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PDF file - 75K, High responsiveness of c-Met-addicted cell lines to treatment with EMD 1214063 and EMD 1204831. The impact of EMD 1214063 and EMD 1204831 on tumor cell viability (IC50) was tested in vitro, using a panel of tumor cell lines specifically characterized for their levels of c-Met expression, c-Met auto-phosphorylation, and HGF production. The human colorectal carcinoma cell line HT29 and the human lung carcinoma cell line A549 displayed low levels of c-Met/phospho-c-Met, while the lung carcinoma cell line EBC-1 and the gastric carcinoma Hs746T and MKN-45 exhibited high levels of both c-Met and phospho-c-Met, due to the described c-Met gene amplification (3). The c-Met-"addicted" cell lines (EBC-1 (4), Hs746T (3) and MKN-45 (3)), exhibited high sensitivity to both EMD 1214063 and EMD 1204831. In contrast, high concentrations of EMD 1214063 were required to induce 50% reduction in the viability of HT29 and A549. Similar to EMD 1214063, EMD 1204831 also showed strong impact on the viability of Hs746T cells (IC50 = 4.72E-09), EBC-1 (IC50 = 1.21E-08), and MKN-45 (IC50 = 4.93E-08). Data are cumulative of three separate experiments; the IC50 represent the mean of three separate experiments.

PDF file - 184K, EMD 1214063 and EMD 1204831 display anti-tumor activity in HGF-dependent and HGF-independent xenograft tumor models. Mice bearing subcutaneous tumors derived from the human gastric Hs746T cell line (A and B) or the glioblastoma U87MG cell line (C and D) were administered daily the indicated doses of EMD 1214063 (A and C), EMD 1204831 (B and D), or vehicle for the indicated treatment duration. Each data point represents the mean � SD of the calculated tumor volumes observed in experimental groups including ten mice. Statistical significance was evaluated by one-way ANOVA (Kruskal-Wallis, Dunn's post test). Asterisks indicate statistically significant differences between treated and control experimental groups (P≤0.05).

PDF file - 255K, Effective inhibition of in vitro tumor cell migration by EMD 1214063 and EMD 1204831. A classical "wound healing" test was performed to assess the impact of EMD 1214063 (A) and EMD 1204831 (B) on tumor cell migration. In brief, H441 lung cancer cells were seeded on day 1 (3 x 105 cells/well in 24-well plate), serum-starved on day 2, and treated on day 3 with the indicated concentrations of the EMD compounds for 1 h. After "scratching" the cell layer, cells were incubated in serum-free medium containing 100 ng/mL HGF and the indicated concentrations of the compounds for 24h. Controls were untreated cells with (left) or without HGF (right). At 0 h and 24 h, cells were washed, fixed with ice-cold methanol, and stained with crystal violet (0.05% in 50% ethanol). Treatment with EMD 1214063 or EMD 1204831 resulted in a strong inhibition of tumor cell migration, preventing wound healing. Pictures were taken at 5x magnification. Data are representative of two independent experiments.

PDF file - 53K, Characteristics of the antibodies used in the study. The specificity, isotype, clone number, and commercial source of the antibodies used throughout the study are indicated. All antibodies were used according to the manufacturer's instructions.

Purpose: The mesenchymal–epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process that is physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with a variety of human malignancies including cancers of the lung, kidney, stomach, liver, and brain. In this study, we investigated the properties of two novel compounds developed to selectively inhibit the c-Met receptor in antitumor therapeutic interventions.Experimental Design: The pharmacologic properties, c-Met inhibitory activity, and antitumor effects of EMD 1214063 and EMD 1204831 were investigated in vitro and in vivo, using human cancer cell lines and mouse xenograft models.Results: EMD 1214063 and EMD 1204831 selectively suppressed the c-Met receptor tyrosine kinase activity. Their inhibitory activity was potent [inhibitory 50% concentration (IC50), 3 nmol/L and 9 nmol/L, respectively] and highly selective, when compared with their effect on a panel of 242 human kinases. Both EMD 1214063 and EMD 1204831 inhibited c-Met phosphorylation and downstream signaling in a dose-dependent fashion, but differed in the duration of their inhibitory activity. In murine xenograft models, both compounds induced regression of human tumors, regardless of whether c-Met activation was HGF dependent or independent. Both drugs were well tolerated and induced no substantial weight loss after more than 3 weeks of treatment.Conclusions: Our results indicate selective c-Met inhibition by EMD 1214063 and EMD 1204831 and strongly support clinical testing of these compounds in the context of molecularly targeted anticancer strategies. Clin Cancer Res; 19(11); 2941–51. ©2013 AACR.

PDF file - 263K, Effects of EMD 1214063 on c-Met phosphorylation and downstream signaling molecules (see Figure 2C). EBC-1 cells were cultured in vitro in control medium (medium + vehicle) (lane 1), or in the presence of increasing concentrations of EMD 1214063 (A-D) or EMD 1204831(E-H) (0.01nM to 30�M, as depicted in lanes 2-15). Upon cell lysis, the levels of total c-Met, AKT, p42/44 MAPK (A, E), Gab1 (C, G), as well as phospho-c-Met, phospho-Akt, phospho-p42/44 MAPK (B, F), and phospho-Gab1 (D, H) were assessed by Western blot analyses. The levels of cofilin were used to ascertain equal loading (A, B, and F). Marked reductions in the levels of c-Met phosphorylation were observed for EMD 1214063 and EMD 1204831 at concentrations ≥0.1 �M (lanes 10-15). Data are representative of two independent experiments; cumulative results are shown in Fig. 2C.

PDF file - 77K, In vivo tumor growth inhibition by EMD 1214063 and EMD 1204831. The effect of EMD 1214063 and EMD 1204831 on tumor growth (T/C) was evaluated in in vivo xenograft models, using a panel of human tumor cell lines. Both inhibitors were highly active on tumor models with different types of aberrant c-Met upregulation, that (i) harbor MET gene amplification, (ii) express high levels of c-Met, or (iii) exhibit an HGF/c-Met autocrine loop, while inactive in models without signs of active c-Met signaling.

PDF file - 74K, Effective inhibition of c-Met phosphorylation in U87MG cells. To assess the pharmacodynamic activity of EMD 1214063 and EMD 1204831 in glioblastoma, U87MG glioblastoma cells were exposed for 2 h to either EMD compound. After lysis, the phosphorylation levels of c-Met Y1234/Y1235 auto-phosphorylation site (A) and of c-Met Y1349 adaptor binding site (B) were assessed and correlated with the levels of total c-Met by a Luminex-based assay. Y1234/Y1235 EC50 values for EMD 1214063 and EMD 1204831 were 0.003 �M and 0.009 �M, respectively. For Y1349, the EC50 values were 0.011 �M and 0.005 �M for EMD 1214063 and EMD 1204831, respectively. While a dose-dependent reduction in the levels of c-Met phosphorylation could be observed upon exposure to both compounds, the levels of total c-Met remained unchanged (C). Data are cumulative of three independent experiments.

PDF file - 45K, ATP competition studies with EMD 1214063. EMD 1214063 inhibited c-Met phosphorylation in an ATP-competitive fashion. In brief, binding competition of EMD 1214063 and ATP to c-Met was analyzed by a kinase reaction followed by Western blotting. ATP in the indicated concentrations was mixed with 1 nM EMD 1214063 and added to 100 ng recombinant human c-Met kinase (Carna Biosciences 08-151). The reaction mixtures were incubated at room temperature for 30 min. The reaction was stopped by addition of 4x LDS Sample Buffer (Invitrogen NP-0007) containing 25 mM DTT and heating at 80{degree sign}C for 20 min. The reaction mixtures were analyzed by Western blotting using 1 ng c-Met kinase per lane in gel electrophoresis. After blotting on nitrocellulose, detection was performed with an anti-phosphotyrosine antibody (clone PY99, Santa Cruz sc-7020) and an anti-GST-tag antibody (clone B-14, Santa Cruz sc-138) as a loading control. Chemiluminescence read out was measured with a VersDoc analyzer (BioRad) and band densities were quantified with the inherent software (ImageLab).

PDF file - 240K, Dose-dependent inhibition of c-Met auto-phosphorylation in vivo. Panel A: Mice bearing established tumors, derived from s.c. injection of Hs746T gastric cancer cells, received a single dose of 3 mg/kg , 10 mg/kg , 30 mg/kg , and 100 mg/kg EMD 1214063. The pharmacokinetic profile of EMD 1214063 was defined by measuring the compound levels in plasma and tumor tissue. The lower limits of detection for EMD 1214063 were 0.007 �M (4 ng/mL) in the plasma and 0.037 �M (20 ng/mL) in tumor samples. The levels of c-Met auto-phosphorylation (Y1234/Y1235) were assessed by Western blot analysis and used as a pharmacodynamic marker of activity. Each data point represents the mean � SD of the values obtained from four mice per experimental group. A data point was not shown if any of the values within the experimental group was below the detection limit. Panel B: A similar experimental approach to that already described for EMD 1214063 was used to define the pharmacokinetic profile and pharmacodynamic effects of EMD 1204831. In brief, mice bearing established Hs746T subcutaneous tumors received a single dose of 3 mg/kg, 10 mg/kg, 30 mg/kg, or 100 mg/kg EMD 1204831. In the pharmacokinetic studies, the lower limits of detection for EMD 1204831 were 0.014 �M (8 ng/mL) in the plasma and tumor samples. The pharmacodynamic effects were determined by Western blot analyses of c-Met auto-phosphorylation (Y1234/Y1235). Each data point represents the mean � SD of the values obtained from four mice per experimental group. A data point was not shown if any of the values within the experimental group was below the detection limit.

PDF file - 54K, Dose-dependent induction of caspase-3 cleavage by EMD 1204831. Mice bearing established (100-200mm3) Hs746T tumors received a single dose of 3, 10, 30, and 100 mg/kg of EMD 1204831. The effects on caspase-3 activation were determined by Western blot analyses, using an antibody (clone 5A1E) specifically recognizing the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp 175. This antibody does not recognize full-length caspase-3 or other cleaved caspases. Treatment with EMD 1204831 resulted in a transient increase in cleaved caspase-3 that peaked at 24 h and declined at 48 h. Data are representative of three separate experiments.

Pharmacological inhibition of the controlling immunity pathway enzymes arginases 1 and 2 (ARG1 and ARG2) is a promising strategy for cancer immunotherapy. Here, we report the discovery and development of OATD-02, an orally bioavailable, potent arginases inhibitor. The unique pharmacological properties of OATD-02 are evidenced by targeting intracellular ARG1 and ARG2, as well as long drug-target residence time, moderate to high volume of distribution, and low clearance which may jointly provide a weapon against arginase-related tumor immunosuppression and ARG2-dependent tumor cell growth. OATD-02 monotherapy had an antitumor effect in multiple tumor models and enhanced an efficacy of the other immunomodulators. Completed non-clinical studies and human pharmacokinetic predictions indicate a feasible therapeutic window and allow for proposing a dose range for the first-in-human clinical study in cancer patients.

Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

Purpose: The mesenchymal–epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process that is physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with a variety of human malignancies including cancers of the lung, kidney, stomach, liver, and brain. In this study, we investigated the properties of two novel compounds developed to selectively inhibit the c-Met receptor in antitumor therapeutic interventions. Experimental Design: The pharmacologic properties, c-Met inhibitory activity, and antitumor effects of EMD 1214063 and EMD 1204831 were investigated in vitro and in vivo, using human cancer cell lines and mouse xenograft models. Results: EMD 1214063 and EMD 1204831 selectively suppressed the c-Met receptor tyrosine kinase activity. Their inhibitory activity was potent [inhibitory 50% concentration (IC50), 3 nmol/L and 9 nmol/L, respectively] and highly selective, when compared with their effect on a panel of 242 human kinases. Both EMD 1214063 and EMD 1204831 inhibited c-Met phosphorylation and downstream signaling in a dose-dependent fashion, but differed in the duration of their inhibitory activity. In murine xenograft models, both compounds induced regression of human tumors, regardless of whether c-Met activation was HGF dependent or independent. Both drugs were well tolerated and induced no substantial weight loss after more than 3 weeks of treatment. Conclusions: Our results indicate selective c-Met inhibition by EMD 1214063 and EMD 1204831 and strongly support clinical testing of these compounds in the context of molecularly targeted anticancer strategies. Clin Cancer Res; 19(11); 2941–51. ©2013 AACR.

Isolated limb perfusion (ILP) with melphalan and tumor necrosis factor (TNF)-α is used to treat bulky, locally advanced melanoma and sarcoma. However, TNF toxicity suggests a need for better-tolerated drugs. Cilengitide (EMD 121974), a novel cyclic inhibitor of alpha-V integrins, has both anti-angiogenic and direct anti-tumor effects and is a possible alternative to TNF in ILP. In this study, rats bearing a hind limb soft tissue sarcoma underwent ILP using different combinations of melphalan, TNF and cilengitide in the perfusate. Further groups had intra-peritoneal (i.p.) injections of cilengitide or saline 2 hr before and 3 hr after ILP. A 77% response rate (RR) was seen in animals treated i.p. with cilengitide and perfused with melphalan plus cilengitide. The RR was 85% in animals treated i.p. with cilengitide and ILP using melphalan plus both TNF and cilengitide. Both RRs were significantly greater than those seen with melphalan or cilengitide alone. Histopathology showed that high RRs were accompanied by disruption of tumor vascular endothelium and tumor necrosis. Compared with ILP using melphalan alone, the addition of cilengitide resulted in a three to sevenfold increase in melphalan concentration in tumor but not in muscle in the perfused limb. Supportive in vitro studies indicate that cilengitide both inhibits tumor cell attachment and increases endothelial permeability. Since cilengitide has low toxicity, these data suggest the agent is a good alternative to TNF in the ILP setting.

In a high-throughput screening campaign for c-Met kinase inhibitors, a thiadiazinone derivative with a carbamate group was identified as a potent in vitro inhibitor. Subsequent optimization guided by c-Met-inhibitor X-ray structures furnished new compound classes with excellent in vitro and in vivo profiles. The thiadiazinone ring of the HTS hit was first replaced by a pyridazinone followed by an exchange of the carbamate hinge binder with a 1,5-disubstituted pyrimidine. Finally an optimized compound, 22 (MSC2156119), with excellent in vitro potency, high kinase selectivity, long half-life after oral administration and in vivo anti-tumor efficacy at low doses, was selected as a candidate for clinical development.

A previously developed capillary electrophoresis method for the simultaneous separation and enantioseparation of thalidomide (TD) and its hydroxylated metabolites was extended to one additional biotransformation product. The dual chiral selector system using native beta-cyclodextrin (beta-CD) and the negatively charged sulfobutyl-beta-CD (SBE-beta-CD) was slightly modified up to a concentration of 12 mg/ml running buffer of each CD. The carrier mode in which these buffer additives transport the neutral compounds to the detector as well as the use of a polyacrylamide-coated capillary were necessary to achieve reproducible enantioseparations of all eight analytes.

Common strategies to optimize prodrugs use either in vitro or rodent in vivo approaches, which do not consider elimination pathways that do not result in the generation of the desired product or might be misleading because of species differences, respectively. As a step forward, we have incorporated a novel application of hepatocytes into our prodrug optimization strategy to increase the bioavailability of a poorly soluble drug candidate by attaching a charged ester linker. The model involves the incubation of hepatocytes from multiple species in serum-containing medium to mimic formation as well as simultaneous metabolism of both prodrug and active drug. Using this strategy, a correlation between the in vitro AUC and the AUC after intravenous administration was obtained for active drug formation in several species. Moreover, hepatocytes correctly predicted the likelihood of undesired exposure with nonhydrolyzed prodrug. This novel approach enabled us to identify several prodrugs, which showed improved exposure over a wide dose range. Furthermore, a strategy was developed resulting in a decision tree that can be used to determine the applicability of the hepatocyte model in the screening process.

Objectives: To evaluate the dose/exposure/target inhibition/tumor growth relationship of MSC2156119J, an orally administered, reversible, ATP-competitive, highly potent and selective c-Met receptor tyrosine kinase inhibitor, in order to determine the recommended phase II dose (RP2D) for the program. Methods: Interim pharmacokinetic (PK) and biomarker (PD) data, i.e. plasma concentrations and c-Met inhibition in tumor biopsies from an ongoing first-in-human (FIM) solid tumor trial of MSC2156119J, were analyzed using the population modeling approach. The exposure/target inhibition relationship established in rich sampled KP-4 xenografted mice was utilized as prior information to develop the human model. Model-based simulations of the c-Met inhibition profiles in humans, aiming for a level that achieved tumor regression in mice, helped to determine the biologically active dose of MSC2156119J. Results: During the FIM study, the maximum tolerated dose of MSC2156119J was not reached at a dose of 1,400 mg/day. Instead, a biologically active dose was selected as the RP2D, based on a translational modeling approach that integrated the quantitative relationship between dose, exposure, and target as well as tumor inhibition in humans and in a preclinical model. In KP-4 xenografted mice, tumor c-Met inhibition (quantified as normalized phosphorylated c-Met expression [pMET]) was described by a turnover full inhibitory Imax model, and tumor growth inhibition was best described by the Simeoni model. Simulations demonstrated that nearly complete pMET inhibition (≥95%) is required for tumor stasis or even regression. In the FIM study, a one-compartment linear model with an absorption transit compartment best described the PK of MSC2156119J at doses of 30-700 mg. The turnover model developed from mice data was applied to evaluate the level of c-Met inhibition in paired human tumor biopsies (taken pre- and on-treatment). System turnover parameters for pMET were set equal to those estimated in mice, while the treatment potency (1/IC50 of the inhibition of build-up) of MSC2156119J in humans was found to be 1.7-fold higher than that in mice. Assuming a 30% inter-individual variability in IC50, human simulations suggested that a 500-mg once-daily dose regimen of MSC2156119J could achieve continuous pMET inhibition of ≥95% in 90% of the population. The model and simulation will be confirmed using additional PK data and further tumor biopsies. Conclusions: c-Met inhibition in human tumor lesions was described by a turnover model developed in KP-4 xenografted mice. Using this translational modeling and simulation approach and human PK and target inhibition data from paired tumor biopsies, a biologically active dose of 500 mg was proposed as the RP2D for MSC2156119J. This dose achieves continuous pMET inhibition of ≥95% in 90% of the population and has been adopted for the current development program for MSC2156119J. Citation Format: Wenyuan Xiong, Samer El Bawab, Friedhelm Bladt, Michael Meyring, Manfred Klevesath, Gerald Falchook, David Hong, Andreas Johne, Pascal Girard. Model-based phase II dose selection of c-Met inhibitor MSC2156119J. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4510. doi:10.1158/1538-7445.AM2015-4510

A previously developed capillary electrophoresis method for the simultaneous separation and enantioseparation of thalidomide (TD) and its hydroxylated metabolites was extended to one additional biotransformation product. The dual chiral selector system using native beta-cyclodextrin (beta-CD) and the negatively charged sulfobutyl ether-beta-CD (SBE-beta-CD) was slightly modified up to a concentration of 12 mg/mL running buffer of each CD. The carrier mode in which these buffer additives transport the neutral compounds to the detector as well as the use of a polyacrylamide-coated capillary were necessary to achieve reproducible enantioseparations of all eight analytes. The optimized method was applied to the analysis of the in vitro biotransformation of TD by rat liver microsomes. The S-enantiomer undergoes metabolism preferentially by hydroxylation in the phthalimide ring, whereas R-(+)-TD is mainly transformed to diastereomeric 5'-hydroxythalidomide (5'-OH-TD) pairs. The chiral capillary electrophoresis of incubation samples of TD enantiomers in combination with X-ray diffraction data allowed us to determine the absolute configuration of all metabolites and furthermore to follow the enantio- and stereoselective effects of metabolism in detail.

The separation of thalidomide (TD) and its hydroxylated metabolites including their simultaneous enantioseparation was studied in capillary electrophoresis (CE) using four different randomly substituted charged cyclodextrin (CD) derivatives, the combinations of some of them with each other, and beta-CD. TD, as well as two metabolites recently found in incubations of human liver microsomes and human blood, 5-hydroxythalidomide (5-OH-TD) and one of the diastereomeric 5'-hydroxythalidomides (5'-OH-TD), are neutral compounds. Therefore, they were resolved using charged chiral selectors in CE. Two different separation modes (normal polarity and carrier mode) and two different capillaries (fused-silica and polyacrylamide-coated) were tested. Based on the behavior of the individual CDs, their designed combinations were selected in order to improve the separation selectivity and enantioselectivity. Under optimized conditions all three chiral compounds and their enantiomers were resolved simultaneously.

High-performance liquid chromatography (HPLC), nano-HPLC, capillary electrochromatography (CEC) and on-line HPLC–atmospheric pressure chemical ionization mass spectrometry (APCI-MS) techniques were used for the identification and detailed characterization of two new metabolites of the former sedative drug thalidomide (TD). The advantages of nano-HPLC and CEC are higher peak efficiency and a drastic decrease in the analysis time, which, together with lower sample dilution during the analyses, allowed to obtain a detection sensitivity that was comparable to HPLC with common-sized columns. Both, nano-HPLC and CEC could be realized in the commercially available capillary electrophoresis system HP3D. On-line HPLC–APCI-MS coupling is a very useful technique for the rapid identification of metabolites without any need for reference compounds.

The mesenchymal-epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with human malignancies, including hepatocellular carcinomas (HCC). The aim of this study was to evaluate the effect of a novel, highly selective c-Met inhibitor, MSC2156119J (EMD 1214063), in hepatocellular cancer models. MHCC97H is a human HCC cell line co-expressing c-Met and HGF, and exhibiting high metastatic potential. The effect of MSC2156119J on MHCC97H was evaluated in a Balb/c nu/nu murine xenograft model in a subcutaneous and orthotopic setting. Tumor-bearing mice were orally treated with 100 mg/kg MSC2156119J (5 days on/2 days off). In the subcutaneous setting, MSC2156119J resulted in tumor regression in 10/10 mice, achieving tumor eradication in 9/10 mice. In the orthotopic model, MHCC97H tumor fragments proliferate in the liver and invariably metastasize to the lung (100%). MSC2156119J treatment started 7 days after tumor fragment implantation. After 5 weeks of treatment, mice were necropsied and a series of parameters were assessed. Primary tumor size and weight, and circulating Alpha-Feto-Protein levels were significantly lower in mice treated with MSC2156119J compared to controls (p To better predict the clinical efficacy of MSC2156119J, 9 patient-derived primary HCC explants models were used. The explants were subdivided into 3 groups, based on their c-Met levels and HGF expression. Each group comprised 3 tumors expressing high, intermediate, and low levels of c-Met and HGF, respectively. Mice were treated with MSC2156119J, sorafenib, or a combination of both. MSC2156119J (100 mg/kg/5 out of 7 days) strongly inhibited tumor growth in 4/9 models (TGD range of 370% to 41%). Analysis of intratumoral c-Met, phospho c-Met and HGF levels indicated that explants with high levels of c-Met and HGF were more sensitive to MSC2156119J than low-expressing models. In 3/4 responsive models, MSC2156119J exhibited a better anti-tumor activity than sorafenib, while sorafenib was more efficacious in a model characterized by intermediate c-Met/HGF expression. MSC2156119J was not efficacious in models exhibiting low or no signs of c-Met signalling. When used in combination, MSC2156119J did not enhance the activity of sorafenib. MSC2156119J monotherapy was well tolerated while sorafenib alone and in combination with MSC2156119J induced significant body weight loss. Overall, these data indicate that MSC2156119J may be a valuable therapeutic option for liver cancers with high levels of c-Met expression/activation. Citation Format: Friedhelm Bladt, Andree Blaukat, Dieter Dorsch, Manja Friese-Hamim, Michael Meyring, Oliver Schadt. The c-Met inhibitor MSC2156119J effectively inhibits growth of liver cancer models. [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 925. doi:10.1158/1538-7445.AM2013-925

c-Met is a receptor tyrosine kinase that has hepatocyte growth factor as its ligand. Evidence from biochemical and human genetic studies indicate that c-Met is one of the most frequently activated tyrosine kinases in human cancer. Dysregulated c-Met signaling can result in the development of tumors highly dependent on c-Met. Furthermore, enhanced c-Met signaling is likely involved in conferring resistance to epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) inhibitors. The orally available EMD 1214063 and EMD 1204831 inhibit c-Met activity in a potent and highly selective fashion, and are currently being evaluated in cancer patients. Here we report the activity of these compounds in combination with inhibitors of EGFR and VEGF in various tumor xenograft models. EMD 1214063 was tested alone and in combination with EGFR inhibitors (erlotinib and cetuximab) in xenografts of erlotinib-resistant tumor cells, namely the lung cancer H1975 and the NCI-H441 non-small-cell lung cancer (NSCLC) cell lines. The H1975 cell line expresses moderate amounts of c-Met, and EMD 1214063 was inactive as a single agent (treatment group/control group [T/C] 78%; tumor growth delay [TGD] 2 days). Cetuximab was active with a T/C of 13% inducing a TGD of 21 days. The combination of EMD 1214063 and cetuximab was active with a T/C of α1% and a TGD of 42 days. The NCI-H441 cell line is characterized by constitutive c-Met overexpression and is sensitive to c-Met inhibitors in vivo. EMD 1214063 (100 mg/kg) was active inducing a T/C of 20%, while erlotinib alone (30 mg/kg) was not efficacious. However, the combination of EMD 1214063 with erlotinib enhanced tumor growth inhibition, induced partial tumor regression in 2/9 mice, and delayed tumor re-growth. These data are compatible with the hypothesis that cMet pathway activation confers resistance to EGFR inhibitors. We additionally investigated the combination of either EMD 1214063 or EMD 1204831 with VEGF inhibitors in NSCLC models. In NCI-H441 xenografts, EMD 1204831 produced a modest T/C (66%) at the suboptimal dose of 25 mg/kg/bid, similarly to aflibercept at 40 mg/kg (T/C 55%). In contrast, the combination of both compounds yielded a T/C of 1% with partial responses in 2/10 mice. In the EBC-1 model, treatment with EMD 1214063 (10 mg/kg) or with the antiVEGF antibody B20-4.1 (20 mg/kg) as single agents resulted in a T/C of β21% (with partial regression in 1/10 mice) and 19%, respectively. In contrast, the combination of EMD 1214063 and B20-4.1 exhibited enhanced anti-tumor activity with a T/C of −72% and partial regressions in 10/10 mice. These data support the hypothesis that EMD 1214063 or EMD 1204831 may be effective in NSCLC patients in combination with VEGF-targeting agents. In conclusion, our findings provide a rationale to evaluate the efficacy of the combination of EMD 1214063 or EMD 1204831 with EGFR and VEGF inhibitors in NSCLC patients. 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 1787. doi:1538-7445.AM2012-1787

The involvement of the mesenchymal endothelial transition factor (c-Met) in the primary event of oncogenic transformation and the secondary ability to mediate metastatic spread has been convincingly demonstrated in preclinical and early clinical settings. The clinical benefits of c-Met kinase inhibitors with various modes of actions and selectivity profiles are currently under investigation hoping that inhibitors of c-Met might emerge as valuable cancer therapeutics in the future. During an HTS run 3-(diethylamino)propyl N-[3-[[5-(3,4-dimethoxyphenyl)-2-oxo-6H-1,3,4-thiadiazin-3-yl]methyl]phenyl]carbamate was identified as an attractive lead structure with an interesting overall profile (clogD (7.4): 2.5, S (pH 7.4): >100 µg/ml, IC50 (cMet enzyme): 30 nM, IC50 (cMet A549): 800 nM) providing a valid starting point. The co-crystal structure of 1 revealed the binding mode and the essential structural features. The initial HTS hit was bound in a DFG-in conformation interacting with the main chain nitrogen atom of Met1160 within the hinge region and with the main chain nitrogen of Asp1222. After subsequent optimization of potency, efficacy, PK properties and the safety profile of thiadiazinone 1, EMD1204831 was identified as development compound. EMD1204831 is currently being investigated in a phase 1 clinical trial. The pyridazinone EMD1204831 inhibits enzymatic and cellular c-Met kinase activity with IC50 values of 12 nM and 15 nM, respectively. EMD1204831 displayed an exquisite selectivity when tested in vitro against a panel of more than 400 potential off-targets, including kinases, GPCRs, ion channels, transporters and various enzymes. EMD 1204831 demonstrated excellent anti-tumor activity in vivo in a variety of xenograft models, including U87-MG glioblastoma cells (autocrine HGF expression), TPR-Met-transformed mouse fibroblasts (oncogenic Met fusion protein) or Hs746T gastric cancer cells (c-Met gene amplification and HGF-independent activation). Depending on the particular model, complete regressions were observed with doses as low as 6 mg/kg/d administered per os. PK/PD analysis revealed efficient, dose- and time-dependent inhibition of c-Met phosphorylation, reduction of IL-8 and cyclin D1 expression as well as an induction of the cell cycle inhibitor p27. The overall profile of EMD1204831 including first time structural disclosure, some structure activity relationships, in vitro potency, selectivity profile and in-vivo data will be presented. 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 2786. doi:10.1158/1538-7445.AM2011-2786

c-Met, the receptor for hepatocyte growth factor (HGF), is a well characterized receptor tyrosine kinase, which is crucial for cell functions, such as survival, proliferation, motility and migration. Activating point mutations of c-Met have been identified in human malignancies, including renal carcinoma and lung cancer and constitutive activation of the HGF/c-Met pathway leads to tumor development and progression in cancer animal models. In the light of these findings, c-Met has become a key target for oncology therapeutics. In order to develop c-Met specific inhibitors, we have conducted a biochemical HTS run with the recombinant kinase domain of c-Met. This approach has enabled us to identify a new class of c-Met inhibitors. Our report will focus on pharmacological and pre-clinical data, characterising EMD1214063, a promising therapeutic candidate currently under clinical testing. Inhibition of the c-Met kinase by EMD1214063 was potent and highly selective. In in vitro kinase reactions and binding assays, EMD1214063 at a dose of 1μM, inhibited only 5 additional kinases in a panel of more than 250 different potential off-targets more than 50%. In vitro, EMD1214063 interfered with survival, anchorage-independent growth and HGF-induced migration of tumor cells, by blocking HGF-dependent as well as constitutive phosphorylation of c-Met. In vivo, oral administration of EMD1214063 resulted in a strong inhibition - ranging from delayed growth to complete regression - of HGF-dependent and -independent c-Met driven tumor xenografts. Such anti-tumor activity was observed at a broad dose range, suggesting that EMD1214063 has a wide therapeutic index. The inhibitor was well tolerated, as indicated by the lack of significant weight loss in treated mice. The mechanism of action of EMD1214063 was investigated in vivo in a series of PK/PD studies. Administration of a single dose of EMD1214063 induced a complete and long-lasting inhibition of c-Met phosphorylation in the tumor xenografts. Furthermore, it strongly reduced the plasma levels of tumor-derived IL-8. c-Met phosphorylation and plasma IL-8 levels decreased as a function of the drug concentration in the tumor and plasma. Taken together, the high c-Met selectivity, potency and in vivo efficacy of EMD1214063 render this compound a very promising candidate for the treatment of patients bearing c-Met-driven tumors. 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 3622.

The role of the receptor tyrosin kinase c-Met in tumor progression, metastasis and aggressiveness has been convincingly demonstrated in preclinical and early clinical settings. Several compounds with different selectivity profiles inhibiting c-Met are currently under preclinical/clinical investigation and might emerge as valuable cancer therapeutics in the future. During an HTS run N-(3-(3,6-Dihydro-5-(3,4-dimethoxyphenyl)-2-oxo-2H-1,3,4-thiadiazin-3-ylmethyl)-phenyl)-carbaminic acid-(3-(N,N-diethylamino)-propylester) (1) was identified as an attractive lead structure with an interesting overall profile (clogD (7.4): 2.5, S (pH 7.4): >100 µg/ml, IC50 (c-Met in vitro): 30 nM, IC50 (c-Met cellular): 800 nM) providing a valid starting point for lead optimization. The co-crystal structure of 1 revealed the binding mode and the essential structural features. The initial HTS hit was bound in a DFG-in conformation interacting namely with the main chain nitrogen atom of methionine 1160 within the hinge region and with the main chain nitrogen of aspartic acid 1222. After subsequent optimization of potency, efficacy, PK properties and the safety profile of thiadiazinone 1, EMD1214063 was identified as candidate for further development and is currently investigated in a phase 1 clinical trial. The pyridazinone EMD1214063 inhibits enzymatic and cellular c-Met kinase activity with IC50 values in the low nanomolar range. This compound displayed an exquisite selectivity when tested in vitro against a panel of more than 250 potential off-targets, including kinases, GPCRs, ion channels, transporters and various enzymes and demonstrated excellent anti-tumor activity in vivo in a variety of xenograft models. Depending on the particular model, complete regressions were observed with doses as low as 6 mg/kg/d administered per os. The overall profile of EMD1214063 including first time disclosure of the precise structure, synthesis, structure activity relationships, in vitro potency, selectivity profile, pharmacokinetic and in vivo data will be discussed. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 5777.

The role of the receptor tyrosin kinase c-Met in tumor progression and metastasis has been shown in preclinical studies and in early clinical settings. Several c-Met inhibitors with different selectivity profiles are currently in preclinical/clinical investigation and might provide effective cancer therapeutics in the future. A biochemical HTS run using the recombinant kinase domain of c-Met enabled us to identify N-(3-(3,6-Dihydro-5-(3,4-dimethoxyphenyl)-2-oxo-2H-1,3,4-thiadiazin-3-ylmethyl)-phenyl)-carbaminic acid-(3-(N,N-diethylamino)-propylester) representing as new chemical class of kinase inhibitors. This HTS hit inhibited c-Met in vitro kinase activity and HGF-induced c-Met phosphorylation in A459 lung cancer cells with IC50 values well below 1 µM. The analysis of co-crystal structures defined the molecular requirements for potency and selectivity and guided the subsequent chemical optimization. As a result of the lead optimization process, two candidate compounds with distinct pharmacological properties were synthesized, EMD 1214063 and EMD 1204831. Both compounds are highly potent and efficient inhibitors of c-Met in vitro kinase activity and c-Met auto-phosphorylation induced in vitro by HGF stimulation or by c-Met overexpression. Both compounds displayed an exquisite selectivity profile when tested in vitro against a panel of more than 250 potential off-targets, including kinases, GPCRs, ion channels, transporters and various enzymes. EMD 1214063 and EMD 1204831 exhibited excellent anti-tumor activity in vivo in a variety of xenograft models, including U87-MG glioblastoma cells (autocrine HGF production), TPR-Met-transformed mouse fibroblasts (oncogenic Met fusion protein) or Hs746T gastric and EBC-1 lung cancer cells (c-Met gene amplification and HGF-independent activation). In c-Met-driven models complete regressions were observed with doses as low as 6 mg/kg/d administered per os. PK/PD analysis revealed efficient, dose- and time-dependent inhibition of c-Met phosphorylation, reduction of IL-8 and cyclin D1 expression as well as an induction of the cell cycle inhibitor p27. The pharmacodynamic effects of EMD 1214063 were longlasting and allowed various schedule variations without compromising its anti-tumor activity, while c-Met inhibition by EMD 1204831 was rather transient, allowing optimal anti-tumor activity when the compound was applied once or twice daily. The profile of EMD 1214063 and EMD 1204831 including synthesis, structure activity relationships, X-ray structures and a comprehensive pharmacological in vitro and in vivo characterization will be presented. Furthermore, potential pharmacodynamic and predictive biomarkers will be discussed. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B252.