WO2017066664A1 - Combination therapy including a raf inhibitor for the treatment of colorectal cancer - Google Patents

Abstract

The present disclosure relates to methods for the treatment of colorectal cancer. In particular, the disclosure provides methods for treatment of colorectal cancer by administering a RAF inhibitor or a pharmaceutically acceptable salt thereof, in combination with one or more of: an EGFR inhibitor or a topoisomerase I inhibitor or pharmaceutically acceptable salt thereof.

Description

COMBINATION THERAPY INCLUDING A RAF INHIBITOR

FOR THE TREATMENT OF COLORECTAL CANCER

Field of the Disclosure

[001] This disclosure relates to methods for the treatment of colorectal cancer. In particular, the disclosure provides methods for treatment of colorectal cancer by administering a RAF inhibitor or pharmaceutically acceptable salt thereof in combination with one or more of an EGFR inhibitor or topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof.

Sequence Listing

[002] This application contains a Sequence Listing which is submitted herewith in electronically readable format. The electronic Sequence Listing file was created on October 14, 2016, is named sequencelisting.txt and has a size of 30 kb. The entire contents of the Sequence Listing in the electronic sequencelisting.txt file are incorporated herein by this reference.

Introduction

[003] Anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies are an effective therapy for a minority of patients with metastatic colorectal cancer (mCRC). An intrinsic mechanism of resistance to anti-EGFR therapy is dysregulation of the MAPK pathway which is common in mCRC. Up to 55% of colon cancers harbor a mutation in KRAS or NRAS with another 8% to 10% with BRAF mutations (Karapetis CS, et al. N Engl J Med 2008;359(17): 1757-65; Douillard JY et al. N Engl. J Med 2013;369(11): 1023-34; Kalady MF, et al. Diseases of the Colon & Rectum 2012;55(2): 128-33). Further, the clinical efficacy of EGFR targeted antibodies is limited by the development of acquired (secondary) resistance, which is often associated with activation (via new activating mutations or amplifications) of the MAPK pathway. Agents that molecularly target the MAPK pathway could potentially provide new treatment options for mCRC patients with intrinsic or acquired resistance to anti-EGFR therapy.

[004] Cellular mechanisms causing resistance to the topoisiomerase I inhibitor irinotecan have been reported for each step of the CPT-11 pathway (Xu JM, et al. Clinical Colorectal Cancer 2002;2(3): 182-8). The MAPK pathway has been implicated in the regulation of apoptosis as well as in the response to chemotherapeutic drugs (Makin G, et al. Trends Cell Biol 2001; 11(1 l):S22-6). Activation of MAPK by SN38, the active metabolite of irinotecan, has been observed in MCF-7 breast cancer cells (Ling X, et al. Am J Transl Res 2009; l(4):393-405) or in response to other chemotherapeutic agents such as 5-FU (Zhao HY, et al. Cancer Res 2008;68(17):7035-41) or oxaliplatin [Chiu SJ, et al. Toxicol Lett 2008; 179(2):63 70]. Preclinical data shows that the phosphorylation status of the MAPK p38a and β isoforms may be a marker of resistance to irinotecan-based chemotherapies in CRC (Paillas S, et al. Cancer Res 2011;71(3): 1041-9). Inhibitors of MAPK as an adjuvant therapy may potentiate the efficacy of irinotecan-based chemotherapies in nonresponder CRC patients. Thus, there is a need for more effective treatment options for colorectal cancer.

[005] Combinations with a RAF inhibitor or pharmaceutically acceptable salt thereof designed to inhibit MAPK kinase signaling could be helpful for the treatment of colorectal cancer and might potentially overcome the resistance to particular anticancer agents. Combinations of a RAF inhibitor or a pharmaceutically acceptable salt thereof with one or more of: (i) an EGFR inhibitor or (ii) a

topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof may have additive or even synergistic therapeutic effects. Thus, there is a need for new colorectal cancer treatment regimens, including combination therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

[006] Figure 1 is a graph that shows the effect of Compound A in combination with irinotecan in COL 205 colon xenograft tumors in NU/NU mice: tumor volume.

[007] Figure 2 is a line graph that shows the effect Compound A, cetuximab, and Compound A in combination with cetuximab in Balb/c nude female mice bearing primary colorectal tumor PHTX-24C xenograft models. The PHTX-24C models are KRAS non-exon 2 mutation positive.

[008] Figure 3 is a line graph that shows the effect Compound A, cetuximab, and Compound A in combination with cetuximab in female Balb/c nude mice bearing primary colorectal tumor CR1530 xenograft models. The CR1530 models are KRAS non-exon 2 mutation positive.

[009] Figure 4 is a bar graph that shows the effect of Compound A alone and in combination with cetuximab on pERK levels in the PHTX-24C colorectal cancer model.

[010] Figure 5 is a bar graph that shows the effect of Compound A alone and in combination with Cetuximab on pERK levels in the CR1530 colorectal cancer model.

DESCRIPTION OF THE DISCLOSURE

[011] The present disclosure realates to new combination therapies for the treatment of colorectal cancer. In particular, the present disclosure relates to methods and compositions for treating a subject suffering from colorectal cancer, comprising administering to the subject a RAF inhibitor or a pharmaceutically acceptable salt thereof; and one or more of: (i) an EGFR inhibitor or (ii) a

topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof; wherein the amount of said inhibitors is such that the combination thereof is therapeutically effective in the treatment of colorectal cancer.

[012] Terms used herein shall be accorded the following defined meanings, unless otherwise indicated.

[013] As used herein, the term "RAF kinase" refers to any one of a family of serine/threonine- protein kinases. The family consists of three isoform members (BRAF, RCRAF (RAF-1), and ARAF). RAF protein kinases are involved in the MAPK signaling pathway consisting of a kinase cascade that relays extracellular signals to the nucleus to regulate gene expression and key cellular functions. Unless otherwise indicated by context, the term "RAF kinase" is meant to refer to any RAF kinase protein from any species, including, without limitation. In one aspect, the RAF kinase is a human RAF kinase.

[014] The term "RAF inhibitor", "inhibitor of RAF" or "RAF kinase inhibitor" is used to signify a compound which is capable of interacting with one or more isoform members (BRAF, CRAF (RAF-1) and/or ARAF) of the serine/threonine-protein kinase, RAF including mutant forms. Some examples of RAF mutant forms include BRAF V600E, BRAF V600D, BRAF V600K, BRAF V600E + T5291 and/or BRAF V600E + G468A.

[015] In some embodiments, the RAF kinase is at least about 50% inhibited, at least about 75% inhibited, at least about 90% inhibited, at least about 95% inhibited, at least about 98% inhibited, or at least about 99% inhibited. In some embodiments, the concentration of RAF kinase inhibitor required to reduce RAF kinase activity by 50% is less than about 1 μΜ, less than about 500 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, or less than about 1 nM.

[016] In some embodiments, such inhibition is selective for one or more RAF isoforms, i.e., the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for one or more of BRAF (wild type), mutant BRAF, ARAF, and CRAF kinase. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for BRAF (wild type), BRAF V600E, ARAF and CRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for RBRAF (wild type), BRAF V600E, ARAF and CRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for BRAF (wild type), BRAF V600D, ARAF and CRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for BRAF (wild type), BRAF V600K, and CRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for more than BRAF V600. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for more than BRAF V600E.

[017] In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for BRAF and CRAF kinases. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for BRAF(wild type), BRAF V600E and RCRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for BRAF (wild type), BRAF V600D and CRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for BRAF (wild type), BRAF V600K and CRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for mutant BRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for mutant BRAF V600E. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for mutant BRAF V600D. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for mutant BRAF V600K.

[018] The term "pan-RAF inhibitor" refers to a RAF inhibitor that inhibits more than the BRAF V600 isoform of RAF proteins.

[019] As used herein, the term "topoisomerase I inhibitor" refers to agents designed to interfere with the action of the enzyme topoisomerase I. Irinotecan is an example of a small molecule

topoisomerase I inhibitor.

[020] As used herein, the term "EGFR inhibitor" refers to agents that inhibit the tyrosine kinase epidermal growth factor receptor (EGFR). Cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab are examples of monoclonal antibody EGFR inhibitors. Gefitinib and erlotinib are examples of small molecule EGFR inhibitors.

[021] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10%.

[022] As used herein, the term "comprises" means "includes, but is not limited to."

[023] As used herein, the terms "treatment," "treat," and "treating" are meant to include the full spectrum of intervention for the colorectal cancer from which the subject is suffering, such as administration of the combination to alleviate, slow, stop, or reverse one or more symptoms of the colorectal cancer and to delay the progression of the cancer even if the cancer is not actually eliminated. Treatment can include, for example, a decrease in the severity of a symptom, the number of symptoms, or frequency of relapse, e.g., the inhibition of tumor growth, the arrest of tumor growth, or the regression of already existing tumors.

[024] The term "therapeutically effective amount" as used herein to refer to combination therapy means the amount of the combination of agents taken together so that the combined effect elicits the desired biological or medicinal response, i.e., either destroys the target colorectal cancer cells or slows or arrests the progression of the colorectal cancer in a subject. For example, the "therapeutically effective amount" as used herein to refer to combination therapy would be the amount of the RAF inhibitor or the pharmaceutically acceptable salt thereof and the amount of one or more of: (i) an EGFR inhibitor or (ii) a topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof that when administered together, either sequentially or simultaneously, on the same or different days during a treatment cycle, has a combined effect that is beneficial. In some embodiments, the combined effect is additive. In some embodiments, the combined effect is synergistic. Further, it will be recognized by one skilled in the art that in the case of combination therapy with a therapeutically effective amount, as in the example above, the amount of the RAF inhibitor or the pharmaceutically acceptable salt thereof and/or the amount of the one or more of: (i) an EGFR inhibitor or (ii) a topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof individually may or may not be therapeutically effective.

[025] "Cytotoxic effect," in reference to the effect of an agent on a cell, means killing of the cell. "Cytostatic effect" means an inhibition of cell proliferation. A "cytotoxic agent" means an agent that has a cytotoxic or cytostatic effect on a cell, thereby depleting or inhibiting the growth of, respectively, cells within a cell population.

[026] In some embodiments, the growth of cells contacted with a combination described herein is inhibited by at least about 50% as compared to growth of non-contacted cells. In some embodiments, cell proliferation of contacted cells is inhibited by at least about 75%, at least about 90%, or at least about 95% as compared to non-contacted cells. In some embodiments, the phrase "inhibiting cell proliferation" includes a reduction in the number of contacted cells, as compare to non-contacted cells. Thus, a combination described herein that inhibits cell proliferation in a contacted cell may induce the contacted cell to undergo growth retardation, to undergo growth arrest, to undergo programmed cell death (i.e., apoptosis), or to undergo necrotic cell death.

[027] The term "subject", as used herein, means a mammal, and "mammal" includes, but is not limited to a human. [028] Unless otherwise stated, structures depicted herein are meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structure except for the replacement of a hydrogen atom by a deuterium or tritium, or the replacement of a carbon atom by a ¹³C- or ¹⁴C -enriched carbon are within the scope of the disclosure.

[029] It will be apparent to one skilled in the art that certain compounds described herein may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure. Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single

stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.

[030] Compounds capable of inhibiting the activity of a RAF kinase maybe be used in the methods of the instant disclosure. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof inhibits more isoforms of RAF kinase proteins than BRAF V600. In some embodiment, the or a pharmaceutically acceptable salt thereof inhibits more isoforms of RAF kinase proteins than BRAF V600E. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof inhibits BRAF, mutant BRAF, ARAF, and CRAF. In some embodiments, the RAF inhibitor or a

pharmaceutically acceptable salt thereof is selective for BRAF, BRAF V600E, ARAF and CRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for BRAF, BRAF V600E, ARAF and CRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for BRAF, BRAF V600D, ARAF and CRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for BRAF, BRAF V600K, and CRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for BRAF, BRAF V600E and CRAF. In some embodiments, the RAF inhibitor or a

pharmaceutically acceptable salt thereof is selective for BRAF, BRAF V600D and CRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for BRAF, BRAF V600K and CRAF. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for mutant BRAF. In some embodiments, the RAF inhibitor or a

pharmaceutically acceptable salt thereof is selective for mutant BRAF V600E. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for mutant BRAF V600D. In some embodiments, the RAF inhibitor or a pharmaceutically acceptable salt thereof is selective for mutant BRAF V600K.

[031] In particular, RAF inhibitors include the compounds and pharmaceutically acceptable salts described herein, as well as compounds and pharmaceutically acceptable salts disclosed in, for example, WO 2006/065703, WO 2010/064722, WO 2011/117381, WO 2011/090738, WO 2011/161216, WO 2011/097526, WO 2011/025927, WO 2011/023773, WO 2011/147764, WO 2011/079133, and WO 2011/063159. BRAF specific inhibitors include vemurafinib (Roche), dabrafenib (GSK), and encoratinib (Novatis). Also suitable for use in the methods of the disclosure are solvated and hydrated forms of any of these compounds. Also suitable for use in the methods of the disclosure are pharmaceutically acceptable salts of any of the compounds, and solvated and hydrated forms of such salts. These RAF inhibitors or pharmaceutically acceptable salts thereof can be prepared in a number of ways well known to one skilled in the art of organic synthesis, including, but not limited to, the methods of synthesis described in detail in the above references.

[032] In some embodiments, the RAF inhibitor or pharmaceutically acceptable salt thereof is a compound that inhibits more isoforms of RAF kinase proteins than BRAF V600. In particular, inhibitors of RAF kinase or pharmaceutically acceptable salts thereof that inhibit more isoforms of RAF kinase proteins than BRAF V600 include for example, Compound A, Compound B, compounds disclosed in WO 2009/006389, and US 2013/0252977 (DP-4978/ LY3009120), and including but not limited to compounds RAF-265, ARQ-736, CEP-32496, CCT 196969, CCT 241161, HM95573, and REDX-04988

[033] RAF inhibitors or a pharmaceutically acceptable salts thereof can be assayed in vitro or in vivo for their ability to bind to and/or inhibit RAF kinases. In vitro assays include biochemical FRET assays to measure the phophorylation of MEK by RAF kinases as a method for quantifying the ability of compounds to inhibit the enzymatic activity of RAF kinases. The compounds also can be assayed for their ability to affect cellular or physiological functions mediated by RAF kinase activity. For example in vitro assays quantitate the amount of phosphor-ERK in colorectal cancer cells. Assays for each of these activities are known in the art.

[034] In some embodiments, the RAF inhibitor is (R)-2-(l-(6-amino-5-chloropyrimidine-4- carboxamide)ethyl)-N-(5-chloro-4-(trifluoromethyl)pyridin-2-yl)thiazole-5-carboxamide (Compound A) or a pharmaceutically acceptable salt thereof:

(Compound A). Compound A is described in WO 2009/006389. [035] In some embodiments, the RAF inhibitor is N-{7-cyano-6-[4-fluoro-3-({[3-(trifluoromethyl)- phenyl]acetyl}amino)phenoxy]-l,3-benzothiazol-2-yl}cyclopropanecarboxamide (Compound B) or a pharmaceutically acceptable salt thereof;

(Compound B). Compound B is described in WO

2010/064722. RAF inhibitors, such as Compound A and Compound B, that can inhibit more isoforms of RAF kinase proteins that BRAF V600 have the ability to inhibit both RAF monomer and dimer-mediated signaling, which is a key feature that distinguishes these RAF inhibitors from recently approved BRAF specific inhibitors (vemurafenib and dabrafenib).

[036] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. A "pharmaceutically acceptable salt" means any non-toxic salt of a compound disclosed herein that, upon administration to a recipient, is capable of providing, either directly or indirectly, the compound or an active metabolite or residue thereof.

[037] Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of compounds described herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethane sulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N⁺(Ci_₄alkyl)₄ salts. This disclosure also provides the quaternization of any basic nitrogen-containing groups. Water or oil-soluble or dispersable products may be obtained by such quaternization. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[038] In some embodiments, the RAF kinase inhibitor inhibits more than the BRAF V600 isoform of RAF proteins.

[039] In some embodiments, the combination comprises both an EGFR inhibitor and a

topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof.

[040] In some embodiments, the combination comprises a RAF kinase inhibitor and an EGFR inhibitor. In some embodiments, the EGFR inhibitor is cetuximab or panitumab. In some embodiments, the EGFR inhibitor is cetuximab. In some embodiments, the EGFR inhibitor is panitumab.

[041] In some embodiments, the combination comprises a RAF kinase inhibitor and a

topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof. In some embodiments, the topoisomerase I inhibitor is irinotecan or a pharmaceutically acceptable salt thereof. In some

embodiments, the topoisomerase I inhibitor is irinotecan.

[042] In some embodiments, the RAF kinase inhibitor is Compound A or Compound B. In some embodiments, the RAF kinase inhibitor is Compound A. In some embodiments, the RAF kinase inhibitor is Compound B.

[043] The present disclosure relates to pharmaceutical compositions comprising a combination of a RAF kinase inhibitor or a pharmaceutically acceptable salt thereof, and one or more of: (i) an EGFR inhibitor or (ii) a topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof.

[044] The combinations described herein may be used to treat a high unmet medical need colorectal cancer. In some embodiments, the combinations described herein are used to treat a genetically defined subset of colorectal cancer. Genes such as BRAF, NRAS, and KRAS are mutated in colorectal cancer. Examples of public databases which include information about mutations associated with colorectal cancers are the Database of Genotypes and Phenotypes (dbGaP) maintained by the National Center for Biotechnology Information (Bethesda, MD) and Catalogue of Somatic Mutations in Cancer (COSMIC) database maintained by the Wellcome Trust Sanger Institute (Cambridge, UK). GenBank or GenPept accession numbers and useful nucleic acid and peptide sequences can be found at the website maintained by the National Center for Biotechnology Information, Bethesda, MD. The content of all database accession records (e.g., from Affymetrix HG133 annotation files, Entrez, GenBank, RefSeq, COSMIC) cited throughout this application (including the Tables) are hereby incorporated by reference.

[045] In some embodiments, the colorectal cancer is BRAF, NRAS, and/or KRAS mutation postitive colorectal cancer.

[046] As used herein, "BRAF" or "B-Raf ' refers to BRAF proto-oncogene, serine/threonine kinase, the gene associated with the mRNA sequence assigned as GenBank Accession No. NM_004333, SEQ ID NO: 1 (open reading frame is SEQ ID NO: 2, nucleotides 62 to 2362 of SEQ ID NO: 1), encoding GenPept Accession No. NP_004324, SEQ ID NO:3). Other names for BRAF include RAFB 1 and Noonan Syndrome 7 (NS7). BRAF functions as a serine/threonine kinase, has a role in regulating the MAP kinase/ERKs signaling pathway and can be found on chromosome 7q. The exons in BRAF are found in SEQ ID NO: l at nucleotide bases 1. 199 (exon 1), 200..301 (exon 2), 302..565 (exon 3), 566..669 (exon 4), 670..772 (exon 5), 773..921 (exon 6), 922..1041 (exon 7), 1042..1201 (exon 8), 1202..1238 (exon 9), 1239..1375 (exon 10), 1376..1493 (exon 1 1), 1494..1578 (exon 12), 1579..1755 (exon 13), 1756..1802 (exon 14), 1803..1921 (exon 15), 1922..2053 (exon 16), 2054..2188 (exon 17), and 2189..2947 (exon 18).

[047] As used herein, "KRAS" or "K-Ras" refers to v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog, the gene associated with the mRNA sequence assigned as GenBank Accession No.

NM_004985, SEQ ID NO:4 (open reading frame is SEQ ID NO:5, nucleotides 193 to 759 of SEQ ID NO: 4), encoding GenPept Accession No. NP_004976, SEQ ID NO: 6, the predominant transcript variant of KRAS gene on chromosome 12. Other names for KRAS include KRAS2, and Noonan Syndrome 3 (NS3). KRAS functions as an oncogene with GTPase activity and can be found on chromosome 12. KRAS interacts with the cell membrane and various effector proteins, such as Akt and Cdc42, which carry out its signaling function through the cytoskeleton and effects on cell motility (Fotiadou et al.

(2007) Mol. Cel. Biol. 27:6742-6755). The exons in KRAS are found in SEQ ID NO: 4 at nucleotide bases 1 .181 (exon 1), 182..303 (exon 2), 304..482 (exon 3), 483..642 (exon 4), and 643..5765 (exon 5).

[048] As used herein, "NRAS" or "N-Ras" refers to neuroblastoma RAS viral (v-ras) oncogene homolog, the gene associated with the mRNA sequence assigned as GenBank Accession No.

NM_002524, SEQ ID NO:7 (open reading frame is SEQ ID NO: 8, nucleotides 255 to 824 of SEQ ID NO: 7), encoding GenPept Accession No. NP_002515, SEQ ID NO: 9). Other names for NRAS include Autoimmune Lymphoproliferative Syndrome type IV (ALPS4), NRAS l, and Noonan Syndrome 6 (NS6). NRAS functions as an oncogene with GTPase activity and can be found on chromosome lp. NRAS interacts with the cell membrane and various effector proteins, such as RAF and RhoA, which carry out its signaling function through the cytoskeleton and effects on cell adhesion (Fotiadou et al. (2007) Mol. Cel. Biol. 27:6742-6755). The exons in NRAS are found in SEQ ID NO: 7 at nucleotide bases 1..237 (exon 1), 238..365 (exon 2), 366..544 (exon 3), 545..704 (exon 4), 705..828 (exon 5), 829..867 (exon 6), and 868..4454 (exon 7).

[049] In some embodiments, the colorectal cancer is KRAS mutation positive colorectal cancer. In some embodiments, the colorectal cancer is a KRAS exon 2 mutation positive colorectal cancer. In some embodiments, one or more of the KRAS exon 2 mutation is in codon 12 or codon 13. In some embodiments, the KRAS exon 2 mutation is in codon 12. In some embodiments, the KRAS exon 2 mutation is in codon 13 Examples include, but are not limited to KRAS protein (SEQ ID NO: 6) mutations G12S, G12R, G12C, G12D, G12A, G12V, G12C, G12F, G13S, G13R, G13C, G13D, G13A, G13V, G13G, V14I, V14G, L19F, and Q22K. In some embodiments, the KRAS has a G12 mutation. In some embodiments, the KRAS has G13 mutation.

[050] In some embodiments, the colorectal cancer is a KRAS non-exon 2 mutation positive colorectal cancer. In some some embodiments, the colorectal cancer is a KRAS exon 3 or exon 4 mutation positive colorectal cancer. In some embodiments, the colorectal cancer is a KRAS exon 3 mutation positive colorectal cancer. In some embodiments, the KRAS exon 3 mutation is in codon 61. Examples include, but are not limited to KRAS protein (SEQ ID NO: 6) mutations A59T, A59E, A59G, A59T, G60G, Q61K, Q61E, Q61R, Q61L, Q61H, E63K. In some embodiments, the KRAS mutation is Q61. In some embodiments, the KRAS mutation is Q61R, Q61L, or Q61H. In some embodiments, the KRAS mutation is Q61R. In some embodiments, the KRAS mutation is Q61L. In some embodiments, the KRAS mutation is Q61H. In some embodiments, the colorectal cancer is a KRAS exon 4 mutation positive colorectal cancer. In some embodiments, the KRAS exon 4 mutation is in codon 1 17 or codon 146. In some embodiments, the KRAS exon 4 mutation is in codon 1 17. In some embodiments, the KRAS exon 4 mutation is in codon 146. Examples include, but are not limited to KRAS protein (SEQ ID NO: 6) mutations Kl 17R, Kl 17N, Kl 17N, C I 18S, P121H, P 121 S, T127I, A130fs* 14, A130V, A134T, A134V, R135K, R135T, S 136N, Y137Y, G138R, G138E, G138G, P 140S, E143K, A146T, A 146P, A146V, K147K, K147N, T148fs*6, R149G, Q 150* . In some embodiments, the KRAS has a Kl 17 mutation or A146 mutation. In some embodiments, the KRAS mutation is Kl 17N. In some embodiments, the KRAS mutation is A 146T or A146V. In some embodiments, the KRAS mutation is A146T. In some embodiments, the KRAS mutation is A146V.

[051] In some embodiments, the colorectal cancer is a BRAF mutation positive colorectal cancer. In some embodiments, the colorectal cancer is a BRAF V600 mutation positive colorectal cancer.

Examples include, but are not limited to BRAF protein (SEQ ID NO: 3) mutations V600R,

V600_W604>DG, V600M, V600L, V600>YM, V600K, V600Q, V600E, V600A, V600G, V600D, V600fs* 1 1, V600_K601>E, V600_S602>DT, V600_S605>D, V600_S605>DV, V600_S605>EK, V600V, V60ODLAT, V600M, V600R, and p.V600_W604del. In some embodiments, the BRAF mutation is V600E. In some embodiments, the BRAF mutation is V600G. In some embodiments, the BRAF mutation is V600A. In some embodiments, the BRAF mutation is V600K. In some embodiments, the BRAF mutation is V600M. In some embodiments, the BRAF mutation is V600R. In some embodiments, the BRAF mutation is V600 K. In some embodiments, the BRAF has a V600 mutation.

[052] In some embodiments, the colorectal cancer is an NRAS mutation positive colorectal cancer. In some embodiments, the colorectal cancer is an NRAS exon 2, exon 3, or exon 4 mutation positive colorectal cancer.

[053] In some embodiments, the colorectal cancer is an NRAS non-exon 2 mutation positive colorectal cancer. In some embodiments, the colorectal cancer is an NRAS exon 3 mutation positive colorectal cancer. In some embodiments, one or more of the NRAS exon 3 mutations is in codon 59 or codon 61. In some embodiments, the NRAS exon 3 mutation is in codon 59. In some embodiments, the NRAS mutation is in codon 61. Examples include, but are not limited to NRAS protein (SEQ ID NO: 9) mutations A59T, A59S, G60E, Q61K, Q61E, Q61R, Q61P, Q61H, Q61L, Q61H, and R68T. In some embodiments, the NRAS has a Q61 mutation.

[054] In some embodiments, the colorectal cancer is an NRAS exon 4 mutation positive colorectal cancer. In some embodiments, the one or more of the NRAS exon 4 mutations is in codon 117 or codon 146. In some embodiments, the NRAS exon 4 mutation is in codon 117. In some embodiments, the NRAS exon 4 mutation is in codon 146. Examples include, but are not limited to NRAS protein (SEQ ID NO: 9) mutations A130D, H131R, E132K, G138R, P140P, A146T, A146V, A146P, A146T, and T148S. In some embodiments, the NRAS mutation is A146T. In some embodiments, the NRAS has a A 146 mutation.

[055] In some embodiments, the colorectal cancer is an NRAS exon 2 mutation positive colorectal cancer. In some embodiments, the one or more of the NRAS exon 2 mutations is in codon 12 or codon 13. In some embodiments, the NRAS exon 2 mutation is in codon 12. In some embodiments, that NRAS codon 13. Examples include, but are not limited to NRAS protein (SEQ ID NO: 9) mutations G12S, G12C, G13S, G13R, G13C, G13D, G13A, G13V, and A18T. In some embodiments,the NRAS has a G12 mutation. In some embodiments, the NRAS has a G13 mutation.

[056] In some embodiments, the colorectal cancer is relapsed, refractory, or advanced colorectal cancer. In some embodiments, the colorectal cancer is refractory. In one aspect, the colorectal cancer is primary colorectal cancer. In one aspect, refractory colorectal cancer does not respond to treatment; it is also known as resistant colorectal cancer. In some embodiments, the tumor is unresectable. In one aspect, an unresectable tumor is unable to be removed by surgery. In some embodiments, the colorectal cancer has not been previously treated. In some embodiments, the colorectal cancer is locally advanced. In one aspect, "locally advanced" refers to a colorectal cancer that is somewhat extensive but still confined to one area. In some instances, "locally advanced" can refer to a small tumor that hasn't spread but has invaded nearby organs or tissues that make it difficult to remove with surgery alone. In some embodiments, the colorectal cancer is metastatic. In one aspect, metastatic colorectal cancer is a cancer that has spread from the part of the body where it started (the primary site) to other parts of the body.

[057] The RAF inhibitor or a pharmaceutically acceptable salt thereof and one or more of: (i) an EGFR inhibitor or (ii) a topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof are administered in such a way that they provide a beneficial effect in the treatment of a colorectal cancer. Administration can be by any suitable means provided that the administration provides the desired therapeutic effect, i.e., additivity or synergism.

[058] The amounts or suitable dosages of the RAF inhibitor or a pharmaceutically acceptable salt thereof depends upon a number of factors, including the nature of the severity of the condition to be treated, the particular inhibitor, the route of administration and the age, weight, general health, and response of the individual subject. In some embodiments, the suitable dose level is one that achieves inhibition of more isoforms of BRAF kinase proteins than BRAF V600. In some embodiments, the suitable dose level is one that achieves inhibition of BRAF, CRAF, ARAF and/or BRAFV600E. In some embodiments, the suitable dose level is one that achieves inhibition of BRAF, CRAF, and/or

BRAFV600E. In some embodiments, the suitable dose level is one that achieves a therapeutic response as measured by tumor regression, or other standard measures of disease progression, progression free survival or overall survival. In some embodiments, the suitable dose level is one that achieves this therapeutic response and also minimizes any side effects associated with the administration of the therapeutic agent.

[059] The present disclosure relates to a method of treating a subject suffering from colorectal cancer, comprising administering to the subject Compound A or a pharmaceutically acceptable salt thereof; and one or more of: (i) an EGFR inhibitor or (ii) a topoisomerase I inhibitor or a

pharmaceutically acceptable salt thereof; wherein Compound A or a pharmaceutically acceptable salt thereof, is administered once weekly (QW) with a rest period of 6 days between each administration in an amount of up to 600 mg per dose.

[060] In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of about 400 mg to about 600 mg per dose. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof is administered in an amount of about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg per dose. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of about 400 mg per dose. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of about 600 mg per dose.

[061] In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of about 400 mg to about 600 mg per dose on days 2, 9, 16 and 23 of a 28-day cycle. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of about 400 mg per dose on days 2, 9, 16 and 23 of a 28-day cycle. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of about 600 mg per dose on days 2, 9, 16 and 23 of a 28-day cycle.

[062] The present disclosure relates to a method of treating a subject suffering from colorectal cancer, comprising administering to the subject Compound A or a pharmaceutically acceptable salt thereof; and one or more of: (i) an EGFR inhibitor or (ii) a topoisomerase I inhibitor or a

pharmaceutically acceptable salt thereof; wherein Compound A or a pharmaceutically acceptable salt thereof, is administered every other day (qod) in an amount of from about 100 mg to about 200 mg per dose. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg per dose. In some

embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of about 100 mg per dose. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of about 160 mg per dose. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of about 200 mg per dose.

[063] In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of from about 100 mg to about 200 mg on days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, and 26 of a 28-day cycle. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of 100 mg per dose on days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, and 26 of a 28-day cycle. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of 160 mg per dose on days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, and 26 of a 28-day cycle. In some embodiments, Compound A or a pharmaceutically acceptable salt thereof, is administered in an amount of 200 mg per dose on days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, and 26 of a 28 -day cycle.

[064] In some embodiments, the EGFR inhibitor is cetuximab. In some embodiments, the cetuximab is administered once weekly (QW) with a rest period of 6 days between each administration in an amount of up to 400 mg/m² per the first dose and up to 250 mg/m² per dose after the first dose. In some embodiments, the cetuximab is administered once weekly with a rest period of 6 days between each administration in an amount of about 400 mg/m² per the first dose and in an amount of about 250 mg/m² per dose after the first dose. In some embodiments, the cetuximab is administered in an amount of about 400 mg/m² per dose on day 1 (cycle 1) and in an amount of about 250 mg/m² per dose on days 1, 8, 15 and 22 (all cycles thereafter) of a 28-day cycle. In some embodiments, the cetuximab is administered over about 1-hour by IV infusion.

[065] In some embodiments, the EGFR inhibitor is panitumumab. In some embodiments, the panitumab is administered in an amount of about 6 mg/kg every 14 days as an intravenous infusion over 60 minutes (< 1000 mg) or 90 minutes (>1000 mg).

[066] In some embodiments, the irinotecan is administered in an amount of up to 180 mg/m² dose. In some embodiments, the irinotecan is administered in an amount of about 180 mg/m² dose. In some embodiments, the irinotecan is administered in an amount of about 180 mg/m² on days 1 and 15 of a 28- day cycle. In some embodiments, the irinotecan is administered over 90 min by IV infusion.

[067] In some embodiments, the method further comprises administering one or more antiemetics selected from dexamethasone, 5HT₃ blocker (ondanestron, granisetron) and prochlorperazine.

[068] The disclosure provides a method for extending duration of response to treatment in subject suffering from colorectal cancer comprising administering to the subject a RAF inhibitor or a pharmaceutically acceptable salt thereof; and one or more of: (i) an EGFR inhibitor or (ii) a

topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof; the amount of said inhibitors being such that the combination thereof is effective for extending the duration of response.

[069] The RAF inhibitor or a pharmaceutically acceptable salt thereof can be administered by any method known to one skilled in the art. For example, the RAF inhibitor or a pharmaceutically acceptable salt thereof can be administered in some embodiments as a pharmaceutical composition of a RAF inhibitor and a pharmaceutically acceptable carrier, such as those described herein. In some

embodiments, the pharmaceutical composition of a RAF inhibitor or a pharmaceutically acceptable salt thereof is a solid dispersion extrudate as described in WO2015148828A1. In some embodiments, the pharmaceutical composition of a RAF inhibitor or a pharmaceutically acceptable salt thereof is a solid dispersion extrudate comprising a vinylpyrrolidinone-vinyl acetate copolymer and one or more pharmaceutical acceptable excipients. In some embodiments, the copolymer is copovidone e.g, Kollidon® VA64. In some embodiments, the pharmaceutical composition of a RAF inhibitor or a pharmaceutically acceptable salt thereof is amorphous. [070] The one or more of: (i) an EGFR inhibitor ir (ii) a topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof can be administered by any method known to one skilled in the art.

[071] If a pharmaceutically acceptable salt of the RAF inhibitor or the one or more of: (i) an EGFR inhibitor or (ii) a topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof is utilized, the salt preferably is derived from an inorganic or organic acid or base. For reviews of suitable salts, see, e.g., Berge et al, J. Pharm. Sci. 66: 1-19 (1977) and Remington: The Science and Practice of Pharmacy, 20th Ed. , ed. A. Gennaro, Lippincott Williams & Wilkins, 2000.

[072] Nonlimiting examples of suitable acid addition salts include the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate,

lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 -phenyl -propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.

[073] Suitable base addition salts include, without limitation, ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine, N-methyl-D-glucamine, t-butylamine, ethylene diamine, ethanolamine, and choline, and salts with amino acids such as arginine, lysine, and so forth.

[074] Also, basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides, such as benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.

[075] The term "pharmaceutically acceptable carrier" is used herein to refer to a material that is compatible with a recipient subject. In one aspect, the subject is a mammal. In one aspect, the subject is a human. In one aspect, the material is suitable for delivering an active agent to the target site without terminating the activity of the agent. The toxicity or adverse effects, if any, associated with the carrier preferably are commensurate with a reasonable risk/benefit ratio for the intended use of the active agent.

[076] The terms "carrier", "adjuvant", or "vehicle" are used interchangeably herein, and include any and all solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington: The Science and Practice of Pharmacy, 20th Ed. , ed. A. Gennaro, Lippincott Williams & Wilkins, 2000 discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of the disclosure, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this disclosure. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as disodium hydrogen phosphate, potassium hydrogen phosphate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium hydroxide and aluminum hydroxide, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, pyrogen-free water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose, sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate, powdered tragacanth; malt, gelatin, talc, excipients such as cocoa butter and suppository waxes, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, glycols such as propylene glycol and polyethylene glycol, esters such as ethyl oleate and ethyl laurate, agar, alginic acid, isotonic saline, Ringer's solution, alcohols such as ethanol, isopropyl alcohol, hexadecyl alcohol, and glycerol, cyclodextrins, lubricants such as sodium lauryl sulfate and magnesium stearate, petroleum hydrocarbons such as mineral oil and petrolatum. Coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[077] The pharmaceutical compositions of the disclosure can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others. Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. Formulations may optionally contain solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, stabilizers and preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. [078] In some embodiments, the compositions of this disclosure are formulated for pharmaceutical administration to a mammal. In one aspect, for pharmaceutical administration to a human being. Such pharmaceutical compositions of the present disclosure may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intravenously, or subcutaneously. The formulations of the disclosure may be designed to be short-acting, fast-releasing, or long-acting. Still further, compounds can be administered in a local rather than systemic means, such as administration (e.g., by injection) at a tumor site.

[079] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, cyclodextrins, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[080] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Compositions formulated for parenteral administration may be injected by bolus injection or by timed push, or may be administered by continuous infusion. [081] In order to prolong the effect of a compound of the present disclosure, it may be desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or

microemulsions that are compatible with body tissues.

[082] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this disclosure with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[083] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert,

pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar—agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paRaffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents such as phosphates or carbonates.

[084] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[085] The active agents can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding

compositions that can be used include polymeric substances and waxes.

[086] Dosage forms for topical or transdermal administration of a compound of this disclosure include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this disclosure. Additionally, the present disclosure contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[087] Compositions for use in the method of the disclosure may be formulated in unit dosage form for ease of administration and uniformity of dosage. The expression "unit dosage form" as used herein refers to a physically discrete unit of agent appropriate for the subject to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. A unit dosage form for parenteral administration may be in ampoules or in multi-dose containers. [088] The disclosure includes a kit, comprising (i) a RAF inhibitor or a pharmaceutically salt thereof; and one or more of: (i) an EGFR inhibitor or (ii) a topoisomerase I inhibitor or a

pharmaceutically acceptable salt thereof; and instructions for administering the RAF inhibitor or a pharmaceutically salt thereof, in combination with the one or more of: (i) an EGFR inhibitor or a pharmaceutically salt thereof or (ii) a topoisomerase I inhibitor or a pharmaceutically salt thereof.

[089] The present disclosure relates to a kit, comprising (i) a RAF inhibitor or a pharmaceutically salt thereof; and one or more of: (i) an EGFR inhibitor or a pharmaceutically salt thereof or (ii) a topoisomerase I inhibitor or a pharmaceutically salt thereof when used to treat colorectal cancer in a subject; and instructions for administering the RAF inhibitor or a pharmaceutically salt thereof, in combination with the one or more of: (i) an EGFR inhibitor or a pharmaceutically salt thereof or (ii) a topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof.

[090] The present disclosure relates to methods for treating a subject suffering from colorectal cancer by administering to the subject a pharmaceutical composition described herein, said method comprising: a) measuring at least one characteristic of at least one or more BRAF, NRAS and/or KRAS markers associated with gene mutation in a subject sample comprising tumor cells; b) identifying whether the at least one characteristic measured in step a) is informative for outcome upon treatment with the pharmaceutical composition; and c) determining to treat the subject with the pharmaceutical composition if the informative characteristic indicates that the tumor cells comprise at least one marker gene with a BRAF, NRAS and/or KRAS mutational status that indicates a favorable outcome to treatment with the pharmaceutical composition.

[091] The present disclosure relates to methods for treating a subject suffering from colorectal cancer by administering to the subject a pharmaceutical composition described herein, said method comprising: subjecting a nucleic acid sample from a colorectal cancer (tumor) sample from the subject to BRAF, NRAS, or KRAS mutational testing or PCR, wherein the presence of at least one mutation in BRAF, NRAS, or KRAS gene indicates an increased likelihood of pharmacological effectiveness of the treatment.

[092] The present disclosure relates to methods of treating a subject suffering from colorectal cancer, said method comprising: i) obtaining a nucleic acid sample from a colorectal cancer sample from said subject; ii) subjecting the sample to BRAF, NRAS, or KRAS mutational testing or PCR and identifying the presence of at least one mutation in BRAF, NRAS, or KRAS gene; and iii) administering an effective amount of a pharmaceutical composition described herein to the subject in whose sample the presence of at least one mutation in BRAF or KRAS gene is identified. [093] In some embodiments, a mutation in a marker can be identified by sequencing a nucleic acid, e.g., a DNA, RNA, cDNA or a protein correlated with the marker gene, e.g., a genotype marker gene, e.g., BRAF or NRAS. There are several sequencing methods known in the art to sequence nucleic acids. A nucleic acid primer can be designed to bind to a region comprising a potential mutation site or can be designed to complement the mutated sequence rather than the wild type sequence. Primer pairs can be designed to bracket a region comprising a potential mutation in a marker gene. A primer or primer pair can be used for sequencing one or both strands of DNA corresponding to the marker gene. A primer can be used in conjunction with a probe, e.g., a nucleic acid probe, e.g., a hybridization probe, to amplify a region of interest prior to sequencing to boost sequence amounts for detection of a mutation in a marker gene. Examples of regions which can be sequenced include an entire gene, transcripts of the gene and a fragment of the gene or the transcript, e.g., one or more of exons or untranslated regions or a portion of a marker comprising a mutation site. Examples of mutations to target for primer selection and sequence or composition analysis can be found in public databases which collect mutation information, such as Database of Genotypes and Phenotypes (dbGaP) maintained by the National Center for Biotechnology Information (Bethesda, MD) and Catalogue of Somatic Mutations in Cancer (COSMIC) database maintained by the Wellcome Trust Sanger Institute (Cambridge, UK).

[094] Sequencing methods are known to one skilled in the art. Examples of methods include the

Sanger method, the SEQUENOM™ method and Next Generation Sequencing (NGS) methods. The Sanger method, comprising using electrophoresis, e.g., capillary electrophoresis to separate primer- elongated labeled DNA fragments, can be automated for high-throughput applications. The primer extension sequencing can be performed after PCR amplification of regions of interest. Software can assist with sequence base calling and with mutation identification. SEQUENOM™ MASSARRAY® sequencing analysis (San Diego, CA) is a mass-spectrometry method which compares actual mass to expected mass of particular fragments of interest to identify mutations. NGS technology (also called "massively parallel sequencing" and "second generation sequencing") in general provides for much higher throughput than previous methods and uses a variety of approaches (reviewed in Zhang et al. (201 1) J. Genet. Genomics 38:95-109 and Shendure and Hanlee (2008) Nature Biotech. 26: 1 135-1 145). NGS methods can identify low frequency mutations in a marker in a sample. Some NGS methods (see, e.g., GS-FLX Genome Sequencer (Roche Applied Science, Branford, CT), Genome analyzer (Illumina, Inc. San Diego, CA) SOLID™ analyzer (Applied Biosystems, Carlsbad, CA), Polonator G.007 (Dover Systems, Salem, NH), HELISCOPE™ (Helicos Biosciences Corp., Cambridge, MA)) use cyclic array sequencing, with or without clonal amplification of PCR products spatially separated in a flow cell and various schemes to detect the labeled modified nucleotide that is incorporated by the sequencing enzyme (e.g., polymerase or ligase). In one NGS method, primer pairs can be used in PCR reactions to amplify regions of interest. Amplified regions can be ligated into a concatenated product. Clonal libraries are generated in the flow cell from the PCR or ligated products and further amplified ("bridge" or "cluster" PCR) for single-end sequencing as the polymerase adds a labeled, reversibly terminated base that is imaged in one of four channels, depending on the identity of the labeled base and then removed for the next cycle. Software can aid in the comparison to genomic sequences to identify mutations. Another NGS method is exome sequencing, which focuses on sequencing exons of all genes in the genome. As with other NGS methods, exons can be enriched by capture methods or amplification methods.

[095] In some embodiments, DNA, e.g., genomic DNA corresponding to the wild type or mutated marker can be analyzed both by in situ and by in vitro formats in a biological sample using methods known in the art. DNA can be directly isolated from the sample or isolated after isolating another cellular component, e.g., RNA or protein. Kits are available for DNA isolation, e.g., QIAAMP® DNA Micro Kit (Qiagen, Valencia, CA). DNA also can be amplified using such kits.

[096] In another embodiment, mRNA corresponding to the marker can be analyzed both by in situ and by in vitro formats in a biological sample using methods known in the art. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from tumor cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987- 1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of

Chomczynski (1989, U.S. Patent No. 4,843, 155). RNA can be isolated using standard procedures (see e.g., Chomczynski and Sacchi (1987) Anal. Biochem. 162: 156-159), solutions {e.g., trizol, TRI

REAGENT® (Molecular Research Center, Inc., Cincinnati, OH; see U.S. Patent No. 5,346,994) or kits {e.g., a QIAGEN® Group RNEASY® isolation kit (Valencia, CA) or LEUKOLOCK™ Total RNA Isolation System, Ambion division of Applied Biosy stems, Austin, TX).

[097] Additional steps may be employed to remove DNA from RNA samples. Cell lysis can be accomplished with a nonionic detergent, followed by microcentrifugation to remove the nuclei and hence the bulk of the cellular DNA. DNA subsequently can be isolated from the nuclei for DNA analysis. In one embodiment, RNA is extracted from cells of the various types of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation to separate the RNA from DNA (Chirgwin et al. (1979) Biochemistry 18:5294-99). Poly(A)+RNA is selected by selection with oligo-dT cellulose (see Sambrook et al. (1989) Molecular Cloning— A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Alternatively, separation of RNA from DNA can be accomplished by organic extraction, for example, with hot phenol or phenol/chloroform/isoamyl alcohol. If desired, RNAse inhibitors may be added to the lysis buffer. Likewise, for certain cell types, it may be desirable to add a protein denaturation/digestion step to the protocol. For many applications, it is desirable to enrich mRNA with respect to other cellular RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA). Most mRNAs contain a poly(A) tail at their 3' end. This allows them to be enriched by affinity

chromatography, for example, using oligo(dT) or poly(U) coupled to a solid support, such as cellulose or SEPHADEX.R™. medium (see Ausubel et al. (1994) Current Protocols In Molecular Biology, vol. 2, Current Protocols Publishing, New York). Once bound, poly(A)+mRNA is eluted from the affinity column using 2 mM EDTA/0.1% SDS.

[098] A characteristic of a marker found in a sample, e.g., after obtaining a sample (e.g., a a tumor biopsy) from a test subject, can be assessed by any of a wide variety of well known methods for detecting or measuring the characteristic, e.g., of a marker or plurality of markers, e.g., of a nucleic acid (e.g. , RNA, mRNA, genomic DNA, or cDNA) and/or translated protein. Non-limiting examples of such methods include immunological methods for detection of secreted, cell-surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, optionally including "mismatch cleavage" steps (Myers, et al. (1985) Science 230: 1242) to digest mismatched, i.e. mutant or variant, regions and separation and identification of the mutant or variant from the resulting digested fragments, nucleic acid reverse transcription methods, and nucleic acid

amplification methods and analysis of amplified products. These methods include gene array/chip technology, RT-PCR, TAQMAN® gene expression assays (Applied Biosystems, Foster City, CA), e.g., under GLP approved laboratory conditions, in situ hybridization, immunohistochemistry,

immunoblotting, FISH (flourescence in situ hybridization), FACS analyses, northern blot, southern blot, INFINIUM® DNA analysis Bead Chips (Illumina, Inc., San Diego, CA), quantitative PCR, bacterial artificial chromosome arrays, single nucleotide polymorphism (SNP) arrays (Affymetrix, Santa Clara, CA) or cytogenetic analyses.

[099] Examples of techniques for detecting differences of at least one nucleotide between two nucleic acids include, but are not limited to, selective oligonucleotide hybridization, selective

amplification, or selective primer extension. For example, oligonucleotide probes can be prepared in which the known polymorphic nucleotide is placed centrally (allele- or mutant-specific probes) and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324: 163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230; and Wallace et al. (1979) Nucl. Acids Res. 6:3543). Such allele specific oligonucleotide hybridization techniques can be used for the simultaneous detection of several nucleotide changes in different polymorphic or mutated regions of NRAS. For example, oligonucleotides having nucleotide sequences of specific allelic variants or mutants are attached to a solid support, e.g., a hybridizing membrane and this support, e.g., membrane, is then hybridized with labeled sample nucleic acid. Analysis of the hybridization signal thus can reveal the identity of the nucleotides of the sample nucleic acid.

[0100] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, devices and materials are herein described. All publications mentioned herein are hereby incorporated by reference in their entirety for the purpose of describing and disclosing the materials and methodologies that are reported in the publication which might be used in connection with the disclosure.

EXAMPLES

[0101] Definitions

QD once daily

QW or Q7D once weekly

SC subcutaneous(ly)

TG treatment group

TGI tumor growth inhibition

WFI water for injection

Example 1: Kinase inhibition assay with purified RAF kinase isoforms

[0102] The kinase activity of Compound A was determined using a biochemical fluorescence resonance energy transfer (FRET) assay as described in WO 2009/006389. The half maximal inhibitory

concentration (IC50) values of Compound A for mutant BRAF V600E, wild-type BRAF, and wild-type CRAF kinases is shown below in Table 1. Compound A binds to the inactive, DFG-out conformation of BRAF kinase.

Table 1.

Example 2: In vivo tumor efficacy of Compound A in colon xenograft model

[0103] A xenograft study was performed using the subcutaneous (SC) human colon xenograft model, COLO205 (harboring BRAF V600E mutation). COLO205 tumor-bearing female CRL NU/NU mice were treated orally with Compound A in combination with irinotecan (Pharmacia & Upjohn). The purpose of the study was to evaluate efficacy of Compound A in combination with irinotecan in the BRAF mutant COLO 205 colon xenograft model (Table 2). COLO 205 tumor-bearing NU/NU mice were dosed orally with Compound A (25 mg/kg, PO, QD) plus irinotecan (10 mg/kg, IP, Monday through Friday for 2 weeks). Cohorts of mice were also administered with irinotecan or Compound A as a single agent treatment.

Table 2

[0104] NU/NU and female mice 6-8 weeks of age with an average weight of 25 grams were purchased from Charles River Laboratories. Vehicle for the test article was Polyethylene Glycol 400 (PEG400).

[0105] Compound A Prep was as follows: 25g of Compound A /vehicle was added to a tarred glass amber vialthat contains 16.67mL of PEG400, capped tightly and the mixture was sonicated until no drug particles remain and a solution is achieved. The result was 6 mg/mL Compound A in PEG400.

[0106] COLO 205 cell lines were obtained from NCI (Bethesda, Maryland). Cells were grown in RPMI 1640 + 10% fetal bovine serum + 1% L-glutamine. All cells were maintained at 37°C in humidified atmosphere equilibrated with 10% CO² and 90% air.

[0107] Subcutaneous xenograft: Tumor cells were prepared (1 x 10⁶ for COLO 205) in 0.2 ml solution containing 1 : 1 mixture of HBSS and Matrigel (BD Biosciences). All cells were injected SC in the flank of mice. Tumor measurements were determined using digital calipers. When the tumor volumes reached a predetermined size, animals were size matched and randomized into treatment and control groups (see Table 2). To calculate volume, a prolate ellipsoid was used to estimate tumor volume (mm³) from two-dimensional tumor measurements: Tumor Volume (mm³) = (Length x Width²) ÷2. Assuming unit density, volume was converted to weight (i.e., one mm³ = one mg). Animal body weights and tumor measurements were collected twice weekly for the duration of the study. [0108] Anti-tumor activity was evaluated by comparing the tumor volume of the control groups with the tumor volume of the treated groups. These values were expressed as a percentage of mean tumor volume of the treated group divided by the mean tumor volume of control group (%T/C). The %T/C reported for each group is calculated for the tumor measurement on the last dosing day or the

measurement following the last dosing day. Percent Tumor Growth Inhibition (%TGI) is calculated by the change in mean treated tumor volume divided by the change in mean control tumor volume, multiplied by 100 and subtracted from 100%.

[0109] Continuous tumor measurements were reported as mean +/- standard error of the mean and plotted over time for xenograft studies. Statistical significance of observed differences between growth curves during and after dosing periods were evaluated by paired repeated measures ANOVA followed by Dunnett's multiple range post comparison test.

[0110] Compound A was administered orally at 25 mg/kg to COLO 205 tumor-bearing mice once daily either as a single agent or in combination with irinotecan (10 mg/kg, IP, Monday through Friday for 2 weeks). Irinotecan demonstrated statistically significant efficacy compared to the combination vehicle controls (P<0.05 from days 18-36) in these COLO 205 tumor-bearing mice following the second cycle of dosing (Figure 1; Data are Mean ± SEM of 9-10 mice per treatment group. *** P<0.001 or *P<0.05 compared to combination vehicle control from Days 18 to 36. C*** P<0.001 comparing Compound A versus irinotecan combination from days 31-56.).

[0111] As shown in Figure 1, administration of Compound A (25 mg/kg, PO, QD for 21 days) in combination with irinotecan (10 mg/kg, IP, Monday through Friday for 2 weeks) to COLO 205 tumor- bearing mice showed additive efficacy compared to either treatment alone from days 18 to 36 (P<0.05 and P<0.001 compared to Compound A and irinotecan respectively). The superiority of the

combination's efficacy was also observed following the second cycle of Irinotecan (days 18 to 36). Efficacy of the combination group was well maintained after the dosing period relative to both single agent groups.

Example 3: Prophetic Example of Methods for Measuring Markers

[0112] BRAF PCR based Assay (Vendor: Qiagen; Catalog#: 870801)

The BRAF RGQ PCR Kit v2 combines two technologies, ARMS® and Scorpions®, to detect mutations in real-time PCR assays. This assay detects BRAF V600 mutations V600E (GAG) and V600E complex (GAA), V600D (GAT), V600K (A AG), V600R (AGG). The kit detects the presence of the V600E (GAG) and V600E complex (GAA) but does not distinguish between them. ARMS

[0113] Specific mutated sequences are selectively amplified by allele specific primer designed to match a mutated DNA.

Scorpions

[0114] Detection of amplification is performed using Scorpions. Scorpions are PCR primer covalently linked to a fluorescently labeled probe (i.e. FAM™ or HEX™) and a quencher. During PCR when the probe is bound to the amplicon, the fluorophore and quencher become separated resulting in an increase in fluorescence signal.

Procedure

[0115] The BRAF RGQ PCR Kit v2 comprises a two-step procedure. In the first step, the control assay is performed to assess the total amplifiable BRAF DNA in a sample. In the second step, both the mutation and control assays are performed to determine the presence or absence of mutant DNA.

• Control assay

[0116] The control assay, labeled with FAM, is used to assess the total amplifiable BRAF DNA in a sample. The control assay amplifies a region of exon 3 of the BRAF gene. The primers and Scorpion probe are designed to amplify independently of any known BRAF polymorphisms.

• Mutation assays

[0117] Each mutation assay contains a FAM-labeled Scorpion probe and an ARMS primer for discrimination between the wild-type DNA and a specific mutant DNA.

Data Analysis: ACt Method

[0118] Scorpions real-time assays uses the number of PCR cycles necessary to detect a fluorescent signal above a background signal as a measure of the target molecules present at the beginning of the reaction. The point at which the signal is detected above background fluorescence is called the 'cycle threshold' (Ct).

[0119] Sample ACt values are calculated as the difference between the mutation assay Ct and control assay Ct from the same sample. Samples are classed as mutation positive if they give a ACt less than the Cut-Off ACt value for that assay. Above this value, the sample either contains less than the percentage of mutation able to be detected by the kit (beyond the limit of the assays), or the sample is mutation negative.

[0120] When using ARMS primers some inefficient priming could occur, giving a very late background Ct from DNA not containing a mutation. All ACt values calculated from background amplification are greater than the cut off ACt values and the sample is classed mutation negative.

[0121] For each sample, the ACt values are calculated as follows, ensuring that the mutation and control Ct values are from the same sample:

ACt = {sample mutation Ct} - {sample control Ct}

Sample control Ct can range between 27-33

Sample mutation Ct can range between 15-40

Acceptable ACt for the mutant call is <6 or 7

[0122] Methods for measuring NRAS mutations are similar to those described above for BRAF. Qiagen NRAS assay for the detection of NRAS Q61 mutations includes:

Q61K (181 OA)

Q61R (182 A>G)

[0123] Methods for measuring KRAS mutations are readily available. For example, Qiagen TheraScreen® KRAS mutation kit.

[0124] Example 4: Pharmacodynamic effect in RAS-mutant colorectal cancer models

[0125] This study evaluated the in vivo pharmacodynamic (PD) effect Compound A, cetuximab, and Compound A in combination with cetuximab in female Balb/c nude mice in various RAS-mutant colorectal cancer (CRC) models as measured by pERK levels. An effect on pERK levels was observed in some but not all RAS mutant CRC models tested. Tumor bearing mice (3 tumors per group) were treated with:

[0126] (1) vehicle (100% PEG400 plus 0.5% HPMC + 0.2% TWEEN 80);

[0127] (2) Compound A (25 mg/kg and 50 mg/kg PO); [0128] (3) Cetuximab (43 mg/kg IP); and

[0129] (4) the combination of Compound A (25 mg/kg and/or 50 mg/kg PO) and cetuximab (43 mg/kg IP).

[0130] Tumors were removed four hours after dosing and pERK (T202/Y204) levels were determined by RPPA (reverse phase protein array). The results for the PHTX-24C xenogRAFt model are provided in Figure 4. The results for the CR-1530 xenograft model are provided in Figure 5. In vivo tumor efficacy was then performed in these two KRAS non-exon 2 mutation positive colorectal cancer xenograft models as described in examples 5 and 6.

Example 5: In vivo tumor efficacy of in the PHTX-24C xenograft model (KRAS non-exon 2 mutation positive primary colorectal cancer)

This study evaluated the in vivo antitumor activity of Compound A, cetuximab, and Compound A in combination with cetuximab in Balb/c nude female mice bearing primary colorectal tumor

PHTX-24C xenografts. The PHTX-24C xenograft is a KRAS exon 4 positive mutation model with mutation at A146T. Tumor bearing mice were treated with:

(1) vehicle (100% PEG400 plus 0.5% HPMC + 0.2% TWEEN 80; orally (PO) once daily for three weeks (QD x 21);

(2) Compound A (25 mg/kg (PO) QD x 21);

(3) Cetuximab (43 mg/kg intraperitoneally (IP) twice weekly for three weeks (ΒΓ\¥ x 3); and

(4) the combination of Compound A (25 mg/kg PO QD x 21) and cetuximab (43 mg/kg IP BIW x

3).

Effects on tumor growth were evaluated by measuring percent tumor growth inhibition (TGI) on Day 21 of the study. Each group contained eight animals. There was no mortality in any of the treatment groups during the treatment period.

[0131] Compound A was formulated in 100% PEG400 as a solution, prepared weekly, and stored at room temperature (18 to 25°C). Cetuximab (ERBITUX^®, 2 mg/mL, Batch Number IMD251, ImClone LLC, Branchburg, NJ, USA) was stored at 4°C and warmed to room temperature (18 to 25°C) just before dosing. Animals in the vehicle group were given 100% PEG400 plus 0.5% HPMC + 0.2% TWEEN 80. The dose volume for vehicle and Compound A administration was 5 mL/kg body weight. Cetuximab was dosed at 0.5 mL/animal (1 mg/animal). Balb/c nude female mice 10 weeks of age with a weight of 22.2 to 23.0 g were purchased from Shanghai SINO-British SIPPR/BK Lab Animal Ltd. Shanghai China Mouse body weight was measured after randomization.

Each animal received SC implantation of the PHTX-24C xenogRAFt (1 x 1 - 2 x 2 mm per implant) in the right flank via trocar. Body weight and the tumor growth were monitored twice weekly. Tumor size was measured to the nearest 0.1 mm using vernier caliper and applying the formula V = W² x L/2, where V = volume, W = width, and L = length for the tumor xenogRAFt. XenogRAFts were allowed to grow until they reached an average size of approximately 150 mm³ after 20 days. Mice bearing the proper size xenografts were randomly assigned into a group and treated with: vehicle (100% PEG400 plus 0.5% HPMC + 0.2% TWEEN 80), Compound A, cetuximab, or the combination of Compound A and cetuximab.

[0132] Tumor size was measured twice weekly beginning on the day of animal grouping (Day 0). See Figure 2. The study was terminated following the last measurement on Day 28. The antitumor activity was determined by calculating the percent TGI on Day 21, see Table 3, using the following equation ("MTV" = mean tumor volume):

Percent TGI = (MTV Vehicle group - MTV Treatment group) ÷ MTV Vehicle group 100.

Table 3

Example 5: In vivo tumor efficacy in the CR1530 xenograft model (KRAS non-exon 2 mutation positive primary colorectal cancer).

This study evaluated the in vivo antitumor activity of Compound A, cetuximab, and Compound A in combination with cetuximab in female Balb/c nude mice bearing primary colorectal tumor CR1530 xenografts. The CR1530 xenograft is a KRAS exon 3 positive mutation model with mutation at Q61H. Tumor bearing mice were treated with:

(1) vehicle (100% PEG400 plus 0.5% HPMC + 0.2% TWEEN 80; orally (PO) once daily for three weeks (QD x 21);

(2) Compound A (25 mg/kg (PO) QD x 21);

(3) Compound A (50 mg/kg PO twice weekly for three weeks (ΒΓν¥ x 3); and

(4) Cetuximab (43 mg/kg intraperitoneally (IP) ΒΓν¥ x 3;

(5) the combination of Compound A (25 mg/kg PO QD x 21) and cetuximab (43 mg/kg IP BIW x

3); and

(6) the combination of Compound A (50 mg/kg PO ΒΓν¥ x 3) and cetuximab (43 mg/kg IP BIW x

3).

Effects on tumor growth were evaluated by measuring percent tumor growth inhibition (TGI) on Day 21 of the study. Each group contained eight animals. There was no mortality in any of the treatment groups during the treatment period.

[0133] Compound A was formulated in 100% PEG400 as a solution, prepared weekly, and stored at 4°C). Cetuximab (Merck KGaA, Lot number 203605) was stored at 4°C and was warmed to room temperature (18 to 25°C) just before dosing. Animals in the vehicle group were given 100% PEG400 plus 0.5% HPMC + 0.2% TWEEN 80. Balb/c nude female mice 10-12 weeks of age with a weight of 19.7 to 23.1 g were purchased from HFK Bio-Technology Co. Ltd. (Beijing, China). Mouse body weight was measured after randomization.

[0134] Animals were inoculated or implanted subcutaneously into the flank with CR1530 tumor fragments and were treated with test materials. See Figure 3. Dosing was initiated on Day 0. Effects on tumor growth were evaluated by measuring percent TGI. Tumor volumes were measured twice per week. The percentage of TGI, see Table 4, was determined on Day 21 of the study using the equation described in Example 4.

Table 4

Compound A

18.1% 50 mg/kg PO BIWx3;

Cetuximab

21.6% 43 mg/kg IP BIW x 3

Compound A (25 mg/kg PO QD

x 21) + Cetuximab (43 mg/kg IP 73.1%

BIWx3)

Compound A (50 mg/kg PO BIW

x 3) + Cetuximab (43 mg/kg IP 27.1%

BIWx3)

Claims

WHAT IS CLAIMED:

1. A method of treating a subject suffering from colorectal cancer, comprising administering to the subject a combination of a RAF kinase inhibitor or a pharmaceutically acceptable salt thereof; and one or more of:

(i) an EGFR inhibitor or

(ii) a topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof;

wherein the amount of said inhibitors is such that the combination thereof is therapeutically effective in the treatment of the colorectal cancer.

2. The method claim 1, wherein the RAF kinase inhibitor inhibits more than the BRAF V600

isoform of RAF proteins.

3. The method of any one of claims 1-2, wherein the combination comprises both an EGFR inhibitor and a topoisomerase inhibitor or a pharmaceutically acceptable salt thereof.

4. The method of any one of claims 1-3, wherein the combination comprises a RAF kinase inhibitor and an EGFR inhibitor.

5. The method of claim 3 or 4, wherein the EGFR inhibitor is cetuximab or panitumab.

6. The method of any one of claims 1-5, wherein the combination comprises a RAF kinase inhibitor and a topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof.

7. The method of claim 6, wherein the topoisomerase I inhibitor is irinotecan or a pharmaceutically acceptable salt thereof.

8. The method of any one of claims 1-7, wherein the RAF kinase inhibitor is

(Compound B) or a pharmaceutically acceptable salt thereof.

9. The method of any one of claims 1-8, wherein the colorectal cancer is KRAS mutation positive colorectal cancer.

10. The method of claim 9, wherein the colorectal cancer is a KRAS exon 2 mutation positive

colorectal cancer.

11. The method of claim 9, wherein the colorectal cancer is a KRAS non-exon 2 mutation positive colorectal cancer.

12. The method of any one of claims 1-11, wherein the colorectal cancer is a BRAF mutation positive colorectal cancer.

13. The method of claim 12, wherein the colorectal cancer is BRAF V600 mutation positive

colorectal cancer.

14. The method of any one of claims 1-13, wherein the colorectal cancer is an NRAS mutation

positive colorectal cancer.

15. The method of claim 14, wherein the colorectal cancer is an NRAS non-exon 2 mutation positive colorectal cancer.

16. The method of claim 14, wherein the colorectal cancer is an NRAS exon 2 mutation positive colorectal cancer.

17. A method of treating a subject suffering from colorectal cancer, comprising administering to the subject Compound A:

or a pharmaceutically acceptable salt thereof; and one or more of:

(ⁱ) an EGFR inhibitor or

(11) a topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof; wherein the amount of said Compound A and inhibitor(s) is such that the combination thereof is therapeutically effective in the treatment of the colorectal cancer.

18. The method of claim 17, wherein Compound A or a pharmaceutically acceptable salt thereof, is administered administered once weekly (QW) with a rest period of 6 days between each administration in an amount of up to 600 mg per dose.

19. The method of claim 17, wherein Compound A or a pharmaceutically acceptable salt thereof, is administered every other day (qod) in an amount of from about 100 mg to about 200 mg per dose.

20. The method of any one of claims 17-19, wherein the EGFR inhibitor is cetuximab, wherein said cetuximab is administered once weekly (QW) with a rest period of 6 days between each administration in an amount of up to 400 mg/m² per the first dose and up to 250 mg/m² per dose after the first dose.

21. The method of any one of claims 17-19, wherein the EGFR inhibitor is panitumumab, wherein said panitumumab is administered in an amount of about 6 mg/kg every 14 days as an intravenous infusion over 60 minutes (< 1000 mg) or 90 minutes (>1000 mg).

22. The method of any one of claims 17-19, wherein the irinotecan is administered in an amount of about 180 mg/m² on days 1 and 15 of a 28-day cycle.

23. A pharmaceutical composition comprising a combination of a RAF kinase inhibitor or a

pharmaceutically acceptable salt thereof, and one or more of:

(i) an EGFR inhibitor or

(ii) a topoisomerase I inhibitor or a pharmaceutically acceptable salt thereof.