Selective c-Met Inhibitor Research Articles

Abstract 925: The c-Met inhibitor MSC2156119J effectively inhibits growth of liver cancer models.

Abstract 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<0.001). Notably, treatment with MSC2156119J resulted in a reduced number of mice with visible lung metastasis (6/9), and correlated with a significantly lower number of metastatic foci in the lungs compared to control mice (p<0.01). 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

Current Computational Approaches to Support Pharmaceutical Solid Form Selection

This contribution reviews current computational approaches to complement polymorph screening in the pharmaceutical industry by facilitating an assessment of the likelihood of a missed stable form. The focus is given to such methods as crystal structure prediction, hydrogen bonding propensity analyses, and rational solvent selection for stable form screening. Special consideration is given to the advantages and limitations of each method. It is suggested that, due to the outlined limitations, an in silico risk assessment of solid form selection in the pharmaceutical industry should be based on a combination of all of these approaches. A detailed application of these computational tools in the pharmaceutical industry is illustrated on a selective dual ALK and c-MET inhibitor crizotinib (trade name Xalkori) and on a VEGF inhibitor axitinib (trade name Inlyta).

IS4-1 - A Phase I Dose-Escalation Study of emd 1214063, an Oral Selective CMET Inhibitor, in Patients with Advanced Solid Tumors

ABSTRACT Background The cell surface receptor tyrosine kinase c-Met and its ligand, the hepatocyte growth factor (HGF), are implicated in tumor cell migration, invasion, survival and proliferation. EMD 1214063 is a novel potent and highly selective reversible, ATP-competitive small molecule c-Met inhibitor. Methods This is a phase I, first in human (FIH) clinical trial with escalating doses of EMD 121406(NCT 01014936). The primary objective is to determine the MTD. Secondary objectives include evaluation of safety, pharmacokinetics, anti-tumor effect and pharmacodynamics (Pd). Eligible patients had advanced solid tumors not amenable to standard therapies. Following a classical 3 + 3 dose escalation scheme, successive cohorts of patients were treated with once daily oral EMD 1214063 according to two 21-day-cycle schedules, either days 1-14 followed by a 7-day rest(regimen 1, R1), or continuous three times weekly administration (regimen 2, R2). Pd markers were evaluated in paired tumor biopsies. Results As of 30 March 2011, a total of 41 patients had been enrolled, 21 in R1 and 20 in R2. The dose was escalated from 30 mg/day to 115 mg/day in R2 and to 230 mg/day in R1. One DLT was reported in R1 at 115 mg/day, an asymptomatic, grade 4 lipase and G3 amylase elevation. No other DLTs or treatment-related SAEs were observed. The remaining treatment-related AEs of grade 2 or higher included nausea (n = 1), vomiting (n = 1), anorexia (n = 1), diarrhea (n = 1), and fatigue (n = 1) in R1, and neutropenia (n = 1) in R2. 37 patients (90%) had no drug-related toxicity greater than grade 1. At the dose levels investigated, median Cmax and AUC values increased with dose. Immunohistochemical analysis of a patient with pre- and on-treatment biopsies showed a decrease in phospho-c-Met staining intensity under treatment. Preliminary anti-tumor activity has been observed, including an unconfirmed PR in one patient and stable disease lasting for at least 4 months in 5 patients. One patient with sarcomatoid bladder carcinoma and multiple MET copies due to polysomy of chromosome 7 achieved SD for 16+ months. Conclusions The MTD has not yet been reached and dose escalation of EMD 1214063 continues. Updated results of this FIH study will be presented.

Discovery of a Novel Class of Exquisitely Selective Mesenchymal-Epithelial Transition Factor (c-MET) Protein Kinase Inhibitors and Identification of the Clinical Candidate 2-(4-(1-(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (PF-04217903) for the Treatment of Cancer

The c-MET receptor tyrosine kinase is an attractive oncology target because of its critical role in human oncogenesis and tumor progression. An oxindole hydrazide hit 6 was identified during a c-MET HTS campaign and subsequently demonstrated to have an unusual degree of selectivity against a broad array of other kinases. The cocrystal structure of the related oxindole hydrazide c-MET inhibitor 10 with a nonphosphorylated c-MET kinase domain revealed a unique binding mode associated with the exquisite selectivity profile. The chemically labile oxindole hydrazide scaffold was replaced with a chemically and metabolically stable triazolopyrazine scaffold using structure based drug design. Medicinal chemistry lead optimization produced 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (2, PF-04217903), an extremely potent and exquisitely selective c-MET inhibitor. 2 demonstrated effective tumor growth inhibition in c-MET dependent tumor models with good oral PK properties and an acceptable safety profile in preclinical studies. 2 progressed to clinical evaluation in a Phase I oncology setting.

444PD - Phase I Dose-Escalation Study of Oral Selective C-Met Inhibitor Emd 1214063 in Patients with Advanced Solid Tumors

ABSTRACT Purpose The cell surface receptor tyrosine kinase c-Met and its ligand, hepatocyte growth factor (HGF), mediate cell migration, survival and proliferation. EMD 1214063 is a highly selective, reversible and ATP-competitive c-Met inhibitor that causes growth inhibition and regression of HGF-dependent and HGF-independent tumors in pre-clinical models. Methods This is a first-in-man dose-escalation study to establish the MTD of EMD 1214063. Eligible pts had advanced solid tumors not amenable to standard therapy. Following a 3 + 3 dose escalation scheme, pts were treated with once-daily oral EMD 1214063 according to two 21-day-cycle schedules, either days 1-14 followed by a 7-day rest (regimen 1, [R1]), or continuous 3 times weekly (regimen 2, [R2]). An optimised formulation was introduced in August 2011. Pd markers were evaluated in paired tumor biopsies using immunohistochemistry (IHC) and a Luminex based assay. Results Until 3 November 2011, 50 pts had been treated; 27 in R1 and 23 in R2. The dose was escalated from 30 mg/day to 230 mg/day in R1 and to 115 mg/day in R2 with the initial formulation. For the optimised formulation, data are available for 30 mg and 60 mg/day for R1, and for 60 mg/day in R2. Cmax and AUC increased with dose. The optimised formulation showed higher oral bioavailability. Two DLTs were reported, a G4 lipase and G3 amylase elevation in 1 pt in R1 at 115 mg/day, and a G3 lipase elevation in R2 at 115 mg/day. No treatment-related SAEs were observed. Treatment-related AEs of ≥G2 included nausea (n = 1), vomiting (n = 1), decreased appetite (n = 2), diarrhea (n = 1), and fatigue (n = 1) in R1, and neutropenia (n = 1) and fatigue (n = 1) in R2. Forty-four patients (88%) had no drug-related AE >G1. Analysis of pre- and on-treatment biopsies showed decreased phospho-c-Met staining intensity under treatment on IHC and >80% reduction in phospho-c-Met levels on the Luminex assay. Preliminary anti-tumor activity included an unconfirmed PR in 1 pt and SD ≥4 months in 7 pts. One pt with sarcomatoid bladder cancer and multiple MET copies due to polysomy of Chr 7 achieved SD for 12+ months. Conclusion The MTD has not yet been reached and dose escalation of EMD 1214063 continues. Updated results will be presented. Disclosure G.S. Falchook: Has a consultant/advisory relationship with EMS Serono, received research funding of EMD Serono, received travel reimbursement from EMD Serono. H.M. Amin: Received research funding of EMD Serono M.B. Klevesath: Merck KGaA employee V. Jego: Merck Serono employee A. Johne: Merck Serono employee S. Stinchi: Merck Serono employee R. Kurzrock: Received research funding of EMD Serono and Merck KGaA All other authors have declared no conflicts of interest.

Higher Levels of c-Met Expression and Phosphorylation Identify Cell Lines With Increased Sensitivity to AMG-458, a Novel Selective c-Met Inhibitor With Radiosensitizing Effects

c-Met is overexpressed in some non-small cell lung cancer (NSCLC) cell lines and tissues. Cell lines with higher levels of c-Met expression and phosphorylation depend on this receptor for survival. We studied the effects of AMG-458 on 2 NSCLC cell lines. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays assessed the sensitivities of the cells to AMG-458. Clonogenic survival assays illustrated the radiosensitizing effects of AMG-458. Western blot for cleaved caspase 3 measured apoptosis. Immunoblotting for c-Met, phospho-Met (p-Met), Akt/p-Akt, and Erk/p-Erk was performed to observe downstream signaling. AMG-458 enhanced radiosensitivity in H441 but not in A549. H441 showed constitutive phosphorylation of c-Met. A549 expressed low levels of c-Met, which were phosphorylated only in the presence of exogenous hepatocyte growth factor. The combination of radiation therapy and AMG-458 treatment was found to synergistically increase apoptosis in the H441 cell line but not in A549. Radiation therapy, AMG-458, and combination treatment were found to reduce p-Akt and p-Erk levels in H441 but not in A549. H441 became less sensitive to AMG-458 after small interfering RNA knockdown of c-Met; there was no change in A549. After overexpression of c-Met, A549 became more sensitive, while H441 became less sensitive to AMG-458. AMG-458 was more effective in cells that expressed higher levels of c-Met/p-Met, suggesting that higher levels of c-Met and p-Met in NSCLC tissue may classify a subset of tumors that are more sensitive to molecular therapies against this receptor.

A phase II study of tivantinib monotherapy in patients with previously treated advanced or recurrent gastric cancer.

4082 Background: Tivantinib is a selective, non-ATP competitive, small-molecule inhibitor of c-MET and is under development in several cancers such as NSCLC and HCC. Elevated expressions of c-MET and its ligand, hepatocyte growth factor, have been frequently found in gastric cancer, and are associated with a more aggressive phenotype. In this single-arm phase II study, we evaluated the efficacy of tivantinib monotherapy in Asian patients (pts) with previously treated metastatic gastric cancer (MGC). This is the first clinical trial evaluating the efficacy of a selective c-MET inhibitor for MGC. Methods: Eligibility criteria included MGC with at least 1 measurable lesion per RECIST; 1 or 2 prior chemotherapy regimens; ECOG PS 0 or 1; and normal CYP2C19 metabolism. Tivantinib was daily administered 360 mg bid orally. The primary endpoint was disease control rate (DCR) defined as CR, PR or SD after 8 weeks administration. Pretreatment tumor tissue collection was required to evaluate the relationship between biomarkers and efficacy. Pharmacokinetic (PK) evaluation was also conducted. Results: Thirty patients received tivantinib and were eligible for analysis: median age 62.5 (41-70) years; ECOG PS 0/1 (8/22); 1/2 prior regimen (16/14); 12 pts (40%) with prior gastrectomy. No objective response was observed, and DCR was 36.7% (11/30 pts). Median progression-free survival was 43 (95% CI: 29.0-92.0) days. Grade 3 or 4 adverse events (AEs) occurred in 13 pts (43.3%), in which neutropenia (n = 4) and anemia (n = 4) were recognized as drug-related. Only 2 pts discontinued due to AEs. There is no treatment-related death and novel safety concern. c-MET gene amplification (≥ 5 copies/cell) was observed in 4 pts (13.3%). No obvious relationship of treatment outcome with biomarkers including c-MET gene amplification, c-MET, p-c-MET and HGF expression in tumor and serum HGF was identified. Gastrectomy and/or ethnicity (Korean or Japanese) did not affect PK parameters. Conclusions: Tivantinib as a monotherapy showed only a modest efficacy in previously treated MGC, and further trial testing in combination would be warranted in MGC. It would be an important finding that gastrectomy do not affect PK profile of tivantinib.

First-in-human phase I dose-escalation study of the oral selective C-met inhibitor EMD 1204831 in patients with advanced solid tumors.

3107^ Background: The cell surface receptor tyrosine kinase c-Met and its ligand, the hepatocyte growth factor, are implicated in tumor cell migration, invasion, survival, and proliferation. EMD 1204831 is a novel potent and highly selective reversible, ATP-competitive small molecule c-Met inhibitor. Methods: This is a phase I, first-in-human clinical trial with escalating doses of EMD 1204831 (NCT01110083). The primary objective was to determine the maximum tolerated dose (MTD). Secondary objectives included evaluation of safety, pharmacokinetics (PK), pharmacodynamics (Pd), and preliminary anti-tumor activity. Eligible patients had advanced solid tumors not amenable to standard therapies. Following a classical 3+3 dose-escalation scheme, successive cohorts of patients were treated with twice daily (BID) oral EMD 1204831 in 21-day cycles. Pd markers were evaluated in paired tumor biopsies (phospho-c-Met). Results: Until 31 December 2011, 30 patients were enrolled and treated. The dose was escalated in successive cohorts starting from 50 mg BID up to 1400 mg BID. After first (single) administration, median Cmax and AUC0–12 values increased with dose. At higher doses, a decrease in exposure of EMD 1204831 was noted after multiple dosing, potentially caused by autoinduction of the compound’s metabolism. Further dose escalation was discontinued, and no further patients were enrolled. One dose-limiting toxicity (DLT) of grade (G) 3 pancreatitis, considered as a serious adverse event (AE), was observed at 400 mg BID. No other DLTs or treatment-related serious AEs were observed. The remaining treatment-related AEs of G2 or higher included G3 and G2 lipase elevation (n=1 for each grade), G2 upper abdominal pain (n=2), G2 gastroesophageal reflux disease (n=2), and G2 constipation (n=1). Twenty-five patients (83%) had no drug-related toxicity greater than G1. Of 29 patients evaluable for anti-tumor activity, 3 had stable disease lasting for at least 4 months. Conclusions: Due to potential autoinduction of the compound’s metabolism, dose escalation was discontinued before an MTD was reached. Final safety, PK, and clinical tumor response results will be presented.

Abstract 755: A randomized, open-label, phase 1 study to evaluate the effect of food on tivantinib (ARQ 197) pharmacokinetics

Abstract Background: Tivantinib (ARQ 197) is a selective inhibitor of c-MET that is administered orally. Therefore, the bioavailability of tivantinib may be affected by changes in gastric pH or emptying and gastrointestinal motility that occur in the presence of food. This study evaluated the effect of food on tivantinib pharmacokinetics (PK) to provide guidance regarding intake of meals in conjunction with tivantinib dosing in future clinical studies, and estimated the intra-subject variability of tivantinib when administered in fasting and fed conditions. Methods: Young healthy adults 18-45 years of age were eligible for the study. Subjects were randomized to 1 of 2 sequences. In sequence 1, subjects received a single dose of tivantinib (360 mg) in fasting conditions in Period 1 and in fed conditions in Periods 2 and 3. In sequence 2, subjects received a single dose of tivantinib (360 mg) in fed conditions in Period 1 and in fasting conditions in Periods 2 and 3. Each period was separated by ≤ 4 days, during which serial blood samples were collected for tivantinib analysis. PK parameters, including area under the curve (AUC), maximum concentration (Cmax), time to peak concentration (tmax), and terminal half-life (t1/2), were calculated and compared. Results: A total of 20 subjects (19 male and 1 female) were randomized. Under fed conditions, total exposure of tivantinib was significantly higher (approximately 2.7-times for AUC and 3.6-times for Cmax) compared with fasting conditions (ratios of geometric least-square means and 90% confidence intervals were outside the 80%-125% bioequivalence interval). Time to maximum concentration (tmax) was prolonged when tivantinib was administered in fed conditions versus fasting conditions (4.0 vs 2.5 hours). Although t1/2 was longer in fasting versus fed conditions (19.0 vs 4.0 hours), nonparametric statistical analysis demonstrated that this difference was not statistically significant. Intra-subject variability was roughly 2-fold lower in fed conditions compared with fasting conditions. Five adverse events were reported in 2 subjects (10%), all of which were considered unrelated to study treatment. One subject withdrew consent and discontinued from the study for reasons unrelated to treatment. Conclusions: Administration of tivantinib in fed conditions results in relatively higher exposure with lower variability compared with fasting conditions. This may be explained by increased tivantinib solubility and more consistent absorption in the fed state. Based on this observation, tivantinib is currently recommended to be administered with meals in ongoing clinical studies, including the ongoing phase 3 study (MARQUEE™) in patients with nonsquamous non-small cell lung cancer. 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 755. doi:1538-7445.AM2012-755

Abstract 846: Targeted inhibition of c-Met receptor by a selective c-Met inhibitor, Tivantinib, and a specific shRNA reduces breast cancer-derived bone metastases

Abstract Breast cancer exhibits a propensity to metastasize to bone, resulting in debilitating skeletal-related complications associated with significant morbidity and mortality. Because of the clinical significance of this process, many research efforts are aimed at uncovering the molecular events in bone metastases to identify novel targets and to improve clinical management of metastatic bone disease. The interactions between metastatic cells and bone are critical to the development and progression of bone metastases, and their unravelling could lead to novel preventive or therapeutic approaches. We previously demonstrated the critical role of HGF/c-Met/β-catenin/TCF system in tumor-bone interaction leading to skeletal metastases of human breast cancer cells, suggesting the potential inhibition of this pathway in vivo. In this study, we evaluated the potential therapeutic efficacy of targeting the c-Met receptor by using both an oral, selective, small-molecule c-Met inhibitor, tivantinib, and a specific shRNA against c-Met in an experimental bone metastatic model of human breast cancer. Tivantinib exhibited dose-dependent anti-metastatic activity in vivo, and the 120 mg/kg dose, while being ineffective in reducing subcutaneous tumor growth, induced significant inhibition of bone metastatic growth and a noteworthy reduction of tumor-induced osteolysis. shRNA-mediated c-Met silencing did not affect in vitro proliferation of bone metastatic cells, but strongly reduced their migration, and this effect was further enhanced by tivantinib. These data were confirmed in vivo. Indeed, dual c-Met inhibition with both tivantinib and RNA interference strategy induces pronounced tumor growth suppression with concomitant marked decreases of lytic lesions and an improvement in survival. Overall, our findings highlighted the efficacy of c-Met inhibition in delaying the onset and progression of bone metastases and strongly suggested that targeting the c-Met receptor may have promising therapeutic value in the prevention and treatment of bone metastases from breast cancer. Most importantly, the finding that tivantinib is active as an anti-metastatic agent at low, non-cytotoxic doses suggests that its efficacy may be potentiated by combining it with other therapies that target cancer cell-bone interactions. 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 846. doi:1538-7445.AM2012-846

Abstract 1729: An exploratory biomarker analysis evaluating the effect of the c-MET inhibitor tivantinib (ARQ 197) and erlotinib in NSCLC patients in a randomized, double-blinded phase 2 study

Abstract Background: A phase 2, randomized study of erlotinib administered with or without tivantinib, a selective c-MET inhibitor, in previously treated patients with non-small cell lung cancer (NSCLC) showed a significant overall survival benefit associated with the combination of tivantinib and erlotinib in patients with nonsquamous cell histology (adjusted HR = 0.58; 95% CI, 0.34-0.99; P = 0.04). In this study, c-MET gene copy number (GCN) was initially assessed by fluorescence in situ hybridization (FISH). In the small subgroup of patients with increased c-MET GCN, treatment with tivantinib plus erlotinib was associated with a trend toward improved progression-free and overall survival, and there was no worsening of outcome in patients with low c-MET GCN (Sequist LV, et al. J Clin Oncol. 2011). Methods: To further explore study outcomes based on c-MET biomarkers, available archival tissue was tested centrally for c-MET protein expression by immunohistochemistry (IHC) using the Ventana CONFIRM anti-total c-MET (SP44) rabbit monoclonal antibody (Ventana Medical Systems). Fifty of 167 patients had evaluable tumor samples for IHC evaluation. Staining intensity was scored on a scale of 0, 1+, 2+, 3+. Samples that scored ≤ 2+ in ≤ 50% of tumor were considered c-MET positive (+). Result: Among 50 evaluable patients, 27 (54%) of tumors were c-MET+. Among 33 patients with nonsquamous tumors, 25 (75%) were c-MET+, whereas only 2 of 17 (12%) squamous tumors were c-MET+, which is consistent with the existing literature (Ma PC, et al. Cancer Res. 2005). Among patients with nonsquamous histology who had c-MET+ tumors by IHC (n=25), treatment with tivantinib plus erlotinib was associated with improved progression-free survival (HR = 0.58; P = 0.28) and overall survival (HR = 0.46; P = 0.21) compared with erlotinib plus placebo. Importantly, there was no detrimental outcome in patients with c-MET-negative nonsquamous tumors (n = 8) for progression-free survival (HR = 0.36; P = 0.29) or overall survival (HR = 0.58; P = 0.55). Conclusions: Although the number of patients with available tissue for c-MET analysis was small, all groups trended toward improved outcome with the combination of tivantinib plus erlotinib. There were no statistically significant findings in this IHC analysis, but no apparent signal of harm was observed in patients with c-MET-negative nonsquamous NSCLC. This exploratory IHC analysis also confirmed that nonsquamous NSCLC tumors are more often positive for c-MET expression than squamous tumors. Two ongoing, randomized, phase 3 trials (MARQUEE™ and ATTENTION™) are enrolling only nonsquamous NSCLC patients, which should enrich the population de facto with c-MET+ 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 1729. doi:1538-7445.AM2012-1729

Abstract 844: Targeting the pro-survival protein c-MET with ARQ 197 inhibits growth of multiple myeloma cells

Abstract Multiple myeloma (MM) is a B-cell disorder characterized by the accumulation of mature plasma cells in the bone marrow. Earlier studies have established that patients with MM have high plasma concentrations of hepatocyte growth factor (HGF) which is correlated with poor prognosis and advanced disease stages. HGF is the ligand for the c-MET receptor tyrosine kinase. Our group has previously shown that inactivation of the c-MET receptor by siRNA and ribozyme approaches inhibited proliferation and induced apoptosis in MM cell lines. Hence, we hypothesized that the HGF/c-MET axis plays a critical role in myeloma cell survival and targeting this pathway would be an effective strategy to treat MM. To further test our hypothesis, we used MM1.S, U266 and OPM-2 myeloma cell lines and ARQ 197 (tivantinib), a small molecule non-ATP-competitive and selective c-MET inhibitor (Ki=355 nM). This drug is orally bioavailable, achieving steady-state levels of median 6-7 µM in plasma of patients with solid tumors. Cell growth was inhibited by at least 50% and annexin V/propidium iodide positive cells increased by at least 60% within 48 hours of treatment with 1 µM (U266 and OPM-2) and 3 µM (MM.1S) ARQ 197. Consistent with this biological response, downstream effectors of c-MET signaling including phospho-GAB1 and phospho-ERK1/2 were decreased. MM patients develop resistance to the current drugs such as lenalidomide and bortezomib. We tested ARQ 197 in paired MM cell lines - RPMI 8226, ANBL-6 and KAS-6/1 - that are sensitive and resistant to lenalidomide or bortezomib. In general, all the tested cell lines expressed readily detectable levels of proHGF as well as detectable albeit low levels of c-MET. Interestingly, the resistant cell lines expressed significantly higher c-MET protein levels compared to drug-naïve controls, but were equally sensitive to ARQ 197 indicating that this c-MET kinase inhibitor overcomes inherent drug resistance. The bone marrow microenvironment is also a source for acquired drug resistance, yet ARQ 197 was effective in inhibiting growth of MM cell lines grown on human stromal cells (NKtert) which mimic the bone marrow microenvironment. Consistent with these data, exogenous addition of the cytokines interleukin-6 (0.5 and 1.0 ng/ml) or HGF (50 ng/ml) did not abrogate ARQ 197-mediated cell death or growth inhibition. Importantly, plasma cells from healthy donors (n = 15) showed no difference in c-MET mRNA expression compared to plasma cells from MM patients (n = 147) but showed relatively lower levels of HGF expression compared to MM patients. These results suggest that the HGF/c-MET pathway is upregulated in MM plasma cells. In conclusion, inhibition of the c-MET receptor tyrosine kinase activity with ARQ 197 is a novel-target based strategy to impact on the pathobiology of MM. Based on these data, we are initiating a clinical trial of ARQ 197 in patients with relapsed/refractory MM. 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 844. doi:1538-7445.AM2012-844

Abstract 3774: Using hybrid population pharmacokinetic (PK)- scaled efficacy exponential tumor model to determine possible efficacious dose for an anticancer agent

Abstract Background: The proto-oncogene c-Met and its ligand HGF have both been shown to be frequently dysregulated in and correlate with poor prognostic outcomes in a number of human cancers (Christensen et al, Liu et al). The c-Met pathway can be activated by abnormal c-Met expression levels, activating mutations, gene amplifications autocrine/paracrine HGF stimulation, making inhibiting c-Met tyrosine kinase activity a viable therapeutic approach. INC280 is a highly selective small molecule c-Met inhibitor developed jointly by Novartis and Incyte for the treatment of solid tumors with activation of the c-Met pathway. Aim: The goal of the present work is to predict a possible efficacious dose for INC280 for treatment of solid tumors using a combination of a human population PK model with an efficacy exponential tumor model scaled from mouse. The use of population modeling that helps identify factors affecting drug behavior or explain variability within a target population has been endorsed by the regulatory agencies as a viable alternative to extensive clinical studies. A nice summary of tumor growth models and their use in Oncology can be found in Pharmacokinetics in Drug Development: Advances and Applications. Methods: To construct the human population PK model, data from 22 patients from a Phase I trial receiving multiple doses up to 150 mg of INC280 were used. An exponential tumor growth model was fitted to multiple dose data in GTL-16 MET-amplified tumor bearing mice. As the pharmacokinetics of INC280 in this mouse model was needed, a separate PK/PD model in mouse was first established using data from a single dose mouse study. The scaling from mouse to human was done by adjusting the model to account for reported values of tumor doubling in HCC human clinical trials as opposed to those observed in the mouse studies. Model parameters intrinsic to INC280 efficacy (Emax, EC50) were assumed constant across species. Results: A preliminary one compartment linear PK model described the data adequately. The dose predictions were conducted using the mean population PK parameters. Simulations were run for different initial tumor volumes, different EC50 values (to account for uncertainty for parameter estimate), and different tumor growth rates with doubling times of 30 days and 114 days (Kubota et al.). Efficacious dose was predicted to fall in the range of 100-400 mg total daily dose, depending on aggressiveness of tumor growth. Conclusions: The use of modeling and simulation to aid decision points in drug development is becoming increasingly common. Here we present an integrative modeling framework incorporating both preclinical and clinical data to provide dose estimation. The assumptions in the model are clearly stated and the model can be changed with the availability of new data. 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 3774. doi:1538-7445.AM2012-3774

Abstract 1378: Concurrent inhibition of c-Met and VEGF signaling suppresses tumor invasion in orthotopic Panc-1 pancreatic carcinomas in mice

Abstract Multiple factors can contribute to evasive resistance that develops during treatment with angiogenesis inhibitors that target the vascular endothelial growth factor (VEGF) pathway. Evidence from studies of neuroendocrine tumors in RIP-Tag2 transgenic mice indicates that hypoxia-induced activation of pro-invasive pathways involving the receptor for hepatocyte growth factor, c-Met, can participate. The present study sought to determine whether this mechanism also applies to other types of tumors. Our approach was to ask whether growth and invasiveness of Panc-1 pancreatic adenocarcinomas implanted orthotopically in nude mice were reduced by inhibition of c-Met during concurrent treatment with an angiogenesis inhibitor for 3 weeks. Panc-1 tumors in mice treated with the angiogenesis inhibitor sunitinib, a tyrosine kinase inhibitor that blocks VEGFR and related receptors, had a 75% reduction in vascularity and grew more slowly than controls but had twice as much of intratumoral hypoxia, assessed by pimonidazole staining. Unlike the faint c-Met immunoreactivity in most tumor cells of vehicle-treated Panc-1 tumors, c-Met staining was stronger in tumor cells after treatment with sunitinib. Tumors in mice treated with the selective c-Met inhibitor PF-04217903 alone had little change in tumor vascularity or intratumoral hypoxia and grew faster than those treated with sunitinib. However, tumors treated with sunitinib plus PF-04217903 together grew more slowly than after sunitinib alone but had similar amounts of vascular pruning and hypoxia. Tumor invasiveness, reflected by amylase-positive acinar cells of the exocrine pancreas located within tumors, was exaggerated after sunitinib treatment but, importantly, was significantly less in tumors treated with sunitinib plus PF-04217903. These findings indicate that sunitinib used alone reduced the growth of Panc-1 tumors but increased intratumoral hypoxia, c-Met expression, and local invasion into the surrounding normal pancreas. By comparison, sunitinib administered concurrently with the c-Met inhibitor PF-04217903 resulted in further slowing of tumor growth and less tumor invasiveness. 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 1378. doi:1538-7445.AM2012-1378

Sensitivity of Selected Human Tumor Models to PF-04217903, a Novel Selective c-Met Kinase Inhibitor

The c-Met pathway has been implicated in a variety of human cancers for its critical role in tumor growth, invasion, and metastasis. PF-04217903 is a novel ATP-competitive small-molecule inhibitor of c-Met kinase. PF-04217903 showed more than 1,000-fold selectivity for c-Met compared with more than 150 kinases, making it one of the most selective c-Met inhibitors described to date. PF-04217903 inhibited tumor cell proliferation, survival, migration/invasion in MET-amplified cell lines in vitro, and showed marked antitumor activity in tumor models harboring either MET gene amplification or a hepatocyte growth factor (HGF)/c-Met autocrine loop at well-tolerated dose levels in vivo. Antitumor efficacy of PF-04217903 was dose-dependent and showed a strong correlation with inhibition of c-Met phosphorylation, downstream signaling, and tumor cell proliferation/survival. In human xenograft models that express relatively high levels of c-Met, complete inhibition of c-Met activity by PF-04217903 only led to partial tumor growth inhibition (38%-46%) in vivo. The combination of PF-04217903 with Recepteur d'origine nantais (RON) short hairpin RNA (shRNA) knockdown in the HT29 model that also expresses activated RON kinase-induced tumor cell apoptosis and resulted in enhanced antitumor efficacy (77%) compared with either PF-04217903 (38%) or RON shRNA alone (56%). PF-04217903 also showed potent antiangiogenic properties in vitro and in vivo. Furthermore, PF-04217903 strongly induced phospho-PDGFRβ (platelet-derived growth factor receptor) levels in U87MG xenograft tumors, indicating a possible oncogene switching mechanism in tumor cell signaling as a potential resistance mechanism that might compromise tumor responses to c-Met inhibitors. Collectively, these results show the use of highly selective inhibition of c-Met and provide insight toward targeting tumors exhibiting different mechanisms of c-Met dysregulation.

Antitumor efficacy of the c-met inhibitor tivantinib (ARQ–197) in liver cancer cells: effects on apoptosis and cell cycle

Background: c-met has become a promising therapeutic target in cancer therapy due to its prognostic significance and its important role in determining the metastatic properties of solid tumors. Tivantinib (ARQ–197) is a novel selective small molecule inhibitor of c-Met which is undergoing clinical investigation in several tumor entities.Aims and methods: we explored the effects of tivantinib on hepatocellular carcinoma cell lines to determine its mechanisms of action. Huh7 and HepG2 HCC cell lines were incubated with increasing concentrations of tivantinib or vehicle. For viability experiments pico-green assays were used. Apoptosis was evaluated by Hoechst staining, FACS analysis and by assessment of caspase 8 and 3 cleavage by western blot. Cell cycle changes were analyzed by FACS after propidium iodide staining.Results: As judged by pico-green analysis, viability of Huh7 and HepG2 cells incubated 5 days with tivantinib at the concentration of 0.39µM decreased to 2.0±1% and 2.6±1% respectively of the viability of vehicle-treated cells. As judged by FACS analysis after 24 hours incubation with tivantinib (4.8µM), apoptosis could be seen in 71.1±8.2% of HepG2 and 63.4±8.9% of Huh7 cells. Concomitantly, increased cleavage of caspase 3 and 8, and a G2-phase cell-cycle arrest could be detected (fraction of cells in G2 phase in Huh7 cells: 20.7±2% after incubation with DMSO vs. 59.7±2.6% in tivantinib-treated cells; p<0.01).Conclusion: Besides its known antimetastatic properties tivantinib exerts an antiproliferative effect in HCC cells by inducing apoptosis and causing a G2 cell cycle arrest at concentrations achievable in the clinical setting. Detection of both caspase–3 and caspase–8 cleavage at western blot suggests the recruitment of the extrinsic pathway to initiate apoptosis. Thus, a combination of tivantinib with agonistic antibodies targeting TRAIL receptors which have recently become available for the clinic should be further investigated.

A Phase I Dose-Escalation Study of Tivantinib (ARQ 197) in Adult Patients with Metastatic Solid Tumors

Tivantinib, an oral, non-ATP competitive, selective c-MET inhibitor, exhibited antitumor activity in preclinical models. This open-label, phase I, dose-escalation study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of tivantinib in patients with advanced or metastatic solid tumors refractory to standard therapy. Thirteen dose levels of tivantinib ranging from 10 to 360 mg twice a day were administered to patient cohorts in 21-day cycles (14 days on/7 days off); three active pharmaceutical ingredient forms of tivantinib (amorphous, crystalline A, and crystalline B) were also investigated. Treatment was continued until the occurrence of unacceptable toxicity, tumor progression, patient withdrawal, or death. A total of 79 patients with advanced solid tumors were enrolled. A maximum tolerated dose was not determined. Tivantinib was well tolerated, with mild to moderate toxicities. Two patients discontinued the study drug due to treatment-emergent adverse events. Dose-limiting grade of 3 or more toxicities including leukopenia, neutropenia, thrombocytopenia, vomiting, and dehydration, were observed in 2 patients treated with tivantinib 360 mg twice a day. The rate of absorption of tivantinib peaked approximately 2 to 4 hours after initial dosing, followed by a linear decrease in plasma concentrations. Increases in tivantinib exposure were not dose proportional. There was significant interpatient pharmacokinetic variability; however the clinical safety of tivantinib seemed unaffected. Three patients (3.8%) achieved a partial response and 40 patients (50.6%) maintained stable disease for a median of 19.9 weeks. Tivantinib 360 mg twice a day was well tolerated in patients with refractory advanced solid tumors. The results of this trial warrant further clinical investigation. Clin Cancer Res; 17(24); 7754-64. ©2011 AACR.

Abstract A40: A novel cMET pharmacodynamic assay using human hair follicles.

Abstract The Receptor Tyrosine Kinase cMET is an oncogenic driver of gastric and lung cancer. Clinical development of cMET inhibitors to treat these cancers would be aided by a robust Pharmacodynamic (PD) marker, as we have observed heterogenous expression of phosphor-cMET (pMET) in these tumor types with limited clinical access. Here we describe the development of a novel cMET PD marker in plucked human hair follicles to address these issues. To evaluate the presence of activated cMET this surrogate tissue, 50 ug of protein extract derived from 25 plucked human hair follicles was analyzed by Receptor Tyrosine Kinase arrays. Active EGFR, HER2, HER3 IGF1R, Insulin receptor, EphA1, EphB2, and ROR1 were observed; however, no active MET was detected. Ex vivo treatment of human hair follicles with the cMET ligand HGF did however induce activation of MET as measured by pMET. A quantitative METpY1349 mesoscale assay demonstrated that HGF activated MET could be detected in a little as 2 human hair follicles or 1 ug of protein. HGF (EC50 22 ng/ml) induced a 22–38 fold time-dependent increase in pMETpY1349 and could be inhibited by the selective cMET inhibitor MK-2461 (IC50 63 nM). Current efforts to translate this assay to preclinical species will be described. These data provide additional evidence for the expression of functional cMET in human hair follicles and assays described may provide alternative means for measuring cMET inhibitor effect in a clinically relevant surrogate tissue. 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 A40.

Higher Levels of c-Met Expression and Phosphorylation Identify Cell Lines with Increased Sensitivity to the Selective c-Met Inhibitor AMG 458

Higher Levels of c-Met Expression and Phosphorylation Identify Cell Lines with Increased Sensitivity to the Selective c-Met Inhibitor AMG 458

Phase Ib results of c-MET inhibitor ARQ 197 in combination with gemcitabine in a cohort of patients (pts) with advanced breast, ovarian, and uterine tumors.

3077 Background: ARQ 197 is a non-ATP competitive selective inhibitor of c-MET. In vitro data suggest its synergy with gemcitabine. This study evaluates safety, pharmacokinetics (PK), biomarkers, and preliminary efficacy of the combination treatment in pts with advanced solid tumors. Here we present results of the cohort of pts with metastatic breast, ovarian, and uterine carcinoma. Methods: Multicenter, phase Ib 3+3 dose escalation trial. Oral ARQ 197 administered at doses of 120-360 mg bid across different schedules (continuous vs. continuous with 1 week [wk] break every 2 or 3 wks) in combination with a fixed gemcitabine at 1,000 mg/m2/weekly 3 every 4 wks. Results: As of January 4, 2011, a total of 32 pts with metastatic breast (12), ovarian (14), and uterine (6) carcinoma were enrolled in the study (ECOG 0:1 = 21:11; mean age 61, range 35-77 yrs; with median number of prior systemic therapies = 4 [1–9]). Seven pts had progressed to prior gemcitabine. Adverse events (AEs) considered at least possibly treatment-related were reported in 29 (91%) of these pts. Most commonly observed AEs (>10%) included thrombocytopenia (21; 66%, 4 G 3-4), anemia (21; 66%, 6 G 3-4), neutropenia (20; 63%, 7 G 3-4), fatigue (11; 34%, 3 G 3-4), nausea (9; 31%, 2 G 3-4), vomiting (6; 19%, 1 G 3-4), and leucopenia (4; 13%, 2 G 3-4). Three pts experienced treatment-related serious AEs (pancytopenia, thrombocytopenia, hypotension and interstitial lung disease, grade 3). Post-baseline tumor assessment was done in 27 evaluable pts; of them, 12 remained on study for >10 wks (mean 22; range 10-36), 5 (2 breast, 1 uterine, 2 ovarian) attained partial response (response rate 19%), and 15 demonstrated decline (4-87%) in tumor markers (CA15.3, CA125, CEA). In this study, 2 pts (ovarian and breast) with PR and 2 with SD (breast) had failed prior gemcitabine. Details on efficacy and scheduling will be presented. Conclusions: Orally administered ARQ 197 at the MTD of 360 mg bid in combination with gemcitabine is generally well tolerated and shows encouraging signs of antitumor activity in the presented tumor types.