Eckhard Guenther

Abstract C214: Disorazol Z: A highly cytotoxic natural compound with antitumor properties.

In search of novel antitumor agents natural macrocyclic polyketides called Disorazols have been isolated from myxobacterium Sorangium cellulosum[1,2,3]. Here we show that Disorazol Z specifically produced by strain So ce427, possesses outstanding cytotoxicity with single digit to even subnanomolar EC50 values in a highly diverse panel of more than 60 different tumor cell lines. Interestingly, the maximum efficacy by which Disorazol Z inhibited cell growth increased with the incubation time, e.g. in the endometrium carcinoma cell line Hec1A reaching a maximum efficacy of about 25% after 24 hours, but about 75% efficacy after 72 hours at a saturating concentration of 100 nM, being approximately 100–200 fold above the respective EC50 values. Based on this observation, it was speculated that the mechanism of action of Disorazol Z is dependent on progressing through cell cycle. Indeed, this was supported by showing that in the cell line RKOp27Kip inducible expression of the cell cycle inhibitor p27Kip led to a complete loss of Disorazol Z cytotoxicity up to a concentration of 100 nM. In contrast, without cell cycle arrest Disorazol Z demonstrated high cytotoxic activity with an EC50 value of 0.54 nM in the same cell line. Furthermore, cell cycle analysis revealed that Disorazol Z arrested KB/HeLa cells in the G2/M phase of the cell cycle with an IC50 value of 0.8 nM. As expected this Disorazol Z-induced G2/M arrest induced apoptotic events, as demonstrated by Caspase 3/7 activation in HCT-116 cells with an EC50 value of 0.25 nM. In agreement with the above described behaviour Disorazol Z could be identified as tubulin binding agent by inhibiting in vitro tubulin polymerisation with a mean IC50 value of 3.3 μM as well as inducing multipolar spindle formation at low nanomolar concentrations in U-2 OS cells. The high divergence of the in vitro tubulin activity compared to the cellular efficacy may point to additional mechanisms of action for Disorazol Z. This working hypothesis may be supported by the fact that synergistic cytotoxic action of Disorazol Z and the tubulin binding agent Colchicin could be observed with combination indices down to about 0.5. Neither Taxol nor Vinblastine showed such synergism, both tubulin binders behaved nearly additive in combination with Disorazol Z. Currently, experiments are under way to identify the tubulin binding site for Disorazol Z. Additional ongoing studies focus on the evaluation of the mechanisms of action of this novel highly potent agent with antitumor properties. Further, we aim at evaluating the utility of Disorazol Z as cytotoxic component in a drug-targeting approach utilizing GPCR ligands as the targeting moieties for the treatment of GPCR overexpressing cancers. References: 1. Jansen et al. (1994), Liebigs Ann. Chem. 1994, 759–773 2. Irschik et al., (1995), Journal of Antibiotics 48, 31–35 2. Elnakady et al. (2004), Biochemical Pharmacology 67, 927–935 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C214.

Abstract 871: Dual inhibition of PI3K and Erk1/2 shows synergy and efficacy in human tumor cells, either by using drug combinations or novel dual PI3K/Erk inhibitors

The Ras/Raf/Mek/Erk and the PI3K/Akt signaling pathways are frequently deregulated in cancer due to activation of upstream receptors, decreased expression of tumor suppressors like PTEN or activating mutations of Ras, B-Raf or PI3K. Several clinical studies with kinase inhibitors targeting single members of the Ras/Raf/Mek/Erk or the PI3K/Akt pathway are ongoing. However, preclinical and clinical trials indicated limited success by single pathway inhibition and different resistance mechanisms were defined. Further clinical trials with drug combinations of Ras/Raf/Mek/Erk and PI3K/Akt suppressors have been initiated, suggesting a more favorable outcome than targeting only one single pathway, Ras/Raf/Mek/Erk or PI3K/Akt. We performed in-vitro combination experiments with Raf, Mek or Erk inhibitors and PI3K or Akt inhibitors in several human tumor cell lines. Strong synergy was achieved with various combinations including the Mek inhibitor CI-1040 and the PI3K inhibitor GDC-0941 (CI = 0.17) or the Erk inhibitor AEZS-131 and the PI3K inhibitor D-117073 (CI = 0.23) in A549 cells. Also Raf inhibitors like Sorafenib and Zelboraf were combined with different PI3K inhibitors, resulting in synergistic anti-proliferative activity. Due to the attractiveness of parallel inhibition of the Ras/Raf/Mek/Erk and PI3K/Akt pathways, we developed AEZS-136 that concurrently inhibits Erk1/2 (IC50 ∼ 50nM) and PI3K (IC50 ∼ 100nM) by an ATP competitive mode of action. Derivatives of the dual PI3K/Erk inhibitor were co-crystallized with Erk2 and PI3Kα enabling an optimization process by SAR driven medicinal chemistry. The anti-proliferative efficacy of AEZS-136 was evaluated in more than 40 human tumor cell lines and physico-chemical as well as in-vitro ADMET properties were widely assessed. Furthermore, the in-vivo pharmacokinetics and anti-tumor efficacy was explored. AEZS-136 was well tolerated and showed dose dependent inhibition of human colon tumor growth of up to 74% in a Hct116 mouse model. Here we present the concept of dual targeting of the Ras/Raf/Mek/Erk and the PI3K/Akt pathways, either by using drug combinations or our novel dual PI3K/Erk inhibitor. AEZS-136 is a small molecule in preclinical development showing a uniquely advantageous kinase inhibition profile. Broad clinical anti-tumor activity is expected for AEZS-136 in tumors with deregulated Ras/Raf/Mek/Erk and PI3K-Akt signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 871. doi:1538-7445.AM2012-871

Abstract 5476: LHRH receptor targeting as mechanism of anti-tumor activity for cytotoxic conjugates of Disorazol Z with the LHRH receptor agonistic peptide D-Lys6-LHRH.

Background For drug-targeting aimed at the treatment of LHRH receptor overexpressing cancers the LHRH receptor agonistic peptide D-Lys6-LHRH has been conjugated to the novel highly cytotoxic natural compound Disorazol Z. As shown previously by early proof of concept in an ovary cancer xenograft model, differentially linked conjugates possess a high potential regarding the treatment of LHRH-R positive tumors [1]. Here we present further characterization of these conjugates with respect to PK/PD parameter and provide evidence that LHRH receptor targeting significantly contributes to their mechanism of action. Materials and Methods LHRH-R competitive binding, calcium release and cytotoxic activity were measured by Tag-Lite technology (Cisbio), and Fluo-4 (Invitrogen) or Resazurin-based detection, respectively. PK parameter were assessed by standard procedures followed by LC-MS/MS analysis. For the xenograft studies, tumor fragments were transplanted subcutaneously in female nude mice and treatment was started at a tumor size of approx. 100 mm3. Results Disorazol Z - D-Lys6-LHRH conjugates showed varying cytotoxic activity from single digit nanomolar to higher submicromolar EC50 values but comparable LHRH receptor binding and activation in the low nanomolar EC50 range. Comparison in ovarian and triple negative breast cancer xenograft models revealed potent inhibition of tumor growth for the conjugates, whereas equimolar dosing of Disorazol Z failed to reach statistical significance. PK analysis showed substantial plasma levels for the conjugates with only minor release of Disorazol Z, pointing to stabilization by conjugation and demonstrating reasonable half-life of the intact conjugates as prerequisite for tumor targeting. In the same tumor models, competition by previous administration of D-Lys6-LHRH provides evidence for LHRH receptor targeting as mechanism of action. Increased sensitivity of LHRH receptor overexpressing cells towards conjugate cytotoxicity, i.e. leading to about 30 fold decreased EC50 values for the conjugate AEZS-125, further supports the LHRH-R dependency of conjugate efficacy. Conclusions The presented LHRH receptor-dependent efficacies of Disorazol Z - D-Lys6-LHRH conjugates in vitro and in mouse xenograft models support the principle of tumor targeting by the LHRH receptor as already employed by the drug candidate AEZS-108, which is currently in phase II clinical studies. Preclinical development of Disorazol Z conjugates will be started in the first half of 2013. Citation Format: Babette Aicher, Tilmann Schuster, Lars Blumenstein, Antje Schubert, Carsten Grundker, Joerg B. Engel, Olaf Ortmann, Rolf Mueller, Eckhard Guenther, Matthias Gerlach, Michael Teifel. LHRH receptor targeting as mechanism of anti-tumor activity for cytotoxic conjugates of Disorazol Z with the LHRH receptor agonistic peptide D-Lys6-LHRH. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5476. doi:10.1158/1538-7445.AM2013-5476

Abstract 2877: Perifosine alone and in combination with antimetabolites interferes with NF-kB pathway activation in colon cancer cell lines

Background: Perifosine, a novel, first-in-class, oral Akt inhibitor, in combination with the antimetabolite Capecitabine is currently in Phase III clinical trials for the treatment of refractory advanced colorectal cancer. We have shown previously that Perifosine in combination with antimetabolites displayed synergistic cytotoxicity and apoptosis in colon cancer and other cell lines [1]. Constitutive NF-kB activation is frequently observed in colorectal cancer and, moreover, is associated with resistance to chemotherapy [2]. Hence we analysed, wether downregulation of the NF-kB pathway contributes to Perifosine mechanism of action and to the observed synergism with antimetabolites in colon cancer cell lines. Methods: Cellular NF-kB activation in nuclear extracts was measured by the TransAm NF-kB p65 ELISA (Active Motif, USA) monitoring binding of NF-kB to its response element. To allow for higher throughput of NF-kB pathway analysis a monoclonal SW620 colon cancer cell line ectopically expressing a NF-kB response element driven reporter gene (Promega, USA) was established. Cellular cytotoxicity and apoptosis assays as well as western blotting were performed according to standard procedures. The analysis of drug combinations was conducted by CalcuSyn software (Biosoft, UK) [3]. Results: Perifosine pretreatment decreased TNFα-induced NF-kB activation in SW620 cells in both independent assay sytems with IC 50 values of about 7-10 µM. This effect seems to be specific, since the results were not mimicked by cytotoxic effects of Perifosine. Some of the antimetabolites alone upregulated NF-kB activity in the reporter gene assay. In the presence of these antimetabolites Perifosine was still capable to downregulate NF-kB activity. Conclusions: In summary, these results demonstrate that downregulation of the NF-kB pathway may contribute to the mechanism of action of Perifosine. Moreover, cotreatment with Perifosine has the potential to interfere with the development of antimetabolite resistance due to NF-kB activation in colon cancer. Further experiments addressing Perifosine9s mechanism of action in combination with antimetabolites in gastric cancer cell lines are ongoing. References [1] Aicher et al. (2010), Perifosine in combination with antimetabolites induces synergistic effects on cytotoxicity and apoptosis in human colon, multiple myeloma, breast, renal, and liver tumor cell lines. AACR-NCI-EORTC International Conference, Berlin, Germany. [2] Sakamoto and Maeda (2010), Expert. Opin. Ther. Targets 14, 593-601 [3] Chou (2006), Pharmacological Reviews 58, 621-681 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2877. doi:1538-7445.AM2012-2877

Abstract 3563: A highly selective Erk1/2 Inhibitor with in-vivo anti tumor potency

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Mutations in receptor tyrosine kinases, Ras or B-Raf frequently manifest constitutive activation of the Ras/Raf/Mek/Erk pathway in a variety of human cancers. The mitogen activated protein kinase Erk1/2 is an important regulator of cell proliferation and was shown to be over-activated by these dysregulated or mutated upstream signals. AEZS-131 selectively inhibits Erk1/2 with an IC50 of 4 nM, blocks cellular Rsk-1 phosphorylation, modulates downstream cellular substrate activation, arrests tumor cells in G1 and inhibits the growth of multiple human tumor cell lines in the nanomolar range. We have studied the response of human cancer cells (Hct116) in in-vivo mouse xenograft experiments. AEZS-131 significantly inhibited tumor growth at daily doses of 30mg/kg. Currently, AEZS-131 is undergoing in-vivo combination experiments with PI3K inhibitors. Since pharmacological inhibition of Erk1/2 reverses Ras and Raf activation also in cells demonstrating resistancy to common Raf inhibitors like GDC-0879 and PLX4720, it appears likely to put more attention on the downstream kinase Erk1/2 as therapeutic target. Furthermore, in instances of coincident activation of the Raf and PI3K pathways, combinations of AEZS-131 and PI3K inhibitors may prove efficacious. Here we present the in-vitro and in-vivo characterisation of the first in-class Erk1/2 inhibitor showing in-vivo efficacy as single therapy agent. Early development of the Erk1/2 inhibitor AEZS-131 by AEterna Zentaris is integral part of our in-house kinase research program comprising the investigation of different compounds for single Erk1/2 inhibition, single PI3K inhibition and dual kinase inhibitions. All compounds are exclusively synthesized by our Drug Discovery Department. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3563. doi:10.1158/1538-7445.AM2011-3563

Abstract 4474: Dual inhibitors for PI3K and Erk induce growth inhibition of tumor cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The Ras/Raf/Mek/Erk and the PI3K-Akt signaling pathways are prime targets for drug discovery in proliferative diseases such as cancer. The results of research to date indicate that both the MAPK and the PI3K signaling pathways represent therapeutic intervention points for the clinical treatment of malignant tumors. Our multi-parameter optimization program for kinase inhibitor selectivity, cellular efficacy, physico-chemical and in-vitro ADMET properties has led to the identification of a small molecular compound class with an uniquely advantageous dual kinase inhibition profile. These ATP competitive compounds inhibit Erk and PI3K in the nanomolar range and exert high selectivity against other serine threonine and tyrosine kinases. The anti-tumor efficacy of these dual kinase inhibitors was evaluated in diverse human tumor cell lines like HCT116, A549, MDA-MB 468, PC-3 and others. Physicochemical and in-vitro ADMET and safety parameters have been widely assessed. Furthermore in-vivo pharmacokinetic experiments showed plasma profiles expected to result in beneficial in-vivo anti-tumor efficacy. Here we present the key characteristics of the compound class that led to the selection of AEZS-132 for in-vivo experiments with tumor bearing nude mice. The optimization of ADME and physicochemical properties such as solubility, permeability and metabolic stability by medicinal chemistry is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4474.