WO2012042421A1 - Method of treating abnormal cell growth - Google Patents

Abstract

The present invention relates to the use of c-Met/HGFR and VEGF inhibitors for treating abnormal cell growth in mammals. In particular, the invention provides methods of treating mammals suffering from cancer. In particular the invention provides methods of treating mammals suffering from metastatic cancer. In particular the invention provides methods of inhibiting metastatic cancer. In particular the invention relates to combination treatment using Sunitinib and crizotinib or PF-04217903.

Description

METHOD OF TREATING ABNORMAL CELL GROWTH

This application claims the benefit of U. S. Provisional Application No. 61/387,934 filed on September 29, 2010, the contents of which is hereby incorporated by reference in its entirety.

Field of the Invention

The present invention relates to the use of c-Met/HGFR and VEGF inhibitors for treating abnormal cell growth in mammals. In particular, the invention provides methods of treating mammals suffering from cancer. In particular the invention provides methods of treating mammals suffering from metastatic cancer. In particular the invention provides methods of inhibiting metastatic cancer.

Background of the Invention

Members of the VEGF (vascular endothelial growth factor) signaling pathway have been shown to play a key role in both physiological and pathological angiogenesis. As a result, the FDA recently approved bevacizumab (an anti-VEGF monoclonal antibody) which opened a new era in cancer therapy (Ferrara N, et al. Angiogenesis as a therapeutic target. Nature 2005; 438 (7070): 967-74). In addition to blocking the ligand as bevacizumab does, several other RTKIs (receptor tyrosine kinase inhibitors), such as sunitinib and sorafenib, which target the VEGF pathway are also FDA-approved or are in various stages of development (Ivy SP, et al. An overview of small-molecule inhibitors of VEGFR signaling. Nat Rev Clin Oncol 2009; 6 (10): 569-79). However, despite demonstrated efficacy and survival benefit in multiple treatment settings, the majority of patients will eventually exhibit disease progression.

Molecular and cellular mechanisms responsible for such unresponsiveness are under extensive investigation. From an angiogenesis standpoint, several studies have suggested that both tumor cells and stroma (non-tumor compartment mainly comprising of fibroblasts, pericytes, myeloid cells and mesenchymal stem cells) contribute to the development of resistance to anti-angiogenic therapy through activation of alternative angiogenic factors such as FGF-2 (fibroblast growth factor (Casanovas O, et al. Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors. Cancer cell 2005;8(4):299-309)), Bv8 (Bombina Variegata (Shojaei F, et al. Bv8 regulates myeloid-cell-dependent tumour angiogenesis. Nature 2007; 450 (7171 ): 825-31 )) and PIGF (platelet derived growth factor (Fischer C, et al. Anti-PIGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels. Cell 2007; 131 (3): 463-75)).

The HGF (hepatocytes growth factor/scatter factor)/c-Met pathway is known to play a significant role in different stages of development and also in tumorigenesis (You WK, et al. The hepatocyte growth factor/c-Met signaling pathway as a therapeutic target to inhibit angiogenesis. BMB reports 2008; 41 (12): 833-9). While HGF is enriched in cells of mesenchymal origin, c-Met is also highly expressed in epithelial cells. c-Met has also been found to express in several other cell types including endothelial cells, neural cells, hematopoietic cells and pericytes. Expression of c-Met and HGF have been identified in several cancer types such as bladder, breast, stomach, colon and renal (Peruzzi B, et al. Targeting the c-Met signaling pathway in cancer. Clin Cancer Res 2006; 12(12): 3657-60). It has been suggested that activation of c-Met results in proliferation, survival and increased invasiveness of tumor cells through signaling pathways such as PIK3/Akt, Src, STAT3 and Ras/Mek (Comoglio PM, et al. Drug development of MET inhibitors: targeting oncogene addiction and expedience. Nature reviews 2008; 7(6): 504-16; Maulik G, et al. Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition. Cytokine & growth factor reviews 2002; 13(1 ): 41 -59). c-Met activation has been shown to induce tumor angiogenesis in the vasculature through paracrine HGF mainly by induction of proliferation, migration and survival of endothelial cells (Birchmeier C, et al. Met, metastasis, motility and more. Nat Rev Mol Cell Biol 2003;4(12):915-25). It has also been shown that HGF promotes angiogenesis through upregulation of VEGF (a key angiogenic inducer) and also via downregulating thrombospondin-1 expression (a potent angiogenic inhibitor) (Zhang YW, et al. Hepatocyte growth factor/scatter factor mediates angiogenesis through positive VEGF and negative thrombospondin 1 regulation. PNAS 2003; 100(22): 12718-23). Similar to VEGF, expression of both c-Met and HGF is induced by HIF-1 a (hypoxia inducible factor) further providing a role for c-Met HGF in adverse microenvironment conditions to assist angiogenesis, cell survival and invasion (Pennacchietti S, et al. Hypoxia promotes invasive growth by transcriptional activation of the met protooncogene. Cancer cell 2003; 3(4): 347-61 ; Wang GL, et al. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J-Biol-Chem 1993 268(29): 21513-8). Consistent with these observations, clinical findings indicate that activation of c-Met pathway is a poor prognostic factor in cancer patients (Abounader R, et al. Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis. Neuro-oncology 2005;7(4):436-51 ; Garcia S, et al. Poor prognosis in breast carcinomas correlates with increased expression of targetable CD146 and c-Met and with proteomic basal-like phenotype. Human pathology 2007; 38(6): 830-41 ; Peghini PL, et al. Overexpression of epidermal growth factor and hepatocyte growth factor receptors in a proportion of gastrinomas correlates with aggressive growth and lower curability. Clin Cancer Res 2002; 8(7): 2273-85). Recent studies have indicated that HGF is highly expressed in bone marrow CD1 1 b+Gr1 + cells isolated from the anti-VEGF-treated resistant tumors (Shojaei F, et al. Tumor refractoriness to anti-VEGF treatment is mediated by CD1 1 b+Gr1 + myeloid cells. Nature biotechnology 2007; 25(8): 91 1 -20). However, a full understanding of the role of HGF in tumor angiogenesis and particularly in mediating resistance to VEGF-inhibitors remains to be determined.

Agents targeting tumor angiogenesis, particularly VEGF inhibitors, have provided benefits for cancer patients. Similar to other anti-cancer agents, tumor recurrence has been one of the major challenges in patients treated with angiogenesis inhibitors. Recent reports in preclinical models suggest that activation of alternative angiogenic pathways in anti-VEGF treated tumors is one of the mechanisms of unresponsiveness to the therapy (Bergers G, et al. Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer 2008; 8(8): 592-603). In addition, two recent independent studies suggested that anti-angiogenic therapy may induce tumor progression and metastasis due to increased invasiveness of tumor cells (Ebos JM, et al. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer cell 2009;15(3): 232-9; Paez-Ribes M, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer cell 2009;15(3):220-31 ). Overall, these observations have emphasized a need to optimize application of this class of agents in the clinical setting. Summary of the Invention

Each of the embodiments of the invention described below can be combined with any other embodiment of the invention described herein not inconsistent with the embodiment with which it is combined.

In one embodiment, the invention provides a method of treating cancer in a mammal in need of such treatment comprising the step of administering a therapeutically effective amount of a VEGF inhibitor and a therapeutically effective amount of c-Met/HGFR inhibitor. In some embodiments, the mammal is a human. In some embodiments, the cancer is selected from the group consisting of colon cancer, non-small cell lung cancer, melanoma, renal carcinoma, hepatocellular carcinoma, glioblastoma multiform, and pancreatic neuroendocrine neoplasms. In some embodiments, the VEGF inhibitor is selected from the group consisting of sunitinib, SU- 14813, PF-337210, bevacizumab, and axitinib. In some embodiments, the VEGF inhibitor is sunitinib. In some embodiments, the VEGF inhibitor is axitinib. In some embodiments, the VEGF inhibitor is bevacizumab. In some embodiments, the VEGF inhibitor is SU-14813. In some embodiments, the VEGF inhibitor is PF-337210. In some embodiments, the c-Met/HGFR inhibitor is crizotinib or PF-04217903. In some embodiments, the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the c- Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is sunitinib and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is sunitinib and the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is axitinib and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is axitinib and the c-Met/HGFR inhibitor is PF- 04217903. In some embodiments, the VEGF inhibitor is bevacizumab and the c- Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is bevacizumab and the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is SU-14813 and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is SU-14813 and the c-Met/HGFR inhibitor is PF- 04217903. In some embodiments, the VEGF inhibitor is PF-337210 and the c- Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is PF- 337210 and the c-Met/HGFR inhibitor is PF-04217903.

In another embodiment, the present invention provides a pharmaceutical composition comprising a VEGF inhibitor and a c-Met/HGFR inhibitor combined in an amount therapeutically effective for the treatment of cancer in a mammal. In still another embodiment, the invention provides a method of treating cancer in a mammal in need of such treatment comprising the step of administering a pharmaceutical composition of the invention. In some embodiments, the cancer is selected from the group consisting of colon cancer, non-small cell lung cancer, melanoma, renal carcinoma, hepatocellular carcinoma, glioblastoma multiform, and pancreatic neuroendocrine neoplasms. In some embodiments, the VEGF inhibitor is selected from the group consisting of sunitinib, SU- 14813, PF-337210, bevacizumab, and axitinib. In some embodiments, the VEGF inhibitor is sunitinib. In some embodiments, the VEGF inhibitor is axitinib. In some embodiments, the VEGF inhibitor is bevacizumab. In some embodiments, the VEGF inhibitor is SU-14813. In some embodiments, the VEGF inhibitor is PF-337210. In some embodiments, the c-Met/HGFR inhibitor is crizotinib or PF-04217903. In some embodiments, the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the c- Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is sunitinib and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is sunitinib and the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is axitinib and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is axitinib and the c-Met/HGFR inhibitor is PF- 04217903. In some embodiments, the VEGF inhibitor is bevacizumab and the c- Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is bevacizumab and the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is SU-14813 and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is SU-14813 and the c-Met/HGFR inhibitor is PF- 04217903. In some embodiments, the VEGF inhibitor is PF-337210 and the c- Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is PF- 337210 and the c-Met/HGFR inhibitor is PF-04217903.

In another embodiment, the invention provides a method of inhibiting angiogenesis in a mammal comprising the step of administering a therapeutically effective amount of a VEGF inhibitor and a c-Met/HGFR inhibitor. In some embodiments, the mammal is a human. In some embodiments, the cancer is selected from the group consisting of colon cancer, non-small cell lung cancer, melanoma, renal carcinoma, hepatocellular carcinoma, glioblastoma multiform, and pancreatic neuroendocrine neoplasms. In some embodiments, the VEGF inhibitor is selected from the group consisting of sunitinib, SU-14813, PF-337210, bevacizumab, and axitinib. In some embodiments, the VEGF inhibitor is sunitinib. In some embodiments, the VEGF inhibitor is axitinib. In some embodiments, the VEGF inhibitor is bevacizumab. In some embodiments, the VEGF inhibitor is SU-14813. In some embodiments, the VEGF inhibitor is PF-337210. In some embodiments, the c-Met/HGFR inhibitor is crizotinib or PF-04217903. In some embodiments, the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is sunitinib and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is sunitinib and the c-Met/HGFR inhibitor is PF- 04217903. In some embodiments, the VEGF inhibitor is axitinib and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is axitinib and the c- Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is bevacizumab and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is bevacizumab and the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is SU-14813 and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is SU-14813 and the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is PF-337210 and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is PF- 337210 and the c-Met/HGFR inhibitor is PF-04217903.

In another embodiment, the invention provides a method of inhibiting angiogenesis in a mammal in need of such treatment comprising the step of administering a pharmaceutical composition of the invention. In some embodiments, the mammal is a human. In some embodiments, the cancer is selected from the group consisting of colon cancer, non-small cell lung cancer, melanoma, renal carcinoma, hepatocellular carcinoma, glioblastoma multiform, and pancreatic neuroendocrine neoplasms. In some embodiments, the VEGF inhibitor is selected from the group consisting of sunitinib, SU-14813, PF-337210, bevacizumab, and axitinib. In some embodiments, the VEGF inhibitor is sunitinib. In some embodiments, the VEGF inhibitor is axitinib. In some embodiments, the VEGF inhibitor is bevacizumab. In some embodiments, the VEGF inhibitor is SU-14813. In some embodiments, the VEGF inhibitor is PF-337210. In some embodiments, the c-Met/HGFR inhibitor is crizotinib or PF-04217903. In some embodiments, the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is sunitinib and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is sunitinib and the c-Met/HGFR inhibitor is PF- 04217903. In some embodiments, the VEGF inhibitor is axitinib and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is axitinib and the c- Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is bevacizumab and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is bevacizumab and the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is SU-14813 and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is SU-14813 and the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is PF-337210 and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is PF- 337210 and the c-Met/HGFR inhibitor is PF-04217903.

In another embodiment, the invention provides a method of treating metastatic cancer in a mammal in need of such treatment comprising the step of administering a therapeutically effective amount of a VEGF inhibitor and a c-Met/HGFR inhibitor. In some embodiments, the mammal is a human. In some embodiments, the cancer is selected from the group consisting of colon cancer, non-small cell lung cancer, melanoma, renal carcinoma, hepatocellular carcinoma, glioblastoma multiform, and pancreatic neuroendocrine neoplasms. In some embodiments, the VEGF inhibitor is selected from the group consisting of sunitinib, SU-14813, PF-337210, bevacizumab, and axitinib. In some embodiments, the VEGF inhibitor is sunitinib. In some embodiments, the VEGF inhibitor is axitinib. In some embodiments, the VEGF inhibitor is bevacizumab. In some embodiments, the VEGF inhibitor is SU-14813. In some embodiments, the VEGF inhibitor is PF-337210. In some embodiments, the c- Met/HGFR inhibitor is crizotinib or PF-04217903. In some embodiments, the c- Met/HGFR inhibitor is crizotinib. In some embodiments, the c-Met/HGFR inhibitor is PF- 04217903. In some embodiments, the VEGF inhibitor is sunitinib and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is sunitinib and the c- Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is axitinib and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is axitinib and the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is bevacizumab and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is bevacizumab and the c-Met/HGFR inhibitor is PF- 04217903. In some embodiments, the VEGF inhibitor is SU-14813 and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is SU-14813 and the c- Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is PF- 337210 and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is PF-337210 and the c-Met/HGFR inhibitor is PF-04217903.

In another embodiment, the invention provides a method of inhibiting metastasis in a mammal in need of such treatment comprising the step of administering a therapeutically effective amount of a VEGF inhibitor and a c-Met/HGFR inhibitor. In some embodiments, the metastasis is in the lymph nodes. In some embodiments, the metastasis is in the colon. In some embodiments, the mammal is a human. In some embodiments, the cancer is selected from the group consisting of colon cancer, non- small cell lung cancer, melanoma, renal carcinoma, hepatocellular carcinoma, glioblastoma multiform, and pancreatic neuroendocrine neoplasms. In some embodiments, the VEGF inhibitor is selected from the group consisting of sunitinib, SU- 14813, PF-337210, bevacizumab, and axitinib. In some embodiments, the VEGF inhibitor is sunitinib. In some embodiments, the VEGF inhibitor is axitinib. In some embodiments, the VEGF inhibitor is bevacizumab. In some embodiments, the VEGF inhibitor is SU-14813. In some embodiments, the VEGF inhibitor is PF-337210. In some embodiments, the c-Met/HGFR inhibitor is crizotinib or PF-04217903. In some embodiments, the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the c- Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is sunitinib and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is sunitinib and the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is axitinib and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is axitinib and the c-Met/HGFR inhibitor is PF- 04217903. In some embodiments, the VEGF inhibitor is bevacizumab and the c- Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is bevacizumab and the c-Met/HGFR inhibitor is PF-04217903. In some embodiments, the VEGF inhibitor is SU-14813 and the c-Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is SU-14813 and the c-Met/HGFR inhibitor is PF- 04217903. In some embodiments, the VEGF inhibitor is PF-337210 and the c- Met/HGFR inhibitor is crizotinib. In some embodiments, the VEGF inhibitor is PF- 337210 and the c-Met/HGFR inhibitor is PF-04217903. Brief Description of the Drawings

Figure 1 : Identification of cell lines resistance or sensitive to Sunitinib treatment.

B16F1 (Fig. 1A) and Tib6 (Fig. 1 B) tumors are sensitive to Sunitinib and EL4 (Fig. 1 C) and LLC (Fig. 1 D) tumors were found to be resistant to such therapy. Fig. 1 E & 1 F) Inhibition of PDGF-R, CSF-1 R and c-Kit pathways does not provide any advantage in tumor growth inhibition compared to inhibition of VEGF alone. Tumor growth inhibition in EL4 (Fig. 1 E) or LLC (Fig. 1 F) is completely similar between Sunitinib vs. axitinib. Figure 2: Endothelial cells, but not tumor cells, are mainly targeted by HGF/c-Met axis. HGF measurement in the sera (Fig. 2A) and tumors (Fig. 2B) in both vehicle- and Sunitinib- treated tumors. Bars represent mean concentration of HGF in Sunitinib or vehicle treated tumors + SEM. (Fig. 2C) HGF is mainly expressed in stromal compartment in the tumor mass. (Fig. 2D) c-Met is mainly expressed in endothelial cells. Images are the representative histograms from each line comparing c-Met expression (red line) vs. isotype control (black line). While tumor cells have minimal expression of c- Met, it is highly enriched in endothelial cells.

Figure 3: Proliferation of endothelial cells, but not tumor cells, is induced by HGF. Tumor cells (B16F1 , Tib6, EL4, LLC; Fig. 3A) and endothelial cells (HUVECs and C166; Fig. 3B) were seeded at 10⁴ in each well of 24 well tissue culture treated plates and were grown in plain media supplemented with 1 % FBS. Cells were treated with three different concentrations (10 ng/ml; 100 ng/ml and 200 ng/ml) of HGF and VEGF. After 4 days incubation, cells were counted using a Coulter Counter Machine. Asterisks are indicative of a significant difference in HGF- or VEGF- treated cells vs. PBS.

Figure 4: Combination of Sunitinib and PF-04217903 has additive effect compared to Sunitinib monotherapy. Efficacy of combination treatment (Sunitinib plus PF- 04217903) in sensitive or resistant tumors. Nude mice (n=6) were implanted with B16F1 (Fig. 4A), Tib6 (Fig. 4B), EL4 (Fig 4C) and LLC (Fig. 4D) cells (1x10⁶ cells per mouse). Treatments started a day after implantation and tumors volumes were measured twice a week. Graphs represent mean tumor volumes and bars represent SEM. Asterisks indicate significant difference (p<0.05) when comparing sunitinib vs. combination treatment.

Figure 5: Inhibition of angiogenesis is one of the mechanisms by which combination treatment affects tumor growth. Vascular quantification in the resistant or sensitive tumors (Fig. 5A). Images of CD31 staining were selected from each section (four image from each section and up to twenty four images in total) and vascular density was calculated by dividing the signal intensity of CD31 + pixels to entire area of the image. Bars represent mean vascular density + SEM. Asterisks indicate significant difference (p<0.05) when comparing Sunitinib to combination treatment (Fig. 5B & 5C). Endothelial cells, but not tumor cells, are sensitive to combination treatment using Sunitinib and PF-04217903. Data are the representative of one of two independent studies. Asterisks indicate significant differences when comparing Sunitinib alone to the vehicle and ♦ indicates significant difference when comparing cell number in combination treatment vs. Sunitinib or PF-04217903 at corresponding concentration.

Figure 6: Effectiveness of single agent and combination treatment on primary versus metastatic tumor growth in H460 lung orthotopic tumors. GFP-images (Fig. 6A) and also terminal tumor weight (Fig. 6B) did not reveal a significant difference between vehicle and any of the single agent or combination treatment. Analysis of metastasis in the lung or in the lymph nodes (Figure 6C) suggested that sunitinib does not inhibit tumor metastasis, however, crizotinib or combination treatment reduced the onset of metastasis. Figure 7: Effectiveness of single agent and combination treatment on primary versus metastatic tumor growth in Colo205 colon orthotopic tumors. GFP images (Fig. 7A) from tumor bearing mice indicated no significant difference between Sunitinib, vehicle and crizotinib, but did indicate a difference in combination treatment with Sunitinib and crizotinib. Combination-treated mice showed a significant reduction in primary tumor volume and primary tumor weight (Fig. 7B). Analysis of metastasis to lymph node and other region in the colon (Fig. 7C) showed that while neither Sunitinib nor PF-02341066 inhibited metastasis, combination treatment completely blocked metastasis. Definitions

As used herein, unless otherwise indicated, the term "abnormal cell growth" refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition).

As used herein, unless otherwise indicated, the term "treating", means reversing, alleviating, or inhibiting the progress of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment", as used herein, unless otherwise indicated, refers to the act of treating as "treating" is defined immediately above.

As used herein the term "angiogenesis inhibitor" includes any substance that inhibits the growth of new blood vessels (i.e angiogenesis). An angiogenesis inhibitor can be any substance that acts by binding/inhibiting VEGF, inhibiting bFGF, inhibiting cell proliferation, cell migration and survival of endothelial cells, inducing apoptosis of endothelial cells, activating of the immune system, downregulating angiogenesis stimulators, stimulating angiogenesis inhibitor formation, inhibiting basement membrane degradation, and the like.

As used herein, the term "metastasis" means the spread of a disease, here cancer or abnormal cell growth, from one organ or part to another non-adjacent organ or part. Metastasis, as used herein, can be local metastasis in which some cancer cells originating from the primary tumor acquire the ability to penetrate and infiltrate surrounding normal tissues in the local area, forming a new tumor (sometimes referred to as the "daughter" tumor). Metastasis, as used herein, also can be lymphatic spread in which some cancer cells originating from the primary tumor acquire the ability to penetrate the walls of lymphnodes or the lymphatic vessels, after which they are able to circulate through the bloodstream (sometimes referred to as "circulating tumor cells") to other sites and tissues in the body. Metastasis, as used herein, also can be hematogeneous spread in which some cancer cells originating from the primary tumor acquire the ability to penetrate the walls of blood vessels, after which they are able to circulate through the bloodstream (sometimes referred to as "circulating tumor cells") to other sites and tissues in the body. The metastasis process can be any known process involving the proliferation of cancer cells in another part of the body, another organ or in another tissue. For example, after tumor cells from a primary tumor come to rest at another site, the cell can re-penetrate through the vessel or walls, continue to multiply, and eventually form another clinically detectable tumor. When tumor cells metastasize, the new tumor is called a secondary or metastatic tumor, and its cells are like those in the original tumor. This means, for example, that, if breast cancer metastasizes to the lungs, the secondary tumor is made up of abnormal breast cells, not of abnormal lung cells. The tumor in the lung is then called metastatic breast cancer, not lung cancer. Through the metastasis processes described herein and those known in the art, sites for metastases include but are not limited to the lungs, liver, brain, and bones.

As used herein "VEGF inhibitor" includes any substance that inhibits or binds to VEGF or VEGF-R. Unless indicated otherwise, all references herein to VEGF inhibitors used in the include references to pharmaceutically acceptable salts, solvates, hydrates and complexes thereof, and to solvates, hydrates and complexes of pharmaceutically acceptable salts thereof, including polymorphs, stereoisomers, and isotopically labeled versions thereof. For example, one preferred VEGF inhibitor is sunitinib which as used herein refers to sunitinib malate (i.e. Sutent^®) and also the freebase of sunitinib and any other pharmaceutically acceptable salt of sunitinib.

A preferred VEGF inhibitor is sunitinib, 5-(5-fluoro-2-oxo-1 ,2-dihydroindol-(3Z)- ylidenemethyl)-2,4-dimethyl-1 H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)-amide, represented by formula

which is a novel oral cancer drug shown to have efficacy in a variety of solid tumor types. Sunitinib targets multiple receptor tyrosine kinase inhibitors, including PDGFR, KIT and VEGFR, and is a potent and selective anti-angiogenesis agent. Sunitinib or its L-malate salt is also referred to as SU1 1248, SU01 1248, sunitinib malate (USAN/WHO designation) or SUTENT™ (L-malate salt). As used herein, the term "sunitinib" includes 5-(5-fluoro-2-oxo-1 ,2-dihydroindol-(3Z)-ylidenemethyl)-2,4-dimethyl-1 H-pyrrole-3- carboxylic acid (2-diethylaminoethyl)-annide, its pharmaceutically acceptable salts, including the L-malate salt, and SUTENT™

Sunitinib, its synthesis, and particular polymorphs are described in U.S. Patent Nos. 6,573,293, 7,435,832 and 7,125,905; U.S. Patent Publication Nos. 2003-0229229, and 2005-0059824, and in J.M. Manley, M.J. Kalman, B.G. Conway, C.C. Ball, J.L. Havens and R. Vaidyanathan, "Early Amidation Approach to 3-[(4-amido)pyrrol-2-yl]-2- indolinones," J. Org. Chem. 68, 6447-6450 (2003). Preferred formulations of sunitinib and its L-malate salt are described in U.S. Patent Publication 2004-0229930 and in PCT Publication No. WO2004/024127. Preferred dosing regimens are described in U.S. Patent Publication 2005-0182122 and in PCT Publication No. WO 2006/120557. The disclosures of these references are incorporated herein by reference in their entireties.

Several references, in addition to those cited above, describe combinations of sunitinib with other agents. For example, U.S. Patent Publication No. 2003-0216410 describes combinations of Compound 1 with cyclooxygenase inhibitors. U.S. Patent Publication No. 2004-0152759 describes combinations of Compound 1 with several agents, such as CPT-1 1 (irinotecan, Camptosar™), docetaxel and 5-fluorouracil (5-FU). Other references to sunitinib and its uses include U.S. Patent Nos. 7,21 1 ,600 and 7,1 19,209, and PCT Publication Nos. WO 2006/101692, WO 2003/015608, WO 2003/035009, WO 2004/045523, WO 2004/075775. The disclosures of these references are incorporated herein by reference in their entireties.

Other preferred VEGF inhibitors include, but are not limited to, axitinib, 6-[2- (methylcarbamoyl)phenylsulfanyl]-3-E-[2-(pyridine-2-yl)ethenyl]indazole, AG-13676, (Pfizer Inc.) having the structure

is a selective VEGF and PDGF inhibitor described in U.S. Patent Nos. 6,534,524, 6,884,890 and 7,141 ,581 , and PCT Publication Nos. WO 2008/122858 and WO 2006/123223, the disclosures of which are incorporated herein by reference in their entireties. SU-14813, 5-[(Z)-(5-fluoro-2-oxo-1 ,2-dihydro-3H-indol-3-ylidene)methyl]-N- [(2S)-2-hydroxy-3-morpholin-4-ylpropyl]-2,4-dinnethyl-1 H-pyrrole-3-carboxamide, (Pfizer Inc.) having the structure

is a potent, selective, oral inhibitor of receptor tyrosine kinases (RTKs) directly involved in signaling cascades that trigger tumor growth, progression, and survival. SU-14813 is an inhibitor of VEGF, PDGFRa and PDGFR , KIT, and FLT3, and is described in U.S. Patent Nos. 6,653,308 and 7,247,627, as well as in some of the references cited for sunitinib, the disclosures of which are incorporated herein in their entireties. PF-337210, N,2-dimethyl-6-(7-(2-morpholinoethoxy)quinoline-4-yloxy)benzofuran-3-carboxamide (Pfizer Inc.) having the structure

is a selective VEGF inhibitor described in US Patent No. 7,381 ,824 and PCT Publication No. WO 2007/017740, the disclosures of which are incorporated herein by reference in their entireties.

Other VEGF inhibitors include, but are not limited to, Avastin (bevacizumab, Genentech), an anti-VEGF monoclonal antibody, and VEGF inhibitors described in, for example, US Patent No. 6,534,524, 5,834,504, 5,883,1 13, 5,886,020, 5,792,783, 6,653,308 and 6,235,764, each of which is incorporated by reference in its entirety for all purposes, and in WO 99/24440, WO 95/21613, WO 99/61422, WO 98/50356, WO 99/10349, WO 97/32856, WO 97/22596, WO 98/54093, WO 98/02438, WO 99/16755, and WO 98/02437, all of which are herein incorporated by reference in their entirety.

As used herein the term "c-l Met/HGFR inhibitor" includes any substance that inhibits or binds to c-Met or its ligand, HGFR. Unless indicated otherwise, all references herein to c-Met/HGFR inhibitors include references to pharmaceutically acceptable salts, solvates, hydrates and complexes thereof, and to solvates, hydrates and complexes of pharmaceutically acceptable salts thereof, including polymorphs, stereoisomers, and isotopically labeled versions thereof.

A particularly preferred c-Met/HGFR inhibitor is crizotinib, (R)-3-(1 -(2,6-dichloro-3- fluorophenyl)ethoxy)-5-(1 -(piperidin-4-yl)-1 H-pyrazol-4-yl)pyridins-2-amine, represented by formula

which is a novel oral cancer drug shown to have efficacy in a variety of solid tumor types. Crizotinib targets protein tyrosine kinases including c-Met/HGFR and ALK. Crizotinib is also referred to as PF-02341066. As used herein, the term "crizotinib" includes (R)-3-(1 -(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(1 -(piperidin-4-yl)-1 H-pyrazol-4- yl)pyridin-2-amine, and its pharmaceutically acceptable salts.

Crizotinib, its synthesis, and particular polymorphs are described in U.S. Patent

No. 7,230,098; U.S. Patent Publication Nos. 2006-0046991 and 2008-0293769. The disclosures of these references are incorporated herein by reference in their entireties. c-Met/HGFR inhibitors useful in the practice of the present invention are described in, for example, WO2007/0265272, WO2009/068955, WO2006/086484, WO2005/030140, WO2008/051808, US Patent 4,923,986, US Patent 7,713,969, US Patent 7,579,473, all of which are herein incorporated by reference in their entirety.

Another particularly preferred c-Met/HGFR inhibitor is PF-04217903, 2-(4-(3- (quinolin-6-ylmethyl)-3H-[1 ,2,3]triazolo[4,5-b]pyrazin-5-yl)-1 H-pyrazol-1 -yl)ethanol, represented by formula

which is a novel oral cancer drug. Crizotinib targets protein tyrosine kinases including c- Met/HGFR and ALK. As used herein, the term "PF-04217903" includes (R)-3-(1 -(2,6- dichloro-3-fluorophenyl)ethoxy)-5-(1 -(piperidin-4-yl)-1 H-pyrazol-4-yl)pyridin-2-amine, and its pharmaceutically acceptable salts including its phosphate salt.

PF-04217903, its synthesis, and particular polymorphs are described in U.S. Patent No. 7,732,604; and U.S. Patent Application No. 12/745,41 1 (corresponding to PCT Publication No. WO 2009/068955). The disclosures of these references are incorporated herein by reference in their entireties.

Other c-Met/HGFR inhibitors useful in the practice of the present invention include but are not limited to AMEP (Bioalliance), EMD-1204831 (Merck KgaA/EMD Serono), INCB-028060 (Incyte/Novartis), ARQ197 (ArQule), AMG102 (Amgen) and RG-3638 (Roche/Genentech), and those described in WO2007/0265272, WO2009/068955, WO2006/086484, WO2005/030140, WO2008/051808, US Patent 4,923,986, US Patent 7,713,969, US Patent 7,579,473, all of which are herein incorporated by reference in their entirety.

As used herein the term "pharmaceutically acceptable salts" includes acid addition and base salts (including disalts). Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2- napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate and trifluoroacetate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. For a review on suitable pharmaceutically acceptable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002), the disclosure of which is incorporated herein by reference in its entirety.

A pharmaceutically acceptable salt of the inventive compounds can be readily prepared by mixing together solutions of the compound and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionization in the salt may vary from completely ionized to almost non-ionized.

The compounds of the invention may exist in both unsolvated and solvated forms. The term 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when the solvent is water. Pharmaceutically acceptable solvates in accordance with the invention include hydrates and solvates wherein the solvent of crystallization may be isotopically substituted, e.g. D₂O, d6-acetone, d6-DMSO.

The invention also includes isotopically-labeled compounds, which are identical to the VEGF inhibitors and c-Met inhibitors described herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶CI, respectively. Compounds of the present invention and pharmaceutically acceptable salts of said compounds, which contain the aforementioned isotopes and/or other isotopes of other atoms, are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, for example those into which radioactive isotopes such as ³H and ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., ³H, and carbon- 14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., ²H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. An isotopically labeled compound can generally be prepared by carrying out the procedures described for the non-labeled compound, substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

Also included within the scope of the invention are complexes such as clathrates, drug-host inclusion complexes wherein, in contrast to the aforementioned solvates, the drug and host are present in stoichiometric or non-stoichiometric amounts. Also included are complexes of the drug containing two or more organic and/or inorganic components which may be in stoichiometric or non-stoichiometric amounts. The resulting complexes may be ionized, partially ionized, or non-ionized. For a review of such complexes, see J Pharm Sci, 64 (8), 1269-1288 by Haleblian (August 1975), the disclosure of which is incorporated herein by reference in its entirety.

Detailed Description

Unless indicated otherwise, all references herein to c-Met HGFR inhibitors or

VEGF inhibitors used in the include references to pharmaceutically acceptable salts, solvates, hydrates and complexes thereof, and to solvates, hydrates and complexes of pharmaceutically acceptable salts thereof, including polymorphs, stereoisomers, and isotopically labeled versions thereof.

In the present study, we tested efficacy of sunitinib (a RTKI targeting VEGF,

PDGFR-β, CSF-1 R, c-Kit and Flt3) in preclinical models and identified tumors that were resistant/sensitive to the therapy. Analysis of protein lysates in the resistant or sensitive tumors found a greater expression for HGF in the former. Combination treatment using highly selective c-Met inhibitors (PF-04217903 and crizotionib (PF-02341066)) and sunitinib provided additive effect in inhibiting tumor growth compared to either single agent. These results indicated a functional role for HGF/c-Met axis in the sunitinib- resistant tumors. We investigated modes of resistance to sunitinib, which is clinically approved for metastatic RCC (renal cell carcinoma) and imatinib-resistant GIST (gastro-intestinal stromal tumor), and is in several trials for multiple tumor types including lung, colon and breast cancers (Smith JK, et al. Emerging roles of targeted small molecule protein- tyrosine kinase inhibitors in cancer therapy. Oncology research 2004;14(4-5): 175-225; van der Veldt AA, et al. Sunitinib for treatment of advanced renal cell cancer: primary tumor response. Clin Cancer Res 2008;14(8):2431 -6). Sunitinib efficacy in murine lines in the current study, and in comparison with recent report (Shojaei F, et al. Nature biotechnology 2007 ; Shojaei F, et al. PNAS 2009, indicate that blocking the receptor- mediated signaling is as efficacious as blocking the ligand in inhibiting tumor growth. Future studies may determine if there are distinct indications in preclinical models in blocking the VEGF pathway vs. inhibiting the ligand.

We focused our investigations on the c-Met pathway as several studies have pointed to a significant role for this pathway in development, tumorigenesis and angiogenesis. There are at least three different mechanisms involved in the activation of c-Met pathway in tumors: i) ligand mediated activation through binding of HGF; ii) amplification of c-Met gene locus and iii) acting mutations in the kinase domain of the c- Met receptor ((Smith JK, et al. Oncology research 2004; van der Veldt AA, et al. Clin Cancer Res 2008). Of note, our data suggest that in the present study c-Met is activated through binding of HGF as none of the cell lines exhibited a notable response to different concentrations of c-Met inhibitor in vitro or in vivo. Therefore in our models, greater concentration of HGF is required to activate c-Met pathway in the tumors. Lack of HGF expression in tumor or endothelial cells signifies the role of stroma in the resistant tumors as a source of HGF. The in vitro data indicate that although both tumor and endothelial cells are exposed to HGF only the latter is capable of proliferation providing a hypothesis that angiogenesis is the main target of HGF in these tumors. Despite greater concentration in the tumors, HGF does not appear to release in the serum. Assessment of myeloid population further confirms a direct angiogenic role for HGF since neither c-Met inhibition nor HGF upregulation in the sensitive tumors did not affect kinetics of myeloid cells in the peripheral blood or in the tumors.

To test if Sunitinib treatment affects metastasis in preclinical models, we investigated metastatic capacity in H460-GFP and Colo205-GFP tumors in orthotopic fashion. GFP transduction allows tracking of tumor growth in the internal organs in live animals. We supervised and conducted the entire procedures in Anti-Cancer Inc which is one of the leading companies in the area of tumor biology and imaging in preclinical models.

Therefore, these studies suggest the unexpected outcome that a c-Met inhibitor can be potentially added to a VEGF inhibitor as part of the therapeutic regimen in any tumor type where sutent treatment results in the activation of c-Met pathway via: i) HGF upregulation and/or; ii) c-Met receptor over expression and/or; iii) c-Met receptor activation. Cancers for which the present invention may find particular application include but are not limited to colon cancer, non-small cell lung cancer, melanoma, renal carcinoma, hepatocellular carcinoma, glioblastoma multiform, and pancreatic neuroendocrine neoplasms.

Administration

Compounds suitable for use in connection with the present invention may be administered in any manner that provides benefit to the patient. Different combinations of c-Met/HGFR inhibitors and VEGF inhibitors may require variation in administration routes and dosing regimens. Different cancer types may also require variation in administration routes and dosing regimens. The invention contemplates all variations in administration based on any combination of factors, such as, the type of cancer being treated, the c-Met/HGFR inhibitor being administered, the VEGF inhibitor being administered, the preferred dosing regimen of the c-Met/HGFR inhibitor being administered, the preferred dosing regimen of the VEGF inhibitor being administered, or the needs of the patient being treated.

Compounds suitable for use in connection with the present invention may be administered by sequential administration. Sequential administration can include administering a VEGF inhibitor followed by administering a c-Met/HGFR inhibitor. Sequential administration can include administering a c-Met/HGFR inhibitor followed by administering a VEGF inhibitor. Sequential administration can be based on the preferred dosing regimen of either the c-Met/HGFR inhibitor or the VEGF inhibitor. The sequential administration can readily be adjusted based on any one or more of the cancer being treated, the c-Met/HGFR inhibitor being administered, the VEGF inhibitor being administered, the preferred dosing regimen of the c-Met/HGFR inhibitor being administered, the preferred dosing regimen of the VEGF inhibitor being administered, or the patient's needs.

Compounds suitable for use in connection with the present invention may be administered by concomitant administration of a VEGF inhibitor and a c-Met/HGFR inhibitor. Concomitant administration can be in the form of a single pharmaceutical composition containing a combination at least one c-Met/HGFR inhibitor and at least one VEGF inhibitor, or simultaneous dosing of at least one c-Met/HGFR inhibitor and at least one VEGF inhibitor administered by different routes of administration, pharmaceutical compositions, or different dosing vehicles.

Whether administration is performed by a single pharmaceutical composition, sequential dosing or concomitant dosing, two or more compounds useful in connection with the present invention or their metabolites may be present in the patient at the same time. Furthermore, whether administration is performed by a single pharmaceutical composition, sequential dosing or concomitant dosing, two or more compounds useful in connection with the present invention or their metabolites may not be present in the patient.

Routes of administration can include, but are not limited to any of oral administration, parenteral administration, topical administration, inhaled/intranasal administration, rectal/intravaginal administration, ocular administration, or any other route of administration known to one of skill in the art. The route of administration may be the same or different for c-Met/HGFR inhibitors and VEGF inhibitors useful in connection with the invention.

Oral Administration

Compounds suitable for use in connection with the present invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth.

Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid- filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films (including muco-adhesive), ovules, sprays and liquid formulations. Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be used as fillers in soft or hard capsules and typically include a pharmaceutically acceptable carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.

The compounds of the invention may also be used in fast-dissolving, fast- disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, H (6), 981 -986 by Liang and Chen (2001 ), the disclosure of which is incorporated herein by reference in its entirety.

For tablet dosage forms, depending on dose, the drug may make up from 1 wt% to 80 wt% of the dosage form, more typically from 5 wt% to 60 wt% of the dosage form. In addition to the drug, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinized starch and sodium alginate. Generally, the disintegrant will comprise from 1 wt% to 25 wt%, preferably from 5 wt% to 20 wt% of the dosage form.

Binders are generally used to impart cohesive qualities to a tablet formulation.

Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.

Tablets may also optionally include surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents are typically in amounts of from 0.2 wt% to 5 wt% of the tablet, and glidants typically from 0.2 wt% to 1 wt% of the tablet.

Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally are present in amounts from 0.25 wt% to 10 wt%, preferably from 0.5 wt% to 3 wt% of the tablet.

Other conventional ingredients include anti-oxidants, colorants, flavoring agents, preservatives and taste-masking agents.

Exemplary tablets contain up to about 80 wt% drug, from about 10 wt% to about

90 wt% binder, from about 0 wt% to about 85 wt% diluent, from about 2 wt% to about 10 wt% disintegrant, and from about 0.25 wt% to about 10 wt% lubricant.

Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tableting. The final formulation may include one or more layers and may be coated or uncoated; or encapsulated.

The formulation of tablets is discussed in detail in "Pharmaceutical Dosage Forms: Tablets, Vol. 1 ", by H. Lieberman and L. Lachman, Marcel Dekker, N.Y., N.Y., 1980 (ISBN 0-8247-6918-X), the disclosure of which is incorporated herein by reference in its entirety.

Solid formulations for oral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

Suitable modified release formulations are described in U.S. Patent No. 6,106,864. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles can be found in Verma et al, Pharmaceutical Technology On-line, 25(2), 1 -14 (2001 ). The use of chewing gum to achieve controlled release is described in WO 00/35298. The disclosures of these references are incorporated herein by reference in their entireties.

Parenteral Administration

The compounds of the invention may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including micro needle) injectors, needle-free injectors and infusion techniques. Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.

The solubility of compounds of the invention used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.

Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Thus compounds of the invention may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug-coated stents and PGLA microspheres.

Topical Administration

The compounds of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol . Penetration enhancers may be incorporated; see, for example, J Pharm Sci, 88 (10), 955-958 by Finnin and Morgan (October 1999). Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and micro needle or needle-free (e.g. Powderject™, Bioject™, etc.) injection. The disclosures of these references are incorporated herein by reference in their entireties. Formulations for topical administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Inhaled/lntranasal Administration

The compounds of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, with or without the use of a suitable propellant, such as 1 ,1 ,1 ,2-tetrafluoroethane or 1 ,1 ,1 ,2,3,3,3- heptafluoropropane. For intranasal use, the powder may include a bioadhesive agent, for example, chitosan or cyclodextrin.

The pressurized container, pump, spray, atomizer, or nebulizer contains a solution or suspension of the compound(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.

Prior to use in a dry powder or suspension formulation, the drug product is micronized to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

Capsules (made, for example, from gelatin or HPMC), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as /-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

A suitable solution formulation for use in an atomizer using electrohydrodynamics to produce a fine mist may contain from 1 g to 20mg of the compound of the invention per actuation and the actuation volume may vary from 1 μΙ_ to 10ΌμΙ_. A typical formulation includes a compound of the invention, propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.

Suitable flavors, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled/intranasal administration.

Formulations for inhaled/intranasal administration may be formulated to be immediate and/or modified release using, for example, poly(DL-lactic-coglycolic acid (PGLA). Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve which delivers a metered amount. Units in accordance with the invention are typically arranged to administer a metered dose or "puff' containing a desired mount of the compound of the invention. The overall daily dose may be administered in a single dose or, more usually, as divided doses throughout the day.

Rectal/lntravaginal Administration

Compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.

Formulations for rectal/vaginal administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

Ocular Administration

Compounds of the invention may also be administered directly to the eye or ear, typically in the form of drops of a micronized suspension or solution in isotonic, pH- adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and nonbiodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as crossed-l inked polyacrylic acid, polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxyethylcellulose, or methyl cellulose, or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.

Formulations for ocular/aural administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted, or programmed release.

Other Technologies

Compounds of the invention may be combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives thereof or polyethylene glycol- containing polymers, in order to improve their solubility, dissolution rate, taste-masking, bioavailability and/or stability for use in any of the aforementioned modes of administration.

Drug-cyclodextrin complexes, for example, are found to be generally useful for most dosage forms and administration routes. Both inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubilizer. Most commonly used for these purposes are alpha-, beta- and gamma-cyclodextrins, examples of which may be found in PCT Publication Nos. WO 91/1 1 172, WO 94/02518 and WO 98/55148, the disclosures of which are incorporated herein by reference in their entireties.

Dosage

The amount of the active compound administered will be dependent on the subject being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compound and the discretion of the prescribing physician. However, an effective dosage is typically in the range of about 0.001 to about 100 mg per kg body weight per day, preferably about 0.01 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.07 to about 7000 mg/day, preferably about 0.7 to about 2500 mg/day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be used without causing any harmful side effect, with such larger doses typically divided into several smaller doses for administration throughout the day.

Kit-of-Parts

Inasmuch as it may desirable to administer a combination of active compounds, for example, for the purpose of treating a particular disease or condition, it is within the scope of the present invention that two or more pharmaceutical compositions, at least one of which contains a compound in accordance with the invention, may conveniently be combined in the form of a kit suitable for coadministration of the compositions. Thus the kit of the invention includes two or more separate pharmaceutical compositions, at least one of which contains a compound of the invention, and means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like.

The kit of the invention is particularly suitable for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit typically includes directions for administration and may be provided with a memory aid.

Examples

General Procedure 1 : Tumor implantations and treatments

Nude mice were purchased from Jackson (ME, USA) or Charles Rivers laboratories (MA, USA) and were maintained under guidelines provided by the Pfizer IACUC (Institutional Animal Care and Use Committee). All the tumor cell lines in the current study were obtained from ATCC (American Tissue Culture Collection; Manassas, VA) and were cultured in RPMI 1640 (Invitrogen, CA) supplemented with glutamine (2 mM) and fetal bovine serum (FBS; 10%). For implantation, tumor cells (1x10⁶ cells per mouse) were resuspended in 100 μΙ media and 100 μΙ matrigel growth factor reduced (BD Biosciences, CA) and were s.c implanted in one of the flanking areas. Tumor bearing mice were treated once a day with Sunitinib at 80 mg/kg or PF- 04217903 (45 mg/kg) or the combination of both compounds using oral route of administration. Tumors volumes were assessed using caliper measurement as described (Zou HY, et al. An orally available small-molecule inhibitor of c-Met, PF- 2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms. Cancer research 2007;67(9):4408-17). HUVECs (human umbilical vein endothelial cells) and C166 cells were purchased from Lonza Inc. (Lonza, CA) and ATCC respectively. For in vitro assays, HUVECs were grown in EBM2 media supplemented with a cocktail of growth factors provided by the supplier (Lonza Inc, Lonza CA) and C166 were grown in DMEM (Invitrogen, CA) supplemented with FBS (10%). General Procedure 2: Histology

Formalin-fixed tumors were embedded in paraffin and were cut (4 μηη) using a cryostat machine. Tumor sections were stained with mouse anti-CD31 antibody (BD Biosciences, CA) using DAB-immunostaining protocol in the laboratory. To quantify vascular surface area (VSA), images of tumors at 20x magnification were taken from each slides and each treatment. Next, areas of CD31 + cells were highlighted and pixels were quantified using Image-Pro software (MediaCybernetics, Bethesda, Maryland). Vascular density was calculated by dividing the CD31 -pixels to the pixels from total area of the image. General Procedure 3: ELISA

Tumors samples were snap frozen in liquid nitrogen and were kept at -70°C. Total protein from each sample was extracted and was quantified using BCA protein assay (Pierce, IL). Levels of HGF and total Met in the tumors and serums were measured by ELISA kits (R&D System, CA) using protocols provided by the manufacturer. Similar methodology was applied to measure levels of HGF in the conditioned-media or in the tumor lysates.

General Procedure 4: Flow cytometry

Single cell suspensions were provided from the tumors by mechanical disruption followed by red blood cell lysis using RBC lysis buffer (eBioscience, CA). Peripheral blood mononuclear Cells (PBMNCs) were also provided by lysing red blood cells using RBC lysis buffer (eBioscience, CA). Both tumor cells and PBMNCs were stained with anti-mouse CD1 1 b-APC (BD Biosciences, CA) and anti-mouse Gr1 -PE (BD Biosciences, CA) for 30 min at 4°C followed by washing twice with PBS-FBS (3%). The stained-samples were analyzed in a FACS calibur machine (BD Bioscience, CA) and frequency of CD1 1 b+Gr1 + cells in the tumors and blood were analyzed using Cell Quest (BD Biosciences, CA) or FCS Express (De Novo Inc, CA) softwares. Similarly, to measure c-Met expression at the cell surface, in vitro-grown cell lines and endothelial cells were stained with PE-conjugated anti-mouse c-Met (eBioscience, CA). To exclude dead cells from the analysis, cells were stained with 7-AAD (7-amino-actinomycin D; BD Biosciences, CA) prior to analysis on the calibur machine. General Procedure 5: In vitro assays

Cell lines including B16F1 , Tib6, EL4, LLC and endothelial cells, HUVECs and C166, were seeded at 10⁴ cells in each well of 24-well tissue culture treated plates. Cells were grown in the standard media as described above. Cells were treated with different concentrations (2 μιτι, 0.2 μιτι and 0.02 μιτι) of Sunitinib, PF-04217903 and combination of both compounds for 4 days. Efficacy of the compounds was measured by counting cells in a Coulter Counter Machine (BD Biosciences, CA). Similar approach was applied to evaluate role of HGF or VEGF on cell proliferation using three different concentrations (10 ng/ml; 100 ng/ml and 200 ng/ml) of each ligand. Example 1 : Identification of cell lines resistance or sensitive to Sunitinib treatment

Sunitinib resistance

Using general procedure 1 , nude mice (n=6) were implanted with murine lines including B16F1 , Tib6, EL4 and LLC. Cells (1 x10⁶ per mouse) were implanted in 100 μΙ media plus 100 μΙ growth-factor-reduced matrigel. Treatment with sunitinib (80 mg/kg) or Vehicle started the day after implantation. Data is representative of one of two independent studies. Similar to recent reports using anti-VEGF Mab (Shojaei F, et al. Tumor refractoriness to anti-VEGF treatment is mediated by CD1 1 b+Gr1 + myeloid cells. Nature biotechnology 2007;25(8):91 1 -20), B16F1 and Tib6 tumors are sensitive to Sunitinib and EL4 and LLC tumors were found to be resistant to such therapy.

As illustrated in Fig. 1 , while B16F1 (Fig. 1A) and Tib6 (Fig. 1 B) tumors were sensitive to Sunitinib, EL4 (Fig. 1 C) and LLC (Fig. 1 D) tumors exhibit resistant to the therapy shortly after the initiation of treatment. These results are in agreement with previous reports investigating efficacy of an anti-VEGF Mab in the same cell lines (Shojaei F, et al. Nature biotechnology 2007; Shojaei F, et al. PNAS 2009) indicating a similar trend in tumor responsiveness in anti-VEGF vs. small molecule inhibitors of VEGF pathway.

Sunitinib v. Axitinib resistance

Using general procedure 1 , nude mice (n=10) were implanted with EL4 or LLC tumors as described. Treatment with vehicle, Sunitinib (80 mg/kg SID) or axitinib (35 mg/kg BID) started one day after implantation. Tumor growth inhibition in EL4 or LLC is completely similar between Sunitinib vs. axitinib. As a result, inhibition of PDGF-R, CSF- 1 R and c-Kit pathways does not provide any advantage in tumor growth inhibition compared to inhibition of VEGF alone.

In addition to VEGF pathway, several other signaling pathways are targeted by Sunitinib such as PDGF-RB (platelet derived growth factor receptor), CSF-1 R (colony stimulating factor) and c-kit (Sun L, et al. Discovery of 5-[5-fluoro-2-oxo-1 ,2- dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1 H-pyrrole-3-carboxylic acid (2- diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived growth factor receptor tyrosine kinase. Journal of Med Chem 2003;46(7):1 1 16-9). To understand if inhibition of these additional pathways by Sunitinib plays a role in responsiveness to the therapy, we compared efficacy of Sunitinib vs. axitinib (a selective VEGF inhibitor (Hu-Lowe DD, et al. Nonclinical antiangiogenesis and antitumor activities of axitinib (AG-013736), an oral, potent, and selective inhibitor of vascular endothelial growth factor receptor tyrosine kinases 1 , 2, 3. Clin Cancer Res 2008;14(22):7272-83)) in resistant tumors (Fig. 1 E&F). Sunitinib and axitinib demonstrated a statistically indistinguishable effect on tumor growth in both EL4 (Fig. 1 E) and LLC (Fig. 1 F) as neither tumors responded to the therapies. Therefore these tumors appear to be resistant to other Sunitinib target such as PDGF-RB, CSF-1 R and c-kit. Example 2: HGF is differentially expressed in the resistant tumors and mainly targets endothelial cells

HGF measurement in the sera and tumors in both vehicle- and Sunitinib- treated tumors Using general procedure 1 , nude mice (n=6) were implanted with B16F1 , Tib6, EL4 and LLC cells and were treated with sunitinib or vehicle as described. At terminal analysis, using General Procedure 3, total protein from each sample was extracted and quantified. ELISA data showed a gradual reduction in HGF concentration in the serum from non-tumor bearing mice to all tumor bearing mice suggesting that HGF is highly concentrated in the tumors and is not released to the blood circulation (Fig. 2A). HGF levels in the tumors (Fig. 2B), however, were significantly (p<0.05) higher in resistant tumors compared to sensitive ones particularly in the Sunitinib-treated mice. These data indicate that greater concentration of HGF might be associated with resistance to Sunitinib.

Identification of the source of HGF in the tumors

Both resistant and sensitive cell lines as well as endothelial cells (HUVECs and C166) and NIH3T3 (positive control for HGF expression) were seeded in each well of 6- well tissue culture treated plates at 5x10⁵ cells per well. Cells were incubated for 72 hrs in the incubators in either normoxic (20% O₂) and hypoxic conditions (1 % O₂) conditions. Conditioned-media was removed from each well and cells were harvested and were kept at -70°C. HGF ELISA was carried out using General Procedure 3. Irrespective of oxygen concentration, neither tumor nor endothelial cells were enriched in HGF suggesting that stromal compartment might be a major source of HGF in the tumor mass (Fig. 2C). Consistent with human fibroblast cell lines (Mrc5) (Jiang WG, et al. Reduction of stromal fibroblast-induced mammary tumor growth, by retroviral ribozyme transgenes to hepatocyte growth factor/scatter factor and its receptor, c-MET. Clin Cancer Res 2003;9(1 1 ):4274-81 ), NIH3T3 cells were found to be highly enriched in HGF in both protein lysates and conditioned media.

HGF mainly targets endothelial cells

Following General Procedure 4, flow cytometry expression of c-Met at the cell surface in both tumor cells and endothelial cells (HUVECs and C166) was investigated. Flow cytometric analysis indicated that while tumor cells express minimal levels of c-Met at the cell surface, it had greater expression (p<0.05) in endothelial cells (Fig. 2D). Example 3: HGF involvement in tumor/endothelial cell proliferation

Following General Procedure 5, tumor cells (B16F1 , Tib6, EL4, LLC) and endothelial cells (HUVECs and C166) were seeded at 10⁴ in each well of 24 well tissue culture treated plates and were grown in plain media supplemented with 1 % FBS. Cells were treated with three different concentrations (10 ng/ml; 100 ng/ml and 200 ng/ml) of HGF and VEGF. After 4 days incubation, cells were counted using a Coulter Counter Machine. Neither VEGF nor HGF affected cellular proliferation in the tumor cells (Fig. 3A). Conversely, both growth factors promoted proliferation of endothelial cells (Fig. 3B). Example 4: Efficacy of combination treatment (sunitinib plus PF-04217903) in sensitive or resistant tumors

Following general procedure 1 , nude mice (n=6) were implanted with B16F1 , Tib6, EL4 and LLC cells (1 x10⁶ cells per mouse). Treatments started a day after implantation and tumors volumes were measured twice a week.

As illustrated in Fig. 4A&B PF-04217903 monotherapy did not inhibit tumor growth in any of the sensitive tumors indicative of lack of activation of c-Met pathway in these tumors. Alternatively, due to the presence of VEGF, lack of response to PF- 04217903 monotherapy may also signify a role for the VEGF pathway in tumor growth. Further, there was no significant difference in tumor growth in the combination treatment vs. Sunitinib monotherapy in the B16F1 (Fig. 4A) and Tib6 (Fig. 4B) sensitive tumors suggesting lack of contribution of c-Met pathway in these tumors. However, growth of both resistant tumors (EL4; Fig 4C and LLC; Fig. 4D) was significantly (p<0.05) inhibited by the combination regimen compared to either single regimen. Example 5:

Vascular quantification in resistant or sensitive tumors

Following General Procedure 2, vasculature was analyzed in both sensitive and resistant tumors (Fig. 5A). Images of CD31 staining were selected from each section (four image from each section and up to twenty four images in total) and vascular density was calculated by dividing the signal intensity of CD31 + pixels to entire area of the image. In the sensitive tumors, Sunitinib significantly (p<0.05) reduced the vascular density, as measured by CD31 positive areas, compared to the vehicle-treated tumors. However, combination treatment did not affect vascular density in sunitinib as single agent vs. the combination regimen. Inhibition of c-Met by PF-04217903 did not significantly alter the vasculature in either sensitive or resistant tumors further signifying role of VEGF in promoting tumor growth and angiogenesis. In the resistant tumors, sunitinib inhibited the vasculature compared to the vehicle-treated tumors. Additionally, combination therapy significantly reduced vascular surface areas compared to sunitinib alone in the resistant tumors.

Endothelial selectivity

Following General Procedure 5, tumor cells (B16F1 , Tib6, EL4, LLC; Fig. 5B) and endothelial cells (HUVECs and C166; Fig. 5C) were seeded (10⁴) in each well of a 12- well tissue culture plate and were grown in the standard media as described. Sunitinib, PF-04217903 or the combination of both compounds were added to each well at three different concentrations including 2 μιτι, 0.2 μιτι and 0.02 μιτι. Cells were incubated in a cell-incubator for 4 days and were counted in Coulter Counter Machine as described. While Sunitinib and the combination significantly inhibited endothelial cells proliferation at pharmacologically relevant concentrations (Fig. 5B), tumor cells were not affected by either agent or the combination (Fig. 5C).

Example 6: Combination of Sunitinib and PF-02341066 inhibits metastasis in orthtotopic tumors in H460 NSCLC (non-small cell lung cancer)

Cells were orthotopically implanted in the lung in nude mice. Briefly, tumor stocks were generated by subcutaneously implanting nude mice with H460-GFP cells (1 x10⁶ cells per mouse). Tumors were harvested from subcutaneous implants at log phase and were maintained in RPMI-1640 medium (Sigma-Alrdrich). Necrotic tumors were removed from the media and viable tissues were cut into pieces of 2-2.5 cm². Recipient mice were anesthetized with isoflurane and the area of surgery was disinfected with iodine and alcohol. Left chest wall of the animals was transversely incised followed by an intercostal incision between the 3^rd and 4^th costal in the left lung. For each mouse, two identical pieces of H460-GFP tumor fragments were implanted to the surface of the lung. Chest was closed after the implantation and lung was re-inflated by intrathoracic puncture. All the surgical procedures were performed in a HEPA filtered laminar flow hoods and using a magnification microscope (Olympus). Mice were allowed to recover from surgical intervention for 72 hrs prior to initiation of treatments. Mice were divided into 4 groups (n=10) to start treatments including Vehicle, Sunitinib, PF-02341066 and combo (combination of Sunitinib and PF- 02341066). Mice were euthanized 23 days after implantation and tumors were harvested for histochemical analyses. Animals were imaged on the last day of the study using open green fluorescent probe imaging (GFP imaging) to evaluate primary tumor growth and metastasis to other organs. As illustrated in Fig. 6, GFP-images (Fig. 6A) and also terminal tumor weight (Fig. 6B) did not reveal a significant difference between vehicle and any of the single agent or combination treatment. Therefore primary tumor growth was not fully inhibited by the Sunitinib or combination regimens suggesting that other angiogenic mediators than VEGF and HGF might be involved in H460 tumor growth. Analysis of metastasis in the lung or in the lymph nodes suggested that sunitinib does not inhibit tumor metastasis, however, PF-02341066 or combination treatment reduced the onset of metastasis (Fig. 6C).

Example 7: Combination of sunitinib and PF-02341066 inhibits metastasis in orthtotopic tumors in COLO205 colorectal tumors

Nude mice were subcutaneously implanted with Colo205-GFP cells (5x10⁶ cells per mouse) to generate stock tumors. Mice were euthanized when tumor growth reached log phase. Tumor chunks were isolated and maintained in RPMI 1640 medium (Sigma-Aldrich) to remove necrotic tumors and to maintain viable tissues for intracecal implantation. The surgical areas in the recipients were sterilized using iodine and alcohol and mice were anesthetized with a mixture of Ketamine, Acepromazine and Xylazine. For intracecal implantation, the left middle abdomen of nude mice were cut for about 1 cm and the colon serosa was removed to allow implantation of two pieces of tumors (each 2-2.5 cm²). To close the abdominal cavity, abdominal muscles and skin were stiched separately using silk sutures. The entire procedure was carried in the laminar flow hood and under a dissecting microscope.

Orthotopically-implanted mice were allowed to recover from surgical intervention for about 72 hrs and treatment started when tumors reached 75-100 mm³. Mice were divided into 4 groups and were administered with therapeutic compounds including Vehicle, Sunitinib, PF-02341066, and a combination of Sunitinib and PF-02341066. Treated-animals were GFP-imaged every week and the study was terminated 14 weeks after tumor implantation. The FluorVivo imaging system (Indec Biosystems) was used to measure tumor size by calculating perpendicular minor dimension (W, width) and major dimension (L, length).

Analysis of GFP images from tumor bearing mice indicated no significant difference between Sunitinib, vehicle and PF-02341066 (Fig. 7A). Combination-treated mice, however, showed a significant reduction in tumor volume and tumor weight (Fig. 7B). Furthermore, analysis of metastasis to lymph node and other region in the colon showed that while neither Sunitinib nor PF-02341066 inhibited metastasis, combination treatment completely blocked metastasis as there was not any mouse to show metastatic lesion to other organs (Fig. 7C).

Claims

We claim:

1 . A method of treating cancer in a mammal in need of such treatment comprising the step of administering a therapeutically effective amount of a VEGF inhibitor and a therapeutically effective amount of c-Met/HGFR inhibitor.

2. The method of claim 1 , wherein the mammal is a human.

3. The method of claim 1 , wherein the cancer is selected from the group consisting of colon cancer, non-small cell lung cancer, melanoma, renal carcinoma, hepatocellular carcinoma, glioblastoma multiform, and pancreatic neuroendocrine neoplasms.

4. The method of any one of claims 1 -3, wherein the VEGF inhibitor is selected from the group consisting of sunitinib, SU-14813, PF-337210, bevacizumab, and axitinib.

5. The method of any one of claims 1 -3, wherein the VEGF inhibitor is sunitinib.

6. The method of any one of claims 1 -3, wherein the VEGF inhibitor is axitinib.

7. The method of any one of claims 1 -3, wherein the VEGF inhibitor is

bevacizumab.

8. The method of any one of claims 1 -3, wherein the VEGF inhibitor is SU-14813.

9. The method of any one of claims 1 -3, wherein the VEGF inhibitor is PF-337210.

10. The method of any one of claims 1 -9, wherein the c-Met/HGFR inhibitor is crizotinib or PF-04217903.

1 1 . The method of any one of claims 1 -9, wherein the c-Met/HGFR inhibitor is crizotinib.

12. The method of any one of claims 1 -3, wherein the VEGF inhibitor is sunitinib and the c-Met/HGFR inhibitor is crizotinib.

13. The method of any one of claims 1 -3, wherein the VEGF inhibitor is axitinib and the c-Met/HGFR inhibitor is crizotinib.

14. The method of any one of claims 1 -3, wherein the VEGF inhibitor is bevacizumab and the c-Met/HGFR inhibitor is crizotinib.

15. The method of any one of claims 1 -3, wherein the VEGF inhibitor is SU-14813 and the c-Met/HGFR inhibitor is crizotinib.

16. The method of any one of claims 1 -3, wherein the VEGF inhibitor is PF-337210 and the c-Met/HGFR inhibitor is crizotinib.