Supplement 2/15

CESAR-Jahrestagung 2015/Annual Meeting 2015 Challenges for Drug Development in Precision Oncology 17.–19. September 2015 Innsbruck

Tagungsort/Venue: Hörsaalzentrum Medizinzentrum Anichstrasse (MZA) Anichstrasse 35 6020 Innsbruck

CESAR-Österreich Verein für neue medikamentöse Entwicklung in der Krebsforschung CESAR Central European Society for Anticancer Drug Research EWIV

memo · Vol. 8 · Suppl 2/15

Published online: 25 August 2015

© Springer-Verlag Wien 35

Supplement 2/15 Meeting Presidents: Günther Gastl (Ö), Heinz Zwierzina (Ö) Präsidium der CESAR: Joachim von Pawel, Gauting (D), Präsident Christoph Ritter, Greifswald (D), Vizepräsident Thomas Gauler (D), Schatzmeister Wissenschaftliche Leitung/Scientific Advisory Board: Jürgen Barth, D Robert Mader, Ö Gerd Bendas, D Ulrich Mansmann, D Heiner Fiebig, D Ulrich Massing, D Claudia Friesen, D Frank Mayer, D Viktor Grünwald, D Rudolf Morant, CH Ralf A. Hilger, D Klaus Mross, D Ulrich Jaehde, D Max E. Scheulen, D Walter Jäger, Ö Daniel Sehrt, D Markus Jörger, CH Dirk Strumberg, D Charlotte Kloft, D Joachim von Pawel, D

Wolfgang Eisterer, Ö Lukas Huber, Ö Christoph Leitner, Ö Georg Pall, Ö Uwe Siebert, Ö Michael Steurer, Ö Gerold Untergasser, Ö

Best Abstract Komitee: Walter Jäger und Team Beiträge zum wissenschaftlichen Programm: CESAR-EWIV und ihre Arbeitsgruppen Arbeitsgruppe für Wirkstoffentwicklung in der Onkologie (CESAR-AWO) Arbeitsgruppe für Pharmakologie in der Onkologie und Hämatologie (CESAR-APOH) Arbeitsgruppe Phase I-Studien (CESAR-Phase I) Arbeitsgruppe Phase II-Studien (CESAR-Phase II) Arbeitsgruppe Phase III-Studien (CESAR-Phase III) onco.expert.net Universitätsklinik für Innere Medizin 5 – Hämatologie & Onkologie, Innsbruck Comprehensive Cancer Center Innsbruck (CCCI) Tirol Kliniken – Universitätsklinikum Innsbruck Medizinische Universität Innsbruck Oncotyrol Center for Personalized Cancer Medicine Cancer Drug Development Forum (CDDF) Kongressbüro/Congress office: Berta Moritz und Maria Rajpal (CESAR Central Office, Wien) & Dr. Eugen Preuß (Innsbruck) Ort der Jahrestagung/Venue: Medizinzentrum Anichstraße Hörsaalzentrum Anichstraße 35, 6020 Innsbruck www.cesar.or.at CESAR Central European Society for Anticancer Drug Research – EWIV c/o Dr. Berta Moritz, Director, Hanglüssgasse 4/1–3, 1150 Wien, Österreich

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Supplement 2/15 Wir danken den Sponsoren für ungebundene finanzielle Unterstützung Goldsponsoren:

Novartis Pharma GmbH Roche Austria GmbH

TAKEDA PHARMA GmbH

Silbersponsoren:

AMGEN GmbH AGEA Pharma GmbH

AOP ORPHAN Pharmaceuticals AG Celgene GmbH

MSD Merck Sharp & Dohme GmbH Pfizer Corporation Austria Gesellschaft m.b.H.

Pharma Mar S.A.

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TEVA-ratiopharm Arzneimittel Vertriebs-GmbH

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Programm Donnerstag, 17. September 2015 Ab 13.00 14.00–14.15

Registrierung Eröffnung und Grußworte Günther Gastl, Tagungspräsident Joachim von Pawel, Präsident der CESAR

14.15–15.00 Keynote Lecture Predictive Molecular Testing in Oncology (L01) Peter Schirmacher, Heidelberg 15.00–15.30 Kaffeepause 15.30–17.00 Session 1: The Search for Druggable Molecular Targets Vorsitz: Robert Mader & Walter Jäger 15.30–16.00 Cancer genomes & drivers (L02) Andreas Wicki, Basel 16.00–16.30 The Cancer-Immune Cycle (L03) Dominik Wolf, Bonn 16.30–17.00 Tumor profiling – The EXACT Study (L04) Gerald Prager, Wien 17.00–18.00 Postersession und Get together 5–6 sessions  Moderatoren: Ulrich Massing, Christoph Ritter & Klaus Mross Ab 18.15 CESAR-Präsidiumssitzung  Hilton Innsbruck, Salurner Straße 15, 6020 Innsbruck

Freitag, 18. September 2015 08.30–09.00 Business Meeting der Arbeitsgruppen CESARAWO und CESAR-APOH Business Meeting der Arbeitsgruppen CESARPhasen I–III Möglichkeit der Vorstellung neuer Mitglieder (nur für Mitglieder der CESAR-Arbeitsgruppen) 09.00–10.30 Session 2:  Preclinical Targeted Drug Development (organisiert von CESAR AWO/APOH & ONCOTYROL) Vorsitz: Lukas Huber & Christoph Ritter 09.00–09.30 Targeted drug development pipelines (L05) Matthias Ocker, Berlin 09.30–10.00 In vitro assay combining tumor & stroma (L06) Lukas Huber, Innsbruck 10.00–10.30 Animal models for personalized treatment options (L07) Iduna Fichtner, Berlin 10.30–11.00 Kaffeepause 11.00–12.00 Ergebnisse aus CESAR-Studien Vorsitz: Berta Moritz & Max Scheulen 12.00–13.00 Generalversammlung der CESAR-DEUTSCHLAND Generalversammlung CESAR-ÖSTERREICH (nur für Mitglieder der CESAR-Deutschland e.V. bzw. CESAR-Österreich)  Generalversammlung der CESAR Central European Society For Anticancer Drug Research-EWIV (nur für Mitglieder CESAR-Deutschland, CESARÖsterreich und für Beiräte der CESAR) 13.00–14.00 Mittagspause in der Klinik-Mensa Innsbruck 14.00–15.30 Session 3:  Clinical Drug Development in Precision Medicine (organisiert von CESAR Phase I–III groups) Vorsitz: Markus Jörger & Frank Mayer

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14.00–14.30 Novel clinical trial designs (L08) Shu-Fang Hsu Schmitz, Bern 14.30–15.00 Targeted therapy for metastatic colorectal cancer (L09) Thomas Winder, Zürich 15.00–15.30 Drug Development for melanoma (L10) Ulrich Keilholz, Berlin 15.30–16.00 Kaffeepause 16.00–17.00 Proffered Presentations (V01 – V04) Moderation: Ulrich Massing & Ulrich Keilholz 16.00–16.15 Novel Zeise-type complexes with aspirin substructure as potential COX-inhibitors (V01) Alexander Weninger, Innsbruck 16.15–16.30 Making use of modelling and simulations: Towards individualised tamoxifen therapy in breast cancer (V02) Lena Klopp-Schulze, Berlin 16.30–16.45 Impact of pharmacological Brk-Inhibition on cell growth of breast cancer cells cultivated under adherent and non-adherent conditions (V03) Markus Oelze, Greifswald 16.45–17.00 Progress in the NanoEFEct project (V04) Paul Debbage, Innsbruck 17.00–19.00 Energietanken/Pause 18.30 Uhr Möglichkeit zur Führung des Bergisel Museums (optional) Ab 19.00 Dinner mit Verleihung des CESAR-Preises 2015 im „Gasthaus Bierstindl“

Samstag, 19. September 2015 09.00–10.30 Session 4: Advances in Cancer Immunotherapy  (organisiert von CESAR & Cancer Drug Development Forum) Vorsitz: Joachim von Pawel & Heinz Zwierzina 09.00–09.30 Proteomic Biomarkers (L11) Heinrich Röder, BIODESIX 09.30–10.30 Harnessing the immune system against epitelial cancers (L12) Hans-Georg Kopp, Tübingen 10.00–10.30 Future Approaches to Cancer Immunotherapy (L13) Philipp Müller, Basel 10.30–11.00 Kaffeepause 11.00–12.30 Podiumsdiskussion: The challenge to balance costs & benefits in precision oncology Moderation: Günther Gastl Impulsvorträge und Diskussion mit: Max Scheulen, Tumorzentrum Essen; Bernhard Wörmann, DGHO, Berlin; Arno Melitopulos, Tiroler Gebietskrankenkasse, Innsbruck Uwe Siebert, Department of Public Health & HTA, UMIT Hall/Center for Health Decision Science, Harvard University, Boston; Horst-Dietmar Müller, Bristol-Myers-Squibb, Wien 12.30–13.00 12.30–12.40 12.40–12.50 12.50–13.00

Best Abstract Award Verleihung Moderation: Walter Jäger Gewinner 1 Gewinner 2 Gewinner 3

13.00 Schlussworte Heinz Zwierzina, Innsbruck Anschließend: Buffet

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Supplement 2/15 Anreise und Unterkunft Tagungsort/Venue Hörsaalzentrum des Medizinzentrums Anichstrasse (MZA) Anichstrasse 35, 6020 Innsbruck

Anreise/How to get to the venue Anreise mit dem Flugzeug/Arrival by plane: Für Teilnehmer aus Deutschland empfehlen wir eine Anreise über den Flughafen München Anreise mit der Bahn/Arrival by train bis „Hauptbahnhof Innsbruck“ Ab Hauptbahnhof Innsbruck/Main Train Station Innsbruck: ●● Bus Linie F – Richtung Flughafen bis Haltestelle „Innsbruck Klinik/Universität“ oder ●● Bus Linie R – Richtung Rehgasse bis Haltestelle „Innsbruck Klinik/Universität“ oder ●● Ca. 5 Minuten zu Fuß ins Areal Anreise mit dem PKW/Arrival by car ●● Von Salzburg Richtung Innsbruck ●● Von Arlberg Richtung Innsbruck ●● Von Brenner Richtung Innsbruck Ausfahrt „Innsbruck West“ → Kreuzung Egger-Lienz-Straße links → geradeaus in die Innerkofler Straße → nach 70 m rechts in die Tiefgarage am Beslerpark (West-Garage) → ca. 3 Min zu Fuß ins Areal

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Abstracts

L07 Animal models for personalized treatment options I. Fichtner Max Delbrück Center for Molecular Medicine and Experimental Pharmacology & Oncology GmbH, Berlin-Buch, Germany The molecular analysis of tumours by highly sophisticated gene expression and sequencing methods allows the comprehensive identification of drugable targets and the increasing chance for personalized treatments. Clinically established examples are the use of Imatinib for BCR-ABL positive leukemias and Vemurafenib for BRAF mutated melanomas. But, in general tumours are characterized by the presence of numerous oncogenes, genetic aberrations and a dynamic growth control. That complex and individual pattern even nowadays cannot fully be understood and mirrored by medical systems biology. Another attractive option to maintain the typical features of a patient tumour is by growing it in a foreign host how it is practised since the invention of the patient-derived xenografts (PDX). This method has been used for more than 30 years but got recently increasing attractivity for the identification of novel biomarkers and as so-called avatars for a patient-related optimization of therapy. Meanwhile, worldwide several thousands of PDX models covering the broad variety of tumour types exist and are used both for fundamental research and for commercial offer.

V01 Novel Zeise-type complexes with aspirin substructure as potential COX-inhibitors A. Weninger, V. Obermoser, R. Gust Institute of Pharmacy, Department of Pharmaceutical Chemistry, University of Innsbruck, Innsbruck, Austria Background:  The development of novel biologically active organometallic compounds with aspirin substructure, that also inhibit cyclooxygenase (COX) enzymes led to the synthesis of appropriate Zeise-type salts. In in vitro studies, it could be shown that Zeise’s salt itself is indeed pharmacologically active and a very potent COX-inhibitor, whereas potassium tetrachloroplatinate(II) and Cisplatin caused no effect on the enzyme activity at comparable concentrations. LC-ESI-Tandem-MS proved that Zeise’s salt binds to the essential amino acids Ser516 and Tyr385 in COX-1, thus impairing the function of the isoenzyme. Aim:  In order to increase selectivity for COX-2 the aspirin substructure was modified resulting in novel Zeise-type complexes. These complexes were investigated for their cytotoxicity and COX-1/2 selectivity and inhibition of the COX isoenzymes. The influence of structural differences in these anionic platinum complexes on cytotoxicity and COX selectivity/inhibition were examined. Methods:  Novel Zeise-type complexes were characterized via nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. Prior to testing these substances

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for pharmacological properties in vitro using modern biological techniques, purity determination and stability tests in water were done via HPLC. Due to the strong trans-effect of the alkene a substitution of a chlorido ligand by water is possible. Another substitution reaction might be the exchange of the alkene in media with high Cl– concentration. Results:  A set of complexes were selected as reference and compared to the new complexes regarding cytotoxicity and COX-1/2 selectivity/inhibition. Structural differences of the aspirin substructure influence the stability of the Zeise-type complexes strongly, but lead to a higher COX-1/2 selectivity. Conclusion:  In conclusion, it could be shown that Zeisetype complexes are a potential class of non-toxic COX-inhibitors, whose COX selectivity and stability can be influenced by the size of the aspirin substructure in the platinum complex.

V02 Making use of modelling and simulations: towards individualised tamoxifen therapy in breast cancer L. Klopp-Schulze1, M. Joerger2, Z. P. Parra-Guillen1, C. Kloft1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany 2 Dept. of Medical Oncology & Hematology, Cantonal Hospital St. Gallen, St. Gallen, Switzerland 1

Background:  Differences in clinical efficacy and treatmentrelated toxicity of tamoxifen (TAM) in oestrogen receptor-positive breast cancer patients have been associated with a high variability in the pharmacokinetics (PK) of TAM and its major metabolite endoxifen (ENDX). Intense discussions are ongoing on how to individualise TAM therapy and whether to guide TAM dosing based on CYP2D6 geno/phenotyping or based on a proposed threshold concentration of ENDX (CTH, ENDX) of 5.97 ng/mL (Madlensky et al. 2011). Aim:  The aim of this study was to evaluate the performance of two recently published PK models of TAM/ENDX focussing on the quantitative impact of a patient’s geno/phenotype on PK and to inform future clinical trials and strategies supporting therapeutic drug monitoring (TDM). Methods:  Concentration-time profiles of TAM and ENDX were simulated (Berkeley Madonna) for 20  mg/day p.o. TAM using two published PK models (Model 1: Dahmane 2013; Model 2: Ter Heine et al. 2014). The impact of genetic covariates (CYP2D6, CYP3A4/5) on PK was implemented (log-normal distribution or discrete probability distribution).The probability of target attainment (PTA) defined as percentage of patients with Cs, min of ENDX > CTH, ENDX was calculated for both models. Results:  Both models reflected the high variability in Cs, TAM and Cs, ENDX as reported in literature. Low fluctuations for Cs, ENDX were observed within a dosing interval for both models. Model 2 compared to Model 1 reached time to steady-state (t97 %Css) considerably faster (TAM: 12 vs. 19 days, ENDX: 17 vs. 47 days). The PTA as simulated from the two models was higher in Model 1 compared to Model 2 (76 % vs. 52 %). Stratification by CYP2D6 activity identified patients at highest risk for subtherapeutic Cs, ENDX (Poor metaboliser: PTA < 3 % in both models). However, patients with higher CYP2D6 activity showed highly variable

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Abstracts PTA values between Model 1 and Model 2 (Intermediate metaboliser: 59 % vs. 27 %; Extensive metaboliser: 93 % vs. 76 %). Conclusion:  Both models were able to describe the quantitative impact of CYP2D6 geno/phenotype on TAM and ENDX PK. Our simulations suggest TAM dose adjustments for individuals at risk (< CTH, ENDX), because information on phenotype alone might not be sufficient to identify all patients at risk. However, a specific time point for Cs, ENDX sampling e.g. for TDM might not be necessary. Our investigations further suggest that for selecting reliable sampling times t97 %Css must be achieved for TDM and clinical trials. Further evaluations with respect to the predictivity of the PK models are currently ongoing and will eventually contribute to a more comprehensive understanding of TAM/ENDX PK.

V03 Impact of pharmacological Brk-Inhibition on cell growth of breast cancer cells cultivated under adherent and non-adherent conditions M. Oelze1, G. Schüler1, K. A. Mahmoud2, W. Sippl2, T. Wersig2, A. Hilgeroth2, C. A Ritter1 Institute of Pharmacy, Clinical Pharmacy, Ernst-Moritz-Arndt-University of Greifswald, Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany 2 Institute of Pharmacy, Pharmaceutical and Medicinal Chemistry, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06100 Halle, Germany 1

Background:  Brk, a soluble tyrosin kinase also known as PTK6 has been shown to be a mediator in relevant cancer cell signaling pathways. Reduced Brk expression was linked to impaired cell proliferation under loss-of-adherence conditions indicating Brk as possible mediator of tumor cell migration, invasion and metastatic progress. The role in pathway interaction depends either on adapter protein characteristics or kinase functionality. Recent findings suggest, that some hallmark effects of cancer may be linked to the active kinase domain whereas inactive Brk seems to be involved in different physiological processes of epithelial cells. Aim:  The aim of this work was to investigate the effects of Brk inhibition on growth of cancer cells under adherent and non-adherent conditions. Methods:  Cell lines were obtained from DSZM cultivated as indicated by distributor. In experiments cell were incubated for 48 h with 4 experimental inhibitors (MK5, MK136, MK138, MK150). Effects of Brk inhibition were examined under adherent and non adherent growth conditions after metabolic conversion of formazan derivatives MTT and XTT, respectively and absorbance measurement at appropriate wavelengths. Dose response was determined by 4-parameter curve fitting of the values normalized to DMSO-treated control. Brk inhibition was determined by Western Blot analyses combined with immuno-staining. Results:  We found a reduction of cell growth under adherent condition only for the highest inhibitor concentrations used in both cell lines (MDA-MB-231: MK5: 97.7 % ± 2.0 to 68.3 % ± 12.2 N = 5; MK136: 84.8 % ± 6.0 to 64.0 % ± 14.2 N = 4; MK138: 84.2 % ± 5.5 to 42.4 % ± 19.2 N = 4; MK150: 97.0 % ± 2.1 to 50.1 % ± 14.5 N = 6; MDA-MB-468: MK5: 82.5 % ± 4.4 to 43.8 % ± 12.5 N = 14; MK138: 95.9 % ± 1,9 to 71.9 ± 9.1 N = 6; MK150: 86.6 % ± 3.0 to 24.9 % ± 11.6 N = 9). When cells were

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forced to grow under loss-of-adherence conditions result were comparable to adherent growth condition in MDA-MB-468 cells (MK5: 94.8 % ± 3.4 to 25.7 % ± 8.5 N = 4; MK138: 82.5 % ± 4.7 to 55.3 ± 15.2 N = 9; MK150: 91.3 % ± 5.1 to 43.9 % ± 16.3 N = 10) Western Blot analysis affirmed Brk expression and verified inhibitor dependent kinase inhibition. Conclusion:  Our data indicates, that pharmacological inhibition of the Brk kinase function does neither influence cell growth under adherent nor under non-adherent conditions. The displayed effect at 10  µM of kinase inhibitor may be due to inhibition of structurally related kinases. Additionally, we showed that the used inhibitors have the ability to constrain Brk kinase functionality in the applied concentrations. Further investigation are needed to portray the impact of Brk inhibition on other aspects of tumor dissemination and metastasis. Acknowledgement:  This work was supported by the German Research Foundation (DFG, Grant No. RI1196/4-1).

V04 Progress in the NanoEFEct project P. Debbage1, G. Thurner1, A. Abdelmoez1, Ý. Mørch2, S. Coelho3, I. Ganzleben4, J. G. Menezes4, M. Roessler5, M. Coelho3, B. Moritz5, A. Hartmann4, M. Waldner4, R. Schmid2 Medical University Innsbruck, Innsbruck, Austria SINTEF, Trondheim, Norway 3 University of Porto, Porto, Portugal 4 University of Erlangen, Erlangen, Germany 5 CESAR, Vienna, Austria 1 2

Background:  The NanoEFEct ERANet project was presented to CESAR during 2014 and has now run for one year. The project aims to carry out translational research to design, create and optimize nanoparticulate materials for clinical use in diagnosis of early stages of colorectal CA. The resulting nanoparticles should render flat lesions visible with high contrast in normal clinical fluoro-endoscopes. The first milestone of the project was therefore to generate nanoparticles capable of delivering extremely intense fluorescence signals. Since earlystage lesions may bind few nanoparticles and the fluorescence may be intrinsically weak, the NanoEFEct consortium decided to generate signals in the red part of the spectrum, where tissue autofluorescence is not a problem. Methods:  3 different types of nanoparticles were either coupled covalently to red fluorochromes or designed to encapsulate the fluorochromes. A custom-built simulacrum of an endoscope was built for each of the participating project partners and compared to a clinical endoscope. Nanoparticles were tested for fluorescence brightness in the red spectrum, and for their size and size dispersity by PCS and by TEM. Their leakiness was tested by TLC and their physical integrity by gel electrophoresis. Results:  Nanoparticles of 2 types (albumin-based and PACA-based) were synthesized that were detectable by fluorescence in the nmol/ml concentration range, and this was achieved by use of two different red dyes. Nanoparticle sizes and dispersities were similar to those we published earlier and the particles exhibited strong physical integrity. The particles based on a dye covalently bound to albumin did not release dye. Those based on PACA did not leak dye under normal storage conditions.

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Abstracts Conclusion:  The first Project aim has been achieved: the nanoparticles fluoresce strongly enough to render flat lesions visible. These nanoparticles were prepared with regard to relevant regulatory criteria. The Project aim of true translational research is within reach; however, further Project milestones remain to be achieved.

Posters

P01 Synthesis and biological investigations of novel NHC gold(I) complexes as potential anticancer agents C. M. Gallati, M. Plangger, I. Würtenberger, R. Gust Department of Pharmaceutical Chemistry, CCB—University of Innsbruck, Innsbruck, Austria Platinum complexes are widely used in cancer treatment since their discovery in the 1960s. However, chemotherapy with platinum drugs is accompanied by severe side effects and development of resistance. Therefore, new metals have been investigated to replace platinum. Several complexes of gold(I) and gold(III) have been synthetized and tested against tumor cells and showed promising results. Their mechanism of action is fundamentally different to the one of platinum drugs, which makes gold such an interesting alternative. Of all known ligands for gold complexes, N-heterocyclic carbenes (NHCs) are especially interesting because of their strong bonding to gold(I), leading to complexes with high stability. A series of NHC gold halide complexes derived from 4,5-diarylimidazoles has been synthesized in the past, showing interesting activity. Based on this work, a new series of NHC ligands with an imidazole core bearing a methoxy-pyridine ring and a substituted phenyl ring in positions 4 and 5, respectively, was designed.

P02 Targeting protein-protein interactions of estrogen receptor alpha: design and synthesis of novel small molecule libraries N. Fahrner, V. Follia, S. Noha, D. Schuster, R. Gust Institute of Pharmacy, Department of Pharmaceutical Chemistry, University of Innsbruck, Innsbruck, Austria Approved Estrogen receptor alpha (ER-alpha) targeting endocrine anticancer drugs such as tamoxifen or fulvestrant act by interaction with the ligand binding domain (LBD) of ER-alpha. In order to overcome limitations of this current approach, alternate-site modulators for several nuclear receptors (NR) have become of particular interest during the last decades. The earliest examples of small molecules that interact with ER alpha outside the LBD were described in 2004. Targeting protein-protein interactions of NRs with small molecules opens the field for the design of “Nuclear Receptor AlternateSite Modulators” (NRAMs). In order to identify essential interactions of NRAMs with the coactivator binding site (CABS) of ER alpha, we applied modern methods of computer-aided drug design. A set of compounds with known activity for the CABS of ER alpha was used as reference. As visualized in Fig.  1, compounds acting as NRAMs should show hydrophobic interactions with the deep hydrophobic groove on the surface of the CABS, and be able to form hydrogen bonds with either Lys 362 or Glu 542, two amino acids of the so called hinge region. Based on these results, a pharmacophore model was derived. This model was applied in combination with docking experiments to identify lead structure from our in-house library. Several derivatives were analyzed in silico and candidates for synthesis were selected according to their estimated binding interactions. Series of candidates were synthesized following established routes and optimizing these when appropriate.

Scheme 1:  General structure of the gold(I) complexes

A versatile synthetic pathway for the preparation of the imidazolium ligand precursors has been developed, enabling the synthesis of a series of derivatives with various substituents. The corresponding gold(I) halide complexes were prepared and characterized. Cytotoxicity of two gold(I) complexes was determined with the MTT assay on three cell lines (MCF-7, MDA-MB-231 and HT-29) and the IC50 values were found to be in the micromolar range. In order to investigate cellular uptake, MCF-7 cells were incubated with a complex whose ligand contains a fluorophenyl residue, and the accumulation of gold and fluorine were determined by means of HR-CS AAS and HR-CS MAS, respectively. The synthesis and biological data will be presented.

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Fig. 1  Cocativator binding site of ER alpha with pharmacophore model showing essential interactions for NRAMs (yellow balls: hydrophobic interactions; arrows: hyrogen bonds)

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Abstracts The newly synthesized compound libraries are currently being characterized in vitro in order to confirm the results obtained in silico.

P03 Biological activity of novel derivatives of the GPR30 agonist, G1, in ovarian and breast cancer cell lines V. Follia1, B. Kircher2,3, S. Alscher4, R. Gust1 Institute of Pharmacy, Department of Pharmaceutical Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria 2 Immunobiology and Stem Cell Laboratory, Department of Internal Medicine V (Hematology & Oncology), Innsbruck Medical University, Innsbruck, Austria 3 Tyrolean Cancer Research Institute, Innsbruck, Austria 4 Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany 1

Background:  The GPR30 receptor is a seven transmembrane Gs protein-coupled receptor, involved in the development of breast and ovarian cancer and it is able to modulate the non-genomic estrogenic signaling in different cell types. Overexpression of the GPR30 is linked to tumor size (> 2  cm) and strongly correlated to proliferation, invasion, metastasis and drug resistance in many cancer cell lines. Moreover, the overexpression of the receptor is associated with lower survival rate. Aim:  As an alternative treatment for breast and ovarian cancer, new derivatives of the GPR30 agonist, G1, were tested for biological activity. Methods:  The antitumor activity was examined in breast and ovarian cancer cell lines. To evaluate the anti-proliferative effects of the new molecules, the metabolic activity assay, EZ4U, and the 3[H]-thymidine proliferation assay were performed. Apoptosis assays and other functional assays were performed in order to evaluate the biological activity of the new compounds in comparison to G1. Results:  The compounds elicited strong concentration-dependent anti-proliferative effects which were highly dependent on their structure. Induction of apoptosis was also dependent on the substitution pattern, but also on the analysed tumor cell line. Importantly, cAMP—as an indirect marker of GPR30 specificity—was only produced by the active compounds, in a concentration-dependent manner. The compounds were mostly more effective than G1. Conclusion:  Our compounds may represent novel drugs targeting aggressive breast and ovarian cancers which overexpress GPR30 and establish a basis for the development of new G1-derivatives.

P04 Antitumour active cobalt alkyne complexes derived from acetylic salicylic acid: studies on impact of fluorination and chlorination of Co-ASS

Institute of Pharmacy, Department of Pharmaceutical Chemistry, University of Innsbruck, Innsbruck, Austria Background:  [(Prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl (Co-ASS), a derivative of the irreversible COX-1/ -2 inhibitor acetylic salicylic acid (ASS) chelated to cobalt, demonstrated high growth-inhibitory potential against various tumour cells with interference in the arachidonic acid cascade as probable mode of action. We modified the ASS moiety esterified by a propargyl group and obtained its chlorinated and fluorinated derivatives, respectively. In a comprehensive structure activity relationship study we investigated the new complexes. Aim:  The aim of our study is to identify the impact of a fluorine or chlorine substitute in different positions of the acetylic salicylic acid moiety on cytotoxic activity, COX inhibition in general and COX isoenzyme selectivity in particular compared to the lead structure Co-ASS. Methods:  Compounds were evaluated for cytotoxicity in breast [MCF-7 (hormone dependent), MDA-MB-231 (hormone independet)] and colon cancer [HT-29] cell lines and for COX-1/-2 inhibitory effects at human recombinant or ovine isoenzymes. For selected compounds with strong COX-1/-2 inhibition, the major COX metabolite prostaglandine E2 (PGE2) was quantified in arachidonic acid-stimulated MDA-MB 231 breast tumor cells via enzyme immunoassay (EIA). Results:  Whereas the ligands only showed an insignificant cytotoxic effect, cobalt complexes were able to inhibit tumor cell growth in all cell lines to a larger extent. Further, complexation of ligands to cobalt led to a tenfold increase in the potency to inhibit COX-1/-2 compared to ligand only. By fluorinating and chlorinating the ASS propargylester moiety, we could achieve a shift in COX selectivity and a cell line specific cytotoxicity comparable to the one of Co-ASS. In general, the position of the halide contributed strongly to these cytotoxic and COX-inhibitory effects. The chlorinated derivatives, however, showed different cyclooxygenase inhibition and cytotoxicity patterns than the fluorinated derivatives. Compounds with a weaker inhibition of COX also showed a lower cytotoxic potential in the cell based assay and vice versa, indicating the interference with the arachidonic acid as a potential mode of action. Conclusion:  Halogenated Co-ASS derivatives are a new approach in the development of new antineoplastic agents with favored inhibition of COX-2, the isoenzyme with greater impact on tumor development compared to COX-1. Due to the low cytotoxicity of cobalt complexes in human in general, also the newly synthesized compounds might be well-tolerated and are, therefore, promising.

P05 Rapid quantification of Lysophosphatidycholine (LysoPC) in plasma of cancer patients by mid-infrared-spectroscopy M. Unger1, I. Schlöffel1, D. Küllenberg2, U. Massing2,3  ETICS Healthcare Technologies GmbH, Schelztorstr. C 54-56, 73728 Esslingen, Germany. 2 Dept. F&E Life Science Applications, Andreas Hettich GmbH & Co KG, Breisacher Str. 117, 79106 Freiburg, Germany 3 Tumor Biology Center Freiburg, Breisacher Str. 117, 79106 Freiburg, Germany 1

V. Obermoser, C. Schuster, S. Lettenbichler, E. Kircher, V. Braun, R. Gust

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Abstracts Background:  Lysophosphatidycholine (LysoPC) is a bioactive phospholipid and a major plasma constituent. Beyond the role of LysoPC as a precursor of lipid biosynthesis, it is implicated in several signal transduction pathways; it can activate a wide range of cell types and events in the vascular system, playing a major role in immunologic and inflammatory events. However, there is no consistent reference level for LysoPC plasma concentrations, and the absolute LysoPC concentration in plasma of healthy individuals is between 300–400 µM. Cancer patients and especially patients with metastasis frequently show dramatic changes in the LysoPC plasma levels. Mostly LysoPC is decreased; however there are also a few studies that show increased levels. Comparable to the observations in cancer patients, markedly reduced LysoPC plasma levels were found in sepsis patients. Aim:  The present study is focused on the rapid quantification of LysoPC. Nowadays, LysoPC is quantified by thin layer chromatography or HPLC-MS methods, for both techniques complex and time-consuming sample preparation is required. Therefore, there is a need for improving the LysoPC detection. Rapid quantification of LysoPC would have also many benefits including early intervention of therapy and reduction in mortality. In this context, mid-infrared spectroscopy, along with chemometrics, is used to establish the analytical method to quantify LysoPC in blood plasma. Methods:  Mid-infrared (FT-MIR) spectroscopy is a widely used method for analysis of a wide range of materials, being simple-to-operate, cost effective and requiring only simple or no sample preparation. The resulting spectrum provides a response from the whole sample and therefore, multiple markers can be selectively targeted. The presented results were obtained on a FT-IR spectrometer based on the AquaSpec® FTMIR technique that measures mid-infrared spectra of liquid biological samples (here blood plasma) in transmission mode. The centerpiece is a flow-through transmission cell, consisting of a path length of approx. 9 µm and therefore optimized for the measurement of aqueous solutions. IR spectra of plasma cancer patients (samples of 300 subjects) were recorded with a measurement time of 3  min (on average, with 300 scans) for each sample. Results:  Distinctive IR-specific absorption bands assigned to LysoPC were detected in the spectra of the plasma cancer patients and used to build up the chemometric model. PLS (partial least squares) regression approach was performed and analyzed for LysoPC with the standard reference technique of HPTLC (high performance thin layer chromatography). The resulting PLS regression model gives a very good linear fit to the data and can be applied for the prediction of LysoPC of unknown plasma patient samples. Conclusion:  This study demonstrates that our proposed IR spectroscopic approach in combination with chemometric applications (PLS regression) has the potential to become a clinical method for very fast determination of LysoPC in blood plasma.

P06

Immunobiology and Stem Cell Laboratory, Department of Internal Medicine V (Hematology & Oncology), Innsbruck Medical University, Innsbruck, Austria 2 Tyrolean Cancer Research Institute Innsbruck, Innsbruck, Austria 3 Institute of Pharmacy, Department of Pharmaceutical Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria 4 Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany 1

Background:  Non-platinum transition metal compounds with anti-tumor and anti-leukemic activity have emerged during the last years. Especially iron-containing complexes strike the medicinal interest. Their redox-active metal centre can interact with different ligand structures and, even more importantly, with the cellular redox state. Thereby, the compounds modify cellular viability and induce various other biological activities. We have previously developed transition metal-based compounds with Schiff base ligands and identified [FeIIIsalophene(Cl)] as a very powerful substance inducing cell-growth inhibition and apoptosis, but it also shows activity against T cells from healthy individuals. Aim:  To obtain compounds with similar good anti-leukemic activity but decreased toxicity against normal control cells [FeIIIsalophene(Cl)] was modified by various substituents. Methods:  Biological activity of the compounds was analysed by proliferation inhibition and apoptosis induction of three leukemic cell lines and of normal control cells (phytohemagglutinin-stimlated T cells from healthy persons). Proliferation of the leukemic cell lines K-562 (chronic myeloid leukemia), HL-60 (acute myeloid leukemia) and SD-1 (acute lymphatic leukemia) and the normal control cells was determined by thymidine uptake. Apoptosis induction was measured indirectly by a metabolic activity assay and directly by annexin V/ propidium iodide staining on a flow cytometer as well as determination of caspase-3 and caspase-7 (luminescent assay). Results:  The modified compounds dose-dependently inhibited the growth of the leukemic cell lines and concomitantly induced their apoptosis. Some of them were effective already at low concentrations and exhibited even higher antileukemic activity than the lead substance, however, were also toxic against normal control cells. Nevertheless, we identified also one compound, [FeIII(5-OMe-salophen)Cl], with strong leukemic cell-growth inhibitory and apoptosis inducing activity, but a nearly non-toxic behaviour against T cells of healthy volunteers. The formation of reactive oxygen species was proved as mode of action. Conclusion:  [FeIII(5-OMe-salophen)Cl] may be a novel anticancer drug candidate.

P07 Red-fluorescing albumin-based nanoparticles designed for use in diagnosis of colorectal CA: translational research in the context of the NanoEFEct EU project

Biological activity of Fe-salophene complexes

G. C. Thurner, A. Abdelmoez, P. Debbage

S. L. Steiner1,2, B. Ma3, A. Hille4, S. Angerer1,2, A. Schraffl1, R. Gust3,4, B. Kircher1,2

Medical University Innsbruck, Innsbruck, Austria

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Background:  NanoEFEct (“Nanoparticle-Enhanced molecular Fluorescence-Endoscopy for detection of early-stage

13

Abstracts colorectal adenocarcinomas”) is a project supported by the EU ERA-NET program on Translational Cancer Research (TRANSCAN). This project, now in Year 2, consisting of 5 partners in Portugal, Norway, Germany and Austria (ourselves and CESAR), aims to increase significantly the sensitivity and specificity of the screening diagnostic procedures for early stages of colon carcinomas. We aim to prepare nanoparticles suitable for preclinical testing by the end of the project. To do so, three different kinds of nanoparticles (NPs) are under development to be used for fluoro-endoscopy: poly(alkylcyanoacrylate) NPs, gold NPs and albumin-based NPs. These nanoparticles should be labelled with a fluorochrome (excitation in the blue-green and emission in the red spectrum) and with a targeting group, and be applied topically during endoscopy using a mouse model. The project has as first milestone the production of adequately bright and stable nanoparticles for this purpose. The choice of fluorochrome was governed by considerations of quantum efficiency, cross-fluorescence with native fluorochromes in the tissues, toxicity, regulatory requirements, fluorochrome chemistry, patentability, cost effectiveness, and fluorochrome ownership. A further important constraint was that the components used to create the nanoparticles should be already approved for clinical use, to minimize complications at the pre-clinical testing stage. In this report, focus is laid on albumin NPs. Methods:  Nanoparticles were synthesized by coacervation of albumin. A red fluorochrome, Protoporphyrin IX, was covalently attached. This fluorochrome is the fluorescent material produced in urotheliomas after injection of aminolevulinic acid, and its detection is now state-of-the-art in modern urological clinics. We measured fluorescence intensity in a simulacrum of a clinical endoscope and checked stability of the particles by gel electrophoresis. We measured particle size by use of Photon Correlation Spectroscopy and Transmission Electon Microscopy, and stability of fluorochrome attachment to the albumin by Thin Layer Chromatography (TLC). Some batches were dialyzed. Results:  Particles were similar in size and size dispersity to those we reported earlier, but exhibited a slight tendency to aggregation during synthesis. They exhibited physical integrity as shown by not entering 7.5 % polyacrylamide gels. TLC results indicated that the fluorochrome was strongly attached to the albumin and was not released under aggressive conditions. The particles fluoresced red so strongly that they could be detected in the endoscope simulacrum at concentrations in the range nmol/ml in water. Conclusion:  As described here, the first milestone in the NanoEFEct project, to create nanoparticles providing strongly detectable fluorescence in a clinical fluoro-endoscope, has been achieved. The synthesis protocol is up-scalable and should be capable of being carried out without problems in GMP. It is therefore now appropriate to focus on later milestones of the NanoEFEct Project, primarily the targeting of the nanoparticles to cell surface proteins overexpressed in early adenocarcinomas. Acknowledgements:  We thank for detailed and valuable discussion: Ruth Schmid, Yrr Morch, Max Waldner, Ingo Ganzleben, Arndt Hartmann, Berta Moritz, Wolfgang Vogel. We are grateful to the firm Unilab for acting as Austrian Project Supplier.

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P08 Development of human albumin-based nanoparticles for diagnostic optical molecular imaging of early inflammation and adenocarcinoma A. Abdelmoez1,2, G. Thurner1, P. Debbage1  ept. Anatomy, Histology & Embryology, Innsbruck Medical D University, Innsbruck, Austria; 2 Dept. Pharmaceutical Organic Chemistry, Assiut University, Assiut, Egypt 1

Background:  Nanoparticles show potential to improve medical treatment by providing higher diagnostic detection sensitivity, with lower toxicity and higher potency in drug delivery. The work reported here focuses on the development of brightly fluorescent, targeted albumin nanoparticles, aiming to image early stages of inflammation or of carcinoma. Since only a small number of blood vessels are expected to show positive signals at early stages of inflammation, and early adenocarcinoma lesions may not express biomarkers strongly, we aimed to create nanoparticles of intense intrinsic brightness for use in relevant optical detection systems. Methods:  Albumin nanoparticles were prepared by conjugating a fluorescent probe to albumin, then preparing nanoparticles by coacervation. Fluorescein isothiocyanate was used as fluorochrome emitting green light and a Nile Red derivative as fluorochrome emitting red light. We varied the synthesis protocols systematically (e.g. molar ratios fluorochrome:albumin, conjugation protocol, inter al.). Each batch was tested for fluorescence intensity by use of either fluorescence spectrometry or by use of a custom-designed imaging apparatus mimicking endoscopes presently in routine clinical use. Molar ratios were assayed in MALDI-TOF spectrometry. Nanoparticle size and dispersity were characterized by use of photon correlation spectroscopy and negative contrast transmission electron microscopy, and particle mechanical integrity was tested by use of thin layer chromatography and gel electrophoresis. Nanoparticle batches were stored in the dark at 4 °C. Results:  The nanoparticles were detectable at fluorochrome concentrations as low as 5–100 nmol/ml; they typically carried 2–4 fluorochromes per albumin molecule. They had a narrow size distribution (60–100 nm diameter), did not disintegrate in sodium dodecyl sulfate and thus did not enter polyacrylamide gels, and were stable during storage at 4 °C for several months, with little aggregation. Conclusion:  Calculation shows that the high fluorescence intensity of these nanoparticles should enable them to visualize early inflammatory or tumorous lesions by use of endoscopes in routine clinical use. The next step will be to attach specific antibody to the nanoparticles to allow their accumulation exclusively at relevant target sites; relevant animal models for the oncological work are already available in collaboration with the University of Erlangen, and we are developing other models for visualization of early inflammation. This work is part of a doctoral thesis supported by a stipend from the Egytian Government.

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P09 The matricellular ligand Cyr61 activates the integrin VLA-4 not via redox modulation in contributing to the metastatic spread of tumors S. G. Hoß, M. Schlesinger, G. Bendas Department of Pharmacy, Rheinische Friedrich-WilhelmsUniversität, Bonn, Germany Background:  Metastatic progress is essentially dependent on the ability of circulating tumor cells to adhere to defined distant sites. We focus our work on the integrin VLA-4, which is strongly expressed by melanoma cells and of key importance for their metastatic spread. We previously showed by a knockdown approach that the matricellular protein Cyr61 exerts a high affinity binding to VLA-4 and thus contributes to adhesive and in respect thereof prometastatic properties [1]. However, the molecular mechanisms of VLA-4 activation by Cyr61 remain elusive. The acronym Cyr is derived from the structural appearance as a “cysteine rich” molecule. Furthermore, VLA-4 binding activity has been shown to be controlled in by redox modulation of thiol groups on the extracellular domain of the α4-subunit. Artificial reduction of surface protein thiols using N-acetyl-L-cysteine (NAC) led to increased adhesion quest, implicating reduced SH-groups as a precondition for a fully active state of VLA-4 [2]. In light of these findings, an interaction of Cyr61 with VLA-4 via redox modulation can be assumed as underlying mechanism for increased affinity. Aim:  The aim of our studies was to elucidate whether the prometastatic properties of Cyr61 are mediated by a direct interaction with thiol groups presented on the extracellular domain of VLA-4 with respect to redox modulation. Methods:  We performed adhesion assays with fluorescence labeled VLA-4 positive cells upon redox modulation. A Surface Acoustic Wave (SAW) biosensor was used to investigate the binding affinity of immobilized VLA-4 to VCAM-1 under redox modulation of the integrin by DTNB, NAC and an interference with rhCyr61. Results/Conclusions:  Although a Cyr61 deficiency by a knock down approach reduced VLA-4 binding, Cyr61 addition to unstimulated wild type cells did not further increase the VLA-4 binding capacity. However, Cyr61 interfered with the VLA-4 activation by Mn2 + since cells stimulated with Mn2 + prior to Cyr61 addition revealed non-significantly lower adhesion compared with those cells only Mn2 + treated. Furthermore, pretreatment of cells with DTNB led to complete loss of adhesion and did not recover upon Mn2 + treatment. These findings were supported by the biosensor approach. VLA-4 binding was disturbed by oxidative effects of DTNB, but reduction of SH groups of VLA-4 by NAC hardly affected the binding towards VCAM1, which was even true in case of subsequently added Cyr61. Thus, the Mn2 + and Cyr61 integrin activation axis appeared independent of redox modulation of VLA-4. In conclusion our data provide that Cyr61 affects the interaction of VLA-4 with its ligand VCAM-1 in a negative direction and therefore points to a decrease in VLA-4 activity. Considering the known data on VLA-4 redox modulation, the loss of activity by Cyr61 was not expected and could be explained by an oxidizing effect of Cyr61 on the thiol groups of the integrin.

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References 1. Schmitz P, et al. Cyr61 is a target for heparin in reducing MV3 melanoma cell adhesion and migration via the integrin VLA4. Thromb Haemost. 2013;110:1046–54. 2. Laragione T, et al. Redox regulation of surface protein thiols: identification of integrin alpha-4 as a molecular target by using redox proteomics. Proc Natl Acad Sci USA. 2003;100:14737–41.

P10 Hedgehog pathway inhibition in combination with lithium chloride: a treatment option for rhabdomyosarcoma? K. A. Boehme1, J. J. Zaborski1, R. Riester1, T. Kluba2, R. Handgretinger3, F. Traub2, S. B. Schleicher3 Department of Orthopaedic Surgery, Laboratory of Cell Biology, Eberhard Karls University Tuebingen, Waldhoernlestraße 22, 72072 Tuebingen, Germany 2 Department of Orthopaedic Surgery, Eberhard Karls University Tuebingen, Hoppe-Seyler-Straße 3, 72076 Tuebingen, Germany 3 Department of Hematology and Oncology, Children’s Hospital, Eberhard Karls University Tuebingen, Hoppe-Seyler-Straße 1, 72076 Tuebingen, Germany 1

Background:  Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, accounting for 5–7 % of all malignancies in children and adolescents. We found inhibitors targeting the glioma-associated oncogene (GLI) transcription factors, downstream effectors of the Hedgehog (Hh)-pathway, to efficiently inhibit proliferation and induce apoptosis of RMS cell lines in vitro. Lithium chloride (LiCl) is used as a mood stabiliser for the treatment of bipolar disorder. Moreover, its antitumorigenic effects have been shown for several cancer types. One of the major targets of LiCl, glycogene synthase kinase 3 beta (GSK-3ß), is also involved in regulation of activity and abundance of the GLI transcription factors. Therefore, LiCl effects on viability and apoptosis induction in RMS cell lines were examined and compared to combinatorial effects with the Hh inhibitors arsenic trioxide (ATO) or GANT61. Methods:  Experiments were performed using four different RMS cell lines, which were treated with increasing concentrations of the GLI inhibitors ATO or GANT61 and LiCl in single and combined application. Furthermore, a triplicate treatment using itraconazole, a smoothened inhibitor, ATO and LiCl was performed. Viability and proliferation were monitored by MTS assay, clonogenic assay and 3-D spheroid assay. Apoptosis induction was assessed by flow cytometry and Western blot. Results:  All RMS cell lines showed a dose-dependent loss of viability after treatment with ATO (IC50: SRH 3.67  µM, RD 1.51  µM, RH30 1.31  µM, ZF 2.00  µM), GANT61 (IC50: SRH 13.32 µM, RD 8.38 µM, RH30 13.13 µM, ZF 9.36 µM) or LiCl (IC50: SRH 32.14 mM, RD: 25.97 mM, RH30 20.26 mM, ZF 11.80 mM), respectively. Moreover, apoptosis induction, indicated by caspase 3 and PARP cleavage, could be detected in three out of four cell lines. Combination of ATO and LiCl or GANT61 and LiCl resulted in additive inhibition of viability in the four RMS cell lines tested, whereas apoptosis induction was increased in three out of four cell lines. Viability of primary skeletal muscle

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Abstracts cells was hardly affected by ATO (IC50 > 10  µM) and LiCl (IC50: 50.12 mM) doses inducing maximal response in RMS cells. Conclusion:  The experiments show, that inhibition of the Hh-pathway in combination with LiCl may be a promising strategy for the treatment of RMS.

P11

P12 Antimetastatic properties of Lysophosphatidylcholine (LysoPC) T. Ross1, B. Jakubzig1, M. Schlesinger1, A. Raynor2, P. Jantscheff2, C. Gorzelanny3, U. Massing2,G. Bendas1  epartment of Pharmaceutical Chemistry, University D of Bonn, Bonn, Germany 2 Tumor Biology Center, Freiburg, Clinical Research, Freiburg, Germany 3 Experimental Dermatology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 1

Are the antimetastatic properties of Lysophosphatidylcholine (LysoPC) based on a deregulation of Proteinkinase C (PKC) activity and/or intracellular calcium levels? B. Jakubzig, T. Ross, M. Schlesinger, G. Bendas Department of Pharmaceutical Chemistry, University of Bonn, Bonn, Germany Background:  A lysophosphatidylcholine (LysoPC) pretreatment of melanoma cells induces an attenuated metastatic spread in experimental metastasis assays, obviously related to a diminished integrin function, without affecting the expression of these proteins. Consequently, integrins appear to be affected at the cellular signaling level by a LysoPC impact on the cell membrane. The Sydecan-4 and Proteinkinase C alpha/delta activation axis appears as a promising candidate to elucidate the molecular mechanisms how LysoPC affects the integrins. Due to the calcium dependency of PKC alpha activity, but not PKC delta we also postulated a probable impact of LysoPC on intracellular calcium levels. Aim and methods:  Using different melanoma cells we want to evaluate whether a shift of PKC alpha, delta as well as Syndecan-4 phosphorylation induced by LysoPC pretreatment is a key for the attenuated integrin activity and whether this is correlated with induced reduction of intracellular calcium levels. We employed immunofluorescence based techniques as well as western blots to analyse Syndecan-4 and Proteinkinase C activity by measuring the amount of protein phosphorylation under the influence of different LysoPC species. Therefore we also established the use of stain-free technology in our work to normalize our western blot results. Cellular calcium levels were detected by employing the calcium sensitive fluorescence probes fura red as well calcium green-1 in a flow cytometric based assay. Results:  LysoPC treatment leads to increased levels of PKC delta and Syndecan-4 phosphorylation as well as to a decrease in PKC alpha phosphorylation in both, B16F10 murine and MV3 human melanoma cells. Unexpectedly, we observed an increase in intracellular calcium concentration under LysoPC pretretament when cells were subsequently exposed to calcium containing media. Conclusion:  LysoPC seems to affect the signaling pathways for integrin activity via interfering with the formation of focal adhesion complexes. Our present data favor Syndecan-4 as a key molecule to deregulate focal adhesion via PKC delta. However, the increase in intracellular calcium observed after LysoPC cell treatment seems to have no functional influence on PKC activation.

Background:  Based on the findings that empty liposomes, applied to tumor bearing mice, possess antimetastatic effects [1] lysophosphatidylcholine (LysoPC) was regarded as the active agent resulting from the liposomal phospholipid degradation within the tumor tissue. Thereupon we demonstrated that a LysoPC pretreatment of human as well as mice melanoma cells strongly attenuated their metastatic spread in a lung invasion model in mice [2]. Aim and methods:  Since LysoPC did not possess apoptotic or cytotoxic effects, the underlying molecular mechanisms of reduced integrin functionality remained open and are the aim of this study. The LysoPC consumption and incorporation of LysoPC-associated fatty acids into tumor cell membranes is monitored by gas chromatography. Membrane rigidity is examined by fluorescence based microscopic and spectroscopic methods as well as atomic force microscopy (AFM). The impact of LysoPC on downstream signaling is observed by immunofluorescence. Cellular migration is observed by a two dimensional wound healing assay. Results:  In vitro studies revealed attenuated integrin functions in cell adhesion as well as in cell migration as the functional consequence of LysoPC for affecting metastasis. LysoPC associated fatty acids were massively incorporated into the cell membrane, affecting the lipid composition of the membrane dramatically. This goes along with morphological changes of the cells observed by electron microscopy. The detection of membrane viscoelasticity by AFM (young’s modulus) confirms that the shift in lipid composition leads to membrane rigidification by saturated LysoPC, which also affects the lateral lipid mobility within the membrane (fluorescence microscopic FRAP technique). The rigidifying effect of LysoPC was further evaluated with respect to LysoPC species (saturated vs. unsaturated) and concentration using trimethylammoniumdiphenylhexatriene as fluorescence anisotropy probe. The increased membrane rigidity corresponds with a diminished receptor-mediated migration capacity of the cells. Conclusion:  LysoPC might seriously affect the signaling pathways essential for integrin activity via interfering with the formation of focal adhesion complexes. Our data shed a new light on liposomal drug carrier approaches in cancer therapy with respect to novel, yet not considered activities of phospholipid degradation products.

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. The authors are thankful for financial support by a “CESAR mini grant”.

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P13 The Aplidin analogs PM01215 and PM02781 inhibit angiogenesis in vitro and in vivo B. Borjan1, N. Steiner1, S. Karbon1, J. Kern2, A. Francesch3, M. Hermann4, W.Willenbacher1, E. Gunsilius1, G. Untergasser1,5  epartment of Internal Medicine V, Innsbruck Medical D University, Innsbruck, Austria 2 Oncotyrol GmbH, Karl Kapfererstrasse 5, Innsbruck, Austria 3 Pharmamar, R&D Department, Avda de los Reyes 1, Colmenar Viejo, Madrid, Spain 4 Department of Anesthesiology & Critical Care Medicine, Innsbruck Medical University, Innsbruck, Austria 5 Tyrolean Cancer Research Institute, Innsbruck, Austria 1

Background:  Tumor development and progression strongly depend on angiogenesis. Thus, inhibition of angiogenesis by “antiangiogenic drugs” represents an important tool for holding tumors in a small avascular state and inhibiting their growth and metastasis. Despite extensive research only few drugs primarily targeting “VEGF signaling” have reached clinical practice and currently face new challenges such as the development of resistances. Therefore, there is an urgent need for novel compounds that act “antiangiogenically” by stopping endothelial cell proliferation without inducing apoptosis in the vascular network of the body and/or affecting coagulation processes. Next to the proteasome inhibitor bortezomib, the cyclodepsipeptide AplidinTM originally isolated from the Mediterranean tunicate Aplidium albicans, has been demonstrated to exert antiangiogenic effects in vitro and in vivo. Aim:  In this study novel synthesized analogs of Aplidin, PM01215 and PM02781, were tested for antiangiogenic effects on primary human endothelial cells in vitro and for inhibition of angiogenesis and tumor growth in vivo. Methods and Results: Both derivatives inhibited angiogenic capacities of human endothelial cells (HUVECs) in vitro at low nanomolar concentrations, as determined by real-time cell proliferation and migration, capillary tube formation and vascular endothelial growth factor (VEGF)-induced spheroid sprouting assays. Antiangiogenic effects of both analogs were observed in vivo in chicken chorioallantoic membrane (CAM) assays. CAM is a simple, highly vascularized extraembryonic membrane. The tissue composition and accessibility of the CAM for experimental manipulation, makes it an attractive preclinical in vivo model for drug screening and for studies of vascular growth. As expected, VEGF induced a strong proangiogenic reaction that was efficiently blocked by simultaneous application of both Aplidin analogs. In addition, growth of human multiple myeloma xenografts in the CAM was significantly reduced after application of both analogs. On the molecular level, both derivatives induced cell cycle arrest in G1 phase, as determined by flow cytometric analysis of endothelial cells. This growth arrest correlated with induction of the cell cycle inhibitor p16INK4A and increased senescence-associated beta galactosidase activity. In addition, Aplidin analogs induced oxidative stress and decreased production of the vascular maturation factors Vasohibin-1 and Dickkopf-3. Conclusion:  From these findings we conclude that both analogs are promising agents for the development of antiangiogenic drugs acting independent on classical inhibition of VEGF signaling.

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P14 Is chemoresistance of human melanoma induced by integrin-mediated cell adhesion phenomena? M. B. R. Piva, M. Schlesinger, G. Bendas Pharmazeutisches Institut, Rheinische Friedrich-Wilhelms Universität Bonn, Bonn, Nordrhein-Westfalen, Germany Background:  Malignant melanoma has an incidence of about 232,000 people worldwide and a 5-year patient survival rate of only 14 %. High mortality results from the lack of understanding of the genetic alterations and signalling pathways of malignant transformation, metastasis, and drug resistance mechanisms. However, chemoresistance in melanoma is far from well-understood and unfortunately, research has been focused on other tumor entities rather than melanoma. Nevertheless, melanoma cells malignancy is often associated with their integrin expression pattern, e.g. the integrin impact on melanoma metastasis. By binding to the ECM, or adjacent cells, integrins regulate not only simple physical attachment of cells, such as cytoskeleton organization, adhesion and migration, but also interfere in complex aspects by activating specific signalling pathways that enhance cell tumour proliferation, survival, apoptosis, invasion and metastasis. Cell-adhesion mediated drug resistance (CAM-DR) frequently appears in cells of hematopoietic origin, but has not yet been reported for melanoma. Aim:  Our project aims to provide insight into the resistance phenomena of melanoma cells, and possibly new therapeutic targets; and to investigate a potential link between integrins and melanoma apoptotic response rate towards chemotherapeutic treatment. Methods:  Cisplatin cytotoxicity in different highly metastatic human melanoma cell-lines (MV3, NW1539 and Mel_1956) was investigated by MTT assays. Cell binding to fibronectin coated surfaces, cell activation by Mn2 + or the chemokine MCP-1 were performed to elucidate the impact of integrin activation status on cytotoxicity response. Results/Conclusion:  Although all three cell lines are derived from highly metastatic human melanomas, they differ in various characteristics, such as integrin expression pattern. Nevertheless, the cell lines display a comparable sensitivity towards cisplatin. Our results show that integrin activation by Mn2 + slightly shifted the sensitivity of cells to higher IC50 values indicating an impact of integrin activity on resistance phenomena. Furthermore, NW1539 and Mel_1956 cells show lower sensitivity when cultivated in contact with fibronectin, a known substrate for integrin binding. These data indicate a potential role of integrin-mediated adhesion processes for cell chemoresistance, which will further be validated in future experiments.

P15 Inhibition of protein disulfide isomerase 1 by PACMA31 reverses cisplatin resistance in ovarian cancer cells M. Kullmann1, G. V. Kalayda1, M. Hellwig1, S. Kotz2, R. A. Hilger3, S. Metzger2,4, U. Jaehde1

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Abstracts Institute of Pharmacy, Clinical Pharmacy, University of Bonn, Bonn, Germany 2 Cologne Biocenter, University of Cologne, Cologne, Germany 3 Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University DuisburgEssen, Essen, Germany 4 IUF-Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany 1

Background:  The clinical use of the antitumor drug cisplatin is often limited by the development of resistance in the course of the chemotherapy. Cisplatin resistance is a multifactorial event, with increased intracellular deactivation of active species being one of the major mechanisms involved. Recently, several intracellular binding partners of the fluorescent cisplatin analogue CFDA-cisplatin have been identified, among them the members of protein disulfide isomerase (PDI) family PDIA1 and PDIA3. Aim:  The aim of this work was to investigate PDIA1 and PDIA3 regarding their contribution to cisplatin resistance using the sensitive ovarian cancer cell line A2780 and its cisplatinresistant variant A2780cis. Methods:  The knockdown of PDIA1 and PDIA3 was performed using the respective small interfering RNA. Cisplatin cytotoxicity was assessed by using the MTT assay. Apoptosis was evaluated using the FITC Annexin V Apoptosis Detection Kit with PI®. The interaction between cisplatin and the PDIA1 inhibitor PACMA31 was studied using the combination index (CI) analysis. Protein expression was studied by Western blot analysis. Results:  Knockdown of PDIA1 led to increased cytotoxicity of cisplatin in resistant A2780cis cells, whereas PDIA3 knockdown had no influence. Sensitivity of the parent A2780 cell line to cisplatin was slightly, however not significantly increased. A tendency for enhanced apoptosis was observed after PDIA1 knockdown in the A2780 and A2780cis cell lines. This was not the case after PDIA3 knockdown. Pharmacological inhibition of PDIA1 with PACMA31 re-sensitized A2780cis cells to cisplatin treatment. In A2780 cells, the cytotoxicity of cisplatin was only marginally increased. Determination of the combination index revealed that cisplatin and PACMA31 act synergistically in both cell lines. Noteworthy, synergism was much more pronounced in the cisplatin-resistant cells. Conclusion:  Our results warrant further evaluation of PDIA1 as promising target for chemotherapy, and its inhibition by PACMA31 as a new therapeutic approach. Furthermore, combination of cisplatin with PACMA31 may be beneficial to overcome resistance in ovarian cancer.

P16

Institute of Pharmacy, Clinical Pharmacy, University of Greifswald, Greifswald, Germany 4 Institute of Medical Virology, Goethe University Hospital Frankfurt, Frankfurt/Main, Germany 5 Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, UK 6 Algorithmic Bioinformatics, University of Bonn, Bonn, Germany 3

Background:  Platinum complexes are still widely used in the treatment of several cancer entities, among them nonsmall cell lung cancer (NSCLC). The treatment outcome is often limited by the development of resistance, with the exact mechanisms still posing many questions. There are hints that altered signalling plays a role in the development of cisplatin resistance. Aim:  This projects aims at revealing signalling differences between cisplatin-sensitive and cisplatin-resistant NSCLC cells. Methods:  Differentially regulated genes in sensitive A549 and cisplatin-resistant A549Pt NSCLC cells were identified by a whole genome array (t-test, p < 0.05)and analysed by PCR and Western Blot to assess their possible contribution to cisplatin resistance. Alterations in apoptosis and cell cycle arrest were explained by building up an interaction network. All results are based on equimolar treatment with 11 µM cisplatin in both cells and equitoxic treatment in resistant cells with 34 µM cisplatin for 24 h after 4 h of serum starvation. Results:  Out of over 3000 differentially expressed genes, 10 genes were identified as key players in various pathways altered after cisplatin treatment, possibly contributing to chemoresistance. Protein and mRNA analysis revealed that reduced levels of HRas and resulting reduced levels of JNK3 after treatment with equitoxic concentrations of cisplatin led to reduced activation of p53 in resistant cells compared to sensitive ones. This is supported by the reduced mRNA expression and protein levels of Mdm2 disturbing the feedback loop to p53. Reduced Gadd45a levels and reduced p21 are likely to account for the loss of the G2/M arrest and less apoptosis in resistant cells. Additionally, increased expression of PP2A and Akt in resistant cells underlined these findings and will be investigated further. Conclusion:  Cisplatin-resistant NSCLC cells exhibit a reduced activity of key players in apoptosis signalling and a loss of signals for a G2/M arrest.

P17 Allometric scaling of the pharmacokinetics of BI 893923, a novel IGF-1 receptor inhibitor M. I. Titze1, O. Schaaf2, M. H. Hofmann2, M. Sanderson2, S. Zahn2, J. Quant2, T. Lehr1

Cisplatin resistance is associated with altered signalling in NSCLC cells

1

N. Sarin1, G. V. Kalayda1, F. Engel2, R. Frötschl2, C. A. Ritter3, J. Cinatl jr.4, F. Rothweiler4, M. Michaelis5, H. Fröhlich6, U. Jaehde1

Background:  The insulin-like growth factor 1 receptor (IGF1R) is involved in tumor establishment and maintenance. BI 893923 is a novel and selective ATP-competitive IGF1R/INSR inhibitor. A population pharmacokinetic (PK) model was developed to describe BI 893923 plasma concentration across different species and to allometrically scale the PK model to humans. Method:  PK models were first developed for each species (mouse, rat, dog, minipig) afterwhich all data was combined

Institute of Pharmacy, Clinical Pharmacy, University of Bonn, Bonn, Germany 2 Federal Institute for Drugs and Medical Devices, Bonn, Germany 1

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 linical Pharmacy, Saarland University, Saarbrücken, C Germany 2 Boehringer Ingelheim RCV GmbH, Vienna, Austria

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Abstracts and fitted to one PK model based on allometric principles. The developed PK model was used to predict the PK of BI 893923 in cynomolgus monkey. The dataset was extended by the monkey data and model parameters were re-estimated. The final PK model was used to predict human exposure using non-linear mixed-effects modeling implemented in NONMEM V7.3.0. Results:  Across all species, BI 893923 PK was best described by a three-compartment model with first-order elimination from the central compartment and parallel fast/slow absorption. All parameters could be scaled well by allometric principles and the monkey PK was predicted accurately. A minimum effective human dose of 4000 mg was predicted. Conclusion:  The scaled human PK model can serve as a useful tool for human therapeutic dose estimation.

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fied on average 25 cell subsets in said samples). Tracking this heterogeneity over a period of 9 weeks, we found that at least under in vitro conditions these subsets are stable, with only minor fluctuations in the absence of drugs. Conversely, treatment with carboplatin and particularly paclitaxel caused the subset distribution to change dramatically, then being skewed towards cell populations bearing stem cell characteristics that originally accounted for only a minor compartment. Conclusion:  Solid cancers are characterized by tremendous cellular complexity that is likely imposed by genetic, epigenetic and microenvironmental means. Small, preexisting cell subsets exhibiting stemness potential are selected during treatment and are ultimately responsible for disease recurrence and drug resistance. Our data show that multicolour flow cytometry can help to discover treatment-refractory cancer cell subsets for which novel targeted therapeutics should be designed for further improvement of cancer care.

Discovering drug-resistant cancer cell subsets through competitive repopulation M. Boesch1,2, A. G. Zeimet3, G. Gastl1, D. Wolf4, S. Sopper1 Internal Medicine V, Medical University Innsbruck, Innsbruck, Austria 2 Institute of Immunobiology, Kantonsspital St. Gallen, St. Gallen, Switzerland 3 Department of Gynecology and Obstetrics, Medical University Innsbruck, Innsbruck, Austria 4 Medical Clinic III, University Clinic Bonn, Bonn, Germany 1

Background:  Cytotoxic and targeted chemotherapies show good response rates in cancer patients and lead to states of remission in a relatively high proportion of cases. However, even patients in complete remission are not tumor-free and harbour residual tumor cells that obviously managed to evade the cytotoxic effects of primary therapy. Although microenvironmental cues are certainly also involved in this clinical phenomenon, a great deal of residual cancer is attributable to cell-intrinsic mechanisms of protection. Aim:  Modern single cell-based technologies as well as sequencing of primary versus metastatic lesions have revealed tremendous heterogeneity and cellular complexity of human tumors that provide a source of drug-resistant disease clones. Despite these technological advances however, the functional and clinical implications of intratumoral heterogeneity remain elusive. Here, we investigated the role of cellular heterogeneity for drug evasion in a gynaecological tumor entity prone to acquisition of chemoresistance. Methods:  We used multicolour flow cytometry to determine the degree of cellular heterogeneity present in both primary ovarian cancer tissue and established ovarian cancer cell lines. Heterogeneous cancer cell fractions were then exposed for ≥ 7 days to selective pressure exerted by the clinically relevant anticancer drugs carboplatin (1–50 µM) and paclitaxel (1–10 nM). The surviving cell fraction was finally screened by flow cytometry for the predominating cell population(s) so as to identify novel drug-resistant cancer cell subsets potentially involved in mediating disease recurrence. Results:  Both primary ovarian cancer samples and established cell lines harbour numerous cell subsets whose ultimate number is primarily determined by the quality and quantity of investigated markers (e.g., the marker combination CD24, CD49d, CD90, CD95, CD140a, CD184, CD338, HLA-ABC identi-

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P19 EuT-PK/PD: a phase IV study on sunitinib and pazopanib pharmacokinetics/ pharmacodynamics in patients with metastasized renal cell carcinoma A. Fritsch1, L. Bergmann2, B. Moritz3, M. Roessler3, F. Sörgel4, M. Diekstra5, H. J. Guchelaar5, U. Jaehde1, for the EuroTARGET consortium  epartment of Clinical Pharmacy, Institute of Pharmacy, D University of Bonn, Bonn, Germany 2 Cancer Center Rhein-Main, Medical Clinic II, University of Frankfurt, Frankfurt, Germany 3 Central European Society for Anticancer Drug Resarch— EWIV, Vienna, Austria 4 Institute for Biomedical and Pharmaceutical Research, Heroldsberg, Germany 5 Leiden University Medical Center, Leiden, The Netherlands 1

Background:  Targeted therapy is of increasing significance in the treatment of metastasized renal cell carcinoma (mRCC) with pazopanib and sunitinib as common first-line therapies. These multi-tyrosine kinase inhibitors provide great benefits in some patients but lack efficacy in others. Therefore, the identification of reliable predictive biomarkers is essential for therapy optimization. Methods:  EuT-PK/PD was conducted as a phase IV substudy of the European-wide non-interventional EuroTARGET project which aims at identifying and characterizing host and tumour-related predictive biomarkers. Main objective of the phase IV study is to develop pharmacokinetic/pharmacodynamics (PK/PD) models to describe the response to targeted therapy and to identify predictors for efficacy in mRCC patients using the software NONMEM®. Results:  In total, 43 patients, either treated with first-line pazopanib or sunitinib, were recruited in 10 German and Dutch study centers. Blood sampling and blood pressure measurements were carried out during the first 18 weeks of treatment, resulting in up to 12 samples per patients. Plasma concentrations of sunitinib, its active metabolite SU12662 and pazopanib were quantified by using validated LC/MS methods. Soluble VEGF receptor (sVEGFR) 2 and 3 levels were determined using commercially available ELISA kits and validated immunoassays.

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Abstracts Conclusion:  Structural models for sunitinib, SU12662 and the respective potential biomarkers developed by Lindauer et al. will serve as basis for modelling. Due to the lack of an established model for pazopanib and the sparse sampling in the underlying study, additional data from phase I and phase II studies provided by GSK is used for model development. Predictive performance of the potential biomarkers will be explored with PK/PD models by linking plasma concentrations of sunitinib, SU12662, and pazopanib with sVEGFR-2/3 levels and blood pressure. Furthermore, the influence of genetic predictors on PK/PD variability will be tested as covariates. This project receives funding from the European Union’s Seventh Framework Program (FP7/2007–2013) under grant agreement no. 259939

Results:  In Arm A, PTX doses were generally higher for both sexes than in Arm B, resulting in a higher risk for severe neutropenia for women in Arm A. While in Arm B, comparable exposure and neutrophil-time course were observed due to dose individualisation considering sex, age and the time that PTX concentrations were above the threshold of 0.05  µmol/L. Despite some misspecification on the population level, good individual predictions of PTX pharmacokinetics was observed. At the PD level, the model overpredicted neutrophil values increasingly over the treatment cycles. This could point towards a plausible bone marrow exhaustion process. Conclusion:  The evaluated population PK/PD model allows dose individualisation of 3-weekly paclitaxel when given in combination with carboplatin. To better describe neutropenia, the model will be refined, accounting for bone marrow exhaustion within the course of multiple chemotherapy cycles. Subsequently, the dosing algorithm will be re-evaluated.

P20 Dose-individualisation of paclitaxel in patients with advanced non-small cell lung cancer: exploiting modelling and simulation to refine dosing algorithm A. Henrich1,2, M. Joerger3, W. Huisinga4, C. Kloft1, Z. P. Parra-Guillen1  ept. Clinical Pharmacy & Biochemistry, Institute D of Pharmacy, Freie Universitaet Berlin, Berlin, Germany 2 Graduate Research Training program PharMetrX, Berlin, Germany 3 Dept. of Oncology & Haematology, Cantonal Hospital, St. Gallen, Switzerland 4 Institute of Mathematics, Universitaet Potsdam, Potsdam, Germany 1

Background:  3-weekly paclitaxel (PTX) combination chemotherapy is complicated by severe neutropenia and/or neuropathy in a substantial proportion of patients. In addition, complex pharmacokinetics (PK) and high interindividual variability suggest a potential benefit of dose individualisation. Concentration-time profiles of paclitaxel and associated neutropenia (pharmacodynamics, PD) have been described before by a population PK/PD model. Based on this PK/PD model, a dosing algorithm (Joerger et al, 2015) has been evaluated in a large prospective clinical trial (CEPAC-TDM, EUDRACT 2010-023688-16). Aim:  The present work investigates the feasibility and suitability of the paclitaxel population PK/PD model to predict the obtained trial data and explore a potential refinement of the model. Methods:  In the trial, patients were randomised into 2 arms, each comprising 183 patients. In Arm A, standard PTX dose (200 mg/m2) was administered 3-weekly for up to 6 cycles, while in Arm B the previously published algorithm was used to select initial and subsequent doses of PTX. All patients received concomittant carboplatin AUC 6 at 3-weekly intervals. PTX plasma concentrations 24 h after drug administration were obtained for Arm B. Neutrophils were quantified for both arms before drug administration and on day 15 of each cycle. Different model evaluation techniques as basic goodness of fit plots and visual predictive checks were used to assess predictive performance of the model (NONMEM 7.3, PsN 4.2 and Xpose4 4.5.3).

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P21 ASCO 2015: a prospective, multi-center study of individualized, pharmacokinetically (PK)guided dosing of 5-fluorouracil (5-FU) in metastatic colorectal cancer (mCRC) patients treated with weekly or biweekly 5-FU/ oxaliplatin containing regimens V. Kunzmann1, K. Link2, M. C. Miller3, S. Holdenrieder4, T. Bertsch2, L. Mueller5, Y.-D. Ko6, O. J. Stoetzer7, I. Suttmann8, J. Braess9, U. Jaehde10, M. Roessler11, B. Moritz11, S. Kraff10, A. Fritsch10, S. J. Salamone3, M. Wilhelm12 Universitätsklinikum Würzburg, Würzburg, Germany Klinikum Nürnberg Nord, Nürnberg, Germany 3 Saladax Biomedical Inc., Bethlehem, PA 4 University of Munich, Munich, Germany 5 Onkologische Schwerpunktpraxis Leer, Leer, Germany 6 Johanniter-Krankenhaus Bonn, Bonn, Germany 7 Hemato-Oncologic Clinic, Munich, Germany 8 Klinikum Dritter Orden Munich, Oncological Center, Munich, Germany 9 Krankenhaus Barmherziger Bruder, Regensburg, Germany 10 Pharmaceutical Institute, University Bonn, Bonn, Germany 11 CESAR Central European Society for Anticancer Drug Research – EWIV, Vienna, Austria 12 University Clinic for Internal Medicine V, Paracelsus Medical University, Nürnberg, Germany 1 2

Background:  Numerous studies have demonstrated that body surface area (BSA)-based dosing leads to under- and overexposure in a large number of patients. PK-guided dosing of 5-FU can optimize 5-FU exposure resulting in higher overall dose intensity with reduced toxicities and improved outcomes. This study was initiated to validate use of PK-guided 5-FU dosing for mCRC patients in clinical practice. Methods:  75 mCRC patients from 8 academic and/or community-based medical centers located throughout Germany received up to 6 cycles of infusional 5-FU according to either the AIO (n = 16), FOLFOX6 (n = 26) or FUFOX (n = 33) regimen. Initial dosing of infusional 5-FU for all patients was based on BSA and subsequent doses were adjusted according to the previous cycle 5-FU area under the concentration-time curve

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Abstracts (AUC) to target an AUC of 20 to 30  mg•h/L. Primary objective was to confirm that PK-guided dosing of 5-FU leads to an increased proportion of patients in the target AUC range at cycle 4 versus cycle 1. Secondary objective was to determine whether PK-guided 5-FU dose adjustment reduced treatment-related toxicities compared to historical patient groups. Results:  Average 5-FU AUC at cycle 1 was 20 + 15  mg•h/L, with 62 %, 32 % and 6 % of the patients below, within or above target AUC range, respectively. By cycle 4, average 5-FU AUC was 25 + 7 mg•h/L (p = 0.007), and a significantly higher proportion of patients were within the target 5-FU AUC range (55 %, p = 0.005). Fewer 5-FU-related grade 3–4 toxicities of diarrhea (5 %), nausea (3 %), fatigue (0 %) and mucositis (0 %) were observed compared to historical data (12 %, 9 %, 12 %, 15 %, respectively). Conclusion:  PK-guided adjustment of 5-FU dosing in routine clinical practice resulted in significantly higher 5-FU exposure, more patients achieving target exposure, and less 5-FU-related toxicities.

P22 ASCO 2015: open-label, randomized study of individualized, pharmacokinetically (PK)-guided dosing of paclitaxel combined with carboplatin in advanced Non-Small Cell Lung Cancer (NSCLC) patients M. Joerger1, J. Von Pawel2, S. Kraff3, J. R. Fischer4, W. Eberhardt5, T. Gauler6, L. Mueller7, N. Reinmuth8, M. Reck9, M. Kimmich10, F. Mayer11, H.-G. Kopp12, D. M. Behringer13, Y.-D. Ko14, M. Frueh15, R. A. Hilger16, M. Roessler17, B. Moritz17, U. Jaehde18 Cantonal Hospital, St. Gallen, Switzerland Pneumology Clinic, Asklepios Fachkliniken, Gauting, Germany 3 Institute of Pharmacy, University of Bonn, Bonn, Germany 4 Klinik Löwenstein, Löwenstein, Germany 5 Department of Medical Oncology, West German Cancer Center, University Hospital Essen, Essen, Germany 6 Department of Medicine (Cancer Research), University Hospital Essen, Essen, Germany 7 Oncological Practice, Leer, Germany 8 Krankenhaus Großhansdorf, Großhansdorf, Germany 9 Lung Clinic Grosshansdorf, Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Grosshansdorf, Germany 10 Klinik Schillerhöhe, Gerlingen, Germany 11 University Hospital, Medical Center II, Tuebingen, Germany 1 2

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 epartment of Oncology and Hematology, University D of Tuebingen, Tuebingen, Germany 13 Augusta-Kranken-Anstalt, Bochum, Germany 14 Johanniter-Krankenhaus Bon, Bonn, Germany 15 Cantonal Hospital St Gallen, St Gallen, Switzerland 16 University Hospital Essen, Essen, Germany 17 CESAR Central Office (CCO), CESAR Central European Society for Anticancer Drug Research—EWIV, Vienna, Austria 18 Pharmaceutical Institute, University Bonn, Bonn, Germany 12

Background:  Variability of chemotherapy exposure may cause severe toxicity or lack of efficacy. Paclitaxel (PTX) exposure (time above a plasma concentration of 0.05µM, Tc > 0.05) has been shown to predict toxicity. Whereas carboplatin dosing is adapted to kidney function, PTX dosing only accounts for body-surface area. We developed a PTX dosing algorithm for avoidance of supra- or subtherapeutic PTX exposure based on Tc > 0.05 determined from a single blood sample drawn 18–30 h after starting PTX infusion. This study was initiated to validate PK-guided PTX dosing in advanced NSCLC patients. Methods:  304 patients with advanced NSCLC were randomly assigned to receive up to 6 cycles of first-line 3-weekly carboplatin AUC 6 combined with PTX either at a standard dose of 200 mg/m2 (Arm A) or at a PK-guided dose (Arm B). Initial PTX dose in Arm B was between 150 to 200 mg/m2 based on age and sex, and subsequent PTX doses were adjusted according to the previous cycle PTX Tc > 0.05 to target a Tc > 0.05 between 26 and 31 h. Dose reductions were permitted in both arms for chemotherapy-associated toxicity. The study had a power of 90 % to detect a 11 % reduction of grade 4 neutropenia with PK-guided PTX dosing. Results:  Major patient characteristics were male gender in 67 %, current smokers in 38 %, ≥ 65 years of age in 50 %, performance status of 2 in 8 %, squamous-cell histology in 21 %. Compared to standard dosing, PK-guided dosing of PTX reduced the incidence of grade 4 neutropenia (measured on day 15 of each cycle) (15 % v 21 %, P = 0.029), grade ≥ 2 neuropathy (14 % v 27 %, P < 0.001), and grade ≥ 3 neuropathy (1 % v 8 %, P < 0.001). Median PTX dose at cycle 6 was significantly lower with PKguided dosing (132 v 197 mg/m2, P < 0.001), and the proportion of patients with supratherapeutic PTX exposure was reduced from 41 % in cycle 1 to 2 % in cycle 6. Objective response rate was 32 % and 29 % in Arms A and B (P = 0.70). Progression-free survival was 5.2 and 4.7 months in Arms A and B (hazard ratio 1.1, 95 % CI 0.8-1.4, P = 0.54). Conclusion:  PK-guided dosing of PTX improves the riskbenefit profile in patients with advanced NSCLC, primarily by a substantial reduction of PTX-associated neuropathy.

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