could
measure the elimination of olsalazine and the appearance and subsequent elimination of its
metabolite mesalazine. The elimination rate was inversely proportional to the molecular
weight and thus faster for mesalazine.
The integration of a redox electrode into the system allowed the continuous measurement
of the intraluminal redox potential. After an initial drop of the potential in the growth phase
of the microbiota(adaptation period of about 16 hours) a strongly reducing environment was
generated (stable ecosystem maintained for at least 48 hours). During this period, the
system is suitable to simulate the metabolization and absorption of the model drug
olsalazine and probably of other actives. In future studies we will further check if these data
can be correlated with in vivo data.
Acknowledgments: G.G. thanks W. Borst, A. Maathuis, J. Lelieve d and M. Minekus (TNO) for their
assistance and GalenusPrivatstiftung for a personal grant. The project was funded by the German federal
ministry of education and research (InnoProfile, FKZ: 03IP612).
References:
1. Minekus, M. et al.:Appl. Microbiol. Biotechnol.1999, 53(1): 108-114.
086
Development of long-term stable hydrogel matrices for a vessel-simulating
flow-through cell
Semmling, B. 1; Seidlitz, A. 1; Nagel, S. 1; Schütt, R. 1; Brand, T. 1; Grabow, N. 2; Sternberg, K. 2;
Weitschies, W. 1
1 Institute of Pharmacy, EMA University of Greifswald, Felix-Hausdorff-Straße 3, 17487 Greifswald,
Germany
2 Institute for Biomedical Engineering, University of Rostock, Friedrich-Barnewitz-Straße 4, 18119
Rostock, Germany
Natural polymeric gels are often unstable and very sensitive to erosion. Therefore synthetic
polymeric materials, gelling agents such as polyvinylalcohol (PVA) or polyacrylamide (PAA)
are interesting due to various properties: stability, elasticity, diffusibility, hydrophilicity, nontoxicity.
Modifications of the composition and the cell entrapment in the hydrogel are
feasible. Drug dissolution as well distribution processes of coated drugs of drug-eluting
stents (DES) were determined in the vessel-simulating flow-through cell (vessel-simulating
FTC) [1]. The current study focuses on the approach of developing long-term stable
hydrogel matrices for the vessel-simulating FTC by replacing the conventional polymer
matrix (2 % (w / w) sodium alginate matrix, AM) by 15 % (w / w) PVA-hydrogel matrices
(PVA-M), 10 % (w / w) PAA matrices (PAA-M) and 2 % (w / w) agarose hydrogel matrices
(Ag-M). The distribution coefficient (DC) of the fluorescent model drug substance
triamterene between the hydrogel and aqueous media, long-term stability of the novel
hydrogel compartments and drug distribution between the three compartments: hydrogel,
dissolution media and stent were investigated.
The distribution coefficient of the model substance between the investigated long-term
stable hydrogel matrices and dissolution media indicate differences in dependency on the
respective hydrogel matrix (data Ag-M and PAA-M not shown): Compared to the reference
matrix (DC of AM after 72 h was found to be about 1.4) the drug delivered into the hydrogel
compartment accumulated up to 2.3 (after 72 h, PVA-M). Test results of the examination of
the long-term stability of novel hydrogel matrices show no signs of gel erosion within 4
weeks perfusion (data not shown) indicating mechanical long-term stability of the novel
hydrogels under typical test conditions.
Release profiles from triamterene coated DES (Eudragit ® RL / RS 30 / 70, dissolution
media PBS 7.4, dissolution temperature 37 °C ± 0.5 °C, flow rate 35 mL / min [2]) obtained
with the vessel-simulating FTC using the novel matrices (PAA-M, PVA-M and Ag-M)
indicate a fast increase in the amount of dissolved drug in the dissolution media (Ag-M:
82 % after 12 h) with a simultaneous fast decrease in residual drug amount in the DES
coatings (Ag-M: 14 % after 12 h). After 12 hours 4 % of the total drug amount was detected
in the agarose matrices. In comparison to release profile of fluorescent model drug using
Ag-M, a slightly different dissolution profile of drug release using the reference hydrogel
matrix (AM) could be observed. After 12 hours about 77 % of the total drug amount eluted
from DES into dissolution media was detected. The residual drug amount in stent coating
increases continuously and was found to be about 18 % after 12 hours. At the same
sampling point the total drug amount in the sodium alginate hydrogel compartment
accumulated up to 5 %. The difference in detected drug amount in the compartments
dissolution media, hydrogel (AM, Ag-M, PAA-M and PVA-M) and DES may be attributed to
different distribution coefficient of triamterene between the hydrogel compartments and
PBS 7.4.
In conclusion, the methods of hydrogel matrix preparation (PAA-M, PVA-M and Ag-M) were
established successfully. The stability studies confirmed sufficient mechanical robustness
of the matrices under long-term test conditions. Additionally, the developed matrices for the
vessel-simulating FTC enable the determination of delivered drug not only in dissolution
media representing the blood, but also in hydrogel matrices simulating the vessel wall as
target organ structure. Consequently, the use of the gel compartment allows the adaption of
the in vitro test closer to the physiological situation and therefore enables the estimation of
the drug distribution of DES under more realistic test conditions.
Poster 113

Acknowledgments: Financial support by the Federal Ministry of Education and Research (BMBF) within
REMEDIS “Höhere Lebensqualität durch neuartige Mikroimplantate” (FKZ: 03IS2081) is gratefully
acknowledged.
References:
1. Neubert, A. et al.: J. Control. Release 2008, 130(1): 2-8.
2. Di Mario, C et al.: Am. J. Cardiol. 1993, 71(14): 54D-61D.
087
New titanium(IV) complexes as potential anticancer drugs
Schur, J. 1; Manna, C.M. 2; Tshuva, E.Y. 2; Deally, A. 3; Tacke, M. 3; Köster, R. 4, Ott, I. 1
1 Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstr. 55, 38106
Braunschweig, Germany
2 The Hebrew University of Jerusalem, Department of Inorganic Chemistry, 91904 Jerusalem,
Israel
3 University College Dublin, UCD School of Chemistry and Chemical Biology, Belfield, Dublin 4,
Ireland
4 Zoological Institute, Department of Cell Physiology, TU Braunschweig, Spielmannstr. 8, 38106
Braunschweig, Germany
Since the serendipitous discovery of the antitumor agent cisplatin in the 1960s, research on
metal-based compounds has made remarkable progress. Nowadays, platinum-based
metallodrugs (e.g. oxaliplatin) play a major role in medical anticancer treatment.
Nevertheless, the activity is limited to a small spectrum of cancers because of increasing
resistance phenomena of these drugs and several side effects caused by high toxicity.
Therefore, in the last two decades, research has been extended to non-platinum
metallodrugs.
An innovative compound in the 1990s was the titanium-based metallodrug titanocene
dichloride that reached clinical trials but failed because of decreased hydrolytic stability and
formulation problems [1]. Based on these promising works and with the aim of improving
cytotoxicity and hydrolytic stability better stabilized titanium-containing compounds e.g.
titanocene Y and a new class of non-Cp diamino bis(phenolato) “salan” Ti(IV) complexes
were recently developed and seem to hold a high potential for anticancer therapy [2,3].
Figure 2: Metallodrugs: (a) Oxaliplatin, (b) Titanocene dichloride, (c) Titanocene Y, (d)
Salan-Ti(IV) complex
In our study the mentioned lead compounds were investigated biologically in comparison to
titanocene dichloride. Very interesting properties for the novel class of salan-Ti(IV)
complexes (see figure 1) and titanocene Y (as a substituted titanocene) could be obtained
and may be a step forward in target identification of titanium-based anticancer drugs.
Figure 3: Zebrafish embryos (94 hpf) exposed to 100 µM Salan-Ti(IV)
The lead compound of new diamino salan-Ti(IV) complexes and titanocene Y have an
improved cytotoxicity in the low micromolar range and a higher accumulation in cancer cells
compared to titanocene dichloride. A relationship between the antiproliferative activity, the
cellular uptake and the protein binding ability of the tested Ti-containing compounds was
noted. In addition, the toxicity of the new titanium-based complexes in zebrafish embryos [4]
was studied and no toxic effects could be observed up to a concentration of 100 µM (see
figure 2). Further investigations were performed on the DNA-binding efficacy, the nuclear
drug content and the drugs accumulation in mitochondria. Our results indicate that novel
titanium(IV) complexes possibly have overcome the disadvantages of the well known
titanocene dichloride, which underlines their high potential as anticancer drugs.
References:
1. Melendez, E., Crit. Rev. Oncol. Hematol. 2002, 42: 309–315.
2. Sweeney, N.J. et al., J. Organomet. Chem. 2005, 690: 4537–4544.
3. Peri, D. et al., Chem. Eur. J. 2009, 15: 2403–2415.
4. Parng, C. et al., ASSAY Drug Dev. Technol. 2002, 1: 41–48.
088
Anti-infectives with novel mode of action: Interruption of P. aeruginosa cell-tocell
communication by PqsD inhibitors
Maurer, C. K. 1; Storz, M. P. 1; de Jong, J. C. 1; Weidel, E. 1; Zimmer, C. 1; Steinbach, A. 1;
Hartmann, R. W. 1 2
1 Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123 Saarbrücken,
Germany
2 Department of Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3,
66123 Saarbrücken, Germany
The opportunistic pathogen P. aeruginosa controls the coordinated production of virulence
factors as well as biofilm formation via a cell density dependent cell-to-cell communication
system known as quorum sensing (QS) [1]. The pqs QS system employs the signal
molecules PQS and its precursor HHQ to activate PqsR [2], a transcriptional regulator that
drives the expression of many genes involved in pathogenicity.
PqsD is a key enzyme in the biosynthesis of HHQ and PQS [3]. Since reduction of HHQ
production is accompanied by reduced mortality in infected mice [4], PqsD proves to be an
interesting target for drug discovery.
Two different drug design strategies were applied: (A) a ligand based approach including
analogues of the natural substrate anthraniloyl-CoA (ACoA) and mimics of the
corresponding transition state as well as (B) the development of compounds derived from
known inhibitors of FabH, an enzyme, which is structurally and functionally related to PqsD
[5,6]. Thereby, inhibitors with IC50 values in the nM to low µM range have been identified.
SPR experiments revealed, that, while (2-nitrophenyl)methanol compounds derived from
approach (A) resulted in time-dependent irreversible PqsD inhibition with binding within the
anthraniloyl binding site, 2-benzamidobenzoic acid derivatives from (B) are reversible
inhibitors that do not occupy the anthraniloyl binding site.
The effect of representative compounds from both classes on the extracellular levels of the
signalling molecules HHQ and PQS in P. aeruginosa PA14 was investigated. While
2-benzamidobenzoic acids had no significant effect, (2-nitrophenyl)methanol derivatives
strongly reduced extracellular HHQ and PQS levels without affecting cell viability.
References:
1. Dubern, J. F.; Diggle S. P.: Mol. BioSyst. 2008, 4: 882.
2. Cao, H. et al.: Proc Natl Acad Sci USA 2001, 98(25): 14613-8.
3. Pistorius, D. et al.: ChemBioChem. 2011, 12(6): 850-3.
4. Lesic, B. et al.: PLoS Pathogens 2007, 3: 1229.
5. Bera, A. K. et al.: Biochemistry 2009, 48: 8644-8655.
6. Nie, Z. et al.: J. Med. Chem. 2005, 48: 1596-1609.
089
A Vascular function of Cyclin dependent kinase 5 (Cdk5)
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.
M. 1, Zahler S. 1
1Center of Drug Research, Ludwig-Maximilians University, Munich, Germany
2Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany
3Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, Texas
4Department of Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, Münster,
Germany
Cyclin-dependent kinase 5 (Cdk5) is essential for CNS development and function. Whereas
its role in neurons has been studied in detail, there still exists only limited knowledge about
its functions in other systems. During recent years, more and more reports indicated
functions of Cdk5 in non-neuronal tissues and we could show that Cdk5 regulates
endothelial cell migration in vitro.
This work elucidates an important function of Cdk5 in the vascular system in vivo. We
specifically disrupted the Cdk5 gene in the mouse endothelium using the Cre-loxP system.
Floxed Cdk5 mice were intercrossed with mice expressing Crerecombinase under control
of the Tie2 promoter (Tie2Cre) or a tamoxifen-inducible VE-Cadherin promoter
(Cdh5(PAC)-CreERT2), respectively. Breedings of Cdk5fl/wtTie2Cre males and Cdk5fl/fl
females resulted in offspring in which only 15% pups harbored the Cdk5fl/flTie2Cre
genotype instead of expected 25% and most of the endothelial-specific Cdk5 knockout
mice did not survive past postnatal day 28. Analysis of the genotypes of embryos at
different stages indicated that Cdk5fl/flTie2Cre embryos frequently died during late
embryogenesis between E16.5 and E18.5. Endothelial-specific Cdk5 knockout leads to a
non-separation phenotype between blood and lymphatic system including edema
formation, blood-filled and dilated lymphatic vessels, defective lymphatic valve formation,
and impaired draining function of the lymphatic system. Concerning blood vessels, we
found a hypervascularization of endothelial-specific Cdk5 knockout mice. This was
indicated by an increased number of branching points in the yolk sacs and skin of E16.5
Cdk5fl/flTie2Cre embryos and increased branching and sprouting in the developing retina
of d6 pups with Cdk5fl/flCdh5(PAC)-CreERT2 genotype after tamoxifen treatment as well
as after pharmacological inhibition of Cdk5 with the small molecule roscovitine. Most
importantly, we found a reduction of tumor growth of subcutaneous implanted wildtype
B16F1 melanoma xenografts in endothelial-specific Cdk5 knockdown mice. Histological
analysis of tumors showed an immature vascularization of tumors grown in
Cdk5fl/flCdh5(PAC)-CreERT2 mice.
To sum up, our results reveal an important vascular function of Cdk5 in angiogenesis and
lymphangiogenesis, suggesting Cdk5 as druggable target for anti-angiogenic therapy.
090
A novel assay for the analysis of inhibitors of ergosterol biosynthesis
Staudacher V; Müller C; Bracher F
Ludwig-Maximilians-Universität München, Department für Pharmazie - Zentrum für
Pharmaforschung, Butenandtstr. 5-13, 81377 München, Germany
Systemic fungal infections are of increasing importance in intensive care, due to the rising
portion of immunocompromised patients (cancer, AIDS, and organ transplantation patients)
in clinical routine. These infections are characterized by a very high mortality, and very
potent and fast-acting antifungal agents are needed for life-saving therapy. Moreover,
resistance to established antifungal drugs (e.g. fluconazole) gains in clinical significance.
Ergosterol is the central sterol in fungal metabolism, and inhibition of ergosterol
biosynthesis represents a classical, but still highly effective strategy in antifungal therapy.
Over the decades, inhibitors of several enzymes in ergosterol biosynthesis have been
marketed, the most prominent ones being the azole-type inhibitors of the enzyme C14demethylase.
The morpholine antifungal amorolfine inhibits two enzymes in ergosterol
biosynthesis, but is not suitable for systemic application, and the squalene epoxidase
inhibitors (naftifine, terbinafine) mainly target dermatophytes.
Notably, the post-squalene part of ergosterol biosynthesis contains a significant number of
additional enzymes which occur only in fungi, but not in mammals, and therefore are
potential targets for new, selective antifungal drugs. In our ongoing research on the
development of new antifungals [1] we focused on new inhibitors of enzymes in ergosterol
biosynthesis. In order to perform effective optimization of first lead structures, there was a
need to work out a screening system which enables us to (a) identify the target enzyme in
ergosterol biosynthesis unambiguously, and (b) gain quantitative data on the effect of the
inhibitors on overall ergosterol biosynthesis.
For this purpose we developed a convenient whole-cell screening assay, which in a first
step enables us to identify substances, which interfere with enzymes of the post-squalene
part of ergosterol biosynthesis, and in a second step to determine IC50 values for inhibition
114 Poster

of ergosterol biosynthesis.
In this assay three different yeasts, Candida glabrata, Saccharomyces cerevisiae and
Yarrowia lipolytica, are treated with test substances. After alkaline cell lysis the sterols are
extracted with an organic solvent, and the crude extract is purified by dispersive solid phase
extraction (dSPE). After silylation the sterols are analyzed by GC-MS.
The obtained sterol patterns provide an indication of the inhibited enzyme(s), since
inhibition of defined enzymes leads to the accumulation of specific sterols. By this way we
can identify inhibitors of any enzyme in the post-squalene part of ergosterol biosynthesis in
one single cellular assay, with no need for having the single purified enzymes in hands.
Furthermore, we extended this qualitative screening method to a quantitative determination
of the effect on total ergosterol biosynthesis. In analogy to a screening system worked out
by us for inhibitors of cholesterol biosynthesis recently [2], incubation with the inhibitors in
the presence of 13C-acetate, followed by GC-MS analysis of the labeled ergosterol isotopes
allows for the convenient determination of IC50 values of the inhibitors.
The system was calibrated with several known antifungals targeting ergosterol
biosynthesis, and meanwhile applied to the identification of the target enzymes of a number
of new inhibitors of ergosterol biosynthesis.
References:
1. Renard D, Perruchon J, Giera M, Müller J, Bracher F: Bioorg. Med. Chem. 2009, 17(23) 8123-8137.
2. Giera M, Plössl F, Bracher F: Steroids 2007, 72(8): 633-642.
091
Cysteine-rich LIM-only protein 4 (CRP4) opposes angiotensin II induced
pathological heart hypertrophy and fibrosis
Straubinger, J.; Majer, M.; Ruth, P.; Lukowski, R.
Institute of Pharmacy, Department of Pharmacology, Toxicology and Clinical Pharmacy, University
of Tübingen, Germany
Background: Cardiac hypertrophy is an adaptive response of the heart to many cardiovascular
disorders including hypertension, infarction and defects of the valves. Although
physiological heart hypertrophy, which refers to an adaption of the heart muscle to healthy
exercise or pregnancy, and pathological hypertrophy share common features, only the
pathological remodelling of the heart leads to fibrosis, increased diastolic stiffness, fatal
failure and death. Clinical studies and functional analysis of genetically modified mice have
established that natriuretic peptides (NP) like ANP and BNP and their cardiac receptors
diminish development of pressure and volume induced heart hypertrophy and fibrosis via
the second messenger cyclic guanosine-3´,5´-monophosphate (cGMP). However, the
signaling events downstream of cGMP and its major effector cGMP kinase I (cGKI) are less
clear. Here, we studied the role of the cysteine-rich LIM-only protein (CRP4) for the cardiac
response to an increase in afterload and healthy exercise training. CRP4 is directly targeted
by cGKI in the cardio-vascular system and a highly related homologue of the muscle LIM
protein CRP3/MLP. CRP/MLP knockout mice develop a dilated cardiomyopathy and
defects in CRP3/MLP are the cause for dilated and hypertrophic cardiomyopathies in
human.
Methods: Gene-targeted CRP4 knockout (KO) mice and their heterozygous (HET) and wild
type (WT) littermates were subjected to a chronic pressure-dose of angiotensin II (AngII).
Cardiac hypertrophy, as defined by the heart-to-body weight ratios (HW/BW), was
determined in AngII treated mice and compared to littermate animals that received saline or
were trained by a duration-controlled swimming exercise protocol to induce a physiological
adaption of the heart muscle. Changes in hypertrophy marker genes, putative effects of
AngII on components of the NP/cGMP pathway and the expression pattern of other
members of the CRP protein family were analyzed in total mRNA and protein isolated from
the ventricles. These experiments were corroborated by the localization of CRP4 in the
myocardium and Sirius red stainings as a quantitative measure of fibrosis.
Results: Under basal conditions and upon AngII infusion CRP4 mRNA and protein were
detectable in the myocardium from WT mice, whereas CRP4 was absent from KO hearts
and significantly reduced in HET mice. HW/BW ratios of all three genotypes were not
different at baseline, but increased in HET and KO animals in response to infusion of AngII.
Sirius red staining and quantitative RT-PCR experiments revealed an increase in interstitial
fibrosis and a diminished production of anti-fibrotic factors such as BNP in HET and KO
hearts. In contrast, neither the increase in cardiac mass nor the extent of fibrosis were
altered upon physiological growth of the heart muscle between the different genotypes.
Conclusion: Hearts of CRP4 KO mice are more susceptible to AngII-induced remodelling,
whereas the lack of CRP4 does not alter physiological cardiac hypertrophy. Therefore, we
conclude that CRP4 is a beneficial mediator in NP/cGMP/cGMP kinase I signaling to
oppose pathological heart growth and fibrosis induced by cardiac Gαq-pathways.
092
Comparison of binding requirements in a series of benzimidazol-2-yl-aminosubstituted
(L)-amino acids by simple QSAR models for the NMDAR glycineB
site and the transporter LAT1
Krauß, A. 1; Rotmann, A. 2 3; Martiné, U. 2; Closs, E. I. 2; Dannhardt, G. 1
1 Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University, Staudingerweg 5,
55128 Mainz, Germany
2 Department of Pharmacology, Johannes Gutenberg-University, Obere Zahlbacher Straße 67,
55101 Mainz, Germany
3 Department of Parasitology, Ruprecht Karls-University, Im Neuenheimer Feld 324, 69120
Heidelberg, Germany
Central bioavailability is a major concern in the development of new ligands targeting the
glycine binding site of the NMDA receptor, as potential applications comprise inter alia
epilepsy, chronic pain, dementia (Alzheimer’s disease, Parkinson’s disease) or
schizophrenia [1]. Uptake by amino acid transporters [2], especially large neutral amino
acid transporters (system L), is therefore considered as a versatile strategy to cross the
blood-brain barrier without disruption. The concept was proven for the low affinity glycineB
site antagonist 4-chlorokynurenine as reviewed in [3], and recently applied to analogues of
pregabalin [4] and analogues of melphalan [5].
This work is based on a congeneric series of benzimidazol-2-yl-amino-substituted (L)amino
acids obtained in a seven-step linear synthesis and assayed for NMDAR glycineB
site binding (displacement of [ 3H]-MDL-105,519 from pig cortical membranes) as well as
system L activity (competition with [ 3H]-L-Phe uptake in C6 rat glioblastoma cells). Two
corresponding QSAR models have been derived, utilizing GraphPad Prism 3.00, GraphPad
Instat 3.06 and NLREG 6.4. for stepwise multiple (non)linear regression. The NMDAR-
QSAR suggests a parabolic relationship with STERIMOL parameter L and, likewise
negative but linear, correlations with Swain and Lupton R and the hydrogen bond indicator
variable HD. The LAT-QSAR discloses an increasing lipophilicity (ClogP) and a decreasing
pka value (of the guanidino group, mainly experimental) as favourable for affinity. Taken
together, within a restricted applicability domain, parallel steric resp. lipophilic demands
dominate a certain opposite trend in the electronic effects. Semi-empirical quantum
mechanical calculations with Spartan 04 (Wavefunction, Inc.) at the HF 3-21G(*) level [6]
may explain these electronic prerequisites partly by =N---HN intramolecular hydrogen
bonding, this proving advantageous for the NMDA receptor, but disadvantageous for LAT.
However, the pronounced conformational flexibility of the scaffold allows for various binding
modes, each with the free amino acid moiety as the most important and common motif.
Moreover, compared to the rather specific and sterically demanding interactions at the
glycineB site of the NMDAR [7], the system L-type transporters are known for their broad
substrate profile, ranging from large neutral (LAT1) to large and small neutral (LAT2) amino
acids [8]. A joint further development of compounds considering both targets is therefore
regarded as possible.
Acknowledgements: The VCCLAB (Virtual Computational Chemistry Laboratory, http://www.vcclab.org) is
appreciated as a fast and user-friendly access to calculated log P values, enabling a comparison of different
methods [9].
References:
1. Bauman, A. et al.: J. Label. Compd. Radiopharm. 2011, 54(10): 645–656.
2. Closs, E. I. et al.: J. Nutr. 2004, 134(10): 2752S–2759S.
3. Kohl, B. K.; Dannhardt, G.: Curr. Med. Chem. 2001, 8(11): 1275–1289.
4. Belliotti, T. et al.: J. Med. Chem. 2005, 48(7): 2294–2307.
5. Matharu, J. et al.: Bioorg. Med. Chem. Lett. 2010, 20(12): 3688–3691.
6. Krauß, A.; Dannhardt, G.: Joint Meeting of the Czech, German and Hungarian Pharmaceutical Societies,
2006, Marburg, Germany Abstract C045, p.115.
7. Jansen, M.; Dannhardt, G.: Eur. J. Med. Chem. 2003, 38(10): 855–865.
8. Verrey, F. et al.: Pflügers Arch. - Eur. J. Physiol. 2004, 447(5): 532–542.
9. Tetko, I. et al.: J. Comput. Aid. Mol. Des. 2005, 19(6): 453–463.
093
A comparative study of in vitro methods analyzing endothelial barrier function:
conventional macromolecular permeability measurements versus
sophisticated impedance sensing
Hornburger, M. C. 1; Mayer, B. A. 1; Vollmar, A. M. 1; Wegener, J. 2; Fürst, R. 1
1 Department of Pharmacy, Center for Drug Research, Pharmaceutical Biology, University of
Munich, Butenandtstr. 5-13, 81377 Munich, Germany
2Institute for Analytical Chemistry, Chemo- & Biosensors, University of Regensburg, Universitätsstr.
31, 93053 Regensburg, Germany
Endothelial barrier dysfunction, i.e. the opening of interendothelial junctions, and the
consequent formation of edema are hallmarks of many severe diseases, such as sepsis,
acute lung injury, or atherosclerosis. Drugs that specifically target endothelial barrierregulating
systems are lacking and there is a strong demand to identify new
pharmacological targets. For the validation of these targets, the barrier function of cultured
endothelial cells is analyzed by two different approaches: 1) the classic measurement of
macromolecular permeability and 2) the modern real-time cell impedance-sensing methods,
which employ alternating currents to analyze changes of cellular morphology. Impedance is
a complex physical quantity consisting of an imaginary capacitive (cell membrane
properties) and a real resistive (intercellular junction stability) contribution.
We aimed at deciphering the most appropriate method for the characterization of
endothelial barrier-modulating agents. Therefore, we compared macromolecular
permeability measurements (two-compartment Transwell ® system, membrane pore size 0.4
μm, tracer: FITC-dextran 40 kDa) with three different commercially available impedance
sensing devices: (i) ECIS ® (Applied Biophysics) and (ii) xCELLigence (Roche), with the
cells directly grown on gold electrodes, and (iii) cellZscope ® (nanoAnalytics), where cells
are placed on a permeable membrane (pore size 0.4 μm) between two electrodes.
First, we tested for the correlation between macromolecular permeability and impedance
measurements. Therefore, we employed the barrier-disruptive mediators TRAP (thrombin
receptor-activating peptide), histamine, and TNF-α, as well as the barrier-stabilizing
compound forskolin (adenylyl cyclase activator) and the Rho kinase (ROCK) inhibitor Y-
27632. The impact of TRAP and forskolin on endothelial barrier function nicely correlated in
all tested systems: forskolin elevated impedance values and reduced dextran flux, whereas
TRAP evoked a fast and sustained decrease of impedance and a significant increase in
macromolecular permeability. Interestingly, the histamine-induced barrier breakdown could
not be detected by the Transwell ® assay, whereas a rapid and transient decrease of
endothelial impedance occurred. The barrier-protecting ROCK inhibitor evoked a massive
and long-lasting decline of impedance, whereas dextran flux was again not affected.
Second, we compared the three impedance systems in order to disclose individual
advantages or limitations. A key feature of the ECIS ® system is the opportunity to break
down impedance in its resistive and capacitive contributions and, moreover, to discriminate
betweenchanges of cell-cell and/or cell-matrix junctions. In contrast, xCELLigence only
provides information on the overall impedance magnitude, internally referenced and
normalized ascell index. Independent of the applied barrier-modulating stimulus, both
systems returned essentially similar results with respect to the time course of the cell
layers’ integral ionic permeability. The filter insert-using cellZscope ® device, however,
reached its limit of sensitivity due to the low overall resistance of the endothelial cells under
study (transendothelial electrical resistance< 5 Ω cm²).
In conclusion, impedance sensing is a very sensitive, label-free and easy-to-use method to
monitor changes in cellular morphology in real-time. Alterations in endothelial permeability
are associated with these morphological changes, which are reflected by different ion fluxes
in impedance sensing and by macromolecular fluxes in the Transwell ® assay. For the
evaluation of new drug targets in endothelial barrier regulation, we therefore recommend to
be aware of these differences and to combine impedance sensing with measurements of
macromolecular permeability, since each method alone cannot fully determine all
consequences of barrier-modulating agents.
094
A novel mouse model for studying the in vivo functions of the sodiumactivated
potassium channel Slack (Slo2.2)
Bausch A1; Hausmann M1; Baranski J1, Meyerdierks N1, Ruth P1 §, Lukowski R1 §,
Poster 115

1 Institute of Pharmacy, Department of Pharmacology, Toxicology and Clinical Pharmacy,
University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany
§ These authors contributed equally to this work.
Background: Sodium-activated potassium channels of the Slack (Slo2.2) subtype are
abundantly present in many neurons [1]. On a cellular level Slack channels might be
involved in action potential repolarisation, setting the resting membrane potential in some
neurons, slow after hyperpolarisation, burst firing and adaptation of firing rate during high
frequency firing thereby shaping neuronal activity and excitability [1]. Although the
molecular properties of Slack channels have been investigated in detail [2] their exact in
vivo-functions remain elusive. Evidence suggests that Slack may play an important role for
physiological processes such as spatial hearing [3], olfaction [4] and nociception [5]. In
addition to physiological functions Slack channels have been indicated to exert control over
pathophysiological processes as cytoprotection following ischemic injury [2,6].
Methods: To investigate the function of the sodium-activated potassium channel Slack
(Slo2.2) in vivo we recently generated knockout mice (KO) with a targeted deletion of the
pore forming exon. Additionally, using the Cre/loxP recombination system, we have
established a mouse line that carries a “floxed” version of the pore forming exon and
therefore enables a conditional (spatial and temporal) inactivation of the Slack gene.
Reverse-Transcription-PCR (RT-PCR) and real-time-PCR were used to analyse the
expression pattern and levels of Slack transcripts and transcripts of highly related
potassium channels in Slack KO and littermate WT animals. Slack protein expression
pattern was assessed by immunohistochemistry (IHC) and Western Blot using our novel
anti-Slack-antibodies. Initial tests of the hypothesis that the Slack KO mice show a motor
phenotype were performed by analysis of footprint patterns and the ability of Slack KO mice
and their WT littermates to maintain balance in the “beam walk test”. Olfactory functions
were investigated using food-finding (“buried pellet test”), innate preference and avoidance
tests.
Results: Slack KO mice are viable, fertile and do not show gross phenoptypical
abnormalities under physiological conditions except for a delayed body-weight gain during
the first 6 to 8 weeks after birth. Using RT-PCR we could confirm the absence of the pore
region in Slack transcripts of Slack KO mice in all brain regions tested (e.g. cerebellum,
neocortex and olfactory bulb). These results were confirmed by IHC analysis, which further
demonstrated the absence of the Slack protein in 12-µm serial cross-sections of Slack KO
brains. Although a high Slack-mRNA-level and Slack immunoreactivity were detected in
brain regions that are involved in motor coordination Slack KO mice did not show an
impaired motor function. All measures in the footprint pattern tests and performance results
of the beam walk test were not different between Slack KOs and WT littermates.
Furthermore, strong Slack-immunoreactivity was detected in the olfactory bulb with the
highest protein density in the mitral cell layer and the outer plexiform layer containing the
dendrites of the mitral cells. Together with the decrease in body weight of young Slack KO
mice these findings raised the possibility that an olfactory disorder caused impaired
suckling of the newborns. Indeed, the retrieval time for Slack KO mice to recover the buried
pellet in the food-finding test was significantly prolonged compared to age- and littermatched
WT mice.
Conclusion: Our preliminary results indicate that Slack deficiency does not cause any
gross abnormalities under physiological conditions. However, Slack ablation in sensory
olfactory neurons and/or second-order neurons (e.g. periglomerular cells, mitral cells) of the
olfactory bulb may be associated with mild olfactory dysfunctions. Together, we provide a
powerful novel tool to study the functions and molecular details of Slack channels in a preclinical
relevant model in vivo. Our ongoing and future investigations of these mice will help
us to address the question whether Slack channels potentially constitute novel drug targets
for stroke, epilepsy, ataxia or olfactory disorders.
References:
1. Bhattacharjee, A. and L.K. Kaczmarek: Trends Neurosci. 2005, 28(8): 422-8.
2. Yuan, A. et al.: Neuron 2003, 37(5): 765-73.
3. Yang, B., R. Desai, and L.K. Kaczmarek, J Neurosci. 2007, 27(10): 2617-27.
4. Lu, S. et al.: J Neurophysiol. 2010, 103: 3311–3319.
5. Tamsett, T.J., K.E. Picchione, and A. Bhattacharjee, J Neurosci, 2009, 29(16): p. 5127-34.
6. Wojtovich, A.P. et al.: PLoS One 2011. 6(12): e28287.
095
Expression analysis of drug transporter proteins in RPMI 2650 cell line and
excised human nasal mucosa
Dolberg, A. M. 1; Reichl, S. 1
1 Institut für Pharmazeutische Technologie, Technische Universität Carolo-Wilhelmina zu
Braunschweig, Mendelssohnstraße 1, 38106 Braunschweig, Germany
The nasal mucosa is an interesting site for application of drugs with systemic action, since it
has a number of advantages as a delivery route, including easy accessibility, extensive
vascular supply and avoidance of gastrointestinal degradation as well as first pass
metabolism. Therefore more and more applications for nasal systemic drug delivery have
been developed during the last decades, and their number is still increasing [1].
In the development of drugs for nasal application, identification and characterization of the
different uptake and efflux systems are necessary. P-glycoprotein (P-gp) and multidrug
resistance-associated proteins (MRP) are classified as ATP binding cassette (ABC)
transporters based on their sequences, organisation of the ATP-binding domains and efflux
function. ABC proteins represent a large family of integral membrane transporters that
utilize the energy of ATP hydrolysis to carry specific substrates across membranes [2]. The
solute carrier gene (SLC) superfamily encodes another large family of membrane-bound
transporters, located in almost every cellular and organelle membranes. Proteins of the
SLC family include passive transporters, ion-coupled transporters and exchangers [3].
A comparison was performed between excised human nasal mucosa from turbinectomy
surgeries and our in vitro model based on immortalized human nasal epithelial cells (RPMI
2650) concerning the mRNA expression of different efflux and uptake transporter proteins.
First investigations to prove functionality of different ABC transporters in RPMI 2650 cells
was carried out using permeation studies and uptake assays.
The mRNA expression of eight ABC transporters as well as 13 SLC transporters of excised
human tissue and RPMI 2650 epithelial model were investigated and compared. The
results showed promising similarity in the possible presence or absence of these efflux and
influx transporters in human tissue and the in vitro model. A similarity between RPMI 2650
models and human nasal mucosa has already been shown for barrier properties to
evaluate passive drug permeation [4].
To verify the functionality of the first transporter protein, P-gp, bidirectional transport studies
using rhodamine 123 with RPMI 2650 cells grown on polyethylene terephthalate filter were
performed. Indirect immunofluorescence staining for P-gp was also carried out. The
findings confirmed the assumption that P-gp is not functionally expressed in these epithelial
cells.
An uptake assay using 5(6)-carboxy-2′,7′-dichlorofluorescein as a substrate for MRP1 and
MRP5 was carried out. The result indicated functional expression of these efflux
transporters in RPMI 2650 cells. Identification and localization of the responsible
transporter proteins have to be proved by immunohistochemistry.
Further investigations will be focused on western blot, immunohistochemical analysis and
permeation as well as uptake studies using specific substrates for active transport to verify
the protein expression and functionality of further drug transporters in human nasal mucosa
and in vitro model based on RPMI 2650 cell line.
Acknowledgments: We are grateful to the German Federal Institute for Risk Assessment (BfR), which
funded this work under grant no. 3-1329-469. Furthermore, the authors thank Prof. Dr. Schroeder, Dr.
Schmidt, Städtisches Klinikum Braunschweig and Dr. Reintjes, HNO-Praxis Schlosscarree Braunschweig for
the supply of human nasal mucosa specimens.
References:
1. Dimova, S. et al.: Toxicology In Vitro 2005, 19: 107–122.
2. Jones, P.M., George, A.M.: Cell. Mol. Life Sci. 2004, 61: 682–699.
3. He, L., Vasiliou, K., Nebert, D.W.: Human Genomics 2009, 3: 195–206.
4. Wengst, A., Reichl, S.: Eur. J. Pharm. Biopharm. 2010, 74: 290–297.
096
A new heterogeneous assay for measuring NAD + -dependent histone
deacetylase (sirtuin) activity
Schiedel, M. 1; Jung, M. 1 2
1 Institute of Pharmaceutical Science, Albert-Ludwigs-Universität, Albertstr. 25, 79104 Freiburg,
2 Freiburg Institute of Advanced Studies (FRIAS)
Background
Sirtuins represent a specific NAD +-dependent class of histone deacetylases (HDACs). By
using NAD + as a cofactor, these enzymes cleave off the acetyl groups from the epsilonamino
group of lysines in histones and other proteins, e.g. p53, FOXO proteins, p300 or
HIV tat. The human family of sirtuins consists of seven members, which are distributed to
different cell compartments and involved in the regulation of various physiological
processes like apoptosis, cell differentiation, metabolism, DNA recombination and HIV tat
transactivation. Thus, sirtuin inhibitors are interesting potential drugs for drug discovery [1].
Inhibitor search is highly dependent on high throughput screening (HTS) which relies on
suitable assay systems. We introduced a new heterogeneous assay based on fluorescence
detection of Ac-p53-TAMRA, a known nonhistone substrate for sirtuins [2].
Material and Methods
Ac-p53-TAMRA is a fluorescence-labelled and biotinylated fragment of the
tumorsuppressor p53. Because the immobilized Ac-p53-TAMRA, which is bound on
streptavidin microplates, is not able to serve as Sirt-substrate, we set up a preincubation in
1 mL Epi-tubes. After three hours at 37 °C the reaction is stopped and trypsin is added,
which hydrolyzes only the deacetylated substrate. In the next step the test samples are
dispensed on a streptavidin-coated microplate in order to immobilize the biotinylated
substrate and to wash away the unbound components, including the fluorescence-tagged
cleavage product of tryptic digestion. Finally the amount of remaining uncleaved but still
biotinylated and TAMRA-labelled substrate is determined by fluorescence readout.
Results
We were able to establish a new nonisotopic, heterogeneous assay for NAD +-dependent
histone deacetylases, which enables in-vitro-testing of library components with intrinsic
fluorescence or quenching properties, that are prone to false-negative or –positive results in
homogeneous fluorescence based assay systems. The presented detection protocol is
suitable for HTS.
References:
1. Trapp, J., Jung, M.: Curr Drug Targets 2006, 7(11): 1553-1560.
2. Milne, J.C. et al.: Nature 2007, 450(7170): 712-716.
097
Influence of different solvents on the applicability of a small intestinal
microdialysis system in vivo
Schönherr, D. 1; Hanke, U. 1; Siegmund, W. 2; Becker, D. 3; Weitschies, W. 1
1University of Greifswald, Center of Drug Absorption and Transport, Institute of Pharmacy, Felix-
Hausdorff-Straße 3, 17487 Greifswald, Germany
2University of Greifswald, Center of Drug Absorption and Transport, Institute of Pharmacology,
Felix-Hausdorff-Straße 3, 17487 Greifswald, Germany
3Novartis Pharma AG, Technical Research & Development, Novartis Campus, WSJ-177.4.14,
4056 Basel, Switzerland
For better understanding the mechanisms of drug absorption after oral drug administration
a new small intestinal microdialysis system was developed. To characterize the influence of
different solvents on the applicability for in vivo studiesseveral experiments were
performedin vitro.
The small intestinal microdialysis system is made up of a minimodule which is built up of 30
hollow fiber capillaries which consist of a polysulfone membrane (Fresenius Polysulfone ®)
and a tubing system with an inflow, an outflow, and a tube for separating samples via air
bubbles. The applicability of the system was tested with paracetamol (acetaminophen) as
model substance using phosphate buffer USP pH 6.8, hydrochloric acid pH 1.2 and a
biorelevant buffer pH 6.8 as solvent.For all experiments phosphate buffer USP pH 6.8
served as perfusion medium as well. Two pumps were used to transport the samples
(300 µL) after an intended equilibration time and to produce air bubbles separating the
samples in the outflow tube. To examine the sensitivity of the system, the minimodule was
inserted into a chamber filled with solvent. After pre-incubation, a solution of the drug
substance was added (resulting theoretical concentration of 0.02 and 2.0 g/L).
Subsequently, solvent was added to dilute the drug solution (resulting theoretical
concentration of 0.01 and 1.0 g/L). Drug concentrations were calculated using UV
spectroscopy and a previously determined minimodule scaling factor.
Drug concentrations as well as concentration changes over time were successfully
monitored for both drug concentrations and all investigated solvents. The functional
applicability of the microdialysis system at both pH values was proven.
The suitability of the new small intestinal microdialysis system to monitor concentration
changes over time in vitro was demonstrated for different solvents. The applicability of the
system in vivo will be examined in the near future.
116 Poster

We thank Fresenius Medical Care GmbH for providing the hollow fiber capillaries. Financial support from
Novartis Pharma AG is gratefully acknowledged.
098
Identification of benzimidazole derivatives as inhibitors of leukotriene
biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein
(FLAP)
Luderer, S. 1, Banoglu, E. 2, Altenhofen, W. 3, Gerstmeier, J. 4, Pergola, C. 4, Werz, O. 4
1 Department of Pharmaceutical Analytics, Pharmaceutical Institute, Eberhard-Karls-University
Tuebingen, Auf der Morgenstelle 8, D-72076 Tuebingen, Germany
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Etiler, 06330
Yenimahalle, Ankara, Turkey
3 Chemical Computing Group, AG, Kaiser-Wilhelm-Ring 11, 50676 Koeln, Germany
4 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University
Jena, Philosophenweg 14, D-07743 Jena, Germany
Leukotrienes (LTs) are lipid mediators formed from arachidonic acid (AA) by the enzyme 5lipoxygenase
(5-LO). 5-LO converts AA from membrane phospholipids to LTA4, aided by
the 5-lipoxygenase-activating protein (FLAP). LTA4 is further metabolized to LTB4 and
cysteinyl-LTs (cys-LTs) by LTA4 hydrolase or LTC4 synthase [1]. LTs are key mediators in
inflammatory, allergic and cardiovascular diseases, but only Zileuton as direct 5-LO
inhibitor (Zyflo CR®) and Cys-LT receptor antagonists (e.g. Montelukast, Singulair ©) are
currently available on the market and used for the treatment of asthma.
One promising target for pharmacological suppression of leukotriene biosynthesis is 5lipoxygenase-activating
protein (FLAP). Here, we applied a virtual screening targeting
FLAP based on a combined ligand- and structure-based pharmacophore model which led
to the identification of 8 compounds (see below) as potential FLAP inhibitors [2].
Compounds 1, 2 and 3 barely inhibited 5-LO product formation with IC50 values > 10 µM
both in a cell-based and a cell-free system. Compounds 4, 5, 6 and 8 reduced 5-LO product
synthesis in intact neutrophils, but also inhibited recombinant 5-LO suggesting a direct
interference with 5-LO. In contrast, the benzimidazole derivative 7 potently suppressed LT
formation in intact neutrophils (IC50=0.31μM) but essentially failed to inhibit 5-LO in cell-free
systems suggesting an interaction with FLAP. Compound 7 was also active in a human
whole blood assay, and acted anti-inflammatory in an in vivo model of acute inflammation,
i.e. the carrageenan-induced pleurisy in rats. For structural optimization, a series of further
benzimidazole-based derivatives were synthesized leading to more potent analogues with
IC50 values between 0.12-0.19 μM. Our results disclose the benzimidazole scaffold bearing
as a new chemotype for further development of anti-leukotriene agents.
References:
1. Radmark, O., et al., 5-Lipoxygenase: regulation of expression and enzyme activity. Trends Biochem Sci,
2007. 32(7): p. 332-41.
2. Banoglu, E., et al., Identification of novel benzimidazole derivatives as nhibitors of leukotriene
biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein (FLAP). Bioorg Med Chem,
2012. 20(12): p. 3728-41.
099
The fourth molybdenum enzyme mARC: Expression and N-reductive activity in
different cell lines
Jakobs H. 1; Reichmann D. 2; Bittner F. 2; Mendel R. 2; Clement B. 1;Havemeyer A. 1
1Department of Pharmaceutical / Medicinal Chemistry, Pharmaceutical Institute, Christian-
Albrechts-University of Kiel, Gutenbergstraße 76, 24118 Kiel, Germany
2Department of Plant Biology, Technical University of Braunschweig, Humboldtstraße 1, 38106
Braunschweig, Germany
mARC (“mitochondrial amidoxime reducing component”) is a mammalian molybdenum
enzyme that was discovered in our lab [1]. The enzyme is located in the outer mitochondrial
membrane and together with the electron transport proteins cytochrome b5 and its NADHdependent
reductase it is capable of reducing N-hydroxylated compounds such as the
model substrate benzamidoxime [2,3].This is actually where the enzyme got its name from.
Besides amidoximes, the enzyme system is also able to reduce other N-hydroxylated
structures such as guanidines and sulphonamides and is thus involved in reductive drug
metabolism[2]. For this reason it can be employed for the activation of
N-hydroxylatedprodrugs using e.g. the prodrug principle of “amidoximes instead of
amidines” [4]. Moreover, the mARC containing enzyme system is also able to reduce
N-hydroxylated DNA-bases[2], which could be a mechanism of detoxification.However, the
endogenous function of mARC is still unknown. The human genome codes for two mARC
proteins, referred to as mARC1 and mARC2 [5].
Our studies upon tissue distribution carried out with porcine material revealed highest
expression levels for mARC in liver, thyroid and kidney [6].
In continuation to our studies with primary hepatocytes, porcine hepatic and extrahepatic
tissue fractions and recombinant human enzymes, we tested extrahepatic cell lines both on
mARC1 and mARC2 expression and N-reductive activities. Cell lines included kidney cell
lines HEK293, RC124, HMCL and Colo357, a pancreatic cancer cell line. Furthermore, we
studied 3T3-L1, a fibroblast-like murine cell line that differentiates under appropriate
conditions into adipocyte-like cells.
N-reductive activity and expression of both mARC1 and mARC2 can be found in all studied
cell lines. N-reduction in all human cell lines obey Michaelis-Menten-kinetics, but show
differences in Vmax and Km. Conversion rates for benzamidoxime correlate with the
expression of mARC1 but not with the expression of mARC2.
All studied human cell lines can therefore be used to study both the enzyme system in
general and the N-reduction of N-hydroxylated compounds.
With regard to murine 3T3-L1,N-reductive activity is enhanced during differentiation into an
adipocyte-like phenotype.
Others found that enzymes and transporters that take part in the TCA cycle, fatty acid
oxidation and ATP synthesis as well as mARC2 show changes in expression during
differentiation of 3T3-L1 into an adipocyte-like cell type [7].Together with the fact that
N-reductive activityis increased during differentiation of 3T3 L1 this could be a hint that
mARC is involved in metabolic processes. Further studies on this topic will be necessary.
References:
1. Havemeyer, A. et al.:J. Bio. Chem.2006, 281(46): 34796–34802.
2. Gruenewald, S. et al.: J. Med. Chem.2008, 51(24): 8173–8177.
3. Neve, E. et al.: J. Biol. Chem.2011, 287(9): 6307–6317.
4. Clement B.:Inventor. Methoden zur Behandlung und Prophylaxe der Pneumocystiscariniipneumonie
(PCP) und anderen Erkrankungen sow e Verbindungen und Formulierungen zum Gebrauch bei besagten
Methoden. 1993, German patent P4321444.4, PCT/DE 94/00756 (1994); U.S. patent 5,786,383 (1998 July
28); European patent 0708640 (1998 September 16)
5. Wahl, B.et al.: J. Biol. Chem.2011, 285(48): 37847–37859.
6. Clement, B. et al.: Drug Metab. Rev.2010,42(S1): 1–323.
7. Newton, B. et al.:J. Proteome Res. 2011,10(10): 4692–4702.
100
QSAR studies of the N-reduction of p-substituted benzamidoximes
Treuer, E. 1; Reichmann, D. 2; Bittner, F. 2; Mendel, R. 2; Clement, B. 1;Havemeyer, A. 1
1Pharmaceutical Institute, Christian-Albrechts-University, Gutenbergstraße 76, 24118 Kiel,
Germany
2 Department of Plant Biology, Technical University of Braunschweig, Humboldtstraße 1, 38106
Braunschweig, Germany
mARC (mitochondrial Amidoxime Reducing Component) is a molybdenum containing
protein, which is localized in the outer mitochondrial membrane [1]. The genome of
mammalians codes for two different forms of this protein (mARC 1 and mARC 2). Together
with the electron transport proteins cytochrome b5 and cytochrome b5reductase, it catalyzes
the N-reduction of different compounds. On the one hand, it transforms toxic and mutagenic
N-hydroxylated base analogues [2]. On the other hand, it is involved in the activation of
several prodrugs like N-hydroxypentamidine and guanoxabenz [3].
For the development of new amidoximeprodrugs, we investigate structure-activity
relationships of the N-reduction of p-substituted benzamidoximes. Therefore, several
compounds were incubated with OMV (outer membrane vesicles) which was isolated from
pig liver mitochondria. The resulting amidine was quantified with HPLC analysis and Km
and Vmax were calculated. The influence of the electronic properties of the substituent in
para-position on the N-reductive activity was measured. There are first hints that the
conversion rates for the N-reduction of benzamidoximes with electron donating groups in
para-position are higher than for compounds with electron withdrawing substituents.
However, the differences are low and all para-substituted benzamidoximes were intensively
reduced. Thus the prodrug principle amidoximes (N-hydroxyamidines) instead of amidines
can be applied to allbenzamidine containing drug candidates irrespective of the
substituents at the aromatic ring.
References:
1. Havemeyer, A. et al.: J. Biol. Chem.2006, 281(46): 34796-34802.
2. Havemeyer, A., Lang, J., and Clement, B.: Drug.Metab.Rev. 2001, 43(4): 524-539.
3. Gruenewald, S. et al.: J. Med.Chem. 2008, 51(24): 8173-8177
101
Development of an ELISA assay for screening of p38δ MAPK inhibitors
Goettert M. a ,Bauer S. a , Shaalan N. b , Graeser, R. c , Laufer, S. a
a Department of Pharmaceutcal and Medicinal Chemistry, Institute of Pharmacy, Eberhard Karls
University of Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany
b Department of Pharmaceutical Biology, Faculty of Pharmacy and Biotechnology, German
University of Cairo, Egypt
c ProQinase GmbH, Breisacher Str. 117, D-79106 Freiburg, Germany
p38 mitogen activated protein kinases (MAPKs) are members of a larger group of serine/
threonine protein kinases.
They contribute in a variety of cellular processes such as gene expression, mitosis,
differentiation, cell survival/apoptosis and biosynthesis/ release of pro- inflammatory
cytokines [1].
In many inflammatory diseases, e.g. rheumatoid arthritis (RA), the role of p38α isoform is
widely investigated.
Activated rheumatoid arthritis synovial fibroblasts (RASFs) are considered as key cells in
the development of RA since they mediate the most relevant pathways of joint destruction
n[2,3].
The p38δ MAPK is activated in RASFs by a cytokine-independent pathway, leading to the
expression of matrix metalloproteinases, e.g. MMP-1 and MMP-3, which contributes to the
destruction of articular cartilage and bone [3,4].
In the RA synovial tissue, all four isoforms- α, β, γ and δ- of the p38 MAPK have been
detected. Particularly, p38δ MAPK occurs predominantly in the site of invasion and bone
destruction. For this, the participation of p38δ MAPK becomes more and more evident
[2,5].
For the purpose of routine screening, we developed a direct 96 Well plate ELISA assay to
identify inhibitors of p38δ MAPK. After incubation with a candidate inhibitor, the activity of
p38δ MAPK is measured by the phosphorylation degree of activation transcription factor 2
(ATF-2). The phosphorylated ATF-2 is directly detected by a monoclonal peroxidase
conjugated antibody. Being a natural substrate of the p38 MAPKs, ATF- 2 phosphorylation
is inversely correlated with the inhibitor potency.
Poster 117

Based on already successfully established ELISA assays for p38α MAPK and JNK3, the
advantages of this assay are its accuracy, easy handling, rapidness and the avoidance of
using radioisotopes.
References:
1. Schindler, J.F., J.B. Monahan, and W.G. Smith, p38 pathway kinases as anti-inflammatory drug targets.
Journal of Dental Research, 2007. 86(9): p. 800-811.
2. Ospelt, C., et al., Toll-like receptors in rheumatoid arthritis joint destruction mediated by two dist nct
pathways. Annals of the Rheumatic Diseases, 2004. 63: p. 90-91.
3. Huber, L.C., et al., Synovial fibroblasts: key players in rheumatoid arthritis. Rheumatology, 2006. 45(6): p.
669-675.
4. Goettert, M., et al., Development of a p38δ mitogen activated protein kinase ELISA assay for the
quantitative determination of inhibitor activity. Journal of Pharmaceutical and Biomedical Analysis, 2012.
5. Korb, A., et al., Differential tissue expression and activation of p38 MAPK alpha, beta, gamma, and delta
isoforms n rheumatoid arthritis. Arthritis and Rheumatism, 2006. 54(9): p. 2745-2756.
102
Is Dimethylarginine-Dimethylaminohydrolase-2 (DDAH-2) involved in
methylarginine metabolism?
Altmann K. S. 1, Havemeyer A. 1, Beitz E. 1, Clement B. 1
1Pharmaceutical Institute, Department of Pharmaceutical and Medicinal Chemistry, Christian-
Albrechts-University of Kiel, Gutenbergstraße 76, 24118 Kiel, Germany
DDAH isoenzymes, discovered in 1989 by Ogawa et al., catalyze the hydrolytic degradation
of free endogenous methylarginines, Nω-monomethyl-L-arginine (L-NMMA) and
Nω,Nω'-dimethyl-L-arginine (ADMA). [1,2] These methylarginines occur as endogenous nitric
oxide synthase (NOS) inhibitors and reduce the formation of nitric oxide (NO).[3,4] By
modulating the rates of L-NMMA and ADMA DDAH enzymes exhibit a therapeutic target to
modulate the bioavailability of NO. [5,6]
The postulated metabolism is evidenced for DDAH-1 but the involvement of DDAH-2 in the
degradation of ADMA and L-NMMA is still a matter of debate.
Experimental Procedures:
Therefore, we determined the isoform specific DDAH protein expression profile in various
porcine tissues by immunoblotting and correlated it with respective DDAH activity levels.
Tissue DDAH activity was measured by the conversion of ADMA alone and in the presence
of a DDAH inhibitor. Furthermore, metabolism of ADMA, L-NMMA and alternative DDAH
substrates, S-methyl-L-thiocitrulline (SMTC) andNω-amino-L-arginine, was studied in
porcine tissues each containing one DDAH isoform.
Results:
DDAH activity was detected in homogenates of porcine kidney, liver, and brain, i.e. tissues
with high expression levels of DDAH-1, but not in those of spleen or thyroid, where DDAH-2
predominates. By the same token neither methylarginines nor alternative DDAH substrates
were converted in the DDAH-2-rich porcine thyroid cytosolic fractions, whereas all
substrates were metabolized by the DDAH-1-rich kidney cytosol preparation.
In addition, L-citrulline formation in porcine tissues was reduced to 50 % in the presence of
1 mM of the DDAH inhibitor Nω-(2-methoxyethyl)-L-arginine (L-257), evidencing DDAH
activity as a major source for the conversion. [7]
Conclusion:
Together, these data show that DDAH-1 and DDAH-2 have distinct tissue distributions as
well as substrate selectivity indicating different roles in NO metabolism. While DDAH-1
metabolizes the endogenous NOS inhibitors L-NMMA and ADMA, a physiological function
of DDAH-2 has yet to be defined.
References:
1. Ogawa, T., Kimoto, M., and Sasaoka, K.: J. Biol. Chem. 1989, 264(17):10205–10209.
2. Leiper, J. M. et al.: Biochem. J. 1999, 343Pt 1: 209–214.
3. Anthony, S., Leiper, J., and Vallance, P.: Vasc Med. 2005, 10Suppl 1: S3-9.
4. MacAllister, R. J. et al.: Br. J. Pharmacol. 1996, 119(8): 1533–1540.
5. Dayoub, H. et al.: Circulation. 2003, 108(24): 3042–3047.
6. Feng, M. et al.: Int. J. Cardiol. 2010, 144(2): 180–186.
7. Rossiter, S. et al.: J. Med. Chem. 2005, 48(14): 4670–4678.
103
New heterocyclic sigma receptor ligands and their role in the activation of
apoptotic pathways
Korpis K1, Weber F2, Wünsch B2, Bednarski P J1 1 Institute of Pharmacy, University of Greifswald, F.-L.-Jahn-Straße 17, 17487 Greifswald, Germany
2 Institute of Pharmacy, University of Münster, Hittorfstraße 58-62, 48149 Münster, Germany
Sigma receptors are present in peripheral organs and in the central nervous system. They
are divided into two distinct subtypes: σ1 and σ2. Both subtypes are involved in
neuromodulatory processes and in particular the σ1 receptor is under evaluation for the
treatment of a number of neurological disorders such as depression or schizophrenia. 1 In
addition to the relevance of these receptors in neurological effects both subtypes are
expressed in a high density in different human tumor cell lines. σ1 receptor antagonists and
σ2 agonists have been shown to be involved in programmed cell death (apoptosis),
whereas σ1 receptor agonists and σ2 receptor antagonists have antiapoptotic and
neuroprotective activity. More specifically, Spruce et al. have shown that σ1 antagonists
induce caspase-dependent apoptosis, which is in accord with recent observations that σ1
agonists prevent caspase-3 activation. On the other hand, σ2 agonists have been shown to
activate a caspase independent apoptotic pathway and have been proposed as putative
anti-cancer drugs. 2
The aim of this work is to determine the role of newly synthesized σ receptor ligands in the
activation of apoptotic pathways.
Flow cytometry with Annexin V and propidium iodide was performed to observe cell
membrane alterations due to apoptosis. Experiments with human cancer cells treated with
our compounds showed apoptosis levels increasing from 1 % in the control to 31 % in the
treated cells. Another apoptosis assay was used to measure the expression of members of
the caspase family to confirm apoptosis as the mechanism of cell death. Treatment of the
multiple myeloma cell line RPMI 8226 caused dose-dependent activation of caspase-3,
caspase-8 and caspase-9, indicating the activation of both the intrinsic and the extrinsic
apoptotic pathway.
However, Western blot analysis showed only cleaved products of caspase-8 and not
caspase-9, evidence for activation of only the extrinsic pathway. These data suggest that
our compounds do not suppress the expression of pro-survival Bcl-2 family proteins, such
as Bcl-2.
Further studies will address the crucial role in the extrinsic apoptotic pathway, which is
initiated by activation of death receptors (i.e. TRAIL) and if there are other apoptosisindependent
pathways of programmed cell death, for example autophagy.
References:
1. Abate, C. et al.: J. Med. Chem. 2011, 54(4): 1022-1032.
2. Riganas, S. et al.: Bioorganic & Medicinal Chemistry 2012, 20(10): 3323-3331.
104
A cell culture based assay for studies on inhibitors of the inducible nitric oxide
synthase (iNOS) and effects of cold atmospheric pressure plasma treatment of
keratinocytes and lymphocytes
Schulz U1, Bundscherer L2, von Woedtke Th3, Masur K2, Morgenstern O1 1 Institut für Pharmazie, Ernst-Moritz-Arndt-Universität Greifswald, Friedrich-Ludwig-Jahn-Str. 17,
17489 Greifswald, Germany
2 Zentrum für Innovationskompetenz plasmatis, AG “Zelluläre Effekte” im INP Greifswald e.V.,
Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany
3 Campus PlasmaMed im INP Greifswald e.V., Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany
The inducible nitric oxide synthase (iNOS) is a target of great interest for development of
drugs for COPD and asthma [1], septic shock [2] and autoimmune diseases e.g. diabetes
type 1 [3] and multiple sclerosis [4]. We synthesized some potential iNOS inhibitors with
general structure 1 based on the fact that substances with thiourea or thiosemicarbazide
structure elements showed iNOS-inhibitory properties before [5].
To investigate the effect of our compounds on iNOS, we established a cell culture based
assay using RINm5F cells that express the target under influence of interleukin-1β (IL-1β)
and interferon-γ (IFN-γ) [6]. We optimized the produced amount of nitric oxide (measured
as nitrite) using various combinations of the two cytokines with two cell counts. Additionally,
we varied the incubation time pre and post addition of IL-1β and IFN-γ.
Here we display the collected data that show that IFN-γ is, as expected, unable to induce
iNOS on its own [7]. IL-1β alone increases nitrite concentration higher in short preincubation.
IFN-γ in addition to IL-1β is able in very little concentrations to push the NO
production to a maximum. We found a combination of low IL-1β and IFN-γ concentrations
after a pre-incubation of 24 h and 48 h incubation after cytokine addition to be most
effective.
With these parameters we screened our derived substances for activity and found 7
compounds to inhibit the target significantly higher than the reference aminoguanidine.
Additionally to the screening of the chemical substances, we used the iNOS test model with
modification to study effects of cold atmospheric pressure plasma treatment of
keratinocytes (HaCaT cells) and lymphocytes (Jurkat cells). We searched for increased
nitrite concentrations due to treatment of RINm5F cells with plasma or with supernatant of
plasma-treated HaCaT and Jurkat cells. The aim was to investigate the influence of the
plasma based secretion of the cytokines IL-1β and IFN-γ in order to induce NO production
from the RINm5F cells. Possible inhibitory effects from produced cytokines e.g. TGF-β were
investigated as well [8].
Acknowledgments: Zentrum für Innovationskompetenz plasmatis, AG “Zelluläre Effekte” im INP Greifswald
e.V., Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany
The authors like to gratefully acknowledge the contribution of Liane Kantz to this work.
References:
1. Sugiura H, Ichinose M: Nitric Oxide 2011, 25(2): 138-144.
2. Fortin C F, McDonald P P, Fulop T, Lasur O: Shock 2010, 33(4): 344-352.
3. Cnop M, Welsh N, Jonas J C, Jörns A, Lenzen S, Eizirik D L: Diabetes 2005, 54(suppl 2): S97-S107.
4. Steinert J R, Chernova T, Forsythe I D: Neuroscientist 2010, 16(4): 435-452.
5. Al-Amiery A A, Al-Majedy Y K, Ibrahim H H, Al-Tamimi A A: Org. Med. Chem. Lett. 2012, 2(1): 4-7.
6. Ankarcrona M, Dypbukt J M, Brune B, Nicotera P: Exp. Cell Res. 1994, 213(1): 172-177.
7. Cetkovic-Cvrlje M, Eizirik D L: Cytokine 1994, 6(4): 399-406.
8. Mabley J G, Cunningham J M, John N, Di Matteo M A, Green I C: J. Endocrinol. 1997, 155(3): 567-575.
105
New Glutathione Peroxidase Inhibitors
Lemmerhirt, H. 1; Schulz, R. 1; Emmrich, T. 1; Wilde, F. 1; Link, A. 1; Bednarski, P. J. 1
1Ernst-Moritz-Arndt-University, Institute of Pharmacy, Friedrich-Ludwig-Jahn-Straße 17, 17489
Greifswald, Germany.
Cancer cells have a variety of mechanisms to protect themselves from anti-cancer drugs.
For example, they express drug efflux transporters like P-gp, increase repair of DNA
damage or mutate targeted enzymes and receptors. A relatively new postulated
mechanism of resistance is the over-expression of enzymes that deal with oxidative stress.
It has been postulated that glutathione peroxidases (GPx, EC 1.11.1.9) is a key enzyme in
the development of resistance. [1] The primary function of GPx is to prevent cells from
oxidative stress by degrading peroxides like hydrogen peroxide to water or organic
hydroperoxides to the corresponding alcohols.
Hydrogen peroxide is postulated to be an important messenger of apoptosis. If cancer cells
treated with anti-cancer drugs were to over-express GPx, then the signals for apoptosis
would be weakened, leading to survival of more cancer cells [2].
The aim of our work is to develop specific and reversible inhibitors of GPx to reverse drug
resistance. Already some inhibitors of GPx are known, such as misonidazole and several
118 Poster

thiol compounds but they have disadvantages; misonidazole shows neurotoxicity and thiols
are unspecific and inhibit GPx irreversible. Furthermore thiols are instable with regard to
oxidation.
R. SCHULZ identified several promising lead structures by means of virtual screening with
AutoDock and determined their GPx inhibitory activity by an enzyme assay [3]. One of them
(1) was chosen for structural modification to improve inhibitory activity.
The synthesized compounds have been tested for enzyme inhibitory activity in a microtiter
based assay. The method is based on the oxidation of glutathione by GPx in the presence
of hydroperoxides. The resulting glutathione disulfide (GSSG) is reduced back to GSH by
glutathione reductase (GR) with consumption of NADPH. The rate of decrease of NADPH,
monitored at λ = 340 nm, corresponds to the catalytic rate of GPx.
We have now identified two new compounds that inhibit GPx with lower IC50 values than the
lead compound. The substances will now be tested in vitro cell culture on human cancer
cells resistant to common anti-cancer drugs. For example, ovarian carcinoma cell lines
resistant to cisplatin will be used to assess whether anti-cancer-drug-resistance can be
reversed with the new compounds.
References:
1. Saga, Y., et al.: Oncol Rep 2008, 20 (6): 1299-303.
2. Gouaze, V., et al.: J Biol Chem 2002, 277 (45): 42867-74.
3. Schulz, R.: Diploma Thesis 2008.
106
Stimulation and Inhibition of Angiogenesis by Non-Thermal Atmospheric-
Pressure Plasma
Haertel B. 1; Eiden K. 1; Deuter A. 1; Wende K. 1; von Woedtke T. 2; Lindequist U. 1
1 Institute of Pharmacy, University of Greifswald, D-17489 Greifswald, Germany
2 Leibniz-Institute for Plasma Science and Technology e.V. (INP), D-17489 Greifswald, Germany
The study of angiogenesis has broad clinical implications e.g. in the fields of wound
healing, oncology, hematology or ophthalmology. The formation of new blood vessels is an
essential feature of tissue remodeling. Further, angiogenesis is a prerequisite for growth of
solid tumors and development of metastasis. However, it is also involved in the
pathophysiology of hematologic malignancies. For improving wound healing angiogenesis
should be promoted, whereas in treating tumors angiogenesis should be inhibited. Inhibition
of angiogenesis is also important in the treatment of macular edema or age-related macular
degeneration. Angiogenesis depends on the expression of mediators initiating events
leading to the formation of new microvessels. Non-thermal plasma induces a variety of
effects on living cells in vitro, including production of fibroblast growth factor-2 (FGF-2) or
reactive oxygen species by endothelial cells. Both FGF-2 and ROS belong to mediators of
angiogenesis. However, up to now there are no quantitative data using non-thermal plasma
in more complex angiogenesis models as the hen’s egg test on the chorioallantoic
membrane (HET-CAM) or the aortic ring test (AOR). It is very important to know whether
and how plasma influences angiogenesis in complex models.
This study focused on the effects of plasma generated by the kINPen 09 [1] on
angiogenesis using two different models: HET-CAM and rat aortic ring (AOR) test. In both
models kINPen 09 treated medium (30 to 300s) was applied. ImageJ was used to analyze
vessel area and fractal dimension after treating the CAM from embryonic day 11 to 13.
Aortic rings were prepared from either LEW.1W or WOK.W rats. They were embedded in
matrigel and treated daily for 4 days starting at day 4 after embedding. We developed a
semi quantitative method to quantify outgrow of microvessels from aortic rings.
In both models natural and spontaneous vessel formation was detected. In the HET-CAM
assay vessel area and fractal dimension were significantly enhanced at embryonic day 14
by the 120s-plasma treated medium compared to untreated controls. There was no effect of
plasma (60 or 120s) on vessel growth of aortic rings prepared from LEW.1W rats. The
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
resolved, many of the distinct interaction sites and the involvement of the C2ld are still
unclear. Therefore the C2ld (amino acids 1-115, fused to maltose binding protein) was
expressed and purified in two steps via amylose affinity chromatography and, after
cleavage, nickel chelating affinity chromatography. Gel filtration studies revealed a high
tendency of the isolated domain to form soluble aggregates. But the single mutation of
tryptophan 75 to alanine led to a dramatic improvement of its biophysical properties leading
to mainly monomeric protein. The proper folding of the tryptophan mutant was successfully
shown by its ability to bind Ca 2+ in a thermal shift assay. Crosslinking of 5-LO or the
isolated C2ld with CLP revealed that the catalytic domain is also involved in the binding of
CLP.
Here we present the purification of a single-mutant that renders the 5-LO C2ld accessible
for interaction studies elucidating the involvement of the regulatory domain to the binding of
small molecules or proteins.
References:
1. Hammarberg T. et al. J Biol Chem. 2000, 275(49): 38787-93.
2. Radmark O. et al. Trends Biochem Sci. 2007, 32(7): 332-41.
3. Rakonjac M. et al. Proc Natl Acad Sci U S A. 2006, 103(35): 13150-5.
4. Dincbas-Renqvist V. et al. Biochim Biophys Acta. 2009, 1789(2): 99-108.
110
Synthesis of annelated and substituted imidazolones and –thiones targeted at
human NAD + -dependent histone deacetylases
Beese K1; El Gaghlab K1; Rumpf T2; Jung M2; Link A1 1Institute of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Friedrich-Ludwig-Jahn-Str. 17,
17489 Greifswald, Germany
2Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstr. 25, 79104
Freiburg, Germany
Histones can be modified enzymatically by insertion and elimination of chemical groups
mainly upon basic amino acids. A very significant histone modification is the acetylation
forming lightly packed, active euchromatin that facilitates the accessibility of genes for
transcription factors, whereas deacetylation leads to tightly packed, inactive
heterochromatin, which results in a repression of transcription.
NAD +-dependent histone deacetylases (HDACs), known as sirtuins, regulate epigenetic
gene expression by deacetylation of histones and non-histone proteins. Thus, an increased
enzyme activity is associated with the development of cancer, HIV and neurodegenerative
diseases, and inhibitors of HDACs might represent promising drugs.
In continuation of our efforts to identify modulators of HDACs with improved activity and
selectivity we designed and synthesized analogs of a novel lead structure [1] reported by
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,
and 1,5-disubstituted1,3-dihydrobenzimidazol-2-ones/thiones,
that can be regarded as non-hydrolyzable analogs of the lactone splitomicin, were prepared
and tested in a new nonisotopic, heterogeneous assay.
introduction
of
hetero atoms
or
substituents
annelation
and /or
substitution
replacement
by
oxygen
introduction
of
aliphatic or aromatic
sidechains
Acknowledgements: Technical assistance of Dr. A. Bodtke and K. Böheim is gratefully acknowledged.
K.E.G. was recipient of grant DFG Li 765/4-2.
References:
1. Freitag, M. et al.: Bioorg Med Chem 2011, 19(12): 3669–3677.
111
ICAM-1 confers the TIMP-1-dependent anti-invasive action of cannabinoids on
human lung cancer cells
Ramer, R. 1; Bublitz, K. 1; Merkord, J. 2; Haustein, M. 1; Borchert, P. 1; Linnebacher, M. 2; Hinz,
B. 1
1 Institute of Toxicology and Pharmacology, University of Rostock, Schillingallee 70, D-18057
Rostock, Germany
2 Section of Molecular Oncology and Immunotherapy, Department of General Surgery, University
of Rostock, Schillingallee 69, D-18057 Rostock, Germany
Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix
metalloproteinases-1 (TIMP-1). This study investigates the role of intercellular adhesion
molecule-1 (ICAM-1) within this action. In the lung cancer cell lines A549, H358, and H460,
cannabidiol (CBD; 0.001-3 μM) elicited concentration-dependent ICAM-1 up-regulation
compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and
p42/44 mitogen-activated protein kinase. Up-regulation of ICAM-1 mRNA by CBD in A549
was 4-fold at 3 μM, with significant effects already evident at 0.01 μM. ICAM-1 induction
became significant after 2 h, whereas significant TIMP-1 mRNA increases were observed
only after 48 h. Inhibition of ICAM-1 by antibody or siRNA approaches reversed the antiinvasive
and TIMP-1-upregulating action of CBD and the likewise ICAM-1-inducing
cannabinoids Δ 9-tetrahydrocannabinol and r(+)-methanandamide when compared to
isotype or nonsilencing siRNA controls. ICAM-1-dependent anti-invasive cannabinoid
effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude
mice, CBD elicited a 2.6- and 3.0-fold increase of ICAM-1 and TIMP-1 protein in A549
xenografts, as compared to vehicle-treated animals, and an antimetastatic effect that was
fully reversed by a neutralizing antibody against ICAM-1 [% metastatic lung nodules vs.
isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM-1
antibody]. Overall, our data indicate that cannabinoids induce ICAM-1, thereby conferring
TIMP-1 induction and subsequent decreased cancer cell invasiveness.
112
Can Heparin influence the Cyr61 pathway for affecting cell adhesion
processes?
Schmitz, P. 1; Gerber, U. 1; Schütze, N. 2; Bendas, G. 1
1 Department of Pharmacy, University Bonn, 53121 Bonn, Germany
2 Orthopedic Center for Musculoskeletal Research, University Würzburg, 97074 Würzburg,
Germany
The cysteine-rich protein 61 (Cyr61, CCN1), a member of the CCN-family, is a matricellular
protein expressed and secreted by various cells, which induce e.g. angiogenetic effects and
enhance the tumorigenicity of solid tumors. Besides a range of functions Cyr61 mediates
adhesion and migration of tumor cells by activating different members of the integrin
adhesion receptor family. Integrins are crucial for different steps in course of the
haematogenous metastasis. Heparin possesses antimetastatic effects by blocking
adhesion receptors; selectins and selected integrins, e.g.VLA-4 (very late activation antigen
4) [1,2], but heparin should also be able to bind Cyr61.
However, neither the molecular mechanisms of Cyr61 affecting VLA-4 mediated adhesion
nor interference by heparin in this pathway have been described. This study focused these
aspects.
To confirm direct binding of heparin to Cyr61, SAW biosensor studies were performed
using a series of modified heparins and recombinant CCN1 proteins. To further focus on
the Cyr61/VLA-4 binding pathway and potential impact on that by heparin, the impact of a
Cyr61 downregulation in MV3 melanoma cells by shRNA technology on VLA-4 mediated
cell adhesion was analysed by microscopic detection.
Our data suggest a reduced VLA-4 mediated cell binding of Cyr61 knockdown MV3 clones
in an adhesion assay, while exogenous recombinant Cyr61 restores the binding
capacities.This confirms that VLA-4 is partly controlled by Cyr61 activity and make Cyr61
as attractive target to interfere with metastasis. Concerning heparin interference, kinetic
binding data suggest a direct interaction between heparin and Cyr61 for the first time.
Binding affinities of fractionated heparin in the micromolar range were intensified by Nacetylation
or 2-O-desulfation of heparin. Furthermore, Cyr61 accumulation at the cell
surface glycosaminoglycan Syndecan-4 could be simulated demonstrating high binding
affinities. However, this binding is non-reversible by adding high concentrations of heparin.
Our data provide evidence for a certain impact of Cyr61 with VLA-4 mediated melanoma
cell binding. The hypothesis of heparin interference in this pathway is strongly supported
providing prospects for therapeutic interference in tumorigenicity and metastasis by
blocking Cyr61 activity.
We gratefully thank the “G.Ronzoni“ Institute, Milano for providing us the modified heparins.
References:
1. Schlesinger, M., et al., Thromb Res. 5 (2012): 603-10.
2. Bendas, G., Borsig, L.; Int J Cell Biol. 2012.
113
Cyclooxygenase-2 and peroxisome proliferator activated receptor gamma
confer cannabidiol-induced apoptosis of human lung cancer cells
Heinemann, K. 1; Ramer, R. 1; Merkord, J. 2; Salamon, A. 2; Linnebacher, M. 3; Hinz, B. 1
1 Institute of Toxicology and Pharmacology, University of Rostock, Schillingallee 70, D-18057
Rostock, Germany
2 Department of Cell Biology, University of Rostock, Schillingallee 69, D-18057 Rostock, Germany
3 Section of Molecular Oncology and Immunotherapy, Department of General Surgery, University of
Rostock, Schillingallee 69, D-18057 Rostock, Germany
The antitumorigenic mechanism of cannabidiol (CBD) is still controversial. This study
investigates the role of cyclooxygenase-2 (COX-2) and peroxisome proliferator activated
receptor γ (PPARγ) in CBD´s proapoptotic and tumor-regressive action. In lung cancer cell
lines (A549, H460) and primary cells from a lung cancer patient CBD elicited decreased
viability associated with apoptosis with the strongest toxic impact on primary tumor cells.
Apoptotic cell death by CBD was suppressed by NS-398 (COX-2 inhibitor), GW-9662
(PPARγ antagonist) and siRNA targeting COX-2 and PPARγ. CBD-induced apoptosis was
paralleled by upregulation of COX-2 and PPARγ expression with a maximum induction of
COX-2 mRNA after 8 h and continuous increases of PPARγ mRNA when compared to
vehicle. In response to CBD tumor cell lines exhibited increased levels of COX-2dependent
prostaglandins (PGs) among which PGD2 and 15-deoxy-Δ12 14-PGJ2 caused a
translocation of PPARγ to the nucleus and induced a PPARγ-dependent apoptotic cell
death. Moreover, in A549-xenografted nude mice CBD caused upregulation of COX-2 and
PPARγ in tumor tissue, and tumor regression that was fully reversible by GW-9662.
Together, our data demonstrate a novel proapoptotic mechanism of CBD involving initial
upregulation of COX-2 and PPARγ and a subsequent nuclear translocation of PPARγ by
COX-2-dependent PGs.
114
Synthesis of Zwitterionic Ligands for PET-Imaging in Melanoma
Kriemen, E. 1; Maison, W. 1
1 Pharmaceutical and Medicinal Chemistry, Bundesstraße 45, 20146 Hamburg, Germany
Malignant melanoma, although it is far less prevalent than non-melanoma skin cancers, is
the major cause of death from skin cancer.[1] This is because of the high aggressiveness of
melanoma metastases and their resistance to conventional chemotherapy and external
beam radiation therapy. Therefore an early diagnosis of melanoma is the key issue for
increasing patient survival.[2]
For our approach we focus on PET (positron emission tomography) as an imaging
technique, which relies on decay characteristics of positron emitting (β+-decay)
120 Poster

adionuclides.[3] Modern PET scanners have high sensitivities, but are still limited by
specific binding/uptake to normal tissues and organs which lead to high background.
Recent data showed that “zwitterionic” tracer molecules lower background binding
dramatically by not interacting with serum proteins due to charge shielding.[4]
With the mentioned principle in mind we designed a melanoma targeted radiotracer 1. It
contains a DOTA moiety for proper chelating a radionuclide such as 89Zr and 68Ga.[5] The
classical DOTA molecule is modified by geometrically balanced charge (positive charge
with quaternary ammonium cations and negative charge with sulfonate groups).[4] This
zwitterionic DOTA derivative is conjugated to CycMSH, a cyclic targeting peptide with high
specificity to the melanocortin receptor MC1. The MC1 receptor is overexpressed in more
than 80% of human metastatic melanoma tumor samples and is thus a valuable tumor
marker.[6,7]
We present the synthesis of a zwitterionic DOTA derivative and it’s conjugation to CycMSH
by a solid phase protocol and evaluate the binding properties of this targeted imaging
reagent to MC1.
References:
1. Siegel, R., Naishadham, D., Jemal, A.: CA Cancer J. Clin. 2012, 62(1): 10–29.
2. Anderson, C. M. et al.: Oncology 1995, 9(1): 1149–1158.
3. Frangioni, J. V.: J. Clin. Oncol. 2008, 26(24): 4012–4021.
4. Choi, H.S. et al.: Angew. Chem. Int. Ed. 2011, 50(28): 6258–6263.
5. Verel, I., Visser, G. W. M., van Dongen, G. A.: J. Nucl. Med. 2005, 46(1): 164–171.
6. Hadley, M. E.: Pigment Cell. Res. 1996, 9(5): 213–234.
7. Guo, H.: J. Nucl. Med. 2011, 52(4): 608–616.
115
Is lysophosphatidylcholine (LysoPC) the key to explain the antimetastatic
effects of empty liposomes?
Ross, T. 1; Schlesinger, M. 1; Raynor, A. 2; Jantscheff, P. 2; Massing U. 2; Bendas, G. 1
1 University of Bonn, Department of Pharmaceutical Chemistry II, An der Immenburg 4, 53121
Bonn, Germany
2 Tumor Biology Center Freiburg, Breisacher Str. 117, 79106 Freiburg, Germany
Liposomes are extensive subject to current anticancer research. However, liposomal
phospholipids (PL) are only regarded as passive carrier constituents. The findings of
Graeser et al. [1] showing that empty liposomes composed of saturated PLs have
antimetastatic effects in different mouse models suggest PL actively interfering in the
metastatic course. Since cellular adhesion receptors are of key importance for the
metastatic route of tumor cells, we proposed affected cell adhesion as the underlying
mechanism. Recently we presumed lysophosphatidylcholine (LysoPC) as key molecule for
this activity. One might assume that, due to the EPR-effect, liposomes accumulate in
neoplastic tissue leading to an increased uptake and metabolisms of saturated PL. Since
phospholipase A2 release is known to be often associated with tumor activity, increased
levels of saturated LysoPC in tumor tissue might result. Jantscheff et al. [2] could show a
reduction of melanoma cell adhesion in vitro as well as an inhibition of metastasis like lung
invasion in vivo of cells pretreated with LysoPC. However, the underlying molecular
mechanisms of LysoPC activity remain to be elucidated. Since any toxic activities of
LysoPC or induction of apoptosis could be excluded, our current research focuses on the
involvement of the plasma membrane affected by LysoPC and consequences for
membrane associated signalling pathways.
Using gas chromatography a rapid change in the lipid composition of the tumor cell
membranes favouring the LysoPC derived fatty acids (FA) was shown under the influence
of different LysoPC derivatives. In order to evaluate the impact of FA saturation on tumor
membrane rigidification and underlying functional consequences, we compared the fully
saturated Lyso-PC C18:0 and the unsaturated C18:1-derivative in MV3 melanoma cells.
Using trimethylammoniumdiphenylhexatriene as a fluorescence anisotropy probe we could
detect a significant rigidification of the cell membrane by C18:0 LysoPC, while C18:1
LysoPC possessed only minor changes. The changes in membrane rigidity correspond with
a diminished receptor-mediated migration capacity of the cells, as shown in microscopic
wound healing assays. Thus, one might assume that LysoPC-induced membrane
rigidification is involved in the reduced receptor mediated cell binding which might be one
reason for the antimetastatic effects of LysoPC.
To further focus on the molecular mechanisms of diminished receptor mediated cell binding
we present preliminary data on the phosphorylation of the proteoglycan syndecan-4, which
is an important element in the formation of focal adhesions complexes. Summarizing, we
propose LysoPC as an active biological molecule affecting the plasma membrane fluidity
with strong consequences for the formation of focal adhesion complexes. These data could
also provide new insights and prospects for liposomal carriers in tumor therapy.
References:
1. Graeser, R. et al.: Pancreas 2009, 38(3): 330-337
2. Jantscheff P. et al.: Mol Cancer Ther 2011, 10(1): 186-197
116
NEFA determination for functional lipidomics: comprehensive UPLC-MS/MS
quantitation, qualification and parameter prediction [1]
Hellmuth C, Weber M, Koletzko B, Peissner W
Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children’s Hospital, Ludwig-
Maximilians-University of Munich, 80337 Munich, Germany
Objectives
Metabolomics is an increasing field in pharmaceutical research and development.
Metabolic profiling depicts abundance changes in endogenous metabolites [2]. E.g. levels
of nonesterified fatty acids (NEFA) are highly associated with obesity, diabetes and
metabolic syndrome. For instance, NEFA are elevated in T2DM adolescents in fasting state
as well as after glucose challenge [3]. Though, straightforward quantitative methods for
determination of NEFA species are still missing.
Methods
Serum samples were prepared by simple protein precipitation, avoiding cumbersome and
solvent-consuming extraction or derivatization procedures. Short run time with high
resolution was achieved by UPLC separation coupled to LC-MS/MS detection.
Results
Accurate quantification of 36 NEFA species in 20 µl of healthy human plasma was
achieved, the highest numbers ever reported for a LC-MS application. The use of qualifier
ions supports unequivocal analyte verification. Sample preparation is fast and nonexpensive.
In combination with automated liquid handling, total assay time per sample is
less than 15 minutes.
Conclusion
The presented method provides a rapid and comprehensive determination of NEFA in
human plasma. This enables application in clinical trials with high sample number analyzed
in short time and with low costs. Usage of 20 µl plasma facilitates application in studies
with children or adolescents, when blood is obtained by finger sticks or heel pricks.
References:
1. Hellmuth C. et al.: Anal Chem. 2012 84(3):1483-90
2. Xu EY, Schaefer WH, Xu Q: Curr Opin Drug Discov Devel. 2009 12(1):40-52
3. Nadeau KJ et al.: J Clin Endocrinol Metab. 2009 94(10):3687-95
117
The impact of DNA methylation on cellular differentiation of immune cells
Flemming S. 1; Grützkau A. 2, Häupl T. 3; Günther S. 1
1 Pharmaceutical Bioinformatics, Institute of Pharmaceutical Sciences, University of Freiburg
2 German Arthritis Research Center, Berlin
3 Department of Rheumatology and Clinical Immunology, Charité University Hospital, Berlin
Epigenetic changes in DNA methylation are associated with regulation of gene expression
and play an important role in cellular differentiation. Such changes might be of importance
in chronic inflammatory diseases where autoreactive immune cells are thought to
perpetuate inflammation and organ destruction. To explore these variations and its
regulatory mechanism, we have gathered genome-wide DNA methylation data in human
immune cells extracted from peripheral blood, specifically T-helper cells (CD4), Tsuppressor
cells (CD8), macrophages (CD14), granulocytes (CD15), B-lymphocytes
(CD19) and natural killer cells (CD56). Furthermore, we have analyzed methylation patterns
associated to cell differentiation of T- and B-cells and the correlation with gene expression
data.
Cells from four healthy donors were sorted by FACS technology and the measurement was
performed with the Illumina HumanMethylation450 BeadChip platform. These arrays
provide a genome-wide coverage of more than 485,000 CpG methylation sites per sample
at single-nucleotide resolution. To evaluate the correlation between methylation patterns
and gene expression, we used the Affymetrix U133P platform.
We observed that both, hyper- and hypomethylation of promoter regions of genes involved
in cell differentiation correlate well with measured gene expression data. Additionally, we
were able to identify a substantial number of common CpG methylation sites, which were
differentially methylated between naive and memory states of T-(CD4, CD8) and B-cells
(CD19).
The impact of these alterations on inflammatory diseases will be a subject to further
research which will include the analysis of autoreactive immune cells from rheumatoid
arthritis patients.
118
Role of Histamine H4 receptors in eicosanoid biosynthesis.
Dos Santos Capelo, R1; Kahnt, AS1; Steinhilber, D1 1 Institute of Pharmaceutical Chemistry, Max-von-Laue-Strasse. 9, 60438 Frankfurt am Main,
Germany
The H4 receptor is a G-protein-coupled receptor (GCPR) expressed in many cells of the
immune system, including monocytes, mast cells, dendritic cells, natural killer cells,
eosinophils and T cells. So far, not much is known about its physiological role, but the
receptor is implicated in the pathology of various diseases such as allergy, autoimmune
disorders, bronchial asthma, atopic dermatitis and pruritus. Upon activation the receptor
couples to Gi/o proteins. This leads to a decrease in cAMP production [1,2,3,4], activates
downstream mitogen-activated protein (MAP) kinase pathways [4] and mobilizes
intracellular Ca2+ [1,5]. 5-lipoxygenase (5-LO) catalyzes the first two steps in leukotriene
(LT) biosynthesis. Upon stimulation (e.g. MAP kinases and Ca2+ influx) the enzyme
translocates to perinuclear membrane compartments where it is activated and starts the
synthesis of LTA4 [6]. LTA4 can then be further metabolized by two terminal synthases.
Soluble LTA4 hydrolase produces the potent chemoattractant LTB4, whereas membrane
bound LTC4 synthase conjugates LTA4 with glutathione (GSH) to form the corresponding
cysteinyl leukotrienes (CysLTs), LTC4, LTD4 and LTE4. Like the H4 receptor, 5-LO is
primarily found in mature leukocytes such as granulocytes, mast cells, dendritic cells,
monocytes / macrophages and B lymphocytes [7]. T cells and erythrocytes are 5-LO
negative. 5-LO derived LTs are lipid mediators which were shown to primarily mediate
inflammatory and allergic reactions. Besides their well studied role in asthma, 5-LO derived
LTs have also been implicated to play a role in cardiovascular diseases and cancer [8].
Recently, a connection between LT biosnythesis and histamine receptors has been drawn.
Flamand et al. have published that histamine is able to inhibit LT formation in human PMN
in a dose-dependent manner via Gs coupled H2 receptor activation. This inhibition was
characterized by cAMP dependent protein kinase A (PKA) activation which in turn led to a
decrease in arachidonic acid release and 5-LO translocation [9]. Based on this, we
postulate that the human histamine H4 receptor might control LT biosynthesis in activated
human leukocytes such as macrophages and eosinophils. Using western blot and
polymerase chain reaction (PCR) technique we have investigated the H4 receptor
expression in various cell lines and primary cells and have chosen suitable cell lines. Next,
we plan to treat these cells with different cytokines plus histamine and monitor 5-LO
activity. After this, we plan to elucidate the underlying mechanism to provide the basis for
Poster 121

the development of potential new anti-leuktriene drugs.
References:
1. Oda, T. et al.: J. Biol. Chem. 2000, 275: 36781–36786.
2. Nakamura, T. et al.: Biochem. Biophys. Res. Commun. 2000, 279: 615–620.
3. Zhu, Y. et al.: Mol. Pharmacol. 2001, 59: 434–441.
4. Liu, C. et al.: Mol. Pharmacol. 2001, 59: 420–426.
5. Morse, K.L. et al.: J. Pharmacol. Exp. Ther. 2001, 296: 1058–1066.
6. Rouzer C.A. et al.: J. Biol. Chem. 1988, 263(22): 10980-10988.
7. Heaggstrom J.Z. et al.: Cell. Mol. Life. Sci. 2002, 59(5):742-753.
8. Werz O. et al.: Pharmacol. Ther. 2006, 112(3):701-718.
9. Flamand N. et al.: Br. J. Pharmacol. 2004, 141(4): 552-561.
119
Quantitative Analysis of Isolated EGFR Adaptor Protein Pattern by NSAF and
SILAC Reveals Cellular Dynamics of Receptor Signaling and Internalization
Foerster, S. 1, Hammer, E. 2; Kacprowski, T. 3, Albrecht, M. 3, Völker, U. 2, Ritter, C.A. 1
1 Clinical Pharmacy, Institute of Pharmacy, Ernst-Moritz-Arndt-University, Friedrich-Ludwig-Jahn-
Str. 17, 17487 Greifswald, Germany
2 Department of Functional Genomics, Interfaculty Institute of Genetics and Functional Genomics,
Ernst-Moritz-Arndt-University, Friedrich-Ludwig-Jahn-Str. 15, 17487 Greifswald, Germany
3 Department of Bioinformatics, Institute of Biometrics and Medical Informatics, University Medicine
Greifswald, Ferdinand-Sauerbruch-Str. 1, 17475 Greifswald, Germany
Overexpression, aberrant or enhanced activation of the epidermal growth factor receptor
(EGFR) are frequent events in human cancers. By recruiting specific adaptor proteins to the
activated intracellular receptor tyrosine kinase domain, EGFR signaling regulates
proliferation, growth and differentiation [1]. We hypothesize that cellular responses are
realized by characteristic patterns of adaptor proteins, which switch respective signal
cascades and therefore developed a method to analyze the EGFR adaptor protein pattern
in relation to its physical state.
The EGFR-adaptor protein-complex was isolated by immunoprecipitation using the specific
EGFR antibody cetuximab,which was biotinylated and bound to magnetic beads. A431 cells
were stimulated with 100 ng/ml EGF for 30 minutes or left untreated and EGFR-associated
protein complexes were precipitated from respective cell lysates. For stable isotope labeling
in cell culture (SILAC), the stimulated cells were first adapted to heavy isotopes ( 13C) of Llysine
and L-arginine and light cells were left untreated or incubated with 10 µM Tyrphostin
AG1478 for 1 h before stimulation with EGF. Lysates of heavy ( 13C) and light ( 12C) cultures
were mixed in a 1:1 ratio for precipitation. After pre-fractionation by 1D-SDS-PAGE and ingel-digestion,
protein complexes were characterized by LC-ESI-tandem high resolution
mass spectrometry (LC-MS/MS). SILAC provides a ratio of heavy/light peptides for
quantification of proteins in stimulated and control samples while the label free complexes
were analyzed with the normalized spectral abundance factor (NSAF), which overcomes
sample to sample variation and enables a semi-quantitative determination of proteins from
different samples [2].
A set of 58 proteins was reproducibly identified in three LC-MS/MS replicates. The proteinprotein
interactions within this set were obtained from the iRefIndex database and the
corresponding protein interaction network was visualized in Cytoscape. The Gene Ontology
annotations of the proteins were clustered and visualized in the network as well.. Most
proteins are involved in intracellular localization and protein transport (27 proteins) or cell
death (13 proteins). The first domain contained structural proteins such as actin and tubulin
subunits as well as importins, exportins and transportins. The cell death regulating category
included serine/threonine-protein phosphatase 2A and heat shock protein-ß1. Furthermore,
several proteins were found which associations to the EGFR are yet unknown, such as
Hook homolog 2 and cancerous inhibitor protein 2A. Quantification by SILAC identified
subunits of the clathrin related adaptor protein complex, which were markedly pronounced
upon EGF stimulation, suggesting increased receptor internalization. These data were
confirmed by Western Blotting and immunofluorescence microscopy. EGFR signaling
related proteins like growth factor receptor bound protein 2 (Grb2) and signal transducer
and activator of transcription 3 (STAT3) were not subjected to stimulation dependent
changes. Further dynamic changes revealed proteins which were affected by stimulation
after inhibition of tyrosine kinase activity. This might indicate that alternative EGFR
signaling pathways occurred. For instance, SHC-transforming protein 1 decreased upon
stimulation and even more when EGFR activity was inhibited. Nevertheless, adaptor
proteins are low abundant proteins, which is why we will work out a protein cross linking
strategy to enrich for specific signaling molecules.
As proof of principle, we were able to identify proteins that are part of the EGFR signaling
pathway as well as proteins that relate to cellular localization and protein trafficking and
whose abundances changed upon EGFR stimulation. Using this approach we will provide
further insights into mechanisms of EGFR signal activation from a systems biology point of
view.
References:
1. Oda, K. et al.: Mol.Syst.Biol. 2005, 1:2005.0010.
2. Gokce, E. et al.: J.Am.Soc.Mass Spectrom. 2011, 22: 2199-2208.
120
Metabolism studies of Ifenprodil, a potent NR2B-selective NMDA receptor
antagonist
Falck, E.; Wünsch, B.
Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-Universität,
Hittorfstr. 58-62, 48153 Münster, Germany
Ifenprodil is known as an antagonist for a special binding site at the NMDA receptor subunit
NR2B, called ifenprodil binding site [1]. NMDA receptors change their concentration and
composition with the development of the brain during the lifetime period [2]. Furthermore,
NMDA receptors containing the NR2B subunit are restricted in specific sites of the human
brain [3]. This makes the NR2B subunit an interesting target for the therapy of
neurodegenerative diseases such as schizophrenia, epilepsy or Parkinson’s disease.
Epilepsy for example causes neuronal cell death which leads to an increased glutamate
concentration. This induces an overactivation of the NMDA receptor inducing further cell
death. Ifenprodil as an antagonist can reduce NMDA receptor activation and protect the
brain against further cell damage. The ifenprodil binding site is located on the surface of the
NR2B subunit and can interact with other binding sites located at the receptor. Ifenprodil
has a high affinity to the receptor but a rather low selectivity. The interaction with other
receptors leads to undesired side effects, e. g. impaired motor function. The ifenprodil
binding site at the NMDA receptor represents an interesting target and ifenprodil a
promising lead compound for the development of innovative NMDA receptor antagonists
with comparable affinity but increased selectivity.
In addition to the poor receptor selectivity fast metabolic degradation and low bioavailability
of ifenprodil have been reported in literature. The precise knowledge about the
biotransformation of ifenprodil is important for the design of metabolically more stable
analogues. However, the metabolism of this compound has not been studied in detail.
Herein we report the metabolism of ifenprodil, regarding the phase I and phase II
metabolites resulting from hydroxylation and conjugation reactions.
Transformations were carried out with rat liver microsomes and the essential co-factors
NADPH/H +, UDPGA, PAPS and SAM. The metabolites were identified by fragmentation
steps carried out using HPLC-ESI-MS n. The supposed structures of the resulting fragments
were confirmed by recording the exact mass yielding from time-of-flight detection. All
metabolites were identified by comparing their fragmentation pattern with the typical
fragmentation pattern observed with the parent compound ifenprodil.
References:
1. Williams, K.: Curr. Drug Targets 2001, 2: 285-298.
2. Chazot, P. L.: Curr. Med. Chem. 2004, 11: 389-396.
3. Childers, W. E., Baudy, R. B.: J. Med. Chem. 2007, 20: 2557-2562.
122 Poster
1
121
11-Keto-β-boswellic acids prevent insulitis in 2 animal models with
autoimmune diabetes
Ammon, H.P.T. 1, Shehata, A.M. 2, Jauch, J. 3, Bettio S. 4, Quintanilla-Fend, L. 4
1Department of Pharmacology, Institute of Pharmaceutical Sciences, University of Tuebingen,
2Department of Pharmacology, Faculty of Pharmacy, University of Beni-Sueif, Beni-Sueif, Egypt,
3Institute of Organic Chemistry II, University of Saarland, Saarbrücken, Germany,
4Institute of Pathology, University of Tuebingen, Germany
Background and aims: Type 1 diabetes and Late onset Autoimmune Diabetes of the Adult
(LADA) are autoimmune diseases where a chronic inflammatory process destroys insulin
producing β-cells. Boswellic acids which derive from gum resin of Boswellia species
(Olibanum) have been shown to possess immunmodulatory properties and suppress
proinflammatory cytokines including NFκB (1). In the model of Multiple Low Dose-
Streptozotocin ((MLD-STZ) diabetes and the genetic Non Obese Diabetic (NOD)mouse,
which are thougt to correspond to human one type diabetes, it was tested whether or not
an extract of the gum resin of boswellia serrata (BE), 11-Keto-β-boswellic acid (KBA) and
Acetyl-O-11-keto-β-boswellic acid (AKBA) could prevent developement of insulitis and
appearance of apoptotic cells in the pancreatic islets, increase of proinflammatory
cytokines in the blood and increase of blood glucose level.
Methods: BK +/+ male mice were i.p. injected with 40 mg/kg STZ for 5 days. A second
group received 150 mg/kg BE, a third group 15 mg/kg AKBA and a fourth group 7.5 mg/kg
KBA simultaneously over a period of 10 days. Infiltration of lymphocytes into pancreatic
islets and apoptosis of islet cells were determined histochemically (CD-3 antibodies and
anticaspase-3) after day 10. In addition, proinflammatory cytokines were determined in
serum. Blood glucose was measured over a period of 35 days.
Four weeks old NOD-mice received daily 7.5 mg/kg KBA i.p. over a period of three weeks.
Thereafter the pancreatica have been taken for histochemical studies.
Results: 10 days after first STZ injection there was infiltration of lymphocytes into
pancreatic islets and appearance of apoptotic cells. Moreover, all tested proinflammatory
cytokines in the serum were significantly increased. There was also a continuous increase
of blood glucose. Simultaneous i.p. administration of BE, AKBA and KBA significantly
reduced cytokines in the serum, infiltration of lymphocytes and apoptosis of islet cells. After
21 days blood glucose levels showed no increase.
In NOD-mice which develope insulitis and first apoptotic cells in the pancreas after four
weeks of age administration of KBA prevented both to a large extent.
Conclusion: An extract of the gum resin of Boswellia serrata (olibanum) and two of its
constituents i. e. AKBA and KBA can prevent developement of insulitis probably through
their inhibitory action on proinflammatory cytokines. 11-Keto-β-boswellic-acids seem to be
promising compounds for the prevention and treatment of autoimmune diabetes.
Supported by: The authors are grateful for support for the Dr. h. c. Bürger-Büsing Foundation and the
German Diabetes Foundation.
References:
(1) Ammon, HPT: Phymed. 2010, 17: 862-867.
122
The Thermal Behavior of Histidine in Frozen Aqueous Solutions: The Effect of
pH, Annealing, Histidine Concentration and Formulation Composition.
Alhussein, A. 1 ;Gieseler, H. 1 2
1Department of Pharmaceutics-University of Erlangen, Cauerstrasse 4, 91058 Erlangen, Germany
2 GILYOS GmbH, Friedrich-Bergius-Ring 15, 97076 Wuerzburg, Germany
Therapeutic proteins are a major focus of research and development activities in the
pharmaceutical industry. Due to their limited stability in aqueous solutions, proteins often
need to be converted into solid state to achieve an acceptable shelf life as pharmaceutical
products [1]. The most commonly used method for manufacture of solid protein
pharmaceuticals is freeze drying [1,2]. In turn, freeze drying process (lyophilization)
generates many stresses during both freezing and drying which may cause the loss of
protein bio-activity. Therefore many excipients are added to the protein formulation to
overcome these stresses and to improve protein stability during freeze drying.
Disaccharides, such as sucrose and trehalose, are widely used as protein stabilizers (cryoand
lyoprotectant), and these sugars have been extensively studied in the literature to
investigate thier stabilizing effect on proteins during the lyophilization. However, formulation
development to achieve the best stabilizing effect on protein is still a great challenge for the
experts of freeze drying.

In the last few years, amino acids are attracting increased attention in the field of the freeze
drying of proteins, and many amino acids were used as protein stabilizer in the freeze dried
products [3,4]. In contrast to the sugars, amino acids can also function as buffers in protein
formulations. Therefore amino acids provide more choices for the design of proteins
formulations. Histidine is one of the amino acids that can be added to the protein
formulations to function as both buffer and protein stabilizer [5].
It is well known that amorphism is an essential property for stabilization of proteins during
freeze drying. Protein stabilizer should remain amorphous (in the same phase with protein)
to be able to interact with the protein molecule by the formation of hydrogen bonds, leading
thereby to the preservation of protein native structure [6]. Since the stabilizing effect of an
amino acid (or other stabilizer) during lyophilization is directly correlated with its physical
state, it is important to evaluate the stabilizing efficiency by studying the changes in the
physical state of the amino acid upon cooling and heating.
The aim of this study was to investigate the thermal behavior of histidine in frozen aqueous
solutions at different conditions using DSC technique (Differential Scanning Calorimetry).
This research focused on tow thermal events: Histidine crystallization and the glass
transition of histidine. The effect of histidine concentration and pH of histidine solution on
the physical state of histidine was investigated. Furthermore, some of popular excipients
which are widely used in the freeze dried protein formulations (such as sucrose, mannitol,
and Tween 80) were added into histidine solutions to evaluate thier effect on histidine
during the DSC measurement. Additionally, the effect of annealing (which is a common
practice applied during the freeze drying to enhance the crystallization of the bulking
agents) was investigated, where all DSC scans of the studied formulations were performed
with/without annealing to detect the possible effect of this step on the thermal behavior of
histidine.
The results showed that histidine crystallization may occur during the heating phase of the
DSC-scan and this event depends strongly on both pH and histidine concentration.
However, the enhancement of histidine crystallization by application of annealing seems
also to be pH-dependent. Furthermore, the glass transition temperature of histidine (Tg )
was greatly varied according to the pH of solution. The addition of sucrose to the histidine
solution resulted in an inhibition of histidine crystallization, while the addition of mannitol or
Tween 80 seems to have no inhibitory effect. The interpretation of histidine thermal
behavior in the systems containing both mannitol and sucrose is more complex and
depends on several factors including the weight ratio of mannitol to sucrose in the
formulation.
References:
1. Wang, W.: Int. J. Pharm. 2000, 203(1): 1-60.
2. Pikal, MJ.: Encyclopedia of Pharmaceutical Technology (Marcel Dekker Publishing Co. New York) 2002.
3. Österberg, T.: Pharm. Res. 1997, 14(7): 892-898.
4. Izutsu, K. et al.: Int. J. Pharm. 2005, 301(1-2): 161-169.
5. Österberg, T.: Eur. J. Pharm. Sci. 1999, 8(4): 301-308.
6. Carpenter, JF. et al.: J. Dairy. Sci. 1990, 73(12): 3627–3636.
123
Biomimetic catecholates for the chemical modification of metal surfaces:
antifouling properties
Khalil, F. 1; Maison, W. 1
1 Pharmaceutical and Medicinal Chemistry, Bundesstraße 45, 20146 Hamburg, Germany
Biofouling is the non-specific attachment of biological material (proteins, carbohydrates and
prokaryotic cells) to surfaces upon their exposure to any biological milieu [1]. Biofouling is
of great concern in many areas ranging from marine technology, biosensors, dentistry to
biomedical implants and devices. The most common approach to prepare non-fouling
surfaces is through the immobilization of polymers such as poly(ethylene glycol) (PEG),
which are able to repel the adhesion of proteins and cells [2].
Methods for the covalent immobilization of molecular monolayers rely on the use of
functionalized anchor molecules, including thiols, silanols, phosphates or phosphonates. All
of these immobilization techniques have drawbacks, such as limited stability, elaborate
syntheses or a limited range of substrate materials. Catecholate, inspired by musseladhesive
proteins (MAPs) [3] and bacterial siderophores [4,5], have received considerable
interest because of their high binding affinity to various surfaces [6].
By following a biomimetic approach, we have synthesized conjugates of catecholates and
bifunctional tetravalent scaffolds based on a trisalkylmethyl core structure. Three
catecholateamines have been conjugated to a central scaffold, resembling the tripodal
metal binding motif of natural metal binders, such as MAPs and siderophores [7]. The
resulting trimeric catecholates are easily coupled to different effector molecules for the
stabile immobilization of effector molecules such as PEG on metal surfaces.
Futher, the immobilization of these multivalent catecholate derivatives on TiO2 surface have
been performed and showed promising antifouling properties.
References:
1. Hall-Stoodley, L., Costerton, J. W., Stoodley, P., Nat. Rev. 2004, 2(2), 95-108.
2. Banerjee, I., Pangule, R. C., Kane, R. S., Adv. Mater. 2011, 23(26), 690-718.
3. Waite, J. H., Tanzer, M. L., Science 1981, 212(4498), 1038-1040.
4. Loomis, L. D., Raymond, K. N., Inorg. Chem. 1991, 30(5), 906-911.
5. Gademann, K., Chimia 2007, 61(6), 373-377
6. Lee, H., Dellatore, S. M., Miller, W. M., Messersmith, P. B., Science 2007, 318(5849), 426-430.
7. Franzmann, E., Khalil, F., Weidmann, C., Schröder, M., Rohnke, M., Janek, J., Smarsly, B., Maison, W.,
Chem. Eur. J. 2011, 17(31), 8596-8603.
124
Quantification of impurities with NMR spectroscopy: Comparison of different
approaches to quantify small signals overlapped with large signals using the
example topiramate.
Wiest , J.; Ilko , D.; Holzgrabe, U.
Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg,
Germany
Molecules without chromophores like topiramate (TOP) pose a challenge to analysts as
they cannot easily be quantified via common photometric detection regimes like UV/VIS or
fluorometry. Our aim was to develop an HPLC-CAD method for the determination of purity
and content of TOP and to compare the results to the existing HPLC refractive index
detection (HPLC-RI) method which has a poor sensitivity and a limited performance. The
newly developed HPLC-CAD method obtained good results for quantification of TOP and
the other related substances, however the main impurity diacetone fructose (DAF) could
not be determined due to its high vapour pressure. To solve this problem, we tried to apply
NMR spectroscopy. Prerequisite of qNMR is a clear separation of the signals of all
components used for quantification. Variation of the solvent resulted in pyridine-d5 which
gave the best signal separation.The spectra were acquired with inverse gated 13C
decoupling using globally optimized alternating phase rectangular pulse (GARP) to remove
disturbing 13C satellites. To avoid rotation sidebands the spectra were measured without
spinning. Processing the spectra has a great influence on the results. Thus we compared
the results of four Topiramate batches with an internal standard using a standard addition
method. The results were obtained with three different approaches: (i) Deconvolution of the
overlapping signals executed with TopSpin 3.0; (ii) Integration after baseline adjustment (iii)
Quantification by signal height. An experimental protocol was found which is particularly
suitable for quantification of DAF residues in TOP samples.
References:
Almeling, S.; Ilko, D.; Holzgrabe, U.: J. Pharm. Biomed. Anal. 2012, Epub ahead of print
www.elsevier.com/locate/jpba , 23.05.2012
Pauli, G.F.: Phytochem. Anal. 2001, 12: 28-42
125
Impact of drug-drug interactions on the INR values of patients treated with
Marcumar ® in the ambulatory care setting
Bertram, L. 1; Krämer, I. 1
1 Department of Pharmacy, University Medical Center of Johannes Gutenberg-University,
Langenbeckstr.1, 55131 Mainz, Germany
PURPOSE: Treatment with the oral vitamin K antagonist phenprocoumon (Marcumar ®) is
associated with a high risk of interactions with additionally prescribed medication and selfmedication.
In consequence the efficacy of Marcumar ® can be increased or reduced and
the resulting INR-values may drift out of the therapeutic range. Thus the risk of thromboembolic
events or bleeding increases.
The aim of this study was to investigate whether patients’ INR-values in the ambulatory
health-care sector in Germany are influenced by additionally prescribed potentially
interacting drugs [1].
METHODS: 50 patients treated with Marcumar ® were evaluated in a retrospective study
over three years (1800 months in total). The INR-values and type of comedication of ten
patients each recruited in five physician offices in Rhineland-Palatinate were retrieved from
the patients’ medical records. The percentage rate of INR values lying in the target range
(e.g. INR 2-3) and extended target range (target range +/- 0.2) was evaluated for individual
patients. Time in therapeutic range (TTR) was calculated by using the Rosendaal-Method
[2]. Potentially interacting drugs associated with serious interactions were chosen from the
literature [1]. The Mann-Whitney-U-test was used to determine any significant relationship.
RESULTS: Patients taking potentially interacting drugs in addition to a permanent
phenprocoumon therapy achieved lower rates of adequate INR values than patients not
taking potentially interacting drugs. The median percentage rate of INR values within the
therapeutic range amounted to 69% without interacting comedication and 53% with using
interacting comedication (p= 0.008) as well as 83% versus 67% (p= 0.076) for the median
extended therapeutic range, and 75% versus 61% (p= 0.002) for median TTR respectively.
CONCLUSION: Results document that the concomitant intake of interacting drugs
significantly affects the rate of INR-values in therapeutic range and TTR. With regard to the
extended target INR range the difference is not significant. However study results clearly
show that the efficacy of phenprocoumon in routine clinical practice is depending on the
potentially interacting concomitant drug therapy. These combinations should be avoided
whenever possible because additional monitoring is necessary and safety of the
anticoagulant therapy is compromised.
References:
1. S. Isstas, „Pharmakovigilanz bei Patienten mit Phenprocoumon-Therapie- Untersuchungen zu
potentiellen Interaktionen und Betreuungsqualität“, dissertation, Johannes Gutenberg-UniversityMainz,
2011.
2. Rosendaal, F.R. et al.: Thromb. Haemost.1993, 69: 236-239.
126
A novel combinative particle size reduction technology for drug nanocrystals
production and further nanosuspension transfer to tablets
Poster 123

Salazar J1, Müller R H1, Möschwitzer J P1 2
1 Institute of Pharmacy, Dept. of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Freie
Universität Berlin, Kelchstrasse 31, 12169 Berlin, Germany
2 Pharmaceutical Development, Abbott GmbH & Co. KG, Knollstrasse 50, 67061 Ludwigshafen am
Rhein, Germany
The aim of this research was to systematically analyze the process parameters affecting
the comminution effectiveness of a novel particle size reduction method. This combinative
method employs spray-drying (SD) as bottom-up step and drug pre-treatment followed by
high pressure homogenization (HPH) as top-down process for nanosuspension production
1 2. The liquid nanosuspensions were then transferred to immediate release (IR) tablets by
applying downstream techniques such as wet granulation (WG), freeze-drying (FD) or SD.
Finally, a dissolution test was performed for drug release assessment.
The poorly water-soluble compound glibenclamide (BCS Class II) was used as model drug.
The influence of drug and surfactant concentrations during SD on the particle size reduction
effectiveness of the HPH was analyzed. The drug was dissolved in ethanol to prepare
solution concentrations of 1%, 2% or 3% (w/v). The amounts of docusate sodium salt
(DSS) in the ethanolic solution were 0%, 0.1%, 0.2%, 0.3% or 0.4% (w/v). The ethanolic
solutions were then spray-dried using a Mini Spray Dryer B-290 coupled to an Inert Loop B-
295 (Büchi Labortechnik AG, Switzerland). The morphology and solid state of the spraydried
powders were analyzed by SEM, DSC and PXRD. Suspensions were produced by
dispersing 1% (w/w) drug (treated/untreated) in demineralized water using an Ultra-Turrax
T-25 (IKA Werke GmbH & Co. KG, Germany). In the case of the drug powders processed
without surfactant DSS 0.2% (w/v) was added as dispersant. The different suspensions
were then homogenized by employing the HPH technique (1500 bar, 20 cycles) using a
Micron LAB 40 homogenizer (APV Systems, Germany). The particle size of the
nanosuspensions was analyzed using photon correlation spectroscopy and laser
diffractometry. The liquid nanosuspensions were down-streamed by WG, FD or SD and
then processed to tablets using lactose monohydrate as filler. Mannitol was used as matrix
former for FD and SD (0%-6% w/v). A dissolution test was performed to determine the
immediate drug release performance of the tablets.
It was found that high drug concentrations and middle DSS concentration resulted in
porous, flowable and amorphous drug powders after SD. These characteristics resulted in
smaller particle sizes after reduction. The drug pre-treatment can significantly improve the
particle size reduction effectiveness of the HPH. The best nanosuspension produced with
the spray-dried powders (2% drug, 0.2% DSS during SD) showed a very small particle size
(236 nm mean particle size, d50% of 0.131 µm and d90% of 0.285 µm) and a narrow size
distribution. The best drug release was 90% after 10 min and it was delivered by SD of the
best nanosuspension with 2% mannitol before tableting. This drug release was markedly
better compared to the one delivered by the tablets processed with micronized drug (26%
drug release after 10 min). The rank order of the tablet release rate employing the
downstream techniques was: SD tablets > FD tablets > WG tablets. The surfactant and
drug concentrations used for the SD process are critical and need to be selected carefully.
The transfer of the drug nanocrystals to tablets was successfully achieved.
References:
[1]: Möschwitzer, J., Method for producing ultrafine submicronic suspensions, WO/002006094808A3, 2006
[2]: Möschwitzer, J., Müller R.H., New method for the effective production of ultrafine drug nanocrystals,
Journal of Nanoscience and Nanotechnology 6(9-10): 3145-3153, 2006
127
Differential scanning calorimetry as a supportive tool for the analysis of
nanomilled fenofibrate suspensions
Komoß, C. 1; Bitterlich, A. 2; Kwade, A. 2; Bunjes, H. 1
1Institute of Pharmaceutical Technology, TU Braunschweig, Mendelssohnstraße 1, 38106
Braunschweig, Germany
2Institute for Particle Technology, TU Braunschweig, Volkmaroder Straße 5, 38104 Braunschweig,
Germany
An important approach to overcome poor dissolution properties and the often resulting bad
biopharmaceutical profile of active pharmaceutical ingredients (API) is to reduce the API´s
particle size, a process also known as “nanosizing” [1]. This leads to a strongly increased
surface-to-volume ratio and, thus, to an increased dissolution rate. However, the large
specific surface area creates a positive gain of free energy. Hence, there is a tendency
towards crystal growth, (re-)agglomeration of the particles and, thus, instability of the
nanosuspensions.
A reduction of particle size can also lead to altered solid state properties like a reduced
melting point [2]. Therefore, in this study, differential scanning calorimetry (DSC) analysis of
nanosuspensions of the lipid lowering agent fenofibrate (kindly provided by Novartis
Pharma AG) was conducted. The suspensions were produced by wet media milling using a
modified planetary ball mill [3] (PM400, Retsch GmbH) or a stirred media mill (Dispermat
SL 5 C nano, VMA Getzmann GmbH) and different polymers as stabilizers.
Nanosuspensions were additionally analyzed using dynamic light scattering (DLS) and
optical methods like transmission electron microscopy (TEM) and polarization microscopy.
The evolution of onset and peak maximum temperature of PVA-stabilized fenofibrate
nanosuspensions over grinding time was investigated via DSC indicating that both values
decreased with longer grinding times [fig.1]. However, particle size determined via dynamic
light scattering passed through a minimum and increased again with extended grinding
times. TEM micrographs revealed that this phenomenon resulted from the formation of
agglomerates containing extremly small particles after very long grinding times. Thus, the
decrease of onset and peak maximum temperature was correlated with decreasing primary
particle size.
Furthermore, DSC was applied to differentiate between different types of instability of the
fenofibrate nanosuspensions: particle growth or agglomeration of very small crystals,
respectively. For this purpose, the onset and peak maximum temperatures of a HPMCstabilized
nanosuspension at day 0 and after 1 week of storage under different conditions
were compared. In combination with TEM investigations, the data clearly showed an
increase in melting temperature for the samples with a pronounced crystal growth. DSC
analysis can thus be a helpful approach not only for getting information about the real
primary particle size but also for differentiating between different kinds of instabilities.
124 Poster
Temperature [ C]
78
77
76
75
74
73
72
71
70
69
68
Onset
Peak
z-avg
4 6 8 10 20 40 60 100 200 400 800
Grinding time [min]
Fig. 1: Onset and peak maximum temperature determined via DSC and particle size (zaverage)
determined via DLS of a PVA-stabilized fenofibrate nanosuspension at different
points of the milling process
References:
1. Merisko-Liversidge, E., Liversidge, G.G.: Adv. Drug Deliv. Rev. 2011, 63(6): 427-440.
2. Bunjes, H., Koch, M. H. J., Westesen, K.: Langmuir 2000, 16(12): 5234–5241.
3. Juhnke, M., Berghausen, J., Timpe, C.: Chem. Eng. Technol. 2010, 33(9): 1412-1418.
128
Hydrogels for drug delivery: hydrolytically cleavable carbamate linkers for time
controlled protein release
Hammer N. 1,Brandl F.P. 1, Kirchhof S. 1 , Meßmann V. 1, Teßmar J.K.V. 1, Göpferich A. 1
1 University of Regensburg, Department of Pharmaceutical Technology, Universitätsstr.31
Regensburg, 93053, Germany
In the past decades, the significance of macromolecular drugs of biological origin has
increased enormously. However, their application is still hampered by short biological halflives.
Hydrogels are one possibility to protect these delicate molecules against
physicochemical instability and proteolytic degradation. Furthermore, hydrogels are
considered biocompatible due to their physicochemical similarity to native extracellular
matrix. Despite these advantages, one frequently observed problem is the rapid release of
incorporated proteins due to the high water content of the hydrogels. To overcome this
problem, proteins can be reversibly attached to the hydrogel backbone by suitable linker
molecules.
We used non-degradable hydrogels to characterize carbamate-mediated protein release.
The gels were formed from star-shaped poly(ethylene glycol) (PEG) alkyne and starshaped
PEG azide; the cross-linking reaction was catalyzed by 0.5 µmol CuSO4 and 2.5
µmol ascorbic acid. For the attachment of proteins to the hydrogel structure, we
synthesized different star-shaped PEG linkers. Two to three of the four end-groups were
modified with an alkynyl groups to allow cross-linking with star-shaped PEG azides. The
remaining end groups were modified with hydrolytically cleavable carbamates for reversible
protein binding.
Gel properties were studied by oscillatory rheology. Swelling and stability of the gel
cylinders in phosphate buffered saline at 37 °C were analyzed by weighing the cylinders at
regular time points. We compared protein release from hydrogels with and without
carbamate linkers. For protein binding, lysozyme was incubated with the carbamate linker
for 2h at room temperature and mixed with additional polymer to form hydrogels. The gel
cylinders were incubated in phosphate buffered saline at 37 °C. The amount of released
protein was determined at regular time points by Bradford assay.
Gelation started after about 16 min depending on the copper concentration.Oscillatory
rheology showed a gel strength of 7600 Pa, which indicates an almost complete
crosslinking reaction. Hydrogels with attached lysozyme still had a gel strength of 5400 Pa.
We determined swelling ratios of about 1.2 (without lysozyme) and 1.28 (with lysozyme),
respectively. The gel cylinders were stable for more than 2 weeks. Without binding,
incorporated lysozyme was released within the first 24 h. Attachment of the protein to the
hydrogel backbone decelerated the release significantly. After cleavage of the carbamate
linker, 22 % of lysozyme was released within 14 days. The study is not completed yet;
further time points will follow.
lysozyme conc. [%]
20%
release of attached lysozyme
0%
0 5 10 15
time [d]
Altogether, covalent attachment of proteins to the gel structure by degradable carbamate
linkers seems to be a promising strategy to control protein release.
Acknowledgments: DFG, grant GO 565/16-1
lysozyme conc. [%]
50%
1000
900
800
700
600
500
400
300
200
100
0
Particle size [nm]
release of lysoyzme without linker
100%
0%
0 20
time [h]
40

129
Production of Resveratrol Nanocrystals by a Novel Combinative Process (H 42
Technology)
Liu, T. 1; Müller, R. H. 1; Möschwitzer, J. P. 1 2
1 Institute of Pharmacy, Dept. of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Freie
Universität Berlin, Kelchstr.31 Berlin, 12169,Germany
2 Pharmaceutical Development, Abbott GmbH & Co. KG, Knollstr. 50 Ludwigshafen am Rhein,
67061, Germany
In order to improve the manufacturing efficiency of drug nanocrystal, a new combinative
particle size reduction technology (H 42) has been developed recently. Spray-drying as a
bottom-up step is applied to modify the properties (e.g. crystallinity and morphology) of the
raw drug particles. The subsequent high pressure homogenization step is used to obtain
the final nanosuspensions. According to the design of experiment (DoE) methodology,
different compositions of resveratrol (API) and sodium cholate (surfactant) were dissolved
in ethanol and the solutions were spray-dried using a mini spray dryer B-290 with inert loop
B-295 (Büchi Labortechnik AG, Switzerland). The crystallinity of spray-dried powders was
analyzed by differential scanning calorimetry (DSC, Mettler Toledo AG, Germany) and
powder X-ray diffraction (PXRD) with Philips PW 1710 diffractometer control unit (Philips
Industrial & Electro-Acoustic Systems Division, The Netherlands). High pressure
homogenization (HPH) for 20 cycles at 1500 bar using a Micron Lab 40 homogenizer (APV
Deutschland GmbH, Germany) was employed to produce nanosuspensions. Laser
diffractometry (LD, Malvern Instruments, UK) and photon correlation spectroscopy (PCS,
Malvern Instruments, UK) were applied to determine the particle size. The PXRD and DSC
results showed that 6 DoE points of spray-dried powder resulted in amorphous drug, which
evidently indicated a relationship between formulation of the feed and final API crystallinity.
After only 1 homogenization cycle at 1500 bar, a particle size of around 200nm (d 0.5) was
achieved by using modified API, which was comparable with the result of unmodified API
after 20 cycles. The production time for the nanocrystals had been significantly reduced. On
the other hand, the largest particle size (d 0.9) obtained from modified API (736 nm) was
much smaller than the unmodified drug (2.2 µm) after 20 cycles. However, no direct
correlation was found between the degree of crystallinity and the particle size of the drug
nanocrystals after HPH. To sum up, due to the short production time and low energy input
the new combination technology (H 42) was confirmed to be an effective method to
produce resveratrol nanocrystals. Further more detailed study will be focused on the
mechanism of this process and influencing factors of particle size reduction.
130
Differentiation of several identically prepared batches of tablets using near
infrared spectroscopy
Kaminski, D; Baumann, K
1 Technische Universität Braunschweig, Institute for Medical and Pharmaceutical Chemistry,
Beethovenstr. 55, 38106 Braunschweig, Germany
Near infrared spectroscopy (NIR) is the first choice method in pharmaceutical industry for
rapid inspection of raw materials. It is also suitable for online process control of the
products as the NIR is a nondestructive analytical technique. Chemical composition as well
as physical properties can be monitored. Even small variations, which are unavoidable
during the production process, lead to significant changes in NIR spectra. This allows for a
close monitoring of the physical properties, such as compaction pressure [1], humidity [2]
and the homogeneity of active pharmaceutical ingredient (API) distribution [3] during the
production process and the storage of tablets.
This leads to the question whether it is possible to differentiate several batches of directtabletted
ibuprofen with exactly the same manufacturing process by means of chemometric
methods. Different methods of data pre-treatment are compared and an outlier detection is
performed by principal component analysis (PCA). Those samples that show outlying
spectra are scrutinized with respect to their content of the AIP which is independently
determined by titration. The tabletting material and the production process are deliberately
varied in order to study the sensitivity of the developed chemometric methods.
The approach presented here attempts to separate the random error and deterministic
variations in the spectral signal. The goal of this work is to define confidence limits based
on a comprehensive characterization of all sources of spectral variations in order to detect
tablets which are likely out of specification for further analysis by orthogonal analytical
techniques.
References:
1. Kirsch JD, Drennen JK: J. Pharmaceut. Biomed. 1999, 19: 351-362
2. Last IR, Prebble KA: J. Pharm. Biomed. Anal. 1993, 11: 1071-1076
3. Plugge W, Van der Vlies C: J. Pharm. Biomed. Anal. 1993, 11: 435-442
131
Development of a medicated chewing gum containing riboflavin phosphate
Wilde L. 1, Wollatz U. 1, Kordaß B. 2, Glöckl G. 1, Weitschies W. 1
1Institute of Pharmacy, Ernst-Moritz-Arndt-University, Department of Biopharmaceutics &
Pharmaceutical Technology, Center of Drug Absorption and Transport (C_DAT), Felix-Hausdorff-
Straße 3, D-17487 Greifswald, Germany
2Greifswald University Hospital, D-17475 Greifswald, Germany
Medicated chewing gums are most often used for the treatment of travel-sickness or for the
dehabituation of smokers. It was our idea to test, whether they might also be applicable to
characterise the chewing motion of different patients by dentists. Therefore, a medicated
chewing gum was developed, which contains an active agent, Riboflavin-5’-phosphate
sodium, that can be dissolved by the saliva. Two different chewing gum matrices were
investigated: Cafosa Gum S/A (Spain) and Gumlink A/S (Denmark). Both products are
lipophilic mixtures consisting of elastomers for elasticity, resins for a cohesive body, waxes
as softening agents and fats as plasticizers. Both gum bases can be compressed directly
with the help of an eccentric tablet punch. For the preparation of the Gumlink gums, 19.9 g
gum base and 100 mg lecithin were mixed in a cube blender for 10 minutes. After addition
of 4 mg riboflavin phosphate and again blending for 10 minutes, the punches had to be
lubricated with magnesium stearate to prevent sticking. The Cafosa gums were prepared in
the same way, but with 19.8 g gum base, 200 mg lecithin and 20 mg riboflavin phosphate.
In this way, we produced biconvex gums with a diameter of 12 mm and a weight of 500 mg.
For the determination of content, a gum was given into an Erlenmeyer flask and 6 mL of
heptane was added. After complete dissolution of the gum 20.0 mL purified water was
added, the two phases were mixed intensely for 2 minutes and then separated by standing
for 1 minute without stirring. For the Cafosa gums, two millilitres of the water phase were
diluted and analysed at a wavelength of 267 and 600 nm (background correction) using a
Cary 50 Scan (Varian). The water phase of the Gumlink gums was analysed without
dilution. The determination of content show a content uniformity for the Gumlink chewing
gums of 0.091 mg ± 0.0006 mg (n = 7) and for the Cafosa gums of 0.886 mg ± 0.070 mg (n
= 7).
For the chewing trials, the gums were chewed 5, 7 or 10 times (n = 6) by one volunteer,
removed with a forceps and the saliva was rinsed off with purified water. Afterwards the
determination of content followed as mentioned above.
The results for the first chewing tests (figure 1) showed that chewing five times dissolved
about 25 % of active agent, while seven chews increased this value to nearly 35 %. Ten
chews released even more than 50 % of riboflavin phosphate out of the chewing gum
matrix.
Figure 1: Amount of riboflavin phosphate (Gumlink) after chewing 5, 7, 10 times; nominal
quantity in each gum: 0.1 mg riboflavin phosphate (n = 6, mean ± SD).
The Cafosa gum base was less suitable for spectrometric analysis because of the opacity
of the solution even after filtration and centrifugation. That was the reason for higher
absorption and thus incorrect content readings.
We succeeded in producing uniform chewing gums with a small amount of hydrophilic
active agent by direct compression and developed a method for the determination of
content. We showed that the number of chews affects the dissolution of this agent, so that
conclusions can be drawn from the chewing motion.
Acknowledgments: The gum bases were a gift donated by Cafosa Gum S/A (Spain) and Gumlink A/S
(Denmark). Financial support by the Federal Ministry of Education and Research (FKZ: 03IP612) is
gratefully acknowledged.
132
Test purchase of slimming products containing unapproved active ingredients
that may cause harmful side effects
Parr MK1 2; Geyer H1; Paul M3, Smolka K4, Schänzer W1 1 Freie Universität Berlin, Pharmaceutical Chemistry, Königin-Luise-Str. 2+4, 14195 Berlin,
Germany
2 German Sport University Cologne, Biochemistry, Am Sportpark Müngersdorf, 50933 Cologne,
Germany
3 Stadt Köln, Gesundheitsamt, Neumarkt 15-21, 50667 Cologne, Germany
4 Zollkriminalamt, Bergisch-Gladbacher-Str. 837, 51069 Cologne, Germany
An increasing trend for attractive body appearance, stronger muscles and slender shape,
coupled with a maximum of convenience is recognized in broad parts of society.
Suggestions from advertisements and the easy access of image enhancing products,
combined with the implication that such products cannot be harmful, make people believe
that they can simply take “pills for everything” [1].
Thus a huge market of dietary supplements offers numerous products containing a variety
of unapproved and partially also harmful ingredients.
Herein we present the results of products obtained by test purchases on the Internet. All
products promised to cause extreme weight losses in a very short time period. The
products were delivered by regular mail after credit card payment. No cautionary note was
found on the label of the products.
They were tested by GC-MS for their active ingredients. One of the products, named DNP
Burn, brand name Body Advance Performance, was found to contain high amounts of 2,4dinitrophenol
(Figure 1(1)). Identity was confirmed by comparison with authentic reference
material. Semi-quantitation yielded an amount of about 200 mg. DNP uncouples the
oxidative phosphorylation in the mitochondria resulting in a drastically increased metabolic
rate. Several fatalities have been recorded after its intake [2].
Other products were tested positive for the presence of methylhexanamine (Figure 1(2)).
Due to its indirect sympathomimetic effects it was tested as locally administered
vasocontrictor for the relief of hyperplasic nasal mucosa. Currently no pharmaceutical
containing methylhexanamine is approved. Nowadays lots of preparations with
methylhexanamine are marketed as supplements for slimming purposes. Increasing
numbers of adverse analytical findings with methylhexanamine in doping control [3] indicate
a growing market. Recently two fatal casualties in physical stress under the influence of
methylhexanamine are investigated. This resulted in a recall of such products by the US
FDA [4].
Labelling of weight loss supplements uses several substitutes such as geranium root
extract, geranium oil extract, Geranamin(e), 4-methyl-2-hexanamin(e), Forthan(e), 4methyl-2-hexylamin(e),
2-hexanamine-4-methyl-, 2-methyl-4-methylhexan, Floradrene, 1,3dimethylamylamin(e),
DMAA, 1,3-dimethylpentylamin(e), 2-amino-4-methylhexan(e), or
pentylamine-1, 3-dimethyl- making the inference almost impossible for the consumers.
Poster 125
OH
NO 2
NO 2
NH 2
(1) (2)
Figure 1: Chemical structure of ingredients of weight loss supplements
(1) 2,4-dinitrophenol, DNP, (2) 4-methylhexan-2-amine
References:
1 Graham MR, Ryan P, Baker JS, Davies B, Thomas NE, Cooper SM, Evans P, Easmon S, Walker CJ,
Cowan D, Kicman AT. Counterfeiting in performance- and image-enhancing drugs. Drug Test Anal,
2009;1(3):135-42.

2 U.S. Dept Health & Human Services/Agency for Toxic Substances & Disease Registry. Hazardous
Substances Data Bank HSDB. Toxicological Profile for Dinitrophenols, 1995, online at
http://www.atsdr.cdc.gov/ToxProfiles/tp.asp?id=729&tid=132, accessed 16.05.2012
3 World Anti-Doping Agency. Adverse analytical findings reported by accredited laboratories, online at
http://www.wada-ama.org/en/Science-Medicine/Anti-Doping-Laboratories/Laboratory-Statistics, accessed
16.05.2012
4 DMAA News: FDA challenges marketing of DMAA products for lack of safety evidence, 2012, online at
http://fdarecalls.wordpress.com, accessed 16.05.2012
133
Coating of collars via fluidised-bed technology: Influence of process
parameters on coating homogeneity
Wentzlaff, M. 1; Seidlitz, A. 1;Nagel, S. 1; Harder, C. 2; Schnittker, C. 3; Trip, E. 3; Bock, R. 3;
Grabow, N. 4; Sternberg, K. 4;Weitschies, W. 1
1Biopharmaceutics and Pharmaceutical Technology, University of Greifswald, Felix-Hausdorff-
Str.3, 17487 Greifswald, Germany
2Biotronik SE & Co.KG, Hartmannstr. 65, 91052 Erlangen, Germany
3Biotronik SE & Co.KG, Woermannkehre 1, 12359 Berlin, Germany
4Institute for Biomedical Engineering, University of Rostock, Friedrich-Barnewitz-Str.4, 18119
Rostock, Germany
Introduction
Collars are polymeric tubes mounted on cardiac pacemaker leads containing an active
substance to reduce inflammatory and proliferative reaction of myocardia after implantation
[1,2]. Currently the active pharmaceutical ingredient is incorporated in collars during its
manufacturing via injection moulding technology. However, this often results in high drug
content variability. The fluidised-bed process is an established method used in galenics for
coating of a large amount of small dosage forms with high uniformity.
The purpose of this research was to adapt the process parameters of fluidized-bed process
for coating of collars.
Materials and Methods
The fluidised-bed apparatus Mini-Glatt (Glatt GmbH, Germany) was used for coating of
placebo-collars made of polyurethane. An aqueous dispersion of Eudragit RS ® 30D
containing the water-soluble model substance fluorescein sodium was usedas the coating
lacquer. Different coating processes parameters like spraying pressure, varied nozzle
inserts and spray rate were investigated for optimisation coating homogeneity. Coating
homogeneity was studied by scanning electron microscopy.
Results
By varying the spraying pressure it is possible to influence the surface morphology. While a
spraying pressure of 0.7 bar created a rough surface morphology the surface became
smoother when increasing the spraying pressure to 1.1 bar. A smoother surface was also
created by reducing the nozzle orifice. It was shown that a nozzle orifice with a diameter of
0.3 mm reduced the surface roughness compared to an orifice with a diameter of 0.5 mm.
A reduction of the spray rate from 2.35 g/min to 0.77 g/min also resulted in a smoother
surface.
Conclusion
Our findings provide a basis for coating of collars via fluidized-bed technology. By adapting
the process parameters it was possible to produce coatings with desired morphologic
characteristics.In order to obtain a sufficient coating thickness with a uniformdrug content,
coating conditions have to be defined precisely. Therefore, further trials with optimization of
coating dispersion concentrations and composition are necessary to ensure minimum
mechanical stress acting on the collars and coatings.
Acknowledgments: This project was partially funded by the Federal Ministry of Education and Research
(BMBF) within the research project REMEDIS
References:
1. A. Wilson et al.: PACE1990, 13: 1876-1878.
2. G. Brewer et al.: PACE 1988, 11: 1760-1769.
134
Evaluation of Interactions between Extractables of Elastomeric Closures and
Protein Therapeutics
Richter, C. 1 2, Lipke, U. 1, Zapf, T. 1, Lamprecht, A. 2, Lipperheide, C. 1
1 Federal Institute for Drugs and Medical Devices, Kurt-Georg-Kiesinger-Allee 3, 53175 Bonn,
Germany
2 Institute of Pharmacy, University of Bonn, Gerhard-Domagk-Str.3, 53121 Bonn, Germany
Parenteral liquid medicinal products are commonly offered in glass containers sealed with
stoppers, which are made of elastomeric materials by vulcanization with the utilisation of
various additives. These additives are predominantly small molecules. As they remain
unlinked in the rubber material after the production process they are capable to migrate into
the product formulation (“leachables”) during product shelf life and to react with the
ingredients of the formulation. Particularly, medicinal products containing proteins as active
ingredients are susceptible to react with substances released from the elastomeric material.
In order to confirm compatibility between container and the product migration studies are
requested prior to marketing authorisation. They should give evidence that quality, safety
and efficacy of the protein containing medicinal product remains unaffected by leachables
until end of shelf-life.
The aim of the present study is to establish an appropriate method for evaluating the impact
of leachables from elastomeric closures on protein stability without time-consuming
migration studies.
For the study various rubber stoppers were purchased from different vendors from the
market. These rubber stoppers were extracted by applying worst case conditions
(extraction with 2-propanol for 3 hours under reflux conditions) in order to receive the
maximum amount of extractable substances (“extractables”) which is equivalent to the
maximum amount of potential leachables. The extraction profiles of the different rubber
stoppers were examined by RP-HPLC-DAD with a method, especially developed for the
simultaneous analysis of polar and rather non-polar extractables. For further investigations,
a chlorobutyl rubber stopper was chosen which showed the highest amount of extractables.
Extractables stock solution was prepared as follows: 16 samples of the chlorobutyl rubber
stopper were extracted in 200ml of 2-propanol for 6h under reflux conditions. After filtration
(PE filter 0.45 µm) and solvent evaporation an oily residue was obtained. The residue was
dissolved in 20ml of medium (20% (w/w) 1.2-propanediol and 0.5% (w/v) polysorbate 80 in
water) for 1h in water bath (60-70°C). The resulting stock solution was filtrated and the
amount of extractables was analyzed by RP-HPLC-DAD to confirm all extractables solved
in the stock solution.
The extractables stock solution was added to solutions of human serum albumin and
different protein therapeutics and stored at 5°C. Samples were taken at several time points
and examined by size-exclusion chromatography and capillary electrophoresis in order to
detect physical and chemical alterations in protein structure as a result of the contact with
the extractables.
The proposed test approach turned out to be a suitable alternative to time consuming
migration studies to investigate potential interactions between rubber stoppers and protein
therapeutics.
135
In vitro dissolution testing of drug-eluting stents: Impact of implantation
procedure
Seidlitz, A. 1; Nagel, S. 1; Semmling, B. 1; Grabow, N. 2; Sternberg, K. 2; Weitschies, W. 1
1 Institute of Pharmacy, EMA University of Greifswald, Felix-Hausdorff-Straße 3, 17487 Greifswald,
Germany
2 Institute for Biomedical Engineering, University of Rostock, Friedrich-Barnewitz-Straße 4, 18119
Rostock, Germany
Drug-eluting stents (DES) are drug/device combination products that are used to physically
and pharmacologically prevent the renarrowing of a previously occluded natural passage.
In the case of coronary DES the drug is intended to suppress intimal hyperplasia which
often occurs after balloon angioplasty and stenting. Due to the placement of the DES in
contact with the vessel wall, which is the site of action for the released drug, and the flowing
blood, not only drug release but also its distribution between these compartments is of
greatest importance. In vitro drug release and distribution can be examined using the
vessel-simulating flow-through cell [1], a method adapted from the compendial flow-through
cell (USP 4) for DES. In this setup the DES is implanted into a hydrogel compartment
simulating the vessel wall and is perfused by PBS pH 7.4 representing the blood which is
recirculated through the system (closed loop). For the studies presented here a
haemostatic valve (Y-connector with rotating adapter and Tuohy-Borst valve) was added to
the setup allowing for the placement of the DES in the gel via balloon catheter under flow
conditions. Release and distribution of fluorescent model substances from coated stents
(hydrophilic fluorescein sodium or hydrophobic triamterene embedded in a
polymethacrylate blend, Eudragit RL/RS ratio 3/7) were examined using this setup. Stents
were introduced via the haemostatic valve and the placement in the gel lumen under media
flow (flow rate 35 mL/min, perfusion time prior to stent expansion 1 min) was compared to
expansion into the air-filled gel lumen and subsequent perfusion. The experiments were
terminated after different perfusion times up to 60 min and drug content in the perfusion
media, in the hydrogel and the amount remaining in the stent coating were determined
fluorimetrically. The results are presented in figure 1.
Figure 1: Normalized amount of fluorescein sodium (A) and triamterene (B) in the
compartment media, hydrogel or stent coating over time as a function of implantation
procedure (without media present in the lumen or with media flowing through hydrogel
during stent placement at 35 mL/min), n = 3 for each data point, mean ± SD.
The initial distribution of released fluorescein sodium between the gel and media was
greatly influenced by the implantation procedure. A much lower concentration in the
hydrogel was observed when implanting under media perfusion of the lumen. This must be
attributed to the fact that a certain amount of the fast released model substance is released
exclusively into the media prior to contacting the hydrogel. When implanting without flow on
the other hand, the initial release occurs solely into the gel for the first seconds. For
triamterene, which is released much slower from the stent coating, this initial difference
concerning the available acceptor compartments does not seem to have an impact on
model substance release and distribution. Based on these results, the implantation
procedure can be expected to have a great influence on release and distribution from fast
releasing stent systems. The methods is also promising for other drug/device combination
products applied in the vascular system such as drug-eluting balloons, which are intended
to deliver their drug load to the vessel wall within very short contact times of approximately
1 min.
Acknowledgments: Financial support by the European Regional Development Fund (ERDF) and the
European Social Fund (ESF) within the collaborative research between economy and science of the state
Mecklenburg-Vorpommern is gratefully acknowledged. The authors thank Biotronik SE & Co. KG, B. Braun
Melsungen AG and Rhoem GmbH for generously supplying raw materials.
References:
1. Seidlitz, A. et al.: J. Control. Release 2008, 130(1): 2-8.
136
A Novel Method to Characterize Screw-Elements in Pharmaceutical Twin-
Screw-Extrusion
Kiene, F. 1; Eeckman, L. 2; Thommes, M. 1
1 Institut für Pharmazeutische Technologieund Biopharmazie, Heinrich-Heine-Universität
Düsseldorf, Germany
2Department of PharmaceuticsLaboratory of Pharmaceutical Technology, Ghent University,
Belgium
Twin screw-extrusion is a commonly used technique in pharmaceutical applications. One of
the relevant parameters in pharmaceutical extrusion is the screw configuration, because it
can be adjusted to achieve desired product properties[1].The aim of this study was to
characterize different types of conveying and kneading elements by their specific mechanic
power and their transportation capacity, using high viscous silicon oil as model compound.
For this, a part (4D) of the barrel of the lab scale extruder Micro 27GL-28D (Leistritz,
Nuremberg, Germany) was separated with a front- and back-plate in order to investigate a
smaller section of the barrel. A pressure sensor,I31-S-6-M-B01C-1-4-D (Gefran,
126 Poster

Provagliod’Iseo Brescia, Italy),was attached to the front-plate. The torque was measured by
a torque-gaugeT22/50 (HBM Messtechnik, Darmstadt, Germany)[2].The barrel load was set
to 75% (Wacker ® AK 100000 Silicon oil, WackerChemie AG, Burghausen, Germany) and
the rotation speed of the screws was varied from 50 to 200 rpm.
Figure 1: Schematic drawing of the extruder setup; A: Back-plate with assembled screw; B:
Isolated Extruder-Barrel
Depending on the screw geometry different torqueand pressure were obtained and showed
a linear correlation to rotation speed. This could be explained by the newtonian behaviour
of silicon oil. Furthermore the specific mechanic power as a function of rotation speed (f)
and torque (τ) for different loadings (m) was calculated as:
. Equation 1
Kneading blocks and conveying elements applied similar specific mechanic power to the
material while differences within the subtypes can be observed. For example a high
staggering angle of the kneading blocks led to lower values for the specific mechanic
power. This could be explained by a higher transportation capacity of elements with lower
staggering angle and can be determined by the pressure. Considering the conveying
elements, a lower pitch resulted in a higher pressure andconsequently in a higher
transportation capacity.
In conclusion the setup was able to measure the specific mechanic power and the
transportation capacity of different screw elements. Based on this, it will be possible to get
deeper insight into the extrusion process.
1. Djuric, D. and P. Kleinebudde, J Pharm Sci 2008, 97(11): 4934-4942.
2. Köster, M. and M. Thommes, Chemical Engineering Journal 2010, 164(2-3): 371-375.
137
Rational Design of Nanoparticle Dissolution Testing for Parenterals: The
Challenge of In Vitro Drug Release
Janas,C. 1; Wacker, M. 1
1Institute of Pharmaceutical Technology, Goethe-University, D-60438 Frankfurt, Germany
Over the past decade significant research efforts have focused on a new generation of
nanocarrier systems. These parenteral drug delivery devices are well-suited for the delivery
of drugs with poor aqueous solubility, unfavourable body distribution and/or toxicity after
intravenous injection. Due to the modified drug release of the active moieties from the
nanocarrier matrix, physiology-based drug release models are needed in order to assure
the quality and safety of these products. Moreover, they provide information about the drug
release under in vivo conditions and are therefore an important tool in formulation
development.
There are several approaches which aimed to adopt standard methods from the European
Pharmacopeia to the dissolution testing of parenteral nanoparticles and liposomes. Most of
these studies showed a number of limitations due to the special requirements of the
nanosized dosage form [1].
In particular the isolation of the dissolved drug from particle bound drug molecules in the
polymeric matrix is one major challenge in the development of an efficient dissolution
method. Further aspects are the selection of dissolution media with regards to physiological
parameters such as a solubility enhancement by human plasma, the colloid stability under
in vivo conditions and the highly specific changes in surface properties by degradation and
decoration with different plasma proteins. In order to ascertain anexperimental setup, that
covers a wide range of different drug substances, the developed model has to be validated
by using drugs with various physico-chemical properties.
In our survey PLGA nanoparticles (NPs) loaded with model compounds intended for
parenteral application are investigated in different release studies using dialysis-based
methods. Buffers for dissolution media are prepared with various additives in order to not
only bridge the gap between drug transport through the dialysis membrane but also to
create conditions related to the in vivo situation.
Initial experiments with the poorly soluble photosensitizer mTHPP were run with several
membrane types to identify the transport rate of the API assuring that the dissolution of the
drug is followed by direct passage through the membrane. The analysis shows acceptable
transport rates in hydro-alcoholic solution and in complex with β-Methylcyclodextrin (0,1%
in phosphate buffer saline, PBS). Based on these results the dissolution behaviour of
mTHPP incorporated into PLGA NPs in PBS pH 7,2 is studied and compared with
dissolution in buffer solutions composed of increasing amounts of foetal calf serum (FCS)
and cyclodextrins (CD) respectively [2].
Furthermore the colloid stability of investigated nanocarriers in phosphate buffer in the
presence and absence of FCS is analysed as a function of time. There was no effect on
particle size and polydispersity index (measured with photon correlation spectroscopy)
during 24 h of incubation at 37°C which might predict no/delayed aggregation in vivo. The
results of dissolution testing of mTHPP demonstrate a release of 100 % in both media (see
Fig.1) and hence the sustained release of the NP formulation depending on media
composition is investigatedin our experiments.
Fig.1: Particle size, polydispersity index, and dissolution of mTHPPNP (n=3)
Acknowledgments: Biolitec research GmbH, Dr. Arno Wiehe and Dr. Volker Albrecht
References:
1. D Souza S, DeLuca P,:Pharm. Res.2006, 23 (3): 460-474.
2. DesRoches M-C et al.: J.Photochem.Photobiol. B. 2006, 85 (1): 56-64.
138
Chemically selective imaging of structured surfaces – a novel complementary
analytical approach for pharmaceutical investigations
Kann, B. 1;Gantzsch, S. 1; Lehr, C.M. 1 2 3; Schaefer, U. F. 1; Windbergs, M. 1 2 3
1Saarland University, Department of Biopharmaceutics and Pharmaceutical Technology, Campus
A4.1, 66123 Saarbruecken, Germany
2PharmBioTec GmbH, Campus C2.2, 66123 Saarbruecken, Germany
3Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123 Saarbruecken,
Germany
Confocal Raman microscopy is an upcoming analytical technique in the field of
pharmaceutics. It is well suited for the contactless and chemically selective characterization
of drug delivery systems and in vitro test systems.However, confocal Raman microscopy
requires a smooth sample surface as spectra are exclusively recorded from the focal plane.
As pharmaceutical samples often exhibit a structured surface such as lyophilisates and
porous materials, confocal Raman analysis is impaired.
We overcome the pitfalls of a confocal setup by combining Raman microscopy with optical
profilometry as complementary analytical tools gaining a full-fledged characterization of the
sample surface independent of its structure.
To evaluate our novel approach, we analyze a protein-loaded lyophilisate. The therapeutic
efficiency of proteins highly depends on their physical structure and molecular
conformation, thus changes can have severe side effects on drug dosing for the patient.
However, maintaining their therapeutically active conformation throughout manufacturing
and within the final dosage form during shelf-life is still a major challenge. We freeze dry an
aqueous solution containing a mixture of native and heat denaturated bovine serum
albumin (BSA) for 48 h with a final drying step for 1 h gaining a solid lyophilisate. Optical
profilometry of the surface is performed prior to Raman analysis (WITec alpha 300/500) as
visualized in Figure 1a. As structural changes within the protein get along with a change in
their Raman spectra, Raman microscopy is a valuable method to detect protein
deconformation. The shift of the amide I peak in the Raman spectrum of the protein
occurring between 1500-1800 cm-1 is used to discriminate between active and heat
denaturated BSA within the dosage form. By overlaying the topography map with the
respective Raman spectra at each measuring point we gain a spatially resolved false color
image of the lyophilisate indicating each protein conformation in a different color.
Furthermore, we investigate a Transwell-based permeation model. Polymeric membranes
provide a suitable alternative to cell culture models overcoming the drawbacks of timeconsuming
preparation and high expenses. For simulation of the intestinal mucosa, the
apical side of the membrane is coated with lipids. Due to its non-destructive and chemically
selective nature, Raman microscopy is a well suited technique for the analysis of chemical
composition and three dimensional morphology of these systems.Raman spectra are
recorded and subsequently converted into chemically resolved images (WITec alpha
300/500). A comprehensive physical characterization of the transwell-based intestinal
model is performed of the pure membrane as well as of the coated membrane before and
after experiments. The pore morphology of the pure membrane is investigated with Raman
spectroscopy in xz and xy-direction resulting in virtual cross-section images and three
dimensional presentations (Figure 1b), respectively. After the coating, the pore morphology
is verified again. Coating itself is achieved by centrifuging the lipid onto the membrane and
a subsequent drying step. To monitor the coating formation on the membrane, a
topography map is created after each coating step followed by subsequent Raman
analysis.
Figure 1. a) Topography map of a lyophilisate. b) Three dimensional image of the pore
morphology of a polymeric membrane.
We successfully introduce chemically selective imaging of structured surfaces for
pharmaceutical applications by combining confocal Raman microscopy and optical
profilometry as complementary analytical techniques.
139
Custom-tailoring microsystems for production intensification of lipid based
colloidal drug delivery systems
Finke, J. H. 1, Niemann, S. 1,Richter, C. 2, Gothsch, T. 3, Kwade, A. 3, Büttgenbach, S. 2, Müller-
Goymann, C. C. 2
1 Technische Universität Braunschweig, Institut für Pharmazeutische Technologie, Mendelssohnstr.
1, 38106 Braunschweig, Germany
2 Technische Universität Braunschweig, Institut für Mikrotechnik, 3 Technische Universität
Braunschweig, Institut für Partikeltechnik
The manufacturing of colloidal drug delivery systems poses big challenges to the
Poster 127

production process. It comprises a variety of different process steps, especially if solid lipid
nanoparticles (SLN) are produced. Commonly, the whole process is broken down to
separate process steps in different devices and performed batch-wise. In terms of process
intensification, a change towards substantially smaller, cleaner and more efficient
equipment or methods is required. Therefore, a continuously working microsystem (MS)
approach was applied for SLN manufacturing. It comprises: (i) dispersion of solid active in
the lipid phase, (ii) initial formation of lipid droplets in the aqueous phase, (iii)
homogenization of these to colloidal, narrowly distributed droplets and (iv) crystallization of
the latter in a defined way (Fig. 1). Additionally, this approach reduces educt batch sizes
and product loss, thus facilitating its application as an instrument for formulation screening.
In this study, the progress of process intensification is presented with regard to the
homogenization step.
MSs for high pressure homogenization were primarily fabricated in silicon/glass [1].
Different geometric design approaches were tested and ranked regarding their droplet
breakup efficiency in emulsions: straight channels < Z-channels < Y-channel ≈ orifice.
Furthermore, various minor structural changes within the main geometric principles were
analysed [2].
According to silicone MS results, orifice channels fabricated in stainless steel [3] were
investigated in detail as the most promising design principle. In general, steel MS supply
lower fragility, higher process pressures and elevated temperatures, as necessary for SLN
production. To elucidate the design parameters which influence the colloidal dispersion
efficiency, orifice widths, number of consecutive orifices, and the distance between orifices
were varied. It was shown that double orifices yield lower particle sizes compared to single
orifices. Nevertheless, triple orifices do not provide further decrease in particle size, but
rank between double and single orifices. These phenomena are attributed to the
development of counter pressure towards the first orifice by a second one causing a more
efficient breakup, on the one hand. On the other hand, the pressure drop is distributed over
a higher number of orifices reducing the stress intensity at each orifice and, thus, the
overall efficiency in case of three orifices. A longer distance between orifices appears more
efficient in droplet breakup likely due to a reduction of counter pressure effects because a
stable flow pattern is not able to recover due to the proximity of two orifices at a low
distance (300 µm, equal to orifice length). For the combination of 80 and 120 µm wide
orifices in one MS, it has been demonstrated more efficient if the smaller orifice is exposed
to the product flow first, yielding the higher pressure drop over the size determining, first
distinct energy input. These findings were confirmed by applying a pressure regulating
valve, facilitating the arbitrary variation of counter pressure within the experimental setup. It
was proved that a moderate counter pressure drastically decreases resulting particle sizes
for a single orifice MS. In double orifices no further decrease in particle size was detected
due to external counter pressure.
Regarding the producibility of SLN, it was shown that particles can be produced in custom
tailored MS with comparable or even better properties (particle size and distribution, melting
behaviour) within one cycle instead of 20 cycles in a conventional homogenization setup.
Conclusively, the effectiveness of MSs for high pressure homogenization in a continuously
perfused microsystem for SLN production was systematically improved and crucial
parameters were identified. This demonstrates a successful process intensification
compared to conventional colloidal dispersion production.
Fig. 1. Schematic representation of SLN production process in an microsystem approach
Acknowledgments: The present study was performed in the context of the Research Group 856
funded by the German Research Foundation (DFG). One of the authors (S. B.) gratefully
acknowledges the financial support of the Volkswagen Foundation.
References:
1. Lesche, C., Büttgenbach, S.: MST Kongress 2009
2. Finke, J. H. et al.: 8th CESPT, Austria, Graz 2010
3. Richter, C., Krah, T., Büttgenbach, S.: Microsyst. Technol. 2012: in press
140
Air drying of drug nanosuspensions as a simple small scale process for
screening and feasibility studies
Laabs, F.; Bunjes, H.
Institute of Pharmaceutical Technology, Mendelssohnstraße 1, 38106 Braunschweig, Germany
The conversion of poorly water soluble pharmaceutical drug substances into
nanosuspensions is a common approach to overcome their poor dissolution and absorption
properties. However, solid dosage forms are often preferred with regard to improved
physicochemical stability and better patient convenience. Established unit operations like
spray drying or freeze drying can be applied to convert nanosuspensions into solid dosage
forms. However, preservation of the particle size during the drying process can be
challenging [1]. Furthermore, the conversion step is often time consuming (e. g. freeze
drying) and/or only a single sample can be processed at a time (e. g. spray drying). This is
especially impedimental for screening studies where processing of many samples is
required.
In order to circumvent the above mentioned issues, a simple small scale air drying process
was established. In this study, the applicability of the air drying process on screening and
feasibility studies was investigated. In particular, the influence of the type and concentration
of different carrier materials and the transferability of the air drying results onto those
obtained by spray drying were assessed for two different API nanosuspensions.
A naproxen nanosuspension (5 %) stabilized with Kollidon VA64 (2.5 %) and a griseofulvin
nanosuspension (5 %) stabilized with Kollidon VA64 (2.5 %) and sodium dodecyl sulfate
(0.25 %) were prepared by wet media milling for 4 h using a stirred media mill (Dispermat
SL 5 C nano). Air drying of the nanosuspensions was performed on a polystyrene well plate
or using an evaporator (EVA EC S) and samples were compared to those obtained by
spray drying (Büchi B-191). After processing, the dried samples were resuspended and the
particle size was determined via photon correlation spectroscopy.
As exemplified in Figure 1 and 2, air drying of the nanosuspensions could successfully be
performed and the results were similar to those obtained by spray drying. As already
observed in previous spray drying studies [2], drying of the griseofulvin nanosuspension
was shown to be less critical compared to the naproxen nanosuspension.
In conclusion, the air drying process seems promising for screening and feasibility studies
and can provide first estimates of results that can be achieved using other drying
processes.
Figure 1: Particle size values
(PCS) of a naproxen
nanosuspension before and after
spray and air drying with Kollidon
VA64 as carrier material at
different carrier:API ratios
Figure 2: Particle size values (PCS)
of a griseofulvin nanosuspension
before and after spray and air
drying with different carrier
materials at a carrier:API ratio of
0.5:1
Acknowledgments: We would like to thank A. Bitterlich and Prof. Dr. A. Kwade (TU Braunschweig, Institute
for Particle Technology) for the cooperation concerning the development and the production of the
nanosuspensions.
References:
Van Eerdenbrugh, B. et al.: Eur. J. Pharm. Sci. 2008, 35(1-2): 127-135
Laabs, F., Komoß, C., Bunjes, H.: 8 th PBP World Meeting 2012, Istanbul, Turkey
141
Tablet disintegration – design of an apparatus to measure the disintegration
force and water uptake of tablets simultaneously
Quodbach J1, Kleinebudde P1 1 Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitaetsstrasse
1, 40225 Duesseldorf, Germany
The underlying mechanisms of action of tablet disintegrants are still mostly unknown. Even
though tablet disintegration is often the first step in the bioavailability cascade, only few
attempts have been made recently to understand how disintegration takes place or is
influenced [1, 2, 3]. An apparatus was built to gain a more profound knowledge about the
disintegration force kinetics, water uptake kinetics, and differences of tablets containing
various super disintegrants. The measuring principle is based on a design from the
University of Pavia [4] and was altered to allow for a higher sample throughput and more
precise results. The experimental setup is schematically shown in Figure .
One beaker is placed on a balance (Sartorius CP224s) another one is placed under the
measuring probe of a Texture Analyser (TA.XTplus). The beakers are connected with a
tube allowing water to flow from one beaker to the other. A tablet holder made of aluminium
with 19 holes (r = 0.5 mm) is placed in the beaker on the Texture Analyser. An additional
filter paper is placed on the tablet holder. The tablet itself is attached to the measuring
probe with adhesive tape. Water is filled into the beakers until the tablet holder is in contact
with the surface. Then the measuring probe with the tablets is slowly moved downwards
until a trigger threshold is reached (0.25 N) when the tablet touches the tablet holder and
the data acquisition begins. The Texture Analyser registers the force the tablet exerts onto
the measuring probe, whereas the balance records the water uptake of the tablets as the
negative mass change. Adhesive tape is fixed around the circumference of the tablet to
ensure that the complete amount of force is transmitted to the Texture Analyser.
As example, three measurements are presented in Figure . The measured tablets consist
of 2 % Kyron T-314 (Corel Pharma Chem), a polacrilin potassium ion-exchange resin also
used as super disintegrant, 97.5 % dibasic calcium phosphate (Chemische Fabrik
Budenheim KG) and 0.5 % magnesium stearate (Merck AG). Initially, little water uptake
results in a strong force development. Afterwards, the water uptake and force behave
almost linear, which is followed by a decrease of absolute force. This can be due to a
collapse of the internal structure after the maximum force is reached.
Further improvements of the apparatus are still necessary to get a more precise resolution
of the mass change, especially at the beginning. Yet, it proves to be an easy to use tool
which allows insights into the relationship between water uptake and force development but
also into kinetic processes during tablet disintegration.
References:
1. Desai P M, Liew C V, Heng P W S, J. Pharm. Sci. 2012, 101(6): 2155-2164.
2. Krausbauer E, et. al. J. Pharm. Sci. 2007, 97(1): 529-541.
3. Massimo G, et. al.: Pharm. Dev. Technol. 2000, 5(2): 163-169.
4. Caramella C: Pharm. Tech. Int. September 1990, (9):30-42.
128 Poster
z-average diame er (nm)
1950
1900
1850
250
200
150
100
50
0
be ore drying
after spray drying
after air drying (well plate)
PdI
10 1 5 1 2:1 1:1 0.5:1 0 1
carrier:API ra io
1.0
0.9
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
PdI
z average d ameter (nm)
300
250
200
150
100
50
0
before drying
after sp ay drying
after air drying (well plate)
after air drying (evaporator)
PdI
mann tol lac ose KVA 6 K30
carr er material
Figure 1: Experimental setup Figure 2: Water uptake versus force
development
0. 0
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
PdI

142
Selection of solvent type affects nanocrystal particle size during precipitation
combined with high pressure homogenization process
Sinha, B. 1; Möschwitzer, J. P. 2; Müller, R. H. 1
1 Institute of Pharmacy, Dept. Pharmaceutics, Biopharmaceutics&Nutricosmetics, Freie University
of Berlin, Berlin, 12169, Germany;
2 Pharmaceutical Development, Abbott GmbH &Co.KG, Ludwigshafen, 67061, Germany
Precipitation combined with high pressure homogenization could be a feasible technique for
production of small size drug nanocrystal. Solvent type plays a critical role in nucleation,
stabilization and particle growth during precipitation process. The aim of this study was to
investigate how various solvents affect the final particle size of drug nanocrystal during this
combination technique.
Precipitation step was combined with high pressure homogenization process to prepare
drug nanocrystal in this study. Ibuprofen (IBP, BASF, Germany) and resveratrol
(E.denkFineChemie GmbH, Germany) were used as two model compounds. Solvent phase
(S) was prepared by solubilizing drug in a water miscible solvent such as alcohol (Ethanol,
Methanol, Isopropanol), acetone, tertrahydrofuran (THF), dimethylformamide (DMF),
dimethylsulfoxide (DMSO) and ethylacetate. Aqueous solution of SDS (0.2%) was mixed
with either 0.1% HPMC (viscosity 5 cps) or with 0.5% PVP (K40) to constitute the antisolvent
phase (AS) for Ibuprofen and Resveratrol, respectively. Using a needle and a pump
system, S phase was injected in the AS phase just before homogenization zone in
Emulsiflex C5 (Avestin Europe GmbH, Mannheim, Germany) operated at 1000-1200 bar.
Particle size of the nanocrystal was measured by laser diffractometry using Mastersizer
2000 (Malvern Instruments, UK). Solvents were rank ordered based on the minimum
particle size obtained using a solvent.
Depending on the solvent type, particle size of drug nanocrystals was found to change.
When isopropanol was used for making ibuprofen nanocrystal, the smallest particle size
obtained was 306 nm [d(0.5)]. It was only possible to reduce particle size further to less
than 200 nm when solvent was changed to acetone, methanol, ethanol, THF or DMSO.
Mean particle size was increased when ethyl acetate and DMF was used. Therefore, the
solvent preference rank order for preparing IBP nanocrystal was found to be
THF~Ethanol>Methanol~DMSO>Acetone>IPA>Ethyl acetate. On the other hand, use of
DMSO produced the smallest size resveratrol nanocrystal, i.e. 153 nm [LD (50%)]. Hence,
the solvent preference rank order for resveratrol was found to be
DMSO~Ethanol~IPA~Acetone>Methanol>DMF>THF. Other factors such as
solvent/antisolvent ratio, drug concentration in the solvent phase and stabilizer composition
was also found to be critical to control the particle size.
Beside other factors, selection of the right solvent for precipitation study is necessary to
achieve smaller particle size. Further optimization of other parameters leads to the smallest
size drug nanocrystal in precipitation coupled with homogenization process.
143
Identification of factors affecting the sorption isotherms of spray dried binary
carbohydrate mixtures containing maltodextrin and sucrose
Tackenberg, M. W. 1 2; Horvat, M. 1;Thommes, M. 2; Kleinebudde, P. 2; Schuchmann, H. P. 1
1 KIT – Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences, Food
Process Engineering (LVT), Kaiserstr. 12, 76131 Karlsruhe
2 Heinrich-Heine-University, Institute of Pharmaceutics and Biopharmaceutics, Universitaetsstr. 1,
40225 Duesseldorf, Germany
Maltodextrins, amorphous starch degradation products, are widely used as excipients for
solid drug formulations. The different types of Maltodextrins are characterized by their
dextrose equivalent (DE), which is correlated to the average molecular size (DE 0: ~ starch;
DE 100: glucose). The DE of common maltodextrins varies between 2 and 20.
Furthermore, maltodextrins can be distinguished by their ratio of amylose to amylopectin
content and carbohydrate composition with respect to the origin. These differences can be
used to tailor-made new matrix formulations with outstanding properties for drug
administration like inclusion compounds and storage stability. This study elucidates the
effect of type and content of the maltodextrins on the sorption isotherms of spray dried
binary carbohydrate matrices.
Various maltodextrins (Glucidex ® with DE 6, Waxy Maize based; Glucidex ® with DE 12 and
17, Maize based, Roquette Frères; Kleptose ® Linecaps, Pea based with DE 12 and 17,
Roquette Frères) were coprocessed with sucrose (Compri-Zucker O, Südzucker AG) using
spray drying (Büchi Minispray Dryer B-191, Büchi Labortechnik AG). Afterwards the
sorption isotherms were determined at 25.0 °C (SPS 11, PMT Analytical GmbH & Co. KG).
Therefore 0.5 g sample were dried to a water activity (aW) < 0.005, followed by a increasing
of the water activity in steps of 0.1 to aW 0.9. The obtained experimental data points
between aW 0.1 and 0.8 were fitted with the Guggenheim-Anderson-de Boer Equation
(GAB) (1). For all isotherms R² > 0.99 was observed.
m����K C a� m=
(1−Ka� )(1 − K a� +CKa�) (1)
• m = water content; aW = water activity
• C = Guggenheim constant; K = correcting factor
• mmono = water content of the saturated molecule monolayer of the surface
The sucrose content increases the water adsorption at aW ≥ 0.4, as shown for the
Glucidex ® DE 17 formulations (Fig. 1). This could be correlated to the highly hygroscopic
amorphous sucrose molecules. At aW ≤ 0.3 the water uptake decreases with increasing
sucrose content, which could be caused by the dominant properties of the high molecular
weight fraction at low aW [1].
Furthermore an effect of the DE to the water adsorption was found, as shown for the
Glucidex ® DE 6 and 17 formulations with 20 % sucrose (Fig. 2). At low aW (< 0.5) the water
uptake decreases with increasing DE. At aW > 0.5 it increases with increasing DE. This
could be explained with the dominant properties of the high molecular weight compounds at
low aW and the increasing lower molecular weight compounds (higher DE) at higher aW, too.
Finally, an influence of the starch source (Maize or Pea) on the sorption isotherm of the
maltodextrin sucrose mixtures was not detected, as shown for the Glucidex ® DE 17 and
Kleptose ® Linecaps (KL) DE 17 (Fig. 2).
Fig. 1: Influence of sucrose content on
sorption behaviour.
Fig. 2: Influence of DE and starch basis on
sorption behaviour.
From the results, it can be assumed that the average molecular size is the most important
factor regarding the sorption behaviour of carbohydrates. Hence, this behaviour can be
customized by a combination of maltodextrin and sucrose.
Acknowledgments: Roquette Frères; Südzucker AG, Max-Buchner-Forschungsstiftung; AIF-ZIM Project
KF2256805
References:
1. Radosta, S. et al.: Starch/Stärke 1989, 41(10): 395-401
144
Improved test on emulsifying properties of lanolin forcommunity pharmacies
Koeck J; Kortmann C; Pein M; Breitkreutz J
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitaetsstraße 1,
40225 Duesseldorf, Germany
Lanolin is a commonly used ointment base in dermal preparations. However, several
problems are known like its sensitization potential, discussed controversially [1, 2],
insufficient emulsification and stability properties [3]. As of its natural origin the emulsifying
properties (EP) change depending on batch and age.
For evaluating EP there have been two tests established: Addition of water and coldemulsifying
according to German Pharmacopoeia [4] and titration, which was introduced by
Reimannusing a solution containing Octoxynol-10 [5]. The first test only differentiates
between “pass” and “fail”, the latter provides the possibility to check the EP more precisely
and faster.As Octoxynol-10 is rarely used in community pharmacies, there is a need to
improve the Reimann method.
Lanolin of two qualities was used: Eucerinumanhydricum (EA) (Beiersdorf, D-
Hamburg,Release date: 2004) and UnguentumAlcoholumLanae (UAL) (Caesar &Loretz, D-
Hilden, Release date: 2010).
Test samples were prepared of cold-emulsifying with stirring by hand 10 g of lanolin with
15 g of water.
Titration solutions (TS) were prepared, containing 4 g Methylene Blue, 1 g Octoxynol-10
and water to 100 ml as prescribed by Reimann (TS1). The other test was performed
exchanging Octoxynol-10 (HLB=13.6, Triton® X-100, Roth, D-Karlsruhe) byPolysorbate 80
(HLB=15.0, Casear&Loretz, D-Hilden) (TS2).
After titration in small steps (approximately 0.1 ml) test sample was stirred by hand until a
blue coloured homogenous semisolid substance resulted. Endpoint was specified when TS
was not further incorporable when stirring.
It was possible to formulate a titration solution with Polysorbate 80 (TS2) which can be
used with comparable results to Octoxynol-10 (Amount of TS to instability of aqueous
lanolin (UAL): TS1 0.9 ml and TS2 1.0 ml). With this optimized method it is possible to use
the proposed test on EP in community pharmacies with conventional and available
excipients.
Further, it was possible to differentiate between lanolin of different qualities and ages (EA
and UAL) with TS1, according to its EP(Amount of TS1 to instability of EA: 0.4 ml and UAL:
0.9 ml). Reimann specified an amount of at least 0.5 ml TS1 to use lanolin for dermal
preparations.
Concluding, it becomes feasible for community pharmacies to easily test EP of lanolin
before preparing drug formulations. Other water-in-oil emulsions will be investigated by this
test in future.
References
1. Kligman, A.M.: Contact Dermatitis.1998, 39(3): 103-107.
2. Aberer, W.: Dermatologie in Beruf und Umwelt.2006, 54(4): 135-139.
3. Eifler-Bollen, R.: Pharmazeutische Zeitung.2002, 147(17): 1898.
4. Deutsches Arzneibuch: Wollwachsalkoholsalbe(Deutscher Apotheker Verlag).1999.
5. Reimann, H.: PharmazeutischeZeitung.1993, 138(2): 114-115.
145
Tailor-made production of quercetin and rutin nanocrystals for investigating
the correlation between the size and effect to cell
Chen,R. 1, Wszelaki, N. 2, Melzig, M. F. 2, Müller, R. H. 1, Keck, C. M. 1 3
1Institute of Pharmacy, Free University of Berlin, Kelchstrasse 31, 12169, Berlin
2Institute of Pharmacy, Free University of Berlin, Königin-Luise-Strasse 2+4, 14195 Berlin
3Applied Pharmacy Division,University of Applied Sciences Kaiserslautern, Campus Pirmasens,
Carl-Schurz-Strasse 10-16, 66953 Pirmasens
Flavonoids are believed having potential to protect neuro cell from neurogenerative
damages caused by endogenous toxins. However, most flavonoids are poorly soluble,
which lead to a low bioavailability and/or cell uptake when they are applied in aqueous
solution form. To formulate the flavonoids into nanocrystals is an effective method to
increase the solubility and to enable the cell uptake. As the solubility enhancing effect and
the ability to be taken up by cells is size dependent, tailor-made production of nanocrystals
is the first required step for investigating the correlation between size and effect to cell in
future.
In this study, quercetin and rutin nanocrystals were produced by high pressure
homogenization (HPH) (LAB 40, APV Deutschland) in sterile condition. For the further
investigation about the influence of size on the performance of nanocrystals in cell culture,
a series of nanocrystals with different sizes were tailor-made with Poloxamer 188 as a nontoxic
surfactant (0.2%, w/w) and glycerol as a osmotic adjuster (2.5%, w/w) by applying
different HPH pressure (up to 1500 bar) and cycles (up to 20 cycles) and choosing different
active concentrations (2%, 0.2%, 0.02%). Particles size was characterized by photon
Poster 129

correlation spectroscopy (PCS), laser diffractometry (LD) and light microscopy. Zetapotential
and short-term stability were examined. For correct size analysis the
measurement method of PCS was optimized by choosing different measuring cuvettes and
using different dilution media and sample concentrations. The ways to adding glycerol in
nanosuspension was also compared.
Via controlling production condition, a series of nanocrystals (from 172 nm to 1500 nm)
were produced. Results showed that an increase in cycles of HPH could lead to a
significant smaller size for quercetin but not for rutin. Lower active concentration in
quercetin had no influence on size but in rutin it provided a smaller size. Zeta-potential in
50μS/cm NaCl solution and original dispersion medium of quercetin nanocrystals were both
above 30 mV, which indicated a better stability compared to rutin nanocrystals holding a
zeta-potential of around 25 mV. The short-term stability also confirmed that the quercetin
nanocrystals in 4 oC had a good stability. Through investigating PCS cuvettes and dilution
methods, measurement condition was fixed as using a low volume cuvette, active powder
saturated diluting medium and 0.02% measuring concentration. Adding glycerol in
suspension before HPH gave a slightly smaller size compared to HPH without glycerol.
However, for precise controlling the concentration of glycerol in final product, it was added
in after HPH.
In conclusion, different sizes of quercetin and rutin nanocrystals for cell culture test can be
tailor-made by applying different pressures and cycles as well as using different active
concentrations. The preliminary requirement for further cell culture research was achieved.
Acknowledgements: The authors would like to thank China Scholarship Council for financial support.
146
Hydrogels for drug delivery: influence of macromer branching on
characteristics of poly(ethylene glycol) based hydrogels
Kirchhof, S. 1, Brandl F. 1, Hammer N. 1, Meßmann V. 1, Teßmar J. 1 and Göpferich A. 1
1 University of Regensburg, Department of Pharmaceutical Technology, Universitätsstr.31, 93053
Regensburg, Germany
Poly(ethylene glycol) (PEG) based hydrogels are interesting materials for a variety of
biomedical applications. In the field of controlled drug delivery, the use of PEG gels has
been promoted due to their excellent biocompatibility. Nevertheless, most of the existing
cross-linking mechanisms are associated with disadvantages, such as the formation of
potentially harmful radicals. A relatively unknown cross-linking mechanism for hydrogels is
the Diels-Alder click reaction. Previously, we have shown the suitability of this reaction for
the formation of suitable PEG based hydrogels. Here, we report the influences of the
macromer branching factor on the characteristics of the resulting hydrogels.
Gelation time / min
250
200
150
100
50
0
4armPEG-hydrogel
8armPEG-hydrogel
5 10 15
Total polymer concentration / w/v
Figure 1: Gelation times of different PEGhydrogels;
gelation time depends on
branching factor and total polymer
concentration
Rea tive mass increase Mt/M0
18
16
14
12
10
8
6
4
2
15% 4armPEG-hydrogel
5% 8armPEG-hydrogel
0
0 1 2 3 4 15 16 17 18 19 20 21
Time / days
Figure 2: Swelling behaviour of different
PEG-hydrogels; 4armPEG-hydrogels were
dissolved within 4 days, whereas
8armPEG-hydrogels swelled on a lower
level even after 3 weeks
PEG with branching factors of four and eight (4armPEG10k-OH and 8armPEG10k-OH)
were used as raw materials. 4armPEG10k-furan, 8armPEG10k-furan and 4armPEG10kmaleimide
were synthesized according to previously established protocols [1]. For the
synthesis of 8armPEG10k-maleimide, 8armPEG10k-amine and Nmethoxycarbonylmaleimide
were reacted to yield the desired product. All compounds were
characterized by 1H-NMR spectroscopy. For hydrogel preperation, defined amounts of
4armPEG10k-maleimide and 4armPEG10k-furan or 8armPEG10k-maleimide and
8armPEG10k-furan were separately dissolved in Millipore water. The two precursor
solutions were mixed to initiate gel formation. Gelation kinetics and gel strength were
studied by performing oscillatory shear experiments on a TA Instruments AR 2000
rheometer with parallel plate geometry. For swelling studies, gel cylinders (Ø 7 mm) were
incubated with 10 ml of phosphate buffer solution (pH 7.4).
Rheological studies showed decreasing gelation times with increasing polymer
concentrations (Fig. 1). This was observed for both degrees of branching. Moreover, the
gelation time dramatically decreased by increasing the branching factor from four to eight.
This is explained by the higher cross-linking density of the resulting hydrogels. 4armPEGhydrogels
(15 % polymer) showed very high swelling and dissolved within one week (Fig.
2). This could be explained by the reversibility of the Diels-Alder reaction. In contrast to
that, 8armPEG-hydrogels showed almost no swelling even at lower polymer concentrations
(5 % polymer). Until now, it is not completely clear why 8armPEG-hydrogels show this
extraordinary stability. Either the retro-Diels-Alder reaction could be unfavorable or the gels
could show self-healing properties, a phenomenon which has been described for other
materials cross-linked by Diels-Alder reactions. Further studies are under way.
In summary, our studies showed a clear influence of the branching factor on gelation time,
gel strength, swelling and degradation. Given their favorable gelation time and long-term
stability, 8armPEG-hydrogels seem to be a promising biomaterial for biomedical
applications.
Acknowledgments: This work has been supported by Deutsche Forschungsgemeinschaft (DFG), grant
number GO565/16-1.
References:
1. Kirchhof, S. et al.: Application of the Diels-Alder Reaction as a New Cross-Linking Mechanism for
Poly(ethylene glycol) Based Hydrogels, ACS Meeting Philadelphia, 2012
147
Chemical stability studies on the antileishmanial drug candidate KuRei300 in
lipid based nanocarrier formulations as important prerequisite for in vivo
experiments
Kupetz, E. 1; Preu, L. 2; Bunjes, H. 1
1 Institute of Pharmaceutical Technology, Technische Universität Braunschweig,
Mendelssohnstraße 1, 38106 Braunschweig, Germany
2 Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig,
Beethovenstraße 55, 38106 Braunschweig, Germany
Visceral leishmaniasis is an infection caused by Leishmania donovani parasites. The WHO
assigned it the status of a neglected tropical disease thus encouraging research for new
active compounds. In a directed synthesis program the paullon chalcon derivative
KuRei300 emerged as a promising drug candidate. It exhibited good antileishmanial activity
in experiments with axenic amastigotes and human macrophages [1]. In vivo studies in
mice are intended and a new parenteral formulation for KuRei300 was developed for this
purpose [2]. This formulation is a mixed micellar solution based on phospholipids and a bile
salt. It meets important quality criteria for parenteral preparations such as the use of
approved excipients, physical stability and the possibility for sterile filtration. Another
fundamental aspect that is, however, often overlooked is the possible decomposition of a
formulated agent during storage. The present study therefore addressed the chemical
stability of KuRei300 in liquid formulations.
Since DMSO is a common solvent for in vivo / in vitro tests, a preliminary experiment was
performed in which KuRei300 containing solutions were stored either under the influence or
protection from light and monitored for signs of decay by HPLC. In the light exposed
solution the original drug content declined rapidly and one predominant new peak
appeared. The corresponding compound was identified as the Z-isomer of KuRei300 by 1H
NMR spectroscopy. As a result of these findings all samples were stored protected from
light in subsequent experiments. In a second set of experiments the stability of KuRei300 in
the mixed micellar carrier solution was assessed under different conditions. To obtain the
mixed micellar solution Lipoid S100 (13.4% w/w) was dispersed in an aqueous phase that
contained sodium glycocholate (7.4% w/w overall) and was buffered to pH 7.4 to avoid
phospholipid hydrolysis. This turbid phospholipid dispersion was then vigorously shaken
until a translucent solution had formed. A KuRei300 solution in ethanol was pipetted into a
vial and the ethanol was evaporated thus forming a substance coat on the walls of the vial.
The micellar solution was then added and the vial placed on a rotator. During this passive
loading procedure the major part of the substance film dissolved in the carrier. After
loading, excess KuRei300 was filtered off and the loaded carrier was transferred into fresh
vials which were crimped [2]. The samples were produced without and with 0.1% EDTA in
the aqueous phase and were or were not flushed with nitrogen. At regular time intervals the
chemical stability of KuRei300 was evaluated by HPLC. In the non-stabilized preparation
(no N2, no EDTA) the substance underwent drastic and quick decomposition. The signals in
the chromatogram differed, however, very much from those in the DMSO solution. Z-
Kurei300 was not formed in significant amounts. Nitrogen flushing of the vial´s head space
considerably slowed down the decay and after 10 weeks additional EDTA demonstrated a
further benefit.
In conclusion, the chemical stability of new drug candidates needs to be given the
appropriate attention from the earliest stages of formulation. Photoisomerization and its
impact on pharmacodynamics have been reported for anti-tumorigenic chalcones
previously [3]. The relevance for the biological activity of KuRei300 has not been studied
yet. Our study demonstrated that simple techniques such as light exclusion, storage under
an inert gas and addition of a metal complexing agent (EDTA) strongly improved the
chemical stability of the drug candidate. Such measures can prevent hard-to-interpret
results from in vitro / in vivo experiments that may result if a potential chemical
decomposition is disregarded.
Acknowledgements: We would like to thank J. Ryczak (Institute of Medicinal and Pharmaceutical Chemistry,
Beethovenstraße 55, 38106 Braunschweig) for the synthesis of KuRei300 and assistance in the
interpretation of NMR spectra.
References:
1. Reichwald, C. et al.: J. Med. Chem. 2008, 51(3): 659-665
2. Kupetz, E. Et al.: 8 th PBP World Meeting 2012, Istanbul, Turkey
3. Iwata, S. et al.: Biol. Pharm. Bull. 1997, 20(12): 1266-1270
148
Development of a microfluidic system for screening of colloidal drug
formulations under flow conditions
Pretor, S. 1; Finke, J.H. 1; Al-Halhouli, A.T. 2; Schmolke, H. 3; Busker, M. 4;Dietzel,
A. 2;Büttgenbach, S. 2;Klages, C.-P. 3;Behrends, S. 4; Reichl, S. 1;Müller-Goymann, C.C. 1
1Institut für Pharmazeutische Technologie, TU Braunschweig, Mendelssohnstraße 1, 38106
Braunschweig, Deutschland
2Institut für Mikrotechnik, TU Braunschweig,
3Institut für Oberflächentechnik, TU Braunschweig;
4Institut für Pharmakologie und Toxikologie, TU Braunschweig
In recent years, research and drug development have focused on microfluidic systems
because they offer miniaturization, parallelization and automation of screening processes
[1]. In biotechnology, these systems are used as microbioreactors, whichhave already
shown to be versatile tools for screening of microorganisms and optimal production
parameters in small scale [2]. Additionally, cultivation of mammalian cell lines is
alsopossible in microscale under flow conditions[3].
Amicrofluidic systemconsisting of apolydimethylsiloxane (PDMS) fluidic channel bonded on
a glass surface, a piezo driven micropump (Bartels Mikrosysteme) and a culture medium
reservoir has been developed for the cultivation of mammalian cells. In a first series of
experiments, immortalized human corneal epithelial cells (HCE-T) and primary human
dermal fibroblasts (HDF)were cultivated in themicrochannel of the microfluidic system and
examined according to their attachment and viability on surface-treated PDMS.
In a second approach, the cellular uptake of fluorescence-labeled solid lipid nanoparticles
was investigated. The aim was to checkthe suitability ofthe microfluidic system for tracing of
nanoparticles, because it maysimulate physiological conditions by applying defined
amounts of shear stress to the cells.
Methods:
Theattachment behavior of HDF on hydrophilizedPDMS plates with positively and
negatively charged surfaces was estimated. The surface modification was done by layerby-layer
deposition of polyelectrolytes.Positively charged surfaces were coated with
130 Poster

fibronectin in addition. Cells were seeded on these plates and the ratio of attached cells
was calculated by counting suspended cells after certain periods of time. Afterwards, the
remaining cells were microscopically observed. Untreated PDMS served as negative
control.
For flow experiments, a constant medium flow of 0.2-1.0 ml/min was applied to the cells for
24 hours. Cell staining was performed with Hoechst 33342, Annexin V and
Ethidiumhomodimer to indicate viability, apoptosis and necrosis, respectively.
Fluorescence labeled solid lipid nanoparticleswere prepared by using a microchannel
emulsification method according to [4].HCE-T and HDF-cells were incubated with
nanoparticles bothon a standard glass well plate and in the flow system.The
attachednanoparticleswerevisualizedwith a confocal laser scanning microscope equipped
with a 60x oil immersion objective.
Results:
Optimal cell growth wasachieved via treatment of PDMS surfaces with a positively charged
coating and an additional fibronectindeposition.This coatingresulted in short attachment
times after cell seeding tomicrochannel surfaces.
Neither PDMS and glass, nor shear stress of 10 dyn/cm² affected growth of HCE-T and
HDF cells in microchannels negatively with respect to cell viability.
Solid lipid nanoparticles were able to transport the fluorescent dyecoumarin-6 as a model
drug to epithelial cells.Its fluorescence intensity was investigated by CLSM both on special
culture plates and in themicrochannels.
Acknowledgement:The present study was performed in the context of the research group 856 mikroPART
funded by the German Research Foundation (DFG). One of the authors (S. B.) gratefully acknowledges the
financial support of the Volkswagen Foundation.
References:
[1] Dittrich, P.S.; Manz, A.: Nature20065: 210-218
[2] Demming et al.: Biomicrofluidics2011 5: 014104
[3] Kim et al.: Lab on a Chip20077: 681-694
[4] Finke et al.:2nd Symposium on Phospholipids in Pharmaceutical Research Heidelberg2011(poster)
149
Optimizing the PCS measurement methods for nanoemulsions to see minimal
changes in the z-average
Harden, D. 1; Müller, R. H. 1; Keck, C. M. 1 2
1 Institute of Pharmacy, Department of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Freie
Universität Berlin, Kelchstraße 31, 12169 Berlin, Germany
2 Applied Pharmacy Division, University of Applied Sciences Kaiserslautern, Campus Pirmasens,
Carl-Schurz-Straße 10-16, 66953 Pirmasens, Germany
The first instrument for the photon correlation spectroscopy (PCS) was introduced to
market in the end of 1970 (Malvern Instruments). Today the PCS is one of the most
frequently used instruments to measure the mean particle size (z-average) of
nanoparticles. PCS is known as a very accurate method with high reproducibility. However,
reproducibility not only depends on the method, but can also be influenced by the sample.
An example is particle interaction, i.e. repulsive or attractive forces, which can lead to faster
or slower diffusion and thus to smaller or larger size results. Particle interaction depends on
the particle concentration within the sample. Hence, if the concentration varies between
different measurements of one sample, the size results might not be reproducible. The aim
of this study was to investigate the reproducibility of PCS measurements (Zetasizer Nano
ZS, Malvern Instruments, UK) using different concentrations of a nanoemulsion (Lipofundin
MCT 20%, B.Braun Melsungen AG, Germany) for the measurements.
Today, modern instruments can analyze samples containing high concentrations of
nanoparticles. In this method (1), the distance of the laser beam in the measuring cell is
automatically adjusted to the particle concentration of the sample. This is done to avoid
multiple scattering effects and to obtain an optimal intensity of the scattered light on the
detector. The results of the PCS measurements using this method did not lead to similar
results. Results show that there was a variation of 8 nm for a particle concentration of
0.005% to 1.0% (228 nm to 236 nm). The variation was random, i.e. with increasing the zaverage
went up and down.
In case of the older PCS instruments, there was no changing in the distance of the laser
beam of measuring point to the cuvette wall, therefore for these measurements a lower
particle concentration must be chosen/ selected, if not there would be multi scattering and
this will cause a smaller z-average. This method (2) was transferred to the Zetasizer Nano
ZS with a constant distance of 4.65 mm from the wall to the measurement point. The same
particle concentrations as in method 1 (1% to 0.005%) were chosen. The results showed
that there was an concentration dependent increase in the z-average from 184 nm (1%) to
236 nm (0.005%), however, the differences in the size when analyzing low concentrations
(0.05% to 0.005%) was only 2nm, whereas it was 8nm with method 1.
The results indicate that the reproducibility the PCS analysis should be investigated for
each sample, which could possess particle interactions. For this different concentrations
should be analysed using either the automatic method 1 or method 2 with the fixed laser
beam position. In case of the nanoemulsion it is best to analyze the samples with method 2,
e.g. using lower concentrations of the nanoemulsion and no automatic adjustment to obtain
reproducible size results. The concentration range for good reproducibility was found be
0.02% to 0.005%.
150
Efficiency increase by transfer of a two-pot to a single-pot fluid bed
granulation
Germer, K. 1; Rockmann, Th. 2; Wolf, B. 1
1 Anhalt University of Applied Sciences, Pharmaceutical Engineering, Strenzfelder Allee 28, 06406
Bernburg, Germany
2 Salutas Pharma Corp., Teamleader Galenics, Otto-von-Guericke Allee 1, 39197 Barleben,
Germany
Traditionally, pharmaceutical granulates are manufactured by spray agglomeration in a
mixing apparatus. In the past, the wet granulates were dried by time consuming tray drying.
Nowadays, fluid bed drying is the preferred procedure. The application of two consecutive
steps in different plants is still connected with material loss and contamination risk for which
reason both agglomeration and drying step should be performed in consecutive steps in
one and the same apparatus [1, 2].
The aim of the investigation was the transfer of a two-step process consisting of spray
mixing agglomeration followed by fluid bed drying of a commercial drug product to a singlepot
procedure in a batch laboratory fluid bed apparatus. Therefore, a fluid bed granulator
and dryer GPCG 1.1 (Glatt Corp., Binzen, Germany) was used (Figure 1). To optimize the
process parameters of fluid bed agglomeration, the spray rate was varied between 20 and
40 g/min, all other process parameters were kept constant. Parameters of the laboratory
fluid bed drying process referred to the production process. Dry granulates were
compressed to tablets. Evaluation criteria were the maintenance of stable and reproducible
fluid bed process, furthermore acceptable yields and high granulate and tablet quality
according to market product specification and pharmacopoeia.
Figure1: Fluid bed granulator and dryer GPCG 1.1
Best products were received with high inlet air temperature (80°C), high spray rate (30-40
g/min) and additional water spraying. Without interruption of the fluid bed process the wet
agglomerates were dried. The whole granulation process was stable and reproducible.
Product yield was above 90%. Granulate and tablet properties fulfilled the specified quality
criteria. Particle size was slightly increased with increasing spray rate compared to the
production process, but there was no significant influence neither on granulate nor on tablet
properties. The two-step granulation process with two different plants was successful
transferred to a single-pot fluid bed process saving process time and equipment and
reducing material loss and contamination risk.
References:
1. K. Giry et al.: Chemical Engineerig and Processing 2009, 48: 1293-1301.
2. J.Z. Gao et al.: International Journal of Pharmaceutics 2002, 237: 1-14.
151
Determination of residence time distribution in hot melt extrusion via image
analysis
Reitz E; Brock D; Thommes M
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine University, Universitaetsstrasse
1, 40225 Duesseldorf, Germany
Hot melt extrusion is an important and modern technology in pharmaceutical applications
and is gaining more and more interest because of its versatile capabilities. One
fundamental parameter for process understanding and process control is the residence
time (RT). This parameter plays an important role especially in hot melt extrusion
processes. The knowledge of the RT is important in critical hot melt extrusion processes,
e.g. in processing of heat sensitive products. For such processes a short RT is desired to
prevent degradation. The knowledge of the RT is also important for those types of solid
dispersions, where a drug shall be grinded or dispersed and for which a long RT is
beneficial.
The aim of this study was to develop a method for an on-line RT measurement. The idea
was to determine the grey value of the extrudates by adding carbon black as tracer to white
sugar alcohol extrudates (figure 1).
Figure 1: colouring of the white sugar alcohol extrudate by carbon black
0 200 00 600 800
time [s]
1000 1200 1 00
Poster 131
black
grey
white
Xy itol 0. mm
Xy itol 0. mm
Manni ol 0. mm
Manni ol 0. mm
Figure 2: residence time distribution for xylitol and mannitol, measured by taking pictures of
the extrudates leaving the die (n=2)

Hot melt extrusion (Mikro 27GL-28D, Leistritz, Nuremberg) was performed with xylitol and
mannitol (n=2). Carbon black in a defined amount (0.5 g carbon black as bolus by 40 g/min
powder feed rate) was added and a series of pictures (Nikon D300, Nikon Corp. Japan)
was taken from the extrudate in the die region in two-second intervals. A software algorithm
(Matlab version R2009a, Mathworks) was developed which enabled the calculation of a
grey value of a defined piece of the extrudate over the time resulting in a residence time
distribution (RTD).
It was possible to calculate the grey value for the extrudate emerging from the die plate at
each time point, describing the load of the tracer. An adjustment of the grey values to a
black and white standard was necessary to exclude any light effects affecting the colour of
the extrudate on the photograph. Therefore on each photograph black and white standards
were recorded as well. The camera software cut a piece of the extrudate as well as of the
black and white standard out of the photographs for each time point. For these three parts
of the photographs, the grey values were calculated and the value for the extrudates was
adjusted by the standard values.
Table 1: important parameters of the RTD
onset [s] midpoint [s] offset [s]
xylitol 317 435
690
328 430
706
mannitol 233 324
523
249 325
525
With this calculation, the RT as well as the RTD were determined (figure 2). This process
was reproducible for both carriers used in this study (table 1). Due to the high sampling rate
of the photos, the residence time could be determined very precisely.
In conclusion, a tool for residence time measurement was developed, which helps to
understand and to control hot melt extrusion processes.
152
Systematic studies of process parameters for wet granulation in a high-shear
mixer
Hoffmann, B. 1; Mosig, J. 1; Kleinebudde, P. 1
1 Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitaetsstr.1,
40225 Duesseldorf, Germany
The aim of this study was to investigate the influence of different process parameters on
wet granulation processes in a high-shear mixer. The statistical evaluation was arranged by
the programme MODDE 9.0 (Umetrics, Sweden). The DOE had a full factorial design of 4
factors with an interaction model and three repetitions of the center point.
Lactose monohydrate (Granulac 200, Meggle, Germany) was used as excipient. For the
granulation polyvinylpyrrolidone (Kollidon 30, BASF, Germany) was added as solid binder
and water as granulation liquid.
It was granulated in a 10 L-bowl of a high-shear mixer (Diosna, Germany). Water addition
time was kept constant at 200 s, the chopper was started every minute for 10 s. Varied
parameters of the process were the impeller speed (imp; 150 rpm to 300 rpm), the liquid
content (liq; 10 % to 12 % (w/w) related to solid mass), the binder content (bin; 4 % to 6 %
(w/w) of total solid mass) and the wet massing time (wmt; 0 to 300 s). Response
parameters were the particle size distribution (x50, x25 and x75), the interquartil range, the
yield (particles < 1250 µm) and the particle size distribution of the yield (x50 of the yield
y50). Further response parameters were bulk density, tapped density and the Hausner factor
(according to Ph. Eur. 2.9.34).
The mean particle size (x50) ranged between 150 µm – 1700 µm. Although most granules
had an x50 about 200 - 300 µm, there were coarse granules obtained in every batch. This
became apparent in the results of the yield, as 94 % in the best and only 10 % in the worst
case could be obtained. Decreasing binder content had the biggest influence on the
increase of the yield. Contrary to expectations a lower influence of the impeller speed in
contrast to the other factors was observed. After backward regression, it became only
significant for the x50 and the y50. This should be exemplified by the coefficient plot of the y50
(fig. 1). The effect of the liquid content and the wet massing time was much higher (imp
106.5 µm; liq 236.7 µm; bin 177.6 µm; wmt 240.2 µm). In contrast to literature1. a
significant effect of the impeller speed on the interquartil range as parameter for the width
of particle size distribution could not be found. For densities and the Hausner factor no
parameter seemed to have a significant effect.
In conclusion, liquid content, wet massing time and binder content had the greatest effect
on analyzed granule properties. Further studies shall investigate if these findings can be
transferred to high-shear granulation processes with further filler-binder combinations.
[µm]
140
120
100
80
60
40
20
0
-20
-40
imp
iq
Figure 1 – Coefficient plot of y50 (R² Adj. = 0.936; Q² = 0.828)
References:
1. Oulahna, D. et al., Powder Technology 2003 130: 238-246.
bin
wmt
imp*bin
imp*wmt
liq*wmt
bin*wmt
153
Fluorescent Nanoparticles capped with Octreotide for Targeting of
Somatostatin Receptors
Abd-Ellatif. A, Hennig. R, Pollinger. K, Tessmar. J .and Goepferich. A
Department of Pharmaceutical Technology, University of Regensburg, Regensburg, Germany
Somatostatin receptor subtype 2 (SSTR2) is widely expressed in normal and tumor cells,
which is clinically used for diagnostic purposes [1].Targeting therapeutic nanoparticles to
the SSTR2 using somatostatin analogue octreotide (OCT), would be a promising approach
for certain cancer cells, since this would reduce side effects and increase drug delivery
efficacy to the target site, highly over-expressing the respective receptors.
To attach OCT to nanoparticles, it was succinylated with succinic anhydride at low pH
selectively at its N-terminus [2]. Succinylated OCT was then conjugated using a watersoluble
carbodiimide (EDC) and N-hydroxy succinimide to photo-stable fluorescent
Quantum dot nanoparticles. These have an emission wavelength of 655 nm and are
functionalized with amine-derivatized polyethylene glycol (PEG).These are known to have a
very low non-specific binding to cellular membranes.
Succinylated OCT, was characterized by mass spectrometry and the formulated
nanoparticles were characterized by size exclusion chromatography (SEC), fluorescence
spectrometry, 1H-NMR,FTIR spectroscopy and for the resulting zeta-potential using
dynamic light scattering. Finally, cellular uptake was studied for SSTR2-positive pancreatic
carcinoid tumor cells (BON1) using confocal microscopy and flow cytometry.
Mass spectrometry indicated mono-succinylation of OCT by succinic anhydride in aqueous
media, as the result showed an increase in molecular mass of about 100 Da. A slight peak
shift was observed in SEC for OCT-Qdots compared to the native nanoparticles. In addition
a slight red shift was observed in the fluorescence emission spectra of Qdots after
conjugation to OCT. The zeta-potentials showed an inversion to positive charge of Qdots
after the conjugation to OCT. FTIR spectroscopy and 1H-NMR confirmed the attachment of
OCT to Qdots, which served as proof for the binding of the peptide to Qdots.
When BON1 cells incubated with the ligand modified particles, confocal microscopy and
flow cytometry showed high binding to BON1cells. Flow cytometry revealed the
displacement of Qdots-OCT, when high concentrations of free OCT were used to displace
it.
We concluded that nanoparticles modified with OCT may serve as promising tool to target
different cells that over-express SSTR2 receptors.
This work has been financially supported by a scholarsh p from the Arab Republic of Egypt for Ahmed
Abdellatif.
References:
1. Reubi, J.C. et al., J. Clin. Endocrinol. Metab. 2010, 95(5): 2343-50.
2. Kinstler, O.B. et al., Pharm Res. 1996, 13(7): 996-1002.
3. Updegrove, T.B. et al., RNA 2011, 17(3): 489-500.
4. Na, D.H. et al., AAPS Pharm. Sci.Tech. 2003, 4(4): E72.
154
An accelerated release method for fast assessment of real time release from
an intravaginal ring
Externbrink A1, Klein S1 1 Ernst-Moritz-Arndt University Greifswald, Department of Pharmacy, Institute of Biopharmacy and
Pharmaceutical Technology, C_DAT, Felix-Hausdorff.Str. 3, 17489 Greifswald
Purpose
Intravaginal rings (IVR’s) are usually designed to deliver the active drug over long periods
of time. Accelerated drug release testing would allow for fast assessment of real time
release during product development and quality control. Therefore, the objective of the
present study was to develop a discriminative accelerated release method for an IVR and
to establish a relationship between real time and accelerated release profiles.
Methods
NuvaRing ®, an approved hormonal contraceptive IVR which releases 120 µg etonogestrel
(ENG) and 15 µg ethinylestradiol per day over 21 days, was selected as a model
formulation. Release experiments were performed with ring segments and the release from
one IVR was calculated with respect to the mass ratio. Prior to the release experiments,
rings were cut into 10 segments which were endcapped with acrylate glue to prevent
uncontrolled diffusion which would affect the overall release. USP apparatus 7 (400-DS)
was used to determine the daily etonogestrel release. 10 mL of a vaginal fluid simulant
were used as the release medium. The first set of experiments was performed at 37 °C
(real time conditions) and automated sampling as well as media replacement was
performed every 12 hours. Subsequent experiments were performed at elevated
temperatures, i.e. 44, 50 and 55 °C (accelerated conditions). Sampling and media
replacement was performed in appropriate intervals.
Results
For all temperatures the zero order release constants were calculated from the slopes of
the cumulative release versus time graph and increased with increase in temperature. The
elevated temperature release constants were plotted according to the arrhenius equation
and a linear correlation between rate constants and temperature was found. The predicted
rate constant at 37 °C was in agreement with the experimental value.
The real time and accelerated release profiles at 50 °C were directly compared by plotting
the cumulative real time release versus the accelerated release and a strong linear
correlation was obtained with a correlation coefficient of 0.999. Additionally, the cumulative
real time and accelerated release profiles at 50 °C were plotted on the same axis after time
scaling. Applying the scaling factor which was determined as the ratio between the release
constants at elevated temperature and 37 °C real time and accelerated release profiles at
50 °C were superimposable.
Reservoir type IVR’s like NuvaRing ® typically show an initial burst release phase followed
by near zero order release kinetics with a slight decrease in daily drug release over time.
Drug release is usually expressed as daily release versus time. In order to be able to
monitor the changes in the daily real time release profile over time with accelerated test
conditions the sampling frequency was adjusted to reflect daily real time release. The
required sampling interval at 50 °C was determined from the ratio between zero order
release constants at 37 °C and 50 °C. A good agreement between the daily real time
release profiles and the elevated temperature release profiles with adjusted sampling
intervals was observed.
Conclusion
An accelerated release method that is predictive of real time release was developed.
132 Poster

Temperature appears to be a valid parameter to accelerate ENG release from NuvaRing ®.
An Arrhenius relationship between temperature and release constants indicates that the
integrity of the dosage form and the release medium is maintained under accelerated
conditions. Comparison of real time and elevated temperature release profiles at 50 °C
revealed a strong correlation between real time and accelerated release. In order to assess
the changes in the daily release profiles over time the sampling intervals at elevated
temperatures can be adjusted to reflect daily real time release. With the proposed method
and a temperature of 50 °C the overall duration of drug release from Nuvaring segments
could be reduced from 21 to less than five days.
155
Sartan-modified quantum dots targeting the Angiotensin II receptor type 1
Hennig R1; Pollinger K1; Wenzel R1; Breunig M1; Tessmar J1; Göpferich A1 1 University of Regensburg, Dept. of Pharmaceutical Technology, Universitätsstr. 31, 93053
Regensburg, Germany
The angiotensin II type 1 (AT1) receptor, which belongs to the class of G protein-coupled
receptors, is a pharmacological target of great interest. Although it is expressed
ubiquitously throughout the body, it is known that this receptor is overexpressed in several
tissues due to pathological conditions and, therefore, represents a promising target for
nanoparticulate drug delivery [1, 2].
To address nanoparticles to AT1 receptors we labelled commercially available fluorescent
quantum dots (Qdots) with a metabolite of losartan, EXP3174, which is largely responsible
for the actions of losartan in vivo [3]. In addition to the increased receptor affinity, EXP3174
has a carboxylic function that allows relatively easy conjugation to nanoparticles.
Furthermore, amides of that carboxylic group are known to retain a high receptor blocking
efficacy in similar derivatives [4]. First, quantitative real-time PCR showed that human
adrenal corticocarcinoma NCI-H295R cells, which served as receptor-positive model cells,
highly expressed the AT1 receptor. Flow cytometry (FACS) experiments with these ontarget
and HeLa cells as off-target cells revealed a strong binding of EXP3174-modified
Qdots to AT1-positive but not to AT1-negative HeLa cells. To further investigate the
distribution pattern of the modified Qdots we performed binding experiments with a confocal
laser scanning microscope (CLSM). The EXP3174-Qdots showed a membrane-associated
staining of the NCI-H295R cells, indicating a binding to the cellular surface. This was
expected for antagonist-modified nanoparticles which do not provoke receptor
internalization upon ligand binding. In competitive displacement experiments a great excess
of free ligand was needed to displace the EXP3174-modified Qdots from the cells, which is
characteristic for nanoparticles that exhibit multivalent binding [5].
In conclusion, we could show that a non-peptide antagonist for the AT1 receptor can be
bound to quantum dots in order to target AT1-positive cells in a multivalent manner.
Although the chemical character is significantly altered by conjugation, a sufficient receptor
affinity remains. This allows for potential targeting of AT1 receptor overexpressing sites
within the body.
Acknowledgements: This work was supported by the Deutsche Forschungsgemeinschaft: DFG grant No
GO565/17-1.
References:
1. Dvir, T. et al.: Nano Lett. 2011, 11(10): 4411–4414.
2. Suzuki, K. et al.: Am. J. Pathol. 2007, 170(6): 1841-1853.
3. Fabiani, M. et al.: Am. J. Hypertens. 2000, 13(9): 1005–1013.
4. Santella, J. B. et al.: Bioorg. Med. Chem. Lett. 1994, 4(18): 2235–2240.
5. Montet, X. et al.: J. Med. Chem. 2006, 49(20): 6087-6093.
156
Biphasic drug release from coated pellets
Priese, F., Wolf, B.
Anhalt University of Applied Sciences, Pharmaceutical Engineering, Neues Labor, Strenzfelder
Allee 28, D-06406 Bernburg, Germany
Pellets used for multiparticulate drug delivery systems may contain the API (active
pharmaceutical ingredient) incorporated into the pellet core or as a coating layer on the
pellet surface. Rapid release occurs when the API is readily soluble. With the help of further
excipients the API release can be varied in a very broad range. Retarded release is
achieved with functional polymers showing swelling capacity but no solubility under
physiological conditions [1]. Additional to the advantage of homogeneous drug release of
multiparticulate systems a biphasic release may be achieved by incorporation of a fast as
well as a slow or retarded released dosage of the API. The therapeutic effect is reached
immediately after drug administration and the patient is supplied with drug over a long time
period by the slow released maintenance dosage.
Inert microcrystalline cellulose pellets (Cellets ® 200, IPC GmbH, Dresden, Germany) were
coated with the model API sodium benzoate as maintenance dosage (Figure) in a first step.
The coating liquid consists of sodium benzoate and furthermore of polyvinylpyrrolidone for
film stability increase and talcum to prevent the pellets from sticking during the coating
process. For retarded release of that dosage the pellets were coated with Surelease ®
dispersion (ethylcellulose, Colorcon Corp.) in a second step. At least the pellets were
coated with the initial sodium benzoate dosage. The coating was performed in a batch
laboratory fluid bed apparatus with Wurster technique (GPCG 1.1, Glatt Corp., Binzen,
Germany). The weight ratio of initial to maintenance dosage was varied (1:2.2 and 1:6).
Evaluation criteria were the coating process stability, the product yield and the pellet
quality. Mean pellet size, sphericity, bulk density and drug release were measured.
Figure: Scheme of threefold coated pellets
The fluid bed coating processes were stable. At the end of the first and the second coating
step the coating liquids were changed so that the process was performed without
interruption. Yield was above 90%, significant material loss at the inner wall of the chamber
or at the partition was never observed. The pellets were received as particles with smooth
surface and sphericity of 0.95-0.96 indicating homogeneous growth of the several coating
layers. The initial sodium benzoate dosage from the external layer was released
immediately after 1 minute. The maintenance dosage was retarded released due to
ethylcellulose film swelling (90% release after 17-25 minutes). In comparison, the release of
sodium benzoate coated pellets without polymer film occurs completely within 1 minute due
to readily solubility and high dissolution rate. The retarding effect of Surelease is improved
with increasing layer thickness. By variation of the ratio of initial to maintenance dosage
and polymer layer thickness the release kinetics can be varied in a wide range. The final
pellets are filled into hard gelatin capsules or compressed into tablets. The influence of the
compression process on the integrity of the pellets and the maintenance of the release
kinetics is under investigation.
Acknowledgement: The technical assistance of the student Markus Seifert in the experimental trials is
grateful acknowledged.
References:
[1] Siepmann, F., Siepmann J., Walther, M., MacRae, R.J., Bodmeier, R.: Polymer blends for controlled
release coatings, Journal of Controlled Release 2008, 125: 1–15.
157
Influence of fluid bed rotor pelletization parameters on pellet properties
Wolf B, Ren X
Anhalt University of Applied Sciences, Pharmaceutical Engineering, Neues Labor, Strenzfelder
Allee 28, D-06406 Bernburg, Germany
API (active pharmaceutical ingredient) loaded pellets are used for the preparation of
multiparticulate drug delivery systems. Actual market products are pellet filled hard gelatine
capsules, in some cases pellets are compressed into tablets. The API may be incorporated
into the pellet or sprayed onto the surface of inert pellets forming an external layer. Enteric
resistance or controlled release can be achieved in both cases with further coating layers.
Pellets are predominantly manufactured by extrusion/spheronization, but fluid bed rotor
agglomeration [1, 2] is of increasing interest because of the advantage of a single pot
process, i.e. mixing of the initial solid components, agglomeration and drying are performed
in one and the same apparatus.
Preliminary studies were performed with a simple binary mixture of microcrystalline
cellulose and lactose monohydrate 1:1 to evaluate the possible process parameter range.
In a second trial, the highest possible content of ternary model substances (benzocaine,
calcium hydrogen phosphate, talcum and maize starch) in the mixture was investigated.
Evaluation criteria were process stability and pellet quality. Finally, by use of a 23 design of
experiments (DoE) the influence of the process parameters spray rate, process air
temperature and rotor disc rate and the influence of the model substances (content 10% in
the formulation) on the pellet formation process and on the mean particle size d50 was
investigated. The pellet manufacturing process was performed in a laboratory fluid bed
granulator and dryer (GPCG 1.1 with rotor disc equipment, Glatt Corp., Binzen, Germany).
Purified water was used as granulation liquid. At the end of the agglomeration process the
product was dried to residual moisture below 5%.
With the binary mixture, the pellet fluid bed manufacturing was possible in a broad range of
spray rate, process air temperature and rotor disk rate. The pellets were received in
satisfactory spherical shape. The maximum content of maize starch was 40%, higher
amount led to overwetting and formation of inacceptable large agglomerates. The content
of insoluble and not swellable calcium hydrogen phosphate was limited to 40%. Above this
value agglomeration did not occur anymore, the fluid bed process broke down and the
product precipitated onto the wall and at the spray nozzle. This was also the case with
more than 15% of coarse-grained benzocaine as well as fine-grained talcum what is
sometimes used in low concentration as anti-sticking agent in coating processes.
Factor and interaction Effect Tendency
Factor Pellet size
A = Spray rate high positive A ↑ ↑
B = Process air temperature high negative B ↑ ↓
C = Rotor disk rate weak negative C ↑ ↓
Interaction A/B and A/C negative A/B, A/C ↑ ↓
Interaction B/C and A/B/C weak positive B/C, A/B/C ↑
Table: Evaluation of DoE, factors, effects and tendency on pellet size
Low and high level of the process parameters were spray rate 15/25 g/min, process air
temperature 50/70°C and rotor disc rate 450/750 rpm. The evaluation of the DoE
experiments is summarized in the table. The influence of spray rate on particle size is high
positive and that of process air temperature high negative as expected. The rotor disk rate
does not significant effect the particle size. Effects by interaction are proved but not
significant. Fine-grained and insoluble calcium hydrogen phosphate does not influence the
particle size. Vice versa coarse-grained benzocaine gives high effect. The flow properties of
the products are influenced by the particle size and the shape. Angle of slope is diminished
with increasing pellet size indicating improved flow properties. On the other hand flow time
is raised with increasing particle size. Large pellets deviate from spherical shape more than
smaller ones deteriorating the flow property. Fluid bed rotor granulation may be used for
pellet manufacturing in a wide range of process parameters but the drug substance
properties i.e. initial particle size, solubility and hydrophilic-lipophilic properties should be
taken into consideration.
References:
[1] Parikh, D.M., Mogavero, M.: Batch fluid bed granulation. In: Parikh, D.M. (editor): Handbook of
pharmaceutical granulation technology (Taylor&Francis) 2005.
[2] Kristensen, J.,Hansen, V.W.: AAPS PharmSciTech 2006,7(1): E153-E162
Poster 133

158
Stability and cellular uptake of a model vaccine containing Resovist ® as
marker for MRI tracking
K. Thom1, K. Schulz1, K. Aurich2, G. Glöckl1, J.-P. Kühn3 and W. Weitschies1 1Institute of Pharmacy, EMA University of Greifswald, Greifswald, D-17489, Germany
2Institute of Immunology and Transfusion Medicine, EMA University of Greifswald, Greifswald, D-
17489, Germany
3Institute of Diagnostic Radiology and Neuroradiology, EMA University of Greifswald, Greifswald,
D-17489, Germany
Aluminum containing adjuvants in vaccines applied for immunopotentiation have been
considered as save and efficacious for decades. However, their mode of action could not
be ascertained in detail to date. Tracking the adjuvant after injection using magnetic
resonance imaging (MRI) may be a new method to investigate the fate of aluminum
hydroxide (AH) adjuvant in vivo. For this purpose the MRI contrast agent Resovist ® (Bayer
Schering Pharma AG) containing ferucarbotran nanoparticles, was combined with
Aluminum hydroxide Gel (Sigma). Prior to MRI analysis this combination was investigated
concerning stability and cellular interaction.
Opposite surface charges (zeta potential Resovist ® -38 mV, Aluminum hydroxide Gel
+13 mV) lead to electrostatic attraction and hence to adsorption by the adjuvant within
30 minutes. Tracking the AH adjuvant requires a strong adsorption of the ferucarbotran
nanoparticles even under in vivo conditions. Therefore the degree of adsorption was
determined in simulated interstitial fluid (sIF) [1] and in plasma for a period of 24 h. At
different timepoints unbound iron content was determined using atomic absorption
spectrometry (AAS).
Investigating the uptake of ferucarbotran and ferucarbotran adsorbed to AH adjuvant into
peripheral mononuclear blood cells (PBMC) gave an indication of the influence of the
nanoparticles on the adjuvants behavior after injection of the model vaccine. For isolating
PMBC a density gradient centrifugation was used (OptiPrep ®, Sigma). Cells were incubated
either with Resovist ®, Aluminum hydroxide Gel or the combination thereof at 37 °C and
300 rpm. One fraction was incubated without additives as negative control. For cell dilution
different media were used (RPMI with 10 %, 50 % or without fetal calf serum = FCS, 100 %
FCS or sIF) to investigate media influence on the uptake into cells. After one hour cells with
adsorbed or incorporated nanoparticles were separated utilizing their magnetic behaviour.
Subsequently magnetic and non-magnetic cells were counted. The iron content of each
separated fraction with sIF as dilution medium was determined using AAS.
In our experiments sIF eluted twice as much iron than plasma during the first hour but after
6 and 24 hours iron content in all samples was nearly the same. Agitation of the samples
during incubation did not have any effect on the elution. Iron amounts in the supernatant
below 7 % of the input indicate high adsorption strength after injection of the model vaccine.
Resovist ® was used in this study to ensure visibility of the AH adjuvant for MRI. The
contrast agent leads to signal loss on MR images if it is accumulated in tissues. Incubation
with ferucarbotran particles led to an iron uptake into cells which allowed separation in a
magnetic field. During this process the dilution medium had an obvious influence. With
increasing FCS concentration in the medium the number of magnetic cells decreased due
to the rising amount of adherent proteins which block the cell surface. The amount of
ferucarbotran which was taken up within sIF medium was similar to the values for RPMI
with 50 % FCS. This correlates with the protein content of 2.5 % in sIF which corresponds
to 5 % proteins in serum respectively. When the Resovist ® particles were adsorbed to AH
adjuvant prior to incubation the magnetic cell fraction in each dilution medium exceeded the
one reached after pure Resovist ® treatment. The enhancement of the iron uptake may
facilitate signal loss resulting in visibility.
The non-magnetic fraction of Resovist ® treated cells contained more iron than the magnetic
fraction propably due to an excess of unbound Resovist ®. Cells incubated with Resovist ®
adsorbed to AH gave opposite results. Here the AH-ferucarbotran aggregates showed
magnetic behaviour by themselves and thus increased the iron content of the magnetic cell
fraction.
In conclusion, adsorption of Resovist ® is most probably stable enough for tracking AH
adjuvant in vivo. AH adjuvant enhanced the iron uptake into PMBC. This might facilitate the
detection on MR images
References:
1. Wolff, L. et al.: Vaccine 2009, 27: 1834-1840.
159
Effects of non-thermal plasma on HaCaT keratinocytes is not mediated by
ozone
Blackert S1, Haertel B1, Talmann L2, Oehmigen K2, von Woedtke T2, Lindequist U1 1 Institute of Pharmacy, University of Greifswald, D-17489, Germany
2 Leibniz-Institute for Plasma Science and Technology e.V. (INP), D-17489 Greifswald, Germany
Non-thermal atmospheric-pressure plasma has been shown to influence as first target the
cell membrane with its embedded proteins. Such cell surface molecules as integrins,
cadherins or the epidermal growth factor receptor (EGFR) are of importance in wound
healing and also for development of cancer metastasis. Cold plasma comprises of
electrons, positive or negative ions, free radicals [e.g. reactive oxygen species (ROS),
ozone] and other excited atoms and molecules. Further, plasma has an optical emission in
the UV-region, especially UVB. Each of these components can affect the cells during
treatment.
Since it is known that during plasma treatment ozone is generated, we wanted to know as
to whether this ozone concentration affects HaCaT keratinocytes.
Adherent HaCaT keratinocytes were treated with plasma by a surface dielectric barrier
discharge in air and argon (1 to 5 min) or with ozone (5 min). Ozone was generated by an
Ozonisator and monitored by FT-IR (100, 400, 900 and 1800 ppm). Intracellular ROS,
apoptosis (annexin V/propidium iodide staining) and cell surface molecules (α2-, α3-, α4-,
α6-, αV-, β1-integrin, E-cadherin, EGFR) were analyzed by flow cytometry 24h after
treatment.
Besides a reduction of cell viability significant intracellular changes were observed. DBD/air
plasma for 5 min caused an increased expression of α2- and β1-integrin whereas Ecadherin
and EGFR expression was not influenced. The effects of DBD/argon plasma were
less pronounced. Apoptosis was only increased by DBD/air plasma (5 min) although the
proportion of apoptotic cells was rather low. Intracellular ROS detected by the fluorescent
dye CM-H2DC-FDA increased from 6.6 ± 1.1% (untreated control cells) to 18.0 ± 3.2% (5
min DBD/air) and 10.9 ± 1.3% (5 min DBD/argon). A concentration of about 100 ppm
ozone was measured above the solid phase during a 5 min DBD/air treatment cycle which
had no influence on integrin, E-cadherin or EGFR expression. 1800 ppm ozone caused an
increase of α2- and β1-integrin whereas all other molecules measured were not affected.
Taken together, the extent of effects depended on the nature of plasma (air vs. argon) and
the exposure time of cells to the plasma. Short (≤ 1 min) treatment cycles did neither
change cell surface protein expression nor induced apoptosis or intracellular ROS. The
lowest concentration of ozone tested (100 ppm = about 200 mg/m 3), which is 1000 fold
higher than the MAC had marginal effect on viability and no influence on integrin
expression of HaCaT keratinocytes. The effects of plasma on cell membrane proteins
observed are rather attributed to induction of intracellular ROS than to generation of ozone.
* Work supported by the German Federal Ministry of Education and Research within the joint research
project Campus PlasmaMed” (grant no. 13N11182)
160
Evaluation of German and International drug interaction databases
Pauly A. 1; Wolf C. 1; Busse M. 2; Strauss A-C. 2; Krebs S. 2; Dörje, F. 2; Leuner, K. 1
1 Professur für Molekulare und Klinische Pharmazie, Cauerstraße 4, 91058 Erlangen, Germany
2 Apotheke des Universitätsklinikums Erlangen, Palmsanlage 3, 91054 Erlangen, Germany
Background
Drug interactions are a common problem in drug therapy, especially if polypharmacy is
needed [1]. There are a number of databases available helping to identify clinically relevant
drug interactions. They vary in scope, accuracy, comprehensiveness, user-friendliness and
cost. So far, there does not exist an evaluation which includes German databases.
Therefore, we compared seven databases in German and English to determine the best
database for daily clinical practice.
Methods
We identified more than 100 drug interaction pairs out of our daily routine and literature
research. These were first validated using three reference databases (Stockley’s, DrugDex,
iFacts). Out of the 100 selected pairs 50 were classified as clinically relevant and 50 as
clinically non-relevant.
A predefined scoring system was used to assess the scope, accuracy and
comprehensiveness of the selected drug interaction databases. ABDA-database, MediQ,
Pharmavista and MMI Pharmindex were selected as German drug interactions
programmes. Lexi-Interact, Epocrates and drugs.com represented the international test
databases.
Results
With its reliable monographs and high accuracy, Lexi-Interact scored 509 of 600 possible
points and ranked first in our evaluation. Lexi-Interact was the most comprehensive
database (180 of 200 possible points) with detailed monographs containing not only
mechanism, severity and management of the drug interaction, but also onset, level of
evidence and discussion of evidence, and literature references. These monograph
components were not provided in all databases.
The free of charge Swiss database Pharmavista (495 of 600 points) provided detailed and
accurate monographs, but only recognized Swiss trade names. The German ABDA-
Database ranked third in the overall score (453 of 600 possible points), because it omitted
the level of evidence and discussion of evidence. The ABDA-Database scored best in
accuracy with 335 out of 400 possible points.
MMI Pharmindex (441 of 600 possible points), MediQ (413 of 600), drugs.com (382 of 600)
and Epocrates (357 of 600) ranked fourth to seventh. These databases failed to distinguish
between clinically relevant and clinically non-relevant interactions in a higher percentage,
revealed more problems identifying clinically non-relevant interactions and suffer from
limitations in the quality and comprehensiveness of their monographs.
Discussion
For the daily clinical routine, an easy to use and accurate system is needed, which
identifies drug interactions without the tendency of overalerting. Whereas most databases
were self-explanatory, MMI pharmindex was more complex to use. Drugs.com displayed
the highest rate of excessive alerts. As frequent alerts of clinically irrelevant drug
interactions lead users to ignore the warnings [2], this might be a major caveat to use this
database in the daily clinical routine.
Conclusion
When used appropriately, drug interaction database can help to identify clinically relevant
drug interactions and thereby increase patient safety. However, we should be aware of the
limitations of drug interaction databases. In our evaluation Lexi-Interact proved to be the
most reliable and comprehensive source of drug interaction information.
References:
1. Storka, A. et al, Wiener Medizinische Wochenschrift, 2009. 159(17-18): 462-469.
2. Payne, T.H., et al., Proceedings / AMIA . Annual Symposium. 2002: 602-606.
161
Applicability of different omeprazole formulations via enteral feeding tubes
Hanke, U. 1, Iber, S. 1, Wedemeyer, R.-S. 2, Blume, H. 2, Weitschies, W. 1
1 University of Greifswald, Center of Drug Absorption and Transport, Institute of Pharmacy,
Department of Biopharmaceutics and Pharmaceutical Technology, Felix-Hausdorff-Street 3, 17487
Greifswald, Germany
2 SocraTec R&D Concepts in Drug Research and Development GmbH, Im Setzling 35, 61440
Oberursel, Germany
For patients with swallowing disorders or other diseases that preclude the oral route of food
intake the use of enteral feeding tubes is necessary. Often the administration of protonpump
inhibitors (PPI) like omeprazole is required for these patients to avoid stress-related
mucosal bleeding or acid pneumonitis [1]. Unfortunately, there is still a lack of knowledge
regarding the handling of drug application via feeding tubes. The aim of this study was to
investigate the ability of three different omeprazole formulations (Nexium ® mups 40 mg,
enteric coated tablets containing micropellets (Astra Zeneca GmbH), Omeprazol Hennig ®
40 mg (Hennig Arzneimittel GmbH & Co. KG) and OMEP ® 40 mg (Hexal AG), both
capsules with enteric coated pellets) to pass enteral feeding tubes in order to obtain
recommendations for an appropriate application of these formulations via this route of
administration.
The feasibility of application was tested using two different feeding tubes (representatively
for percutaneous endoscopic gastrostomy (PEG): Freka ® PEG-Set gastral CH 9, length:
30 cm, 1 terminal port; representatively for nasogastric tubes: Freka ® Intestinale Sonde
134 Poster

CH 9 for PEG CH 15, length: 120 cm, 2 lateral, 1 terminal port; both Fresenius Kabi AG).
As application media served tap water, boiled tap water and different commercially
available still mineral waters. The used media were analysed regarding pH, buffer capacity,
and electrical conductivity. Diameter, swelling behaviour, and stability of the pellets over
480 min were also investigated. To study the application of the pellet formulations via
feeding tubes an appropriate application protocol had to be established. Briefly, the tubes
were washed with 30 mL test fluid before and after the application. Pellets from one opened
capsule or one tablet were administered together with 30 mL of the test fluid by using
50 mL catheter syringes with included Luer adaptors. The contents were slowly injected
through the tubes while gently shaking the syringe to avoid obstructions. For a complete
removal of the Omeprazol Hennig ® and OMEP ® pellets out of the syringe the use of further
60 mL of additional test fluid was necessary. The contents of the syringes were injected into
the tubes either directly or after 1 h of rest. After tube passage the administered
suspensions were collected, decanted, solved in 100 mL 0.1 M NaOH and further diluted.
The analyses of the diluted probes (1:20) and calibration measurements were performed at
304 nm using an UV spectrophotometer (Varian Cary 50, Varian Inc., Palo Alto, USA).
Pellets of Nexium ® mups were small enough to pass all tubes without any problems. Pellets
of OMEP ® only were able to pass the PEG without using the Luer adaptor. The application
through the nasogastric tube immediately resulted in obstructions. Omeprazol Hennig ®
pellets passed the PEG without any difficulties, the nasogastric tube could only be passed
immediately after preparation of the suspension without using the Luer adaptor. This may
be attributed to the swelling of the pellets during 1 h of rest. The swollen pellets had a
higher tendency to agglomerate. For all media comparable results were obtained. For the
latter two formulations nasogastric tubes with CH > 9 need to be tested.
In conclusion, the ability of omeprazole formulations to pass through a feeding tube is
extremely dependent on the properties of the formulation and the used liquid as well as on
the type of feeding tube. Micropellets as used in Nexium ® mups are small enough to pass
even small feeding tubes like nasogastric tubes without any problems. The bigger the pellet
diameter, the higher is the risk of obstruction. Also the shape of the pellets has an impact
on the administration as non spheric formed pellets more often tend to agglomerate inside
the tube than spheric ones. Short tubes with only on terminal port like PEGs showed less
obstructions compared to long tubes with lateral ports like nasogastric tubes. Generally, all
omeprazole formulations should be administered slowly and under continuous shaking of
the syringe immediately after preparation of the solution. Tube washing should be
performed before and after the administration of drugs.
References:
1. Johnson, J. L. et al.: Am. J. Health-Syst. Pharm. 2008, 65: 2324-2325.
2. Devlin, J. W. et al.: Ann. Pharmacother 200, 39: 1667-1677.
162
Nanostructured Lipid Carriers (NLC): The optimum concentration and
production condition
Junmahasathien T. 1; Müller R. H. 1; Keck, C. M. 1 2
1 Institute of Pharmacy, Department of Pharmaceutics, Biopharmaceutics & NutriCosmetics, Freie
Universität Berlin, Kelchstrasse 31, 12169, Berlin, Germany
2 Applied Pharmacy Division, University of Applied Sciences Kaiserslautern, Campus Pirmasens,
Carl-Schurz-Strasse 10-16, 66953 Pirmasens, Germany
It is interesting that there is a finished product (lipid matrix) which is the mixture of oils, fatty
alcohol and vegetable waxes (Polyglyceryl-2 Dipolyhydroxystearate, Octyldodecanol,
Carnauba Wax, Candellila Wax, Beeswax, Cetearyl Glucoside and Cetearyl Alcohol) It can
be used as NLC.
Nanostructured lipid carriers (NLC) have been developed to overcome the disadvantages
of solid lipid nanoparticles (SLN). Solid lipid mixed with liquid lipid, to decrease the degree
of perfect crystal of lipid matrix.[1,2] The aim of this study was to develop a new
formulation, e.g. the NLC-preconcentrate, containing high amounts of small sized NLC. For
this propose, a new lipid matrix was used and the influence of the lipid concentration (10-
30%) including the number of high pressure homogenization cycles (3, 5 and 10 cycles) on
the particle size and physical stability were investigated. The formulation consisted of a new
lipid matrix, Plantacare® 2000UP 2% as a stabilizer and water. Each formulation was
premixed by an Ultra-Turrax at 8,000 rpm for 30 seconds and produced at 80°C using high
pressure homogenization (HPH) at 500 bar. The results showed that the increase in the
lipid concentration led to a larger particle size. More cycles led to smaller particles. The
smallest particle size (145 nm) was obtained from the formulation containing 10% lipid
which produced by applying 5 cycles of HPH. The maximum lipid concentration was 25%.
Samples containing higher concentration, e.g. 30% lipid content, solidified during the
production. The physical stability was affected by the lipid content, e.g. a higher lipid
content led to a faster increase in particle size over time. Formulations containing 10% lipid
remained physically stable over time. In conclusion, a new NLC formulation was
successfully developed and optimized in this study. The optimum lipid concentration for
producing this NLC is 10%.The optimal production conditions are 5 cycles of HPH, 500 bar
at 80°C.
Acknowledgments: UPM Handelsgesellschaft mbH, Haupstrasse 47 D-21447 Handorf Germany, PharmaSol
GmbH, Blohmstrasse 66A 12307 Berlin Germany
References:
1. Teeranachaideekul V., et al.: Eur. J. Pharm. Biopharm. 2007, 67: 141-148.
2. Müller R. H., et al.: Adv Drug Deliv Rev. 2007, 59: 522-530.
163
Regulations of Preclinical Studies and Clinical Trials in the Former GDR
Retzar A., Friedrich C. 1
1 Institute for the History of Pharmacy, Philipps-Universität Marburg, Roter Graben 10, 35032
Marburg, Germany
The poster deals with the legal conditions of preclinical studies and clinical trials in the
former German Democratic Republic.
Since 1949 it was determined that introducing pharmaceuticals was not permitted without
registration. The decision on registration was made by the Ministry of Health based on the
recommendation of the so-called ‘Zentraler Gutachterausschuss für Arzneimittelverkehr
(ZGA)’, a commission of clinicians, pharmacologists, pharmacists and representatives from
industry.
In 1960 a directive regulated the procedure of registration, which required the submission of
pharmacological and clinical surveys.
In 1964 the Medicinal Products Act was established containing the obligation to prove the
effectiveness and inoffensiveness of new drugs in preclinical studies and clinical trials
before they were launched. Under the new regulations approval by the ZGA was necessary
to conduct clinical trials. Therefore, an application had to be filed, which provided
information about extent, methods and responsibilities of the trial.
Due to increasing requirements in the field of drug studies the regulations had been
revised. Consequently, the 12th implementing rule of the Medicinal Products Act came into
effect in 1976. From now on drug studies were subdivided into 4 levels. Measures of postmarketing
surveillance in level no. 4 were determined to detect adverse drug reactions.
Acknowledgments: Institute for the History of Pharmacy, Ariane Retzar.
References:
Richter, J., Keune, H. G.: Arzneimittelrecht der DDR. Kommentar Teil I (Akademie-Verlag Berlin) 1972.
Hackenberger, F., Koch H.: medicamentum 1977, 18(5): 130–135.
164
Pharmacy under the sea – The medical supply of the German „U-Boot-Waffe“
during the World Wars
Vongehr, F. 1; Friedrich, C. 1;
1 Institut für Geschichte der Pharmazie der Philipps-Universität Marburg, Roter Graben 10, 35032
Marburg, Germany
Despite the fact that German submarines have been a common subject of military-historical
research and literature, the medical service aboard which was determined by the special
environmental conditions is a rather neglected aspect. However, it is known that
complicated surgery such as amputations could be performed even in dived submarines,
although the crews had to deal with severe problems like climate, humidity and narrowness.
The question arises how the medical supply was organized.
Beginning with the first German submarine „U 1“ commissioned in 1906 the new boats
were only equipped with a few remedies, such as disinfecting ointment and acetylsalicylic
acid, but during Wolrd War I the experience lead to the conclusion to extend the selection
of drugs on the boats. Therefore, the navy issued a compendium, the so-called „Ärztlicher
Ratgeber für Unterseeboote“, containing a more detailed medical supply and several
instructions for the commanders, who mostly had to operate their boat without a physician.
During World War II the „Kriegsmarine“ had learned from the experiences of the
„Kaiserliche Marine“ and published revised editions of the „Ärztlicher Ratgeber“ in the
1940s. The medical supply was now significantly augmented and several boats even had
physicians among the crew. Especially submarines designed for tactical operations in
remote areas carried an extensive supply of drugs, including anti-malaria drugs and
sulfonamides.
The allies confiscated important documents regarding the German „U-Boot-Waffe“ including
logfiles and medical data for their own use after victory. It had lasted several decades until
the documents were returned and opened for research. The historical sources regarding
pharmaceutical questions aboard the German submarines are now an object of detailed
research.
165
Market surveillance study to medical products with pyridostigmine:
tetramethylurea contamination in tablets and injections relevant to regulatory
legislation for toxicological concerns
Bogan R; Zimmermann T
Central Institute of the Bundeswehr Medical Service, Ingolstädter Landstraße 102, 85748 Garching
Hochbrück
Pyridostigmine is a well known Active Pharmaceutical Ingredient (API) described in
monographs of the European, the British and the United States Pharmacopeia.
Nevertheless in previous studies we elucidated a hitherto new degradation pathway of
pyridostigmine leading to formation of tetramethylurea (TMU) [1,2]. Based on these data we
analysed marketed medical products with API pyridostigmine for the occurrence of TMU.
The samples tested were within and after expiry date. Dosage forms of tablets and
injections were considered. As shown before the compendial method of the European
Pharmacopeia could be used for detection and quantification of TMU.
We examined four batches of tablets in two dosage strengths and two batches of injections
in one dosage strength from two marketing authorization holders. Samples were analysed
two times in a distance of 24 months; in between they were stored at the recommended
condition below 25°C. At the start of the study the remaining time to the expiry date of 5
batches was between 5 and 48 months. For comparison reason one batch 7 months after
expiry date was included. At the retest time 5 batches exceeded the expiry date for 4 to 31
months whereas 1 batch was still 24 months before expiry date. At the first examination
TMU was detected in one batch of tablets 9 months before expiry date and in the one batch
of injections which had exceeded the expiry date for 7 months. The second examination
revealed an additional batch with TMU and a marked increase of TMU content in those
batches which were already contaminated. The highest contamination was found in one
batch of tablets with 0.36 % (area percent related to API) 9 months before expiry date and
0.51 % resp. 15 months after expiry date.
The study proved that the predicted degradation pathway also occurs in market authorized
medical products. TMU contamination seems to be in particular a problem at the end or
after expiry date. Nevertheless the occurrence of TMU even within expiry date was shown
and has to be considered. The values measured exceed the threshold values for
degradation products of the ICH [3]. Toxicological evaluation does not suggest acute
toxicity in the relevant dose range [4,5], but in terms of chronic toxicity the risk of a
teratogenic effect of TMU can not be excluded [6,7].
As a conclusion of our findings efforts on a revision of the relevant monograph
pyridostigmine in the European Pharmacopeia as well as regulatory provisions are in
process.
References:
1. Bogan, R. Loch, M.: Wehrmed. Mschr. 2010, 54: 242-247
2. Bogan, R.: Joint meeting of the austrian and german pharmaceutical societies, abstract volume, 2011, PS
001: 86
3. International conference on harmonization of technical requirements for registration of pharmaceuticals
for human use, ICH Harmonised Tripartite Guideline Q3B, Impurities in new drug products, 2006
4. Dixon, R.L. et al.: Arch. Int. Pharmacodyn. Ther. 1966, 160: 333
5. Edler, L. et al.: Food and chemical toxicology. 2002, 40: 283-326
Poster 135

6. Munley, S.M. et al.: Drug. Chem. Toxicol. 2001, 24: 259-271
7. Teramoto, S. et al.: Teratology. 1981, 23: 335-342
166
Improved taste evaluation of pharmaceutics using food sensors?
Eckert, C., Pein, M.
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine University, Duesseldorf,
Germany
Introduction: Two electronic taste sensing systems are commercially available. Whereas
one sensor set with seven sensors related to specific gustatory stimuli is offered for the
Japanese taste sensing system (Insent), Alphamos (France) offers two sensor sets
containing seven cross-selective sensors for the αAstree. The two sensor sets are
recommended for different applications. While sensor set 2 is marketed for pharmaceutical
applications, sensor set 5 is recommended for taste evaluation of food. So far, this
differentiation has not been questioned. Therefore, two active pharmaceutical ingredients
(APIs) and two different lozenges were characterised using these two sensor sets.
Materials and methods: Caffeine citrate (DAC) was purchased from Fagron (Barsbuettel,
Germany), quinine hydrochloride (Ph. Eur.) from Caesar&Loretz (Hilden, Germany). Both
APIs were determined in concentration ranges from 0.05 mM to 50 mM in 12 levels. Both
lozenges were evaluated using different sample weights (0.5-12 g/100 ml).The samples
were dissolved in aqua purificata and measured using the αAstree equipped with sensor
sets 2 and 5. The measurement setup was used according to Pein [1].
Results and discussion: Each sensor of both sensor sets fulfils the repeatability
requirements [2] with RSD values

34 patients (BMI < 20: 4, 20-25: 17, > 25: 13). In 18 patient charts the documentation of
bodyweight and height was missing. As an index for renal function the GFR was evaluated
from serum creatinine. 25 (48.1%) patients showed a GFR > 60 ml/min/1.73 m², 21 (40.4%)
patients between 30-59 ml/min/1.73 m² and 6 (11.5%) patients < 30 ml/min/1.73 m².
Potassium levels as an important electrolyte for cardiac functions were for almost all
patients in a normal range (3.5-5.1 mmol/l).
CONCLUSION: Our results confirm the study of Groth-Tonberge et al. concerning fallpromoting
drugs on a cardiological ward [2]. Average age, medication per patient and most
prescribed medication groups (diuretics, beta blockers, angiotensin-converting-enzyme
(ACE) inhibitors, antibiotics) were in good agreement and a potential relation between falls,
polypharmacy and higher age could be shown. Additionally, considering the fact that more
than 50% of the patients also had an impaired kidney function (GFR < 60 ml/min/1.73 m²)
the importance of pharmaceutical care focused on both polypharmacy and adequate drug
dosing is evident and could help to prevent fall events.
References:
[1] Pierobon, A., Funk, M. Sturzprävention bei älteren Menschen. Risiken–Folgen–Maßnahmen (Georg
Thieme Verl.) 2007.
[2] Groth-Tonberge, C., Feuchtinger, J., Strehl, E. Krankenhauspharmazie 2011, 32(11): 565.
[3] Levey, A. S. et al. Ann Intern Med 2009, 150(9): 604.
170
Development of lipid micropellets with metformin hydrochloride using solventfree
cold extrusion/spheronization
Petrovick, G.F.; Breitkreutz, J.
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, 40225 Duesseldorf,
Universitaetsstrasse 1, Germany
The diabetes mellitus type 2 (DM2) constitutes 85 to 95% of all diabetes cases in
developed countries and has reached epidemic proportions in several nations [1, 2].
Metformin is the drug of choice to treat DM2 in many countries [3, 4]. But the relatively large
size of available tablets (doses form 500 to 1000 mg) can cause difficulty to swallow
(especially in elderly patients) which may lead to discontinuation of treatment. Considering
this problem, microparticulate systems have advantages when compared to tablets or
capsules [5]. Solid lipid extrusion in a twin-screw extruder is an innovative preparation
method for matrices in which lipids are treated below their melting ranges in a thermomechanical
process. The drug is dispersed in a lipid matrix in which the lipid does not melt
but softens [5, 6].
A hard fat (Witocan ® 42/44) was used as lipidic binder. The extrusion was performed at 33
°C in a Micro 27GL-28D extruder (Leistritz, Nuremberg, Germany) with co-rotating twin
screws, using a screw plate with 91 dies of 0.5 mm and 1.35 mm length, with a powder
feed rate of 40 g/min. The extrudates were spheronized (Schlueter spheronizator RM 300,
Schlueter, Neustadt am Ruebenberge, Germany) for 15 min at 1500 rpm and 33 ± 1 °C.
Metformin hydrochloride lipid micropellets (figure 1) with a high drug load of 80% presenting
narrow particle size distribution and aspect ratio under 1.2 could be obtained. The process
parameters such as pressure and product final temperature were also evaluated (figure 2).
Spheronization was difficult as the process temperature range was difficult to control.
Friction and heat conveying were major issues as they influence processability and product
quality.
Solvent-free cold extrusion followed by spheronization present itself as a robust method to
obtain high dosage micropellets of metformin with adequate size and shape characteristics
which are mainly impacted by the spheronization step.
Figure 1: Micropellets with 80%
metformin hydrochloride
Figure 2: Process parameters
References:
1. Zimmet, P.Z. et al., J Diabetes Complicat 1997, 11(2): 60-68.
2. Giannella-Neto, D.; Gomes, M.B. Diabetol Metab Syndr 2009, 1(1): 1-3.
3. Gregorio, F. et al., Arch Gerontol Geriat 1996, 22(1): 161-170.
4. Soto, A. et al., Endocrinol Nutri 2008, 55(2): 39-52.
5. Breitkreutz, J.; Boos, J., Eur J Pharm Biopharm 2007, 56, 255-260.
6. Witzleb, R. et al., Powder Technol 2011, 207, 407-413.
171
Development of a simple, rapid and reliable assay to count enterococcus
faecium with a CASY coulter counter to assess antibiotic therapy
Goebgen E; Wicha SG; Kloft C
Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin,
Kelchstraße 31, 12169 Berlin, Germany
OBJECTIVES: Determination of bacterial counts plays a key role when the interaction
between bacteria and antibiotic are investigated, i.e. to assess the potency of a drug,
evaluate drug combinations or improve dosing schedules. Cell counts of bacteria are
usually determined by densitometry or plate counting methods. As densitometry is highly
imprecise and plate counting methods are time-consuming, cost intense and require long
preparation times, there is a clear need for an easier method to quantify bacteria. In 1953,
W. Coulter developed the technique of electronic cell counting by measuring the change in
resistance for blood cell counting. About 50 years ago, Kubitschek [1] and Swanton et al.
[2] demonstrated the utility of the Coulter Counter in cell counting of bacterial cells and the
determination of cell volumes in growing cultures of bacteria. In comparison to the standard
methods, electronic cell counting promises to save time, to reduce costs whilst providing a
higher content of information (e.g. cell counts, cell volume and distribution of cell volume)
[1,2]. The objective of the current analysis was to establish an assay on a CASY Coulter
Counter as a fast and simple electronic cell counting method for E. faecium to assess
antibacterial effects in vitro.
METHODS: The Coulter Counter (CASY 1, Roche Diagnostics) included a pump, an
aperture tube (60 µm) and the electronic components: one electrode inside the tube, one
outside and the voltage pulse measuring unit. The number of bacteria is determined by
measuring the change in resistance when a particle passed the aperture. Additionally, the
amplitude of the pulse is proportional to the size and the volume of the particle. To
decrease background-counts, all fluid media were filtered through a 0.2 µm filter. An overnight
subculture of E. faecium (ATCC 700221) was used as stock suspension with an initial
inoculum size equal to McFarland 3 in 0.9% NaCl. Serial dilutions in phosphate buffered
saline with peptone (PBSP) were prepared. Dependent on the bacterial concentration of the
suspension, samples of 10, 100 or 1000 µL were measured in 10 mL filtered, ready-to-use
measuring liquid (Casy ton, Roche Diagnostics). The electronic counting method was
compared to the standard plate count method [3]: 100 µL of each suspension was diluted
and poured on Columbia agar plates. Plates containing 30 – 500 colony forming units
(CFU) were read.
RESULTS: The mean diameter range of E. faecium correlated well with the physical
measuring range of the capillary from 1.2 µm to 45 µm. McFarland standard 3 was
equivalent to 2.2x10 8 colony forming units (CFU)/mL. Analogues to a bioanalytical method,
the performance of this electronic cell count assay was evaluated according to the EMA
guideline [4]. Current investigations suggest agreement with the guideline with respect to
linearity, accuracy and precision of the bacterial cell counts with a lower limit of
quantification of 2.2x10 4 CFU/mL.
CONCLUSIONS: A rapid, precise and simple assay for electronic cell counting was
established and allows for bacterial cell counting of E. faecium in PBSP with a sample
volume up to 1 mL. Thus further studies using the Coulter Counter with a smaller aperture
tube for other bacterial species such as E. coli or P. aeruginosa are indicated. Bacterial
growth experiments, in-process controls and the measurement of postantibiotic effects
could be possible applications for the more efficient Coulter Counter principle so as to
streamline the clinical and preclinical development of an antibacterial agent and to facilitate
decision taking in the development of dosing schemes.
References:
1. Swanton EM, Curby WA, Lind HE: Appl. Environ. Microbiol. 1962, 10(5): 480-485.
2. Kubitschek HE: Nature. 1958, 182: 234-235.
3. Schwalbe R, Steele-Moore L, Goodwin AC: Antimicrobial susceptibility testing protocols (CRC Press)
2007.
4. EMEA/CHMP/EWP/192217/2009
172
Cost of drugs in the ambulatory health care sector – how much do we really
spend?
Aßmann, G. 1 2; Fiß, T. 1; Marschall, P. 2; Fleßa, S. 2; Hoffmann, W. 3
1 German Center for Neurodegenerative Diseases (DZNE), site Rostock/Greifswald; Ellernholzstr.
1/2; 17487 Greifswald, Germany
2 University of Greifswald, Faculty of Law and Economics, Friedrich-Loeffler-Str, 70, 17489
Greifswald, Germany
3 University of Greifswald, Institute for Community Medicine; Ellernholzstr. 1/2 , 17487 Greifswald,
Germany
Introduction: As a result of demographic change the prevalence of age-related diseases
increases. Due to the associated intensive drug therapy even in this part the German health
system faces increasing costs and major challenges. Currently there is a lack of knowledge
about daily drug costs in the ambulatory setting. Our aim was the calculation of daily costs
for regularly taken drugs (prescribed and over-the-counter) in the AGnES-home (AGnES:
GP-supporting, community-based, e-health-assisted systemic intervention) visit cohort.
Additionally we focussed on associations between medication costs and sociodemographic
parameters.
Methods: Basis of our analysis was a subsample of the AGnES-home visit cohort who
received a home medication review between 2006 and 2008 (n=778 patients with n=4985
regularly taken drugs). Current drug costs were added automatically at the time of data
recording based on the German drug index. Missing prices were completed by mean DDDcosts
which were extracted from the German Drug Report. Sociodemographic criteria were
taken from the initial proband interview.
Results: Mean age of all participants was 78.8 years, women were significantly older
(women n=555; 71.3%, 79.9 years, p

Herbal medicinal products containing ethanol are widely used in children [1], since ethanol
it is an excellent extraction solvent for the phytochemical components of herbal drugs and
contributes to the stability of fluid preparations. Toxicological and pharmacokinetic
evaluations [2] have shown that the small amounts of ethanol applied with therapeutic
doses of these medicines are safe even in this age group. Despite these medicines have
been used safely since many decades, they occasionally are subject of discussion in the
public, triggered by the increasing problem of recreational misuse of alcoholic beverages by
children and young persons [3, 4]. Therefore, there is a growing need of a systematic
evaluation of epidemiological data on these medicines.
For an evaluation of the experience gained from the therapeutic use of these medicines, 17
pro- and retrospective studies with 10 different herbal medicinal products containing
ethanol at doses of 40 to 240 mg per single application, depending on the age group, have
been analyzed. These studies cover 50,425 patients between 0 and 12 years of age. In
these studies, altogether 15 adverse drug effects have been described, none of which was
attributable to the ethanol content of the medicines.
In a worldwide survey including these and further 6 herbal medicinal products it was shown
that during the past few years, more than 764 millions daily doses have been sold,
corresponding to more than 33 millions patients (according to data obtained from
manufacturers; figures are available partly from 1993 onwards, partly from 2003/4
onwards). From the packages sold in Germany in the years between 2005 and 2009, 48.1
millions were attributable to self-medication, and 10.8 millions to prescriptions reimbursed
by health insurance (IMS, Frankfurt). As non prescription medicines are reimbursed in
Germany only in children, at least the latter part of the prescriptions can be attributed to
children to an extent of at least 90 %. All of these medicines are registered or licensed by
regulatory authorities, so that adverse effects are covered by the pharmacovigilance
system. No adverse effects attributable to the ethanol content have been reported.
This set of pharmacovigilance data supports the conclusion drawn from the experience of a
safe use over decades, i.e. that the ethanol content of herbal medicinal products does not
give any causes for concern regarding their safety even in children.
Dedication in memoriam: This contribution is dedicated to Prof. Dr. Hilke Winterhoff, Institute for
Pharmacology and Toxicology, University of Münster, Germany, who died on 9 May 2010. She has initiated
and supported this work.
References:
1. Gundermann, K.-J. et al., Neurogastroenterol Motil 2010, 22(S1): 69.
2. Kelber, O. et al., Pharm Ind 2008, 70: 1124-1128.
3. Gemeinsamer Bundesausschuss (G-BA), Bundesanzeiger 21 vom 08. Februar 2011.
4. HMPC, 2010. Reflection paper. EMA/HMPC/85114/2008.
174
Oral mesalazine treatment of ulcerative colitis – a review of current dosage
forms
Kersten E1, Klein S1 1Ernst-Moritz-Arndt University Greifswald, Department of Pharmacy, Institute of Biopharmacy and
Pharmaceutical Technology, C_DAT, Felix-Hausdorff.Str. 3, 17489 Greifswald
Ulcerative colitis (UC) is a chronic and debilitating illness prevalent in the Western
population characterized by chronic inflammation of the colon that is continuously
distributed in the colonic mucosa. Most of the currently available agents used in UC
treatment, e.g. mesalazine, are aimed to downregulate the chronic inflammation in the
intestinal mucosa. The main principle of mesalazine action is a topical reduction of
inflammation in the mucosa. Thus, oral mesalazine dosage forms should release the active
substance selectively at the inflamed areas in the GI tract. For a long time marketed enteric
coated formulations containing 250 – 500 mg of the active drug represented the
formulations of choice for this purpose. However, since average daily mesalazine doses are
typically > 3g/ multiple daily dosing is required. Thus, more recently, several new
formulations including coated multiparticulates and an extended release tablet formulation
containing much higher drug loads were released to the market to improve both
effectiveness and patient compliance.
To get an idea, if the newer types of formulations do represent a benefit with regard to
improving UC therapy, we studied drug release of the various formulations with a pHgradient
method that has been established as a predictive in vitro model in earlier studies
(Klein 2005, 2008). Experiments were performed with a BioDis ® Release Rate Tester. In
contrast to simulating only the recommended dosing conditions, i.e. fasted gastrointestinal
(GI) pH-conditions, as has been done in previous studies, in the present study both fasted
and fed state dosing conditions were addressed. Experiments were also performed with the
official mesalazine test methods described in the United States Pharmacopoeia (USP) to
screen, if the could also come along with a in vivo predictivity.
Results obtained in our experiments clearly show that the site and extent of drug release
are strongly dependent on the type of formulation whereas the drug load has a minor
impact on the release pattern. Unexpectedly, drug release from all enteric dosage forms
was not much affected by the different pH-conditions in the fasted and fed GI lumen.
However, as expected, results from the compendial test setup were different from those
obtained with the biorelevant pH-gradients and therefore will not really be predictive for in
vivo drug release.
Results indicate that none of the marketed dosage forms releases the active drug
selectively in the colon. Moreover, as the marketed formulations show significant
differences in their release profiles, the choice of the dosage form should be based on an
individual basis. The enteric formulations were not prone to the pH-conditions used to
simulate fed state gastric residence. However, it should be kept in mind that beside the pHconditions,
other physicochemical and mechanical aspects can affect drug release,
particularly when comparing fasted vs. fed state dosing conditions. As particularly, the
tablet formulations are expected to be sensitive to a variety of these factors, this will be
addressed future studies.
References:
Klein, S., J. Stein, and J. Dressman, J Pharm Pharmacol, 2005. 57(6): 709-19.
Klein, S., et al. J Contr Release, 2008. 130(3): 216-219.
175
ATR-IR-Imaging of Irbesartan Generic Tablets - Comparison with the Originator
Norwig, J.; Jansen, R.
Bundesinstitut für Arzneimittel und Medizinprodukte (BfArM), Kurt-Georg-Kiesinger-Allee 3, D-
53175 Bonn, Germany
According to the CTD format applicants for a marketing authorisation are requested to
declare the exact composition of the dosage form and of the whole product.[1] Modern
ATR-IR-Imaging allows a full identification of the constituents without any sample
preparation for a chromatographic method in the conventional sense.[2]
Neither an extraction, nor a separation, nor any chromatographic method is required. In
case of Irbesartan tablets manufacturers from Far East could be checked whether the
composition complies. Moreover it is estimated which content and which particle size the
active substance shows in comparison with the originator.
The imaging is an impressive powerful method to check the compliance with the
declaration. The time consumption is estimated to be 1 h.
However impurities in the 0.1 percentage range could not be identified. Therefore a trace
analytical procedure should be combined with the method for full compliance with the
Pharmacopoeia.[3, 4]
Further images show the whole range of excipients.
References:
1. Notice to Applicants, Presentation and format of the dossier CTD in EudraLex, VOLUME 2B, Medicinal
products for human use, Editor: European Commission Directorate General III, Brussels, July 2003.
2. ATR Imaging of Pharmaceutical Tablets, PerkinElmer Life and Analytical Sciences.
3. European Pharmacopoeia 7th Edition 2010, Monograph on Irbesartan.
4. Irbesartan tablets, Monograph US Pharmacopoeia 35, Rockville (2012).
176
Lipopeptide-modified carrier systems – the composition determines cell
selectivity
Sydow, K1, Nikolenko, H1, Taraschewski, A1, Dathe, M1 1 Leibniz Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125 Berlin,
Germany
Liposomal und micellar delivery systems have become one of the most promising options
for the improvement of transport efficiency of poorly soluble drugs and for drug targeting.
To increase selective uptake, target recognizing and transmembrane transport-mediating
motifs are used. To achieve these requirements we developed lipopeptides, among them
P2A2[1]. Because of their amphiphilic character, the peptides self-assemble to micelles and
integrate into the lipid bilayer of liposomes. Interestingly, P2A2-micelles and -liposomes
differ in cell selectivity and activate different modes of cellular uptake. To investigate the
role of particle size and surface charge/charge distribution as potential determinants of
particular uptake mechanisms, we synthesized lipopeptides of varying amino acid
composition. Zeta potential measurement and dynamic light scattering were used to
characterize the different micellar and liposomal systems. Cell selectivity was investigated
by uptake studies with endothelial cells of the human aorta (HAoEC) and human brain
microvessels (HBMEC).
All lipopeptides (P2A2, LP1, LP2) differ in amino acid composition but are identical in the
number charged residues. P2A2 and LP1 have both 12 cationic residues, LP2 bears 12
anionic amino acids. All lipopeptides form micelles with an hydrodynamic diameter (d[Vol])
of about 10 nm. The zeta potential of the micelles correlates with the charge properties of
the lipopeptide: P2A2/ LP 1 – positive; LP 2 - negative surface potential. Additionally, three
other peptide-bearing carrier-systems, different in particle size and their molar lipid to
peptide ratio (c l/p) were prepared. Large unilamellar vesicles (LUV, d[Vol] about 90 nm, c
l/p = 1000) and small unilamellar vesicles (SUV d[Vol] about 25 nm, c l/p = 1000 or 20)
have a distinct size distribution. At cl/p = 1000, the zeta potential does poorly distinguish
from the potential of control liposomes. With higher lipopeptide concentration, c l/p = 20, the
zeta potential changes in accordance with the amino acid motif. Uptake studies were done
by using Laser Scanning Microscopy (LSM) and Fluorescence Activated Cell Sorting
(FACS).
All positively charged lipopeptide carriers were internalized into HAoEC and HBMEC.
Negatively charged LP 2 particles were preferentially adsorbed on the cell surface. The
overall interaction efficacy followed the order: micelles > SUV (c l/p = 20) > LUV (c l/p =
1000) > SUV (c l/p = 1000). The uptake of P2A2-micelles into HBMEC was two times
enhanced compared to HAoEC. In comparison to micelles (no cell specificity), LP 1
incorporation into SUV (c l/p = 20) seems to improve cell specificity to HAoEC. A lower lipid
to peptide ratio could enhance uptake of P2A2- and LP 1 - SUV.
Self-aggregation of lipopeptides and binding to liposomes results in efficient cellrecognizing
and uptake mediating systems. Cell-selectivity depends on both the molecular
138 Poster

structure of peptides, the particle size and on the lipid to peptide ratio in liposomal
formulations. The investigated lipopeptides provide a keytool for the development of drug
delivery systems.
References:
1. Leupold, E., Nikolenko, H. & Dathe, M., Biochim. Biophys. 2009, 1788(2): 442-449.
177
Standard doses of intravenous and enteral moxifloxacin provide insufficient
drug exposure for nosocomial pneumonia in intensive care unit patients
Schaeftlein, A. 1 2; Kees, M. 3; Heininger,A. 4; Kloft, C. 1
1Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, FreieUniversitaet Berlin,
Kelchstraße 31, 12169 Berlin,Germany
2and Graduate Research Training program PharMetrX, Germany,
3 Dept. of Anesthesiology and Intensive Care, Charité University Hospital – Campus Benjamin
Franklin, Hindenburgdamm 30, 12200 Berlin, Germany
4 Dept. of Anesthesiology and Intensive Care, University Hospital of Tuebingen, Hoppe-Seyler-
Str.3, 72076 Tübingen, Germany
Background: Moxifloxacin (MOX), a fourth-generation fluoroquinolone,is approved for
treatment of community acquired pneumonia. Due to a broad spectrum and high enteral
bioavailability (>91%) of MOX in healthy volunteers (HV) [1], intravenous/enteral sequential
therapy may also be a suitable treatment option for hospital acquired pneumonia (HAP),
possibly resulting in reduced costs and complications [2].In the current population
pharmacokinetic analysisi.v. and enteral administration of MOX in intensive care unit
patients (ICUP) were compared to determine the clinical feasibility of sequential therapy in
ICUP on a pharmacokinetic basis.Additionally,patient specific characteristics (covariates)
explaining interindividual variability (IIV) on pharmacokinetic (PK) parameters were
investigated, and the simulated exposure of MOX was linked to the efficacybreakpoint of
fluoroquinolonesfor HAP.
Methods: Based on MOX concentration measurements after multiple i.v. and enteral
administrations of 400 mg MOX in 25 ICUP,population PK analysis was performed using
the nonlinear mixed-effect modelling approach (NOMEMTM).Covariates[demographics, coadministered
drugs, state of disease markers] were tested for relevanteffectson the IIV of
absorption rate constant (ka), enteral bioavailabilty, volumes of central and peripheral
distribution and clearance (CL).Based on the final population PK model, 1000
concentration-timecourses were simulated and the AUCtau were usedforprobability of target
attainment functions(fAUC/MICHAP>75[3]), which is indicative for the efficacy and treatment
response of MOX for ICUP with HAP.
Results: Measured MOX concentrations in ICUP were best described and predicted by a
first-order absorption and eliminationtwo compartment PK model. Estimates of CL (11.3
L/h) and volume of distribution(114 L) were in accordance with the literature [4] butF (76%,
95% confidence interval [59%; 94%]) was reduced in ICUP in comparison to HV [1]. IIV on
ka (1.09 1/h) was high (135%) due to a lagging increase of MOX concentrations after
enteral dosing observed in some ICUP which could not be integrated into the final model.
Body weight, age, height, sex, administration of opioids or catecholamines, plasma
proteins, presence of shock, application via gastric tube and sequential organ failure
assessment score [5] had no significant effect on the IIV of MOX PK in ICUP. Only
creatinine clearance (CLCR) effects the elimination of MOX and explained 1/4 of the IIVin
CL, reducing CL by 0.43% for every 1 mL/min decline of CLCR. An acceptable probability
(>0.9) to attain the PKPDtarget for HAP was achieved for only lowMICvalues of0.125
µg/mL or less.
Conclusions: Based on our PK/PD analysis, enteral bioavailability of MOX was little reliable,
limiting the suitability of a switch to enteral therapy in ICUP. In addition, a favourable
outcome of MOX treatment for HAP cannot generally be expected in the investigated
setting, since MIC90values for typical pathogens of HAP (e.g E. coli) exceed 0.125 mg/L.
Hence, unless a highly susceptible HAP pathogen is confirmed, an antibiotic regimen in
ICUP with additional or alternative agents or a dose escalation might be considered.
References:
1. Burkhardt O, Stass H, Thuss U et al.:ClinPharmacokinet 2005, 44(9):969-76.
2. Ramirez JA, Vargas S, Ritter GW et al.:Arch Intern Med1999; 159(20):2449-2454.
3. Ambrose PG,Bhavnani SM, Rubino C et al.:Clin Infect Dis2007, 44(1):79-86.
4. Jeffry AF, Tornøe CW, Brundage R et al.:J ClinPharmacol2011, 51(8):1152-1162.
5. Vincent JL, Moreno R, Takala J et al.: Intensive Care Med1996; 22(7):707-710.
178
Antistatic effect of different silica types on free flowing direct compression
excipients
Knop K.; Martens A.;Sölter V.
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitätsstr. 1,
40225 Düsseldorf, Germany
When powders are handled, e.g. during sieving, mixing, or powder flow through a
hopper,electrostatic charging may occur. Problems in the process of capsule filling or
tableting can be a result of this electrostatic charge [1]. Antistatic agents such as
magnesium stearate, talc, polyethylene glycolsor silicon dioxide are added to prevent these
problems. The aim of the present study was to evaluate the influence of different types of
silicon dioxide on the electrostatic charging of free flowing powders which are used for
direct compression.
Three free flowing direct compression excipientsFlowlac ® 100 (lactose monohydrate),
Parteck ® M 100 (mannitol), and Ludiflash ® (co-processed mannitol with crospovidone,
polyvinylacetate andpovidone) were investigated using the flowability test apparatus no. 1
equipped with nozzleno. 3 (25 mm opening) described in Ph.Eur. 2.9.16 “Flowability”.The
excipients were equilibrated to environmental conditions (21°C, 45% rel. humidity) for at
least 48 h. Six different types of silicon dioxide were added in concentration of 0.5 and 1%:
Hydrophilic (Aerosil ® 200 and 300), hydrophobic (Aerosil ® R972), and granulated fumed
silica (Aeroperl ® 300) as well as porous silica (Syloid ® 244FP and Sylysia ® 350FCP).All
types were tested in undried and dried status (105°C) to take the different water content
into account. The powders were mixed in a Turbula ® mixer for 5 min. The flow rate was
registered by the weight gain per time on a balance and the electrostatic potential was
measured by an electrostatic sensor, focussed on the powder-stream in a well defined
distance. All measurements were carried out in fivefold in an air-conditioned room at 21°C
and 45% relative humidity.
Pure Flowlac ® showed a high flowability and a negative electrostatic charge of about -0.5
kV. The flowability was only slightly improved by the addition of silica. The electrostatic
charge could not be reduced by the addition of the powdered fumed silica but by the
granulated fumed silica and the porous silica types.
Parteck ® M without additives was negatively charged to -1.5 kV during flow. This high
voltage could be lowered to about -0.3 kV by the fumed silica types. The addition of the
porous silica types led to a change in polarity and a charge of about +0.3 kV. The flowability
of Parteck ® was worse than that of Flowlac ® but could be improved by the addition of each
type of silica.
Ludiflash ® was positively charged during flow up to +0.5 kV. All silica types with the
exception of the granulated type reduced the charging to values around zero. Ludiflash ®had
a good flowability like Flowlac ®. The addition of fumed silica led to a decline in flow whereas
the porous silica had no influence.
Neither the concentration of the silica in the powder mixture (0.5 or 1%) nor the water
content of the silicon dioxides (dried or undried) showed a consistent influence onthe
electrostatic charge and the flowability of the powder mixtures. In the investigated rage,
both factors were only of minor influence.
The choice of the best antistatic agent from the group of silicon dioxides has to be made
individually for every powder or powder mixture. The porous silica types showed a charge
reduction for all three investigated systems whereas the powdered fumed silica failed in the
case of Flowlac ®.
References:
1. Szendeleit S.M., Elektrostatische Aufladung im Prozess der Tablettierung, Diplomathesis,
Halle/Düsseldorf 2010.
179
Taste sensing system for the selection of drug candidates in early
pharmaceutical development stage and rational development of taste masked
orodispersible dosage forms
Guhmann M1 2, Gerber F2, Poellinger N2, Breitkreutz J3, Weitschies W1 1 Department of Biopharmaceutics and Pharmaceutical Technology, University of Greifswald,
17489 Greifswald, Germany
2 Glatt Pharmaceutical Services, Technology Centre, Glatt GmbH, 79589 Binzen, Germany
3 Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University of Düsseldorf, 40225
Düsseldorf, Germany
The ability of an electronic taste sensing system to guide the selection of drug candidates
and rationalize the development of taste masked orodispersible formulations was
evaluated.
Diclofenac was used as drug model investigating the free acid, the sodium and the
potassium salt forms. Calibrations curves were established with different concentration
ranges. Measurements were performed using the electronic taste sensing system TS-
5000Z (Insent Inc., Japan) qualified based on ICH guideline Q2 [1]. The system was
equipped with 7 lipid membrane sensors representing bitterness (1,2 and 3), sourness,
saltiness, umami, and astringency and corresponding aftertaste qualities. Sensor
responses (mV) of three consecutive measurements were analyzed by univariate data
evaluation. Taste masked Orally Disintegrating Tablets (ODTs) were developed using the
most suitable diclofenac drug form considering taste modalities, and evaluated by the
electronic tongue versus placebos using Principal Component Analysis (PCA).
Clear differences between the free acid form of diclofenac and its salt counterparts were
detected by the electronic tongue. Diclofenac salts induced bitter (taste and aftertaste) and
astringent (taste) responses with different detection intensities; saltiness stimuli was
additionally recorded for the potassium salt. In contrast, only a bitter aftertaste from sensor
3 was detected for the acid form. Based on these results, diclofenac acid was selected for
manufacturing taste masked ODTs. Taste masking strategies used granules produced by
coating of single drug crystals or wet granulation, further compressed into ODTs. The PCA
map showed that intermediated products (granules) and finished products (ODTs) could be
distinguished by the electronic tongue. All formulations differed from the pure diclofenac
acid. Using PC-1 (77% of the information), the taste reduction was 33% and 54% for drug
coated granules and matrix granules respectively; 67% and 71% for their corresponding
ODTs.
Investigating diclofenac as a drug model, the electronic tongue proved to be a valuable tool
for the selection of different drug candidates in the early pharmaceutical development
phase and the rational development of taste masked orally disintegrating dosage forms
Acknowledgments: The authors gratefully acknowledge the Institute of Pharmaceutics and
Biopharmaceutics of the Heinrich-Heine University Düsseldorf, especially Maren Preis, for providing support
and advice.
Reference:
1. Woertz, K et al.: J. Pharm. Biomed. Anal. 2010, 51: 497-506.
180
Complexation of Itraconazole with various cyclodextrin derivatives: Solubility
and 1H-NMR studies
Taupitz, T. 1; Bodtke; A. 2; Böheim, K. 2; Link, A. 2; Klein, S. 2
1 Goethe University Frankfurt am Main, Institute of Pharmaceutical Technology, Max-von-Laue-Str.
9, 60438 Frankfurt (Main)
2 Ernst-Moritz-Arndt University Greifswald, Department of Pharmacy, Institute of Biopharmacy and
Pharmaceutical Technology, C_DAT, Felix-Hausdorff.Str. 3, 17489 Greifswald
In the present study we wanted to investigate the complexation behaviour of different
cyclodextrin derivatives with itraconazole and moreover elucidate the impact of these
derivatives on the solubility of this poorly soluble compound. For this purpose binary
cyclodextrin complexes of itraconazole, a weakly basic BCS class II drug, were prepared
using the following cyclodextrin derivatives: I) 2-hydroxypropyl-β-cyclodextrin (HP-β-CD),
II) sulfobutylether-β-cyclodextrin (SBE-β-CD) and the recently developed hydroxybutenylβ-cyclodextrin
(HBen-β-CD). The main objective was to obtain formulations that show an
improved solubility superior to that of the pure drug and further to investigate the
interactions in the different itraconazole cyclodextrin complexes in detail.
A freeze drying procedure was used to prepare all inclusion complexes of itraconazole. The
pure drug and all formulations were then subject to pX-RD analysis and solubility tests. To
elucidate the complex formation of each cyclodextrin derivative with the compound, 1H-
NMR studies were performed. Finally, solubility tests were performed in compendial and
biorelevant media simulating conditions in the stomach and the upper small intestine.
Poster 139

PX-RD diagrams of the complex formulations indicated that amorphous inclusion
complexes were obtained. Spectra obtained from the 1H-NMR studies revealed chemicals
shifts of the triazol- and the aromatic protons indicating interactions between each
cyclodextrin derivative and these areas of the drug molecule. Results of the solubility
experiments showed a significant increase of itraconazole aqueous solubility. Particularly,
the recently developed HBen-β-CD derivative could tremendously improve the solubility
under gastric and small intestinal conditions.
Based on the results of the present study, we assume that the interactions between each of
the cyclodextrin derivatives and itraconazole lead to an improvement of the compounds
solubility. However, the extent of this effect varied as a result of the derivative used for
complex formation. Overall, it can be said, that the investigated cyclodextrin derivates were
very useful to increase the solubility of itraconazole in compendial and biorelevant test
media and therefore represent a promising group of excipients for future use in solubility
(and bioavailability) enhancement of poorly soluble compounds.
140 Poster

Autorenverzeichnis
Abdel-Aziz H 98, 103, 137
Abd-Ellatif A 132
Abrahmsén-Alami S 32
Abrahmsson B 32
Abromeit H 102
Abstiens K 80
AbulAzm S 104
Achenbach J 56
Adams R H 114
Adler M 137
Ahmad K 113
Ahmed M 82
Alban S 58
Albrecht M 122
Al-Halhouli A T 130
Alhazmi H 33
Alhussein A 122
Ali H 69
Alresly Z 106
Altenhofen W 117
Altmann K S 118
Ammon H P T 122
Andermark V 111
Anschütz M 32
Arnold N 106
Aßmann G 137
Astner I 91
Aurich K 134
Baaske P 80
Bäcker C 106
Bakowsky U 70
Banoglu E 117
Bantscheff M 25
Baranski J 115
Bartusch A 136
Barzen S 105
Bauer J 102
Bauer K 103
Bauer S 103, 117
Baumann K 79, 104, 125
Bausch A 115
Becker D 116
Bednarski P J 65, 106, 118, 119
Beese K 120
Behrends S 130
Behrendt C T 99
Beißner N 101
Beitz E 118
Below A 110
Bendas G 120, 121
Berg S 43
Bernat V 23, 105
Bernhardt G 28, 98
Bertram L 123
Bertram N 35
Bertsche T 87
Besheer A 80
Bettio S 122
Bibb J A 114
Bickmann D 110
Biller A 137
Bischoff F 100
Bitterlich A 78, 124
Bittner F 117
Blackert S 134
Block S 60
Bloßfeld M 112
Blume H 32, 134
Bock R 126
Bodem J 95, 96, 97
Bodtke A 106, 110, 139
Boeckler F M 55
Bogan R 135
Böheim K 139
Borchert P 120
Böttger S 107
Bracher F 114
Braig S 100
Brand T 113
Brandl F 86, 130
Brandl F P 124
Brandt S 60
Brandt W 109
Breitkreutz J 83, 110, 129, 137, 139
Breunig M 133
Brock D 131
Brückner A 111
Brückner C 102
Brüßler J 70
Bublitz K 120
Bui T H 105
Bundscherer L 118
Bunjes H 75, 78, 124, 128, 130
Buschauer A 28, 98
Busker M 130
Busse M 134
Butterweck V 111
Büttgenbach S 127, 130
Chahar M 101
Chamseddin C 105, 106
Chbeib M 107
Chen R 129
Cheng X 63
Cianciulli C 33, 110
Claes D 108
Clement B 117, 118
Closs E I 115
Collnot E M 69
Crüsemann M 106
Daily A 38
Damm G 42
Dannhardt G 115
Dathe M 138
Dayyoub E 70
de Jong J C 100, 114
Deally A 114
Decker M 36
Dehm F 101
Delves M 98
Dengler M 78
Deuter A 119
Dhein S 94
Diel P 112
Dietzel A 130
Dolberg A M 116
Donath F 32
Dörje F 134
Dorn A 101, 104
Dos Santos Capelo R 121
Dötsch V 119
Duhr S 80
Dzikowski R 61
Eckert C 136
Eeckman L 126
Efferth T 37, 45
Eiden K 119
El Gaghlab K 120
Elz S 102
Emmrich T 65, 106, 118
Engel A 43
Engels B 95, 96
Enzensperger C 103, 104
Erdmann D 28
Externbrink A 132
Fahrmayr C 40
Falck E 34, 122
Ferandin Y 61
Fersht A R 55
Fey P 95
Finke J H 127, 130
Fischer D 73
Fischer M 99
Fischer S 104
Fiß T 89, 137
Flemming S 121
Fleßa S 137
Foerster S 122
Förster F 100
Fossen T 105
Franzmann E 27
Fréchet J M J 72
Freichel M 48
Freitag A 107
Frentsch M 71
Freyse E-J 43
Friedrich C 135
Frieß W 20, 81
Fromm M F 40
Fuchs S 97
Fugel W 61
Funke A 90
Fürst R 97, 114, 115
Gantzsch S 127
Garbacz G 30, 109
Gehringer M 95
Gerber F 139
Gerber U 120
Germer K 131
Gerstmeier J 117
Geyer H 125
Gieré R 74
Gieseler H 122
Glöckl G 113, 125, 134
Gminski R 74
Gobleder S 102
Goebgen E 137
Goettert M 103, 117
Gohlke H 22
Goos K-H 105
Autorenverzeichnis 141

Göpferich A 86, 124, 130, 132, 133
Görke K 109
Gothsch T 127
Grabow N 113, 126
Graeser R 117
Greinacher A 50, 59, 60
Grez M 22
Groll M 99
Grotefend S 94
Grün J 96
Grüning B A 107
Grützkau A 121
Guhmann M 139
Gündisch D 103
Günther S 54, 107, 121
Gust R 52
Guthoff R 94
Ha K 101
Hackbarth C 60
Hackenberg F 68
Haertel B 119, 134
Häfner A-K 119
Hager B 114
Hähn S 99
Hahne T 33, 110
Hamacher A 66
Hamburger M 97
Hammer E 122
Hammer N 86, 124, 130
Hammerschmidt S 60
Hanke T 101
Hanke U 116, 134
Hansen F K 101
Hansen S 31
Harden D 131
Harder C 126
Hare E 69
Härter A 111
Hartmann R W 65, 100, 114
Hartung E 97
Hasenpusch D 107
Haug K G 111
Häupl T 121
Haupt O 112
Hausmann M 115
Häussler S 100
Haustein M 120
Havemeyer A 117, 118
Heilmann J 98
Heinemann K 120
Heininger A 139
Held J 98, 99
Heller E 25
Hellmuth C 121
Henn C 100
Hennig L 111
Hennig R 132, 133
Hentschel-Humeida U 97
Hessler N 73
Hiddemann L 109
Hinz B 111, 120
Hipler U-C 73
Hirt C 65
Hochhaus G 111
Hoffmann B 132
Hoffmann C 25
Hoffmann W 89, 137
Hofmann B 105, 119
Holl K 99
Holloway S 95
Holzgrabe U 25, 96, 103, 123
Hornburger M C 115
Hörold M 136
Horvat M 129
Hoser S 103, 137
Humar M 54
Iber S 134
Ihling C 106
Ihling C H 47
Ilko D 123
Illarionov B 99
Jaax M 60
Jaehde U 87
Jäger C 54
Jain A K 32
Jakobs H 117
Janas C 127
Jansen R 138
Jantscheff P 121
Jauch J 122
Jedlitschky G 50
Jenett-Siems K 106
Jenner D 96
Jensen A A 103
Jira T 105
Joerger A C 55
Juchum M 96
Juli C 96
Jung M 53, 112, 116, 120
Junmahasathien T 135
Kabisch J 109
Kacprowski T 122
Kage H 95
Kahnt A S 121
Kaminski D 125
Kann B 127
Karcher S 104
Kaske M 28
Kassack M U 66
Katritzky A R 101
Kaufeld A M 98
Keck C M 108, 129, 131, 135
Keck P R W E F 100
Kees M 139
Keiser M 42, 43
Kelber O 98, 103, 137
Keller M 28
Kersten E 138
Kesselring J 95
Kessner S 136
Khalil F 27, 123
Kiefer T 65
Kiefer W 96
Kiene F 126
Kipping T 136
Kirchhof S 86, 124, 130
Kirsch B 100
Klages C-P 130
Klein C 95
Klein S 30, 132, 138, 139
Kleinebudde P 128, 129, 132
Kloft C 109, 136, 137, 139
Klos S 103
Knop K 139
Knopke C 32
Kobilka B K 23
Koeberle A 100, 102
Koeck J 129
Kolb P 23, 105
Kölbel K 47, 106
Koletzko B 121
Kölln C 112
Kolodziej H 98, 99
Komoß C 78, 124
Könczöl M 74
König J 40
Konzuch S 99
Kordaß B 125
Korpis K 118
Kortmann C 129
Köster R 114
Kovacevic A 108
Koziolek M 30, 109
Kraft K 137
Kralisch D 73
Krämer I 123
Krauel K 60
Kraus B 36
Krauß A 115
Krauth F 136
Krebs S 134
Kressirer C 100
Kreutzer M F 95
Kriemen E 120
Kroemer H K 50
Kruggel S 61, 109
Kuehnl S 102
Kühn J-P 134
Kummer J 94
Kumpfmüller J 109
Kunfermann A 99
Kunick C 61, 109
Kupetz E 130
Kurz T 98, 99
Kwade A 78, 124, 127
Laabs F 128
Lalk M 106, 109
Lally G 68
Lamers C 56
Lämmerhofer M 101
Lamprecht A 126
Lang C 26
Lange C 106
Laufer S 95, 103, 104, 117
Laufer S A 100, 101, 107
Lehmann J 103, 104
Lehr C M 69, 127
Lehr T 29
Lemcke T 61, 107, 108, 109, 110
Lemmerhirt H 65, 106, 118
Lendlein A 71
Leonard F 69
Lestari M L A D 104
Leuner K 134
Leven M 98
Liebeke M 46
Liebl J 114
Liedl K 21
Liedtke A J 100
Lienau C 99
Liening S 101
Lill A 105
Lindequist U 106, 119, 134
Link A 65, 106, 118, 120, 139
Linnebacher M 120
Lipke U 126
Lipperheide C 126
142 Autorenverzeichnis

Liu T 125
Loch C 94
Löhr J H 110
Lohse M J 25
Lu C 100
Lucas X 107
Luderer S 117
Lukowski R 49, 115
Lütnant I 98
Maedler K 19
Mai A 63
Maison W 27, 108, 120, 123
Majer M 115
Manna C M 114
Marschalek R 113
Marschall P 137
Martens A 139
Martiné U 115
Martz K E 101
Marxer E 70
Masch A 64
Massing U 121
Masur K 118
Mathew S 71
Matz F 97
Matz M 79
Maurer C K 100, 114
Mayer B A 115
Meijer L 61, 100, 109
Mell N 69
Melzig M F 94, 107, 129
Mendel R 117
Menrath C 97
Menzen T 81
Merfort I 54, 74
Merkenschlager A 112
Merkord J 120
Mersch-Sundermann V 74
Messmann V 86
Meßmann V 124, 130
Metz A 22
Meyer A 111
Meyerdierks N 115
Michael S 98
Michel A 119
Minichmayr I 109
Moll H 95
Monbaliu J-C M 101
Mordmüller B 98, 99
Morgenstern O 118
Moritz S 73
Möschwitzer J P 104, 124, 125, 129
Moscow J A 38
Moser M 114
Mosig J 132
Müller A 73
Müller C 114
Müller F A 73
Müller R 18, 100, 109
Müller R H 104, 108, 124, 125, 129,
131, 135
Müller-Goymann C C 127, 130
Mundt S 105, 110
Münsterberg M 108
Munteanu M 73
Müsken M 100
Muth F M 103
Nagaraj M 82
Nagel S 94, 109, 113, 126
Nagel-Steger L 111
Nauert C 137
Neidhardt I 110
Nett M 95
Neumann M 84
Nieber K 98, 103, 105, 112, 137
Niedermeyer T H J 38, 110
Niemann S 127
Nikolenko H 138
Nimtz M 105
Northoff H 102
Norville I H 96
Norwig J 138
Nowotny B 97
Nüssler A 42
Oak P 37
Oberholzer A E 61
Oehmigen K 134
Oetjen E 107
Okpanyi S N 137
Olausson B E S 106
Oli S 97
Oliferenko A A 101
Oswald S 41, 43
Ott I 67, 111, 114
Parr M K 112, 125
Patel H 54, 107
Patil S 68
Paul M 125
Pauly A 134
Pein M 129, 136
Peissner W 121
Perfahl S 119
Pergola C 117
Pertz H H 98
Peters K 111
Petrovick G F 137
Pettelkau J 106
Pfeifer B 109
Pham T L H 110
Plouffe D 98
Pluym N 28
Poellinger N 139
Pohl J 42
Poll B 109
Pollinger K 132, 133
Popella S-D 101
Porzel A 106
Potterat O 97
Pradel G 96
Preisitsch M 110
Pressburger N 61
Pretor S 130
Preu L 130
Priese F 133
Proschak E 56
Puerstinger J 110
Quintanilla-Fend L 122
Quodbach J 128
Quosdorf S J 99
Radebold J 42
Rakow T 119
Ramer R 111, 120
Raszek M 105
Ratin M 61
Rau O 56
Rauch B 50
Rauh D 62, 64
Rauwald H J 111
Rauwald H W 94
Raynor A 121
Redweik S 33, 94
Reichl S 51, 101, 112, 116, 130
Reichmann D 117
Rein H 77, 136
Reitz E 131
Ren X 133
Rethwilm A 97
Retzar A 135
Richter C 126, 127
Rieger M 96
Rischer M 85
Ritter C 82
Ritter C A 122
Robaa D 103, 104
Roch T 71
Rockmann T 131
Rödl C B 105
Roessler C 64
Rollinger J M 102
Rösch P 96
Rosenbaum D M 23
Ross T 121
Rothenhöfer M 98
Rotili D 63
Rotmann A 115
Rumpf T 120
Runge D 42
Rustenbeck I 79
Ruth P 115
Salamon A 120
Salazar J 124
Sarkar-Tyson M 96
Savtschenko A 94
Schad C 95
Schaefer U 31
Schaefer U F 127
Schaeftlein A 139
Schäfer J 70
Schaible A M 102
Schanda J 22
Schänzer W 125
Scherer O 102
Scherübl R 98
Scheuch E 43
Schiedel M 116
Schirmeister T 95, 96, 97, 107
Schlesinger M 121
Schmidt D 23, 105
Schmidt H 136
Schmidt I 96
Schmitz P 120
Schmolke H 130
Schmuhl E 111
Schneider F 84
Schneider G 56
Schneider T 95, 96
Schnittker C 126
Schollmeyer D 100
Schönherr D 116
Schramek N 92
Schröder G 105
Schröder M 119
Schröder T 106
Schubeis T 82
Schubert-Zsilavecz M 56, 101
Schuchmann H P 129
Schug B 32
Autorenverzeichnis 143

Schulz K 134
Schulz R 65, 106, 118
Schulz S 37
Schulz U 118
Schulze M 103
Schumacher K 79
Schümmelfeder J 70
Schur J 114
Schurigt U 95, 96
Schutkowski M 64
Schütt R 113
Schütze N 120
Schwarz R 47
Schweder T 109
Schweimer K 96
Scriba G K E 102
Seeling A 110
Seibel J 96
Seidlitz A 94, 113, 126
Semmling B 113, 126
Seufert F 96
Shaalan N 117
Shehata A M 122
Shoichet B K 23
Siegmund W 42, 43, 116
Siems K 106
Sinha B 129
Sinz A 34, 47, 106
Smolka K 125
Snitko M 95
Söderlind E 32
Sölter V 139
Sotriffer C 95, 96, 97
Sotriffer C A 107
Spitzer G 21
Stark H 105
Staudacher V 114
Steinbach A 100, 114
Steinhilber D 56, 57, 105, 113, 119, 121
Steinhoff B 137
Steri R 56
Sternberg K 113, 126
Stintzing F C 39
Storz M P 114
Straßer A 24, 102
Straubinger J 115
Strauss A-C 134
Streciwilk W 68
Strunz A K 99
Stuppner H 102
Sutter A 90
Swiatecka-Hagenbruch M 38
Sydow K 138
Tabares P 97
Tacke M 68, 114
Tackenberg M W 129
Tajarobi F 32
Talmann L 134
Tänzler D 47
Taraschewski A 138
Taupitz T 139
Tchoukouegno Ngueu S 112
Tessmar J 132, 133
Teßmar J 130
Teßmar J K V 124
Thiessen A 35
Thom K 134
Thommes M 76, 126, 129, 131
Thürmann P A 88
Tränkle C 103
Trauner D 37
Treuer E 117
Trip E 126
Tschammer N 23, 105
Tschan S 98
Tshuva E Y 114
Ullrich A 42
Venema K 113
Viridé A 32
Vissiennon C 105
Vogel S 107
Völker U 44, 122
Vollmar A 100
Vollmar A M 37, 97, 100, 114, 115
von Schwarzenberg K 37, 100
von Woedtke T 118, 119, 134
Vongehr F 135
Voß U 98
Wacker M 127
Wagner S 107
Wagner T 112
Waltenberger C 107
Wanner G 37
Wätzig H 33, 94, 110, 119
Weber B 111
Weber C 60
Weber F 118
Weber M 121
Wedemeyer R-S 134
Wegener J 115
Weidel E 114
Weiser D 98, 103
Weitschies W 30, 32, 43, 84, 94,
109, 113, 116, 125, 126, 134, 139
Weiwad M 96
Welinder A 32
Welker A 95, 96, 97
Wende K 105, 119
Wendling F 97
Wentsch H 104
Wentzlaff M 126
Wenzel R 133
Wenzel S 109
Werz O 100, 101, 102, 117
Wesar F 73
Wessjohann L A 106
Wich P R 72
Wicha S G 136, 137
Wichmann C 22
Wiedmann R M 37
Wiegand C 73
Wiest J 123
Wilcken R 55
Wilde F 106, 118
Wilde L 125
Willbold D 111
Willer E A 97
Wilson C G 113
Windbergs M 127
Winkelmann V 103
Winter G 80
Wischke C 71
Witt S 136
Wittmann H-J 24, 102
Wolber G 21
Wolf B 131, 133
Wolf C 134
Wolff H 36
Wolff M 111
Wollatz U 125
Wollenhaupt S 104
Wray V 105
Wszelaki N 129
Wu H 95
Wünsch B 34, 97, 98, 99, 118, 122
Wurster M 106
Xu Y 33
Zahler S 100, 114
Zapf T 126
Zhang S 114
Zimmer A 82
Zimmer C 100, 114
Zimmermann T 135
Zischka H 37
Zlotos D P 103
Zwernemann G 136
144 Autorenverzeichnis

Notizen
145

Notizen
146

Notizen
147

Notizen
148

Notizen
149

Notizen
150

151

152
Greifswald Innenstadt
1 Tierpark
2 Botanischer Garten
3 Credneranlagen
4 Amtsgericht/Finanzgericht
5 Bahnhof
6 Einrichtungen der Universität
7 Stadtbefestigung
8 Kirche St. Jacobi
9 Historische Sternwarte
10 Rubenowplatz und –denkmal
11 Einkaufszentrum Dompassage
12 Hauptgebäude der Universität
13 Sozio-kulturelles Zentrum St. Spiritus
14 Audimax
15 Caspar-David-Friedrich-Zentrum
16 Musikschule
17 Alte Universitätsbibliothek
18 Dom St. Nikolai
19 Museumshafen
20 Kirche St. Joseph
21 Fußgängerbrücke
Geschäftführer und Leiter der Geschäftsstelle Apotheker Dr. Michael Stein
DPhG Geschäftsstelle
Hamburger Allee 26 - 28
60486 Frankfurt/Main
Tel.: 069-7191596-0
Fax: 069-7191596-29
Email: info@dphg.de
http://www.dphg.de
25.09.2012
22 Caspar-David-Friedrich-Denkmal
23 Alfried Krupp Wissenschaftskolleg Greifswald
24 Volkshochschule
25 Fischmarkt
26 Rathaus
27 Kunstwerkstätten
28 Literaturzentrum „Wolfgang Koeppen“
29 Stadtbibliothek
30 Stadthaus (im Bau)
31 Gotische Giebelhäuser
32 Fangenturm
33 Internationales Begegnungszentrum
34 Kirche St. Marien
35 Museumswerft
36 Pommersches Landesmuseum
37 Stadtarchiv
38 Mensa
39 Fallada-Geburtshaus
40 Stadthalle/Theatercafé
41 Landesarchiv Vorpommern
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
1 Biotechnikum (Targets 11./12.10.)
2 Center of Drug Absorption and Transport C_DAT
3 Mathematisch-Naturwissenschaftliche Fakultät mit
Institut für Pharmazie
4 Leibniz-Institut für Plasmaforschung und Technologie
(INP Greifswald e.V.)
5 Zentrum für Zahn-, Mund- und Kieferheilkunde
6 Institut für Biochemie (Moleküle 11./12.10.)
7 Klinik und Poliklinik für Hals-, Nasen-, Ohrenkrankheiten,
Kopf- und Halschirurgie
8 Institut für Physik (Tabletten 11./12.10.)
9 Arboretum des Botanischen Gartens
10 Universitätsbibliothek
11 Klinik und Poliklinik für Hautkrankheiten
12 Johannes-Kirche
13 BDH-Klinik Greifswald GmbH
14 Volksstadion
15 Kleine Mensa
16 Universitätsklinikum Greifswald
mit Hörsaal Nord (13.10.)
A EUROPA HOTEL Greifswald