WO2017106492A1 - Combinations for the treatment of cancer - Google Patents

Combinations for the treatment of cancer Download PDF

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Publication number
WO2017106492A1
WO2017106492A1 PCT/US2016/066919 US2016066919W WO2017106492A1 WO 2017106492 A1 WO2017106492 A1 WO 2017106492A1 US 2016066919 W US2016066919 W US 2016066919W WO 2017106492 A1 WO2017106492 A1 WO 2017106492A1
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WIPO (PCT)
Prior art keywords
agent
leu
inhibitor
patient
ser
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PCT/US2016/066919
Other languages
French (fr)
Inventor
Gang Li
Ge Wei
Zachary D. HORNBY
Jonathan Ee-Ren LIM
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Ignyta, Inc.
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Filing date
Publication date
Priority to JP2018531120A priority Critical patent/JP7061068B2/en
Priority to KR1020187016957A priority patent/KR20180096621A/en
Priority to MX2018007266A priority patent/MX2018007266A/en
Priority to CN201680078474.5A priority patent/CN108697661A/en
Priority to US16/063,170 priority patent/US10869864B2/en
Priority to AU2016370846A priority patent/AU2016370846B2/en
Application filed by Ignyta, Inc. filed Critical Ignyta, Inc.
Priority to CA3008663A priority patent/CA3008663A1/en
Priority to EP16876677.2A priority patent/EP3389645A4/en
Priority to BR112018012255A priority patent/BR112018012255A2/en
Publication of WO2017106492A1 publication Critical patent/WO2017106492A1/en
Priority to IL259996A priority patent/IL259996A/en
Priority to US17/116,929 priority patent/US20210113555A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present disclosure relates to methods of treating cancer in a patient, wherein the patient is known to possess at least one molecular alteration in ALK, ROS1, NTRK1, NTRK2, NTRK3, TrkA, TrkB, or TrkC, or a combination thereof, comprising
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor or ALK, ROS1, TrkA, TrkB, or TrkC, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • PKs protein kinases
  • Anaplastic lymphoma kinase is a tyrosine kinase receptor belonging to the insulin receptor subfamily of RTKs: the ALK gene is located on chromosome 2 and is expressed mainly in neuronal cells, especially during development. Many data from the literature have demonstrated that the ALK fusion proteins have strong oncogenic potentials.
  • ROS1 belongs to the insulin-receptor superfamily. Like other tyrosine kinase receptor molecules, it plays a role in relaying growth signals from the environment outside the cell into the cell's nucleus. It is 1 of 2 orphan receptor tyrosine kinase family members with no known binding ligand. Genetic changes in ROS1, such as gene rearrangements, mutations, or copy number increases, create oncogenes, which can lead to cancer. ROS1 was discovered in NSCLC patients in the form of a fusion protein (6 different partners for ROS 1) and is found in approximately 2% of patients with NSCLC.
  • ROS1 gene rearrangements have been detected in a variety of other cancers, including glioblastoma multiforme, cholangiocarcinoma, ovarian cancer, gastric adenocarcinoma, colorectal cancer, inflammatory myofibroblastic tumor, angiosarcoma, and epitheloid hemangioendothelioma.
  • Trks are the high affinity receptor tyrosine kinases activated by a group of soluble growth factors called neurotrophins (NT).
  • the Trk receptor family has three members-- TrkA, TrkB and TrkC.
  • the neurotrophins are (i) nerve growth factor (NGF) which activates TrkA, (ii) brain-derived neurotrophic factor (BDNF) and NT-4/5 which activate TrkB and (iii) NT3 which activates TrkC.
  • Trks are widely expressed in neuronal tissue and are implicated in the maintenance, signaling and survival of neuronal cells (Patapoutian et al , Current Opinion in Neurobiology, 2001, 11, 272-280, incorporated by reference in its entirety herein).
  • NTRK1 encodes the TrkA receptor tyrosine kinase. TrkA activates the PI3K/AKT, PKC and ERK1/2 pathways which promote cell growth and survival.
  • Trks are associated with many cancers including neuroblastoma (Brodeur, G. M., Nat. Rev. Cancer 2003, 3, 203-216, incorporated by reference in its entirety herein), ovarian cancer (Davidson. B., et al , Clin. Cancer Res. 2003, 9, 2248-2259, incorporated by reference in its entirety herein), breast cancer (Kruettgen et al , Brain Pathology 2006, 16: 304-310, incorporated by reference in its entirety herein), prostate cancer (Dionne et al , Clin. Cancer Res.
  • pancreatic cancer (Dang et al, Journal of Gastroenterology and Hepatology 2006, 21(5): 850-858, incorporated by reference in its entirety herein), multiple myeloma (Hu et al , Cancer Genetics and Cytogenetics 2007, 178: 1-10, incorporated by reference in its entirety herein), astrocytoma and medulloblastoma (Kruettgen et al , Brain Pathology 2006, 16: 304-310, incorporated by reference in its entirety herein) glioma (Hansen et al, Journal of Neurochemistry 2007, 103: 259-275, incorporated by reference in its entirety herein), melanoma (Truzzi et al, Journal of Investigative Dermatology 2008, 128(8): 2031-2040, incorporated by reference in its entirety herein), thyroid carcinoma (Brzezianska et al.
  • Trk inhibitors are efficacious in both inhibiting tumor growth and stopping tumor metastasis.
  • Trk A, B and C and Trk/Fc chimeras were efficacious in both inhibiting tumor growth and stopping tumor metastasis (Nakagawara, A. Cancer Letters 2001, 169: 107-114; Meyer et al , Leukemia 2007, 1-10; Pierottia and Greco, Cancer Letters 2006, 232:90-98; Eric Adriaenssens et al, Cancer Res 2008, 68:(2) 346-351; Truzzi et al, Journal of Investigative Dermatology 2008, 128(8): 2031-2040, each of which is incorporated by reference in its entirety herein).
  • an inhibitor of the Trk family of kinases is expected to have utility in the treatment of cancer.
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof; and the second agent is a MEK inhibitor or an ERK inhibitor.
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in ALK.
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the ALK receptor tyrosine kinase polypeptide.
  • the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G1202 or G1269 of the ALK polypeptide set forth in SEQ ID NO: 4.
  • the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G1202 of the ALK polypeptide set forth in SEQ ID NO: 4. In some embodiments, the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G1269 of the ALK polypeptide set forth in SEQ ID NO: 4.
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a ROSl inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in ROSl.
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a ROSl inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the ROSl receptor tyrosine kinase polypeptide.
  • the at least one mutation in the ROSl receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G2032 or G2101 of the ROSl polypeptide set forth in SEQ ID NO: 5.
  • the at least one mutation in the ROSl receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G2032 of the ROSl polypeptide set forth in SEQ ID NO: 5. In some embodiments, the at least one mutation in the ROSl receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G2101 of the ROSl polypeptide set forth in SEQ ID NO: 5.
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkA inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK1.
  • the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkA inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkA receptor tyrosine kinase polypeptide.
  • the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G595 or G667 of the TrkA polypeptide set forth in SEQ ID NO: 1.
  • the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G595 of the TrkA polypeptide set forth in SEQ ID NO:
  • the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G595R). In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G667 of the TrkA polypeptide set forth in SEQ ID NO: 1. In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G667C).
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkB inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK2.
  • the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkB inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkB receptor tyrosine kinase polypeptide.
  • the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G639 or G709 of the TrkB polypeptide set forth in SEQ ID NO: 2.
  • the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G639 of the TrkB polypeptide set forth in SEQ ID NO:
  • the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G639R). In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G709 of the TrkB polypeptide set forth in SEQ ID NO: 2. In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G709C).
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkC inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK3.
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkC inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkC receptor tyrosine kinase polypeptide.
  • the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G623 or G696 of the TrkC polypeptide set forth in SEQ ID NO: 3.
  • the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G623 of the TrkC polypeptide set forth in SEQ ID NO: 3. In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G623R). In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G696 of the TrkC polypeptide set forth in SEQ ID NO: 3. In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G696C).
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • the second agent is a MEK inhibitor.
  • the MEK inhibitor is an inhibitor of MEKl , MEK2, or a combination thereof. In some embodiments, the MEK inhibitor is selected from the group consisting of
  • the MEK inhibitor is PD0325901.
  • the MEK inhibitor is selumetinib.
  • the MEK inhibitor is cobimetinib.
  • the MEK inhibitor is refametinib. In some embodiments, the MEK inhibitor is trametinib. In some embodiments, the MEK inhibitor is pimasertib. In some embodiments, the MEK inhibitor is binimetinib. In some embodiments, the MEK inhibitor is AZD8330. In some embodiments, the MEK inhibitor is R04987655. In some embodiments, the MEK inhibitor is R05126766. In some embodiments, the MEK inhibitor is WX-554. In some embodiments, the MEK inhibitor is E-6201. In some embodiments, the MEK inhibitor is GDC-0623. In some embodiments, the MEK inhibitor is TAK-733. In some embodiments, the MEK inhibitor is RG-7304.
  • the MEK inhibitor is CKBP-002. In some embodiments, the MEK inhibitor is RDEA-436. In some embodiments, the MEK inhibitor is sorafenib. In some embodiments, the MEK inhibitor is PD-184352. In some embodiments, the MEK inhibitor is GSK-2091976A. In some embodiments, the MEK inhibitor is AS-703988.
  • the second agent is an ERK inhibitor. In some embodiments, the second agent is an ERK inhibitor.
  • the ERK inhibitor is an inhibitor of ERK1, ERK2, or a combination thereof.
  • the ERK inhibitor is selected from the group consisting of TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • the ERK inhibitor is TG-02.
  • the ERK inhibitor is MK-8353.
  • the ERK inhibitor is ulixertinib.
  • the ERK inhibitor is HE-3235.
  • the ERK inhibitor is AEZS-134.
  • the ERK inhibitor is AEZS-136.
  • the ERK inhibitor is IDN-5491.
  • the cancer is selected from non-small cell lung cancer, papillary thyroid cancer, neuroblastoma, pancreatic cancer, melanoma, and colorectal cancer.
  • the cancer is non-small cell lung cancer.
  • the cancer is papillary thyroid cancer.
  • the cancer is neuroblastoma.
  • the cancer is pancreatic cancer.
  • the cancer is melanoma.
  • the cancer is colorectal cancer.
  • the combination is a pharmaceutical composition comprising the therapeutically effective amount of the first agent, the therapeutically effective amount of the second agent, and at least one pharmaceutically acceptable carrier.
  • the combination is concurrent administration of a first pharmaceutical composition comprising the therapeutically effective amount of the first agent and a second pharmaceutical composition comprising the therapeutically effective amount of the second agent.
  • the combination is sequential administration of a first pharmaceutical composition comprising the therapeutically effective amount of the first agent and a second pharmaceutical composition comprising the therapeutically effective amount of the second agent.
  • FIG. 1 is a Western Blot analysis from Example 9 demonstrating that p-MEKl/2 (Ser217/221) and total MEK1/2 are upregulated in the Ba/F3 cells expressing TPM3-TrkA- G595R versus Ba/F3 cells expressing wild-type TPM3-TrkA.
  • FIG. 2 is a plot of the growth of live cells from Example 10, demonstrating that Ba/F3 cells expressing TPM3-TrkA-G595R that are treated with the combination of entrectinib (300 nM) and trametinib (30 nM) displayed significant inhibition of growth (up to 40 days) compared to the cells that were untreated (DMSO), those treated with entrectinib (300 nM) alone and those treated with trametinib (30 nM) alone.
  • DMSO untreated
  • FIG. 3 is a plot of the results from Example 11, which demonstrates that the growth of tumors in mice implanted with cells expressing TPM3-TrkA-G595R was reduced in those mice to which a combination of entrectinib (RXDX-101) (60 mg/kg) and trametinib (1 mg/kg) was administered versus tumor growth in those mice in the control group (Vehicle), those mice treated with entrectinib (60 mg/kg) alone, and those mice treated with tremetinib (1 mg/kg) alone.
  • RXDX-101 60 mg/kg
  • trametinib (1 mg/kg
  • N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l- yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide means the compound having the chemical structure
  • N-[5 -(3 ,5 -Difluorobenzy 1)- 1 H-indazol-3-yl] -4-(4-methy 1-piperazin- 1 -y l)-2-(tetrahy dro- pyran-4-ylamino)-benzamide may be prepared as described in United States Patent No. 8,299,057, the disclosure of which is hereby incorporated by reference in its entirety.
  • N-[5- (3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l -yl)-2-(tetrahydro-pyran-4- ylamino)-benzamide is associated with CAS Registry Number 1 108743-60-7.
  • the N-[5- (3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l -yl)-2-(tetrahydro-pyran-4- ylamino)-benzamide compound may also be described as "entrectinib" and/or "RXDX- 101.”
  • the term "acidulant,” as used herein, means a chemical compound that is acidic in nature.
  • the term “organic acidulant” means an acidulant the chemical composition of which contains carbon.
  • the term “inorganic acidulant” means an acidulant the composition of which does not contain carbon.
  • administration and “administering,” as used herein, refer to the delivery of a bioactive composition or formulation by an administration route including, but not limited to, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, topically, or combinations thereof.
  • AUC means the area under the curve of a plot of the concentration of a compound in the plasma of a patient versus time.
  • anaplastic lymphoma kinase and "ALK,” as used herein, mean the ALK tyrosine kinase receptor or CD246 (cluster of differentiation 246), which is an enzyme that in humans is encoded by the ALK gene and also has the UniProt identifier
  • AZD8330 Alternative names for AZD8330 include AZD-8330 and 2-(2-fluoro-4-iodophenylamino)- N-(2-hydroxyethoxy)-l,5-dimethyl-6-oxo-l,6-dihydropyridine-3-carboxarnide.
  • AZD8330 is associated with CAS Registry Number 869357-68-6.
  • betaine hydrochloride means a compound having CAS Registry Number 590-46-5 and the common names l-carboxy-NNN- trimethylmethanaminium chloride and (carboxymethyl)trimethylammonium hydrochloride.
  • binimetinib means the compound having the chemical structure
  • binimetinib Alternative names for binimetinib include MEK162, ARRY-162, ARRY-438162, and 5-((4- bromo-2-fluorophenyl)amino)-4-fluoro-N-(2-hydroxyethoxy)-l-methyl-lH- benzo[d]imidazole-6-carboxamide. Binimetinib is associated with CAS Registry Number 606143-89-9.
  • the term "biological sample,” as used herein, encompasses a variety of sample types obtained from an organism and can be used in a diagnostic or monitoring assay.
  • the sample may be of a healthy tissue, diseased tissue, or tissue suspected of being diseased tissue.
  • the sample may be a biopsy taken, for example, during a surgical procedure.
  • the sample may be collected via means of fine needle aspiration, scraping or washing a cavity to collects cells or tissue therefrom.
  • the sample may be of a tumor such as, for example, solid and hematopoietic tumors as well as of neighboring healthy tissue.
  • the sample may be a smear of individual cells or a tissue section.
  • the term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
  • the term encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components.
  • the term encompasses clinical samples, and also includes cells in cell culture, cell supernatants, cell lysates, cell extracts, cell homogenates, subcellular components including synthesized proteins, serum, plasma, bodily and other biological fluids, and tissue samples.
  • the biological sample can contain compounds that are not naturally intermixed with the cell or tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like.
  • the sample is preserved as a frozen sample or as formaldehyde- or paraformaldehy de-fixed paraffin-embedded (FFPE) tissue preparation.
  • FFPE paraffin-embedded
  • the sample can be embedded in a matrix, e.g., an FFPE block or a frozen sample.
  • biomarker means a molecule whose level of nucleic acid or protein product has a quantitatively differential concentration or level with respect to an aspect of a biological state of a patient.
  • Biomarker is used interchangeably with “marker” herein.
  • the level of the biomarker can be measured at both the nucleic acid level as well as the polypeptide level.
  • a nucleic acid gene or a transcript which is transcribed from any part of the patient's chromosomal and extrachromosomal genome, including for example the mitochondrial genome, may be measured.
  • an RNA transcript Preferably an RNA transcript, more preferably an RNA transcript includes a primary transcript, a spliced transcript, an alternatively spliced transcript, or an mRNA of the biomarker is measured.
  • a pre-propeptide, a propeptide, a mature peptide or a secreted peptide of the biomarker may be measured.
  • a biomarker can be used either solely or in conjunction with one or more other identified biomarkers so as to allow correlation to the biological state of interest as defined herein. Specific examples of biomarkers covered by the present disclosure include ALK, ROS1, TrkA, TrkB, and TrkC.
  • cancer refers to any malignant and/or invasive growth or tumor caused by abnormal cell growth.
  • cancer refers to solid tumors named for the type of cells that form them, cancer of blood, bone marrow, or the lymphatic system. Examples of solid tumors include but are not limited to sarcomas and carcinomas. Examples of cancers of the blood include but are not limited to leukemias, lymphomas and myeloma.
  • cancer includes but is not limited to a primary cancer that originates at a specific site in the body, a metastatic cancer that has spread from the place in which it started to other parts of the body, a recurrence from the original primary cancer after remission, and a second primary cancer that is a new primary cancer in a person with a history of previous cancer of different type from latter one.
  • These terms also mean the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell, such as a leukemia cell.
  • cancer includes premalignant, as well as malignant cancers.
  • the cancer is a solid tumor, a soft tissue tumor, or a metastatic lesion.
  • chemotherapeutic agent means a chemical substance, such as a cytotoxic or cytostatic agent, that is used to treat a condition, particularly cancer.
  • Cmax means the peak concentration that a compound achieves in the plasma of a patient after the compound, or a pharmaceutical composition comprising the compound, has been administrated to the patient.
  • the compound, or a pharmaceutical composition comprising the compound is administered orally to a patient to achieve a particular Cmax.
  • cobimetinib as used herein, means the compound having the chemical structure
  • cobimetinib alternatives names include GDC-0973, XL-518, RG7420, CotellicTM, and ( ⁇ [3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl]3-hydroxy-3-(piperidin-2- yl]azetidin-l -yl)methanone.
  • Cobimetinib is associated with CAS Registry Number 934660- 93-2.
  • combination and “in combination with,” as used herein, mean the administration of a compound provided herein together with an at least one additional pharmaceutical or medicinal agent (e.g., an anti-cancer agent), either sequentially or simultaneously. It includes dosing simultaneously, or within minutes or hours of each other, or on the same day, or on alternating days, or dosing the compound provided herein on a daily basis, or multiple days per week, or weekly basis, for example, while
  • an additional pharmaceutical or medicinal agent e.g., an anti-cancer agent
  • N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l -yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof could be dosed every day or several days a week while the chemotherapeutic agent is dosed on alternating days or alternating weeks or other periods of time, such as every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1. 12, 13, 14 or more days.
  • E6201 means the compound having the chemical structure
  • E6201 Alternative names for E6201 include (3S,4R, 5Z, 8S,9S, l l£)-14-(ethylamino)-8,9,16- trihydroxy-3,4-dimethyl-3,4,9,10-tetrahydro-lH-2-benzoxacyclotetradecine-l,7(8H)-dione.
  • E6201 is associated with CAS Registry Number 603987-35-5.
  • ERK means the family of extracellular signal- regulated kinases (ERKs), and are protein kinase intracellular signaling molecules. ERKs include, but are not limited to, ERKl and ERK2.
  • ERKl means extracellular signal-regulated kinase 1 having the UniProt identifier MK03_HUMAN and encoded by the MAPK3 gene.
  • ERK2 means extracellular signal- regulated kinase 2 having the UniProt identifier MK01_HUMAN and encoded by the MAPKl gene.
  • ERKl and ERK2 are also referred to by those having ordinary skill in the art as MAPK3 and MAPKl, respectively.
  • a reference to ERKl is a reference to MAPK3.
  • a reference to ERK2 is a reference to MAPKl. Unless otherwise indicated, use of the term "ERK” may refer to ERKl, ERK2, or a combination thereof.
  • the term "food effect,” as used herein, means a change in the rate and/or extent of absorption of a compound in a patient when the compound is administered to the patient shortly after a meal (fed conditions) as compared to the rate and/or extent of absorption of the compound when the compound is administered to the patient under fasting conditions.
  • no food effect means that there is no significant difference in the rate and/or extent of absorption of a compound in a patient when the compound is administered to the patient in fed conditions compared to fasting conditions.
  • GDC-0623 means the compound having the chemical structure
  • GDC-0623 Alternative names for GDC-0623 include 5-[(2-fluoro-4-iodophenyl)amino]-N-(2- hydroxyethoxy)-imidazo[l,5-a]pyridine-6-carboxarnide. GDC-0623 is associated with CAS Registry Number 1168091-68-6.
  • HE-3235 as used herein, means the compound having the chemical
  • HE-3235 Alternative names include HE3235, H E3235, Apotone®, and
  • IDN-5491 means the compound having the chemical structure
  • IDN-5491 alternatives names include hyperforin triemthoxybenzoate and ( ⁇ , ⁇ , ⁇ )- l-isobutyryl-8-methyl-3,5,7-tris(3-methyl-2-buten-l-yl)-8-(4-methyl-3-penten-l-yl)-4,9- dioxobicyclo[3.3. l]non-2-en-2-yl 2,3,4-trimethoxybenzoate.
  • MEK mitogen/extracellular signal-regulated kinases
  • MEKs mitogen/extracellular signal-regulated kinases
  • MAPK mitogen- activated protein kinase
  • MEK is also known as MAP2K and MAPKK. Isoforms of MEK include, but are not limited to, MEK1 and MEK2.
  • MEK1 means mitogen/extracellular signal-regulated kinase-1 having the UniProt identifier MP2K1_HUMAN and encoded by the MAP2K1 gene.
  • MEK2 means mitogen/extracellular signal-regulated kinase-2 having the UniProt identifier MP2K2 HUMAN and encoded by the MAP2K2 gene. Unless otherwise indicated, use of the term “MEK” may refer to MEK1, MEK2, or a combination thereof.
  • microarray is an ordered arrangement of array elements (for example, small samples of a biological sample from a patient such as tissue samples) mounted on a solid support capable of binding other molecule species or antibodies. The array elements are arranged so that there are preferably at least one or more different array elements.
  • molecular alteration and “genetic alteration,” as used herein, mean any variation in the genetic or protein sequence in or more cells of a patient as compared to the corresponding wild-type genes or proteins.
  • One or more molecular alterations include, but are not limited to, genetic mutations, gene amplifications, splice variants, deletions, gene rearrangements, single-nucleotide variations (SNVs), insertions, and aberrant RNA/protein expression.
  • SNVs single-nucleotide variations
  • insertions and aberrant RNA/protein expression.
  • patient means a mammal, including, but not limited to, a human, a dog or a cat. In some embodiments, the patient is a human. In some embodiments, the patient is a human. In some
  • the patient is a dog. In some embodiments, the patient is a cat.
  • PD0325901, means the compound having the chemical structure
  • PD0325901 Alternative names include PD 0325901, PD325901, and JV-[(2R)-2,3- dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide.
  • PD0325901 is associated with CAS Registry Number 391210-10-9.
  • PD184352 means the compound having the chemical structure
  • PD 184352 Alternative names for PD 184352 include CI- 1040 and 2-(2-chloro-4-iodophenylamino)-N- cyclopropylmethoxy-3,4-difluorobenzamide.
  • PD184352 is associated with CAS Registry Number 212631-79-3.
  • Altemative names for pimasertib include AS-703026, AS 703026, AS703026, and
  • MSC1936369B N-[(25 -2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pyridine-4- carboxamide, and N-(2,3-dihydroxypropyl)-l-((2-fluoro-4- iodophenyl)amino)isonicotinamide.
  • Pimasertib is associated with CAS Registry Number 1236699-92-5.
  • refametinib Altemative names for refametinib include BAY-86-9766, RDEA119, and N-[3,4-difluoro- 2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl]-l-[(25)-2,3-dihydroxypropyl]- cyclopropanesulfonamide.
  • Refametinib is associated with CAS Registry Number 923032-
  • R04987655 means the compound having the chemical structure
  • R04987655 Alternative names for R04987655 include RO 4987655, RO-4987655, RG 7167, CH- 4987655, CH 4987655, CH4987655, and 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-N-(2- hydroxyethoxy)-5-[(3-oxooxazinan-2-yl)methyl]benzamide.
  • R04987655 is associated with CAS Registry Number 874101-00-5.
  • R05126766 means the compound having the chemical structure
  • R05126766 includes RO-5126766 and CH5126766.
  • R05126766 is associated with CAS Registry Number 946128-88-7.
  • ROS1 means the ROS 1 receptor tyrosine-protein kinase having the UniProt designation ROS 1 HUMAN and encoded by the ROS 1 gene.
  • selumetinib alternatives names include AZD6244, ARRY- 142886, and 6-(4-bromo-2- chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxarnide.
  • Selumetinib is associated with CAS Registry Number 606143-52-6.
  • solid support means the well-understood solid materials to which various components such as, for example, proteins and nucleic acids, are physically attached, thereby immobilizing the components.
  • solid support means a non-liquid substance.
  • a solid support can be, but is not limited to, a membrane, sheet, gel, glass, plastic or metal. Immobilized components may be associated with a solid support by covalent bonds and/or via non-covalent attractive forces such as hydrogen bond interactions, hydrophobic attractive forces and ionic forces, for example
  • the term "sorafenib,” as used herein, means the compound having the chemical structure
  • sorafenib alternatives names include BAY 43-9006, Nexavar®, and 4-[4-[[4-chloro-3- (trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide. Sorafenib is associated with CAS Registry Number 284461-73-0.
  • TAK-733 means the compound having the chemical structure
  • TAK-733 Altemative names for TAK-733 include (i?)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro- 4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione.
  • TAK-733 is associated with CAS Registry Number 1035555-63-5.
  • a therapeutically effective amount means that amount of the compound or compounds being administered which will relieve to some extent one or more of the symptoms of the disorder being treated.
  • a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of a cancer tumor, (2) inhibiting (that is, slowing to some extent, preferably stopping) cancer tumor metastasis, (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) cancer tumor growth, and/or, (4) relieving to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer.
  • trametinib as used herein, means the compound having the chemical structure
  • trametinib alternatives names include GSK1 120212 and Mekinist®. Trametinib is associated with CAS Registry Number 871700-17-3.
  • tropomyosin receptor kinase means the family of tropomyosin receptor kinases (Trks) that are activated by peptide hormones of the neurotrophin family and include, but are not limited to, TrkA, TrkB, and TrkC.
  • TrkA means wild-type tropomyosin receptor kinase A having the UniProt identifier NTRK1_HUMAN and encoded by the NTRK1 gene.
  • TrkB means wild-type tropomyosin receptor kinase B having the UniProt identifier NTRK2 HUMAN and encoded by the NTRK2 gene.
  • TrkC means wild-type tropomyosin receptor kinase C having the UniProt identifier
  • TrkA, TrkB and TrkC are also referred to by those having ordinary skill in the art as Trkl, Trk2 and Trk3, respectively.
  • a reference to TrkA is a reference to Trkl .
  • a reference to TrkB is a reference to Trk2.
  • a reference to TrkC is a reference to Trk3.
  • ulixertinib alternatives names include BVD-523, VRT752271, and 4-(5-chloro-2- (isopropylamino)pyridin-4-yl)-N-((5 - l -(3-chl orophenyl)-2-hydroxy ethyl)- lH-pyrrole-2- carboxamide.
  • Ulixertinib is associated with CAS Registry Number 869886-67-9.
  • kits for treating cancer in a patient in need thereof comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof.
  • kits for treating cancer in a patient in need thereof comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof; and the second agent comprises a MEK inhibitor or an ERK inhibitor.
  • kits for treating cancer in a patient in need thereof comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof; and the second agent is a MEK inhibitor or an ERK inhibitor.
  • kits for treating cancer in a patient in need thereof comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in ALK.
  • kits for treating cancer in a patient in need thereof comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the ALK receptor tyrosine kinase polypeptide.
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a ROS1 inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in ROS1.
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a ROSl inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the ROSl receptor tyrosine kinase polypeptide.
  • kits for treating cancer in a patient in need thereof comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkA inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK1.
  • a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkA inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkA receptor tyrosine kinase polypeptide.
  • kits for treating cancer in a patient in need thereof comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkB inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK2.
  • kits for treating cancer in a patient in need thereof comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkB inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkB receptor tyrosine kinase polypeptide.
  • kits for treating cancer in a patient in need thereof comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkC inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK3.
  • kits for treating cancer in a patient in need thereof comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkC inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkC receptor tyrosine kinase polypeptide.
  • knowledge of the at least one genetic alteration is acquired from an antibody-based assay.
  • the antibody-based assay can generally be any antibody- based assay, and can be, for example, ELISA, immunohistochemistry, western blotting, mass spectrometry, flow cytometry, protein-microarray, immunofluorescence, and a multiplex detection assay.
  • the antibody-based assay includes an immunohistochemistry analysis.
  • methods of the present disclosure are to treat, reduce the symptoms of, ameliorate the symptoms of, delay the onset of, or otherwise
  • the first agent is N-[5-(3,5-difluorobenzyl)- lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)- benzamide, or a pharmaceutically acceptable salt thereof.
  • the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof.
  • the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX- 554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD- 184352, GSK-2091976A, and AS-703988.
  • the ERK inhibitor is an ERK1 inhibitor, an ERK2 inhibitor, or a combination thereof.
  • the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS- 136, and IDN-5491.
  • methods of the present disclosure are to treat, reduce the symptoms of, ameliorate the symptoms of, delay the onset of, or otherwise
  • a condition in a patient selected from non-small cell lung cancer, papillary thyroid cancer, neuroblastoma, pancreatic cancer and colorectal cancer associated with a ALK, ROSl, TrkA, TrkB, or TrkC down-regulation defect for example a null mutation such as a ALK, ROSl, TrkA, TrkB, or TrkC deletion by identifying a ALK, ROS l, TrkA, TrkB, or TrkC down-regulation defect, for example a null mutation such as a ALK, ROS l, TrkA, TrkB, or TrkC deletion in a cancer or precancerous cell in the patient, and administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a
  • the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof.
  • the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK- 2091976A, and AS-703988.
  • the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof.
  • the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • methods for treating cancer in a patient in need thereof comprising: (a) acquiring knowledge of the presence of at least one genetic alteration in a biological sample from the patient, wherein the at least one genetic alteration is detected by an assay comprising one or more antibodies that bind to one or more of ALK, ROS1, TrkA, TrkB, and TrkC biomarkers; (b) and administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS 1, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof. In some embodiments, the MEK inhibitor is selected from
  • the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof.
  • the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • methods for treating cancer in a patient in need thereof comprising: (1) testing one or more cells comprising a tumor from the patient for the presence of at least one of ALK, ROS1, TrkA, TrkB, or TrkC; and (2) if the one or more cells tests positive for at least one of ALK, ROS 1, TrkA, TrkB, or TrkC, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof.
  • the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988.
  • the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof.
  • the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • methods for treating cancer in a patient in need thereof comprising: (1) testing one or more cells comprising tumor tissue from the patient for the presence of at least one of ALK, ROS1, TrkA, TrkB, or TrkC; and (2) if the one or more cells tests positive for at least one of ALK, ROS1, TrkA, TrkB, or TrkC, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof.
  • the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988.
  • the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof.
  • the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • methods for treating cancer in a patient in need thereof comprising: (a) acquiring knowledge of the presence of at least one genetic alteration in at least one target gene in the cancer patient, wherein the at least one target gene is selected from ALKl, BDNF, NGF, NGFR, NTF3, NTF4, ROS 1, SORT1, NTRK1, NTRK2, and NTRK3; and (b) administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor based on the recognition that the combination is effective in treating cancer patients having the at least one genetic alteration in the at least one target gene.
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH- indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide ⁇ or a pharmaceutically acceptable salt thereof.
  • the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof.
  • the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK- 2091976A, and AS-703988.
  • the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof.
  • the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • methods for treating cancer in a patient in need thereof comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor, and wherein prior to the administration of the combination, the patient is known to possess at least one genetic alteration in at least one target gene selected from ALKl, BDNF, NGF, NGFR, NTF3, NTF4, ROS 1, SORT1, NTRK1, NTRK2, and NTRK3.
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof.
  • the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK- 2091976A, and AS-703988.
  • the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof.
  • the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • methods for treating cancer in a patient in need thereof comprising administering to the patient known to possess at least one genetic alteration in at least one target gene selected from ALK1, BDNF, NGF, NGFR, NTF3, NTF4, ROSl, SORTl, NTRKl, NTRK2, and NTRK3 a therapeutically effective amount of a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS l, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof. In some embodiments, the MEK inhibitor is selected from
  • the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof. In some embodiments, the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • methods of treating a cancer patient comprising administering to the patient a therapeutically effective amount of a combination comprising a therapeutically effective amount of a first agent and a
  • the first agent is an inhibitor of ALK, ROS l, TrkA, TrkB, or TrkC activity, or a combination thereof;
  • the second agent is selected from a MEK inhibitor and an ERK inhibitor;
  • the cancer patient is has at least one genetic alteration in at least one target gene; and the target gene is selected from ALK1, BDNF, NGF, NGFR, NTF3, NTF4, ROS1, SORT1, NTRK1, NTRK2, and NTRK3.
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4- methyl-piperazin-1 -yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a
  • the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof.
  • the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK- 2091976A, and AS-703988.
  • the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof. In some embodiments, the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • methods of treating a cancer patient comprising (a) acquiring knowledge of the presence of at least one genetic alteration in at least one target gene selected from ALK1, BDNF, NGF, NGFR, NTF3, NTF4, ROS1, SORT1, NTRK1, NTRK2, and NTRK3 in the patient; and (b) administering to the patient a therapeutically effective amount of a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a
  • the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof.
  • the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK- 2091976A, and AS-703988.
  • the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof.
  • the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN- 5491. [0096] In some embodiments are provided methods wherein the tumors are caused by the presence of non-small cell lung cancer, papillary thyroid cancer, neuroblastoma, pancreatic cancer or colorectal cancer in the patient.
  • one or more of the cells comprising the tumors in the patient test positive for the presence of a gene that expresses at least one of ALK, ROSl, TrkA, TrkB, or TrkC kinase or one or more of the cells comprising the tumors in the patient demonstrates at least one of ALK, ROSl, TrkA, TrkB, or TrkC kinase activity.
  • the cells test positive for at least one of ROSl, TrkA, TrkB, or TrkC.
  • the cells test positive for ALK In some embodiments are provided methods wherein the cells test positive for ROSl . In some embodiments are provided methods wherein the cells test positive for at least one of TrkA, TrkB and TrkC. In some embodiments are provided methods wherein the cells test positive for TrkA. In some embodiments are provided methods wherein the cells test positive for TrkB. In some embodiments are provided such methods wherein the cells test positive for TrkC.
  • methods of treating cancer in a patient in need thereof comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of at least one of ALK, ROSl, TrkA, TrkB, or TrkC; and (2) if the one or more cells tests positive for at least one of ALK, ROS l, TrkA, TrkB, or TrkC activity, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor, a ROS l inhibitor, a TrkA inhibitor, a TrkB inhibitor, or a TrkC inhibitor, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • methods of treating cancer in a patient in need thereof comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of ALK; and (2) if the one or more cells tests positive for ALK activity, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5-difluorobenzyl)- lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)- benzamide, or a pharmaceutically acceptable salt thereof.
  • methods of treating cancer in a patient in need thereof comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of ROS1 ; and (2) if the one or more cells tests positive for ROS1 activity, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ROS1 and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • methods of treating cancer in a patient in need thereof comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of TrkA, TrkB, or TrkC, or a combination thereof; and (2) if the one or more cells tests positive for TrkA, TrkB, or TrkC activity, or a combination thereof, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of TrkA, TrkB, or TrkC, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro- 2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • methods of treating cancer in a patient in need thereof comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of TrkA; and (2) if the one or more cells tests positive for TrkA activity, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of TrkA, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • methods of treating cancer in a patient comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of TrkB; and (2) if the one or more cells tests positive for TrkB activity, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of TrkB, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • methods of treating cancer in a patient comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of TrkC; and (2) if the one or more cells tests positive for TrkC activity, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of TrkC, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • the patient has at least one mutation in the TrkA receptor tyrosine kinase polypeptide.
  • the at least one mutation in the TrkA receptor tyrosine kinase polypeptide may be a genetic alteration of SEQ ID NO: 1.
  • the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G595 or G667 of the TrkA polypeptide set forth in SEQ ID NO: 1.
  • the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G595 of the TrkA polypeptide set forth in SEQ ID NO: 1. In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G595R). In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G667 of the TrkA polypeptide set forth in SEQ ID NO: 1.
  • the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G667C).
  • the patient at least one mutation in the TrkB receptor tyrosine kinase polypeptide.
  • the at least one mutation in the TrkB receptor tyrosine kinase polypeptide may be a genetic alteration of SEQ ID NO: 2.
  • the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G639 or G709 of the TrkB polypeptide set forth in SEQ ID NO: 2.
  • the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G639 of the TrkB polypeptide set forth in SEQ ID NO:
  • the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G639R). In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G709 of the TrkB polypeptide set forth in SEQ ID NO: 2. In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G709C).
  • the patient has at least one mutation in the TrkC receptor tyrosine kinase polypeptide.
  • the at least one mutation in the TrkC receptor tyrosine kinase polypeptide may be a genetic alteration of SEQ ID NO: 3.
  • the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G623 or G696 of the TrkC polypeptide set forth in SEQ ID NO: 3.
  • the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G623 of the TrkC polypeptide set forth in SEQ ID NO:
  • the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G623R). In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G696 of the TrkC polypeptide set forth in SEQ ID NO: 3. In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G696C).
  • the patient has at least one mutation in ALK receptor tyrosine kinase polypeptide.
  • the at least one mutation in the ALK receptor tyrosine kinase polypeptide may be a genetic alteration of SEQ ID NO: 4.
  • the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G1202 or G1269 of the ALK polypeptide set forth in SEQ ID NO: 4.
  • the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G1202 of the ALK polypeptide set forth in SEQ ID NO: 4. In some embodiments, the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G1269 of the ALK polypeptide set forth in SEQ ID NO: 4.
  • the patient has at least one mutation in the ROS1 receptor tyrosine kinase polypeptide.
  • the at least one mutation in the ROS1 receptor tyrosine kinase polypeptide may be a genetic alteration of SEQ ID NO: 5.
  • the at least one mutation in the ROS1 receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G2032 or G2101 of the ROS1 polypeptide set forth in SEQ ID NO: 5.
  • the at least one mutation in the ROS1 receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G2032 of the ROS1 polypeptide set forth in SEQ ID NO: 5. In some embodiments, the at least one mutation in the ROS1 receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G2101 of the ROS 1 polypeptide set forth in SEQ ID NO: 5.
  • the combination is a pharmaceutical composition comprising the therapeutically effective amount of the first agent, the therapeutically effective amount of the second agent, and at least one pharmaceutically acceptable carrier.
  • the combination is concurrent administration of a first pharmaceutical composition comprising the therapeutically effective amount of the first agent and a second pharmaceutical composition comprising the therapeutically effective amount of the second agent
  • the combination is sequential administration of a first pharmaceutical composition comprising the therapeutically effective amount of the first agent and a second pharmaceutical composition comprising the therapeutically effective amount of the second agent.
  • the first pharmaceutical composition is administered prior to the second pharmaceutical composition.
  • the first pharmaceutical composition is administered after the second pharmaceutical composition.
  • any of the methods described herein wherein the patient is suffering from cancer selected from non-small cell lung cancer, papillary thyroid cancer, neuroblastoma, melanoma, pancreatic cancer, and colorectal cancer.
  • neuroblastoma neuroblastoma, melanoma, pancreatic cancer and colorectal cancer.
  • neuroblastoma neuroblastoma, melanoma, and colorectal cancer.
  • neuroblastoma and colorectal cancer.
  • the second agent comprises one or more chemotherapeutic agents or radiotherapy, such as radiotherapy as commonly administered to treat, ameliorate the symptoms of, or prevent or delay the onset of cancer.
  • agents include, but are not limited to, antihormonal agents such as antiestrogens, antiandrogens and aromatase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, agents that target microtubules, platin-based agents, alkylating agents, DNA damaging or intercalating agents, antineoplastic antimetabolites, other kinase inhibitors, other anti-angiogenic agents, inhibitors of kinesins, therapeutic monoclonal antibodies, inhibitors of mTOR, histone deacetylase inhibitors, farnesyl transferase inhibitors, and inhibitors of hypoxic response.
  • a product or kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy.
  • a product or a kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy, wherein the first agent comprises an ALK inhibitor, a ROS 1 inhibitor, a TrkA inhibitor, a TrkB inhibitor, or a TrkC inhibitor, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • a product or a kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy, wherein the first agent comprises an ALK inhibitor, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • a product or a kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy, wherein the first agent comprises a ROS 1 inhibitor, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • a product or a kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy, wherein the first agent comprises a TrkA inhibitor, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • a product or a kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy, wherein the first agent comprises a TrkB inhibitor, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • a product or a kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy, wherein the first agent comprises a TrkC inhibitor, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
  • the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • the first agent comprises N- [5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H- pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
  • the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer in an amount ranging from about 200 mg/m 2 to about 1600 mg/m 2 , or from about 200 mg/m 2 to about 1200 mg/m 2 , or from about 200 mg/m 2 to about 1000 mg/m 2 , or from about 400 mg/m 2 to about 1200 mg/m 2 , or from about 400 mg/m 2 to about 1000 mg/m 2 , or from about 800 mg/m 2 to about 1000 mg/m 2 , or from about 800 mg/m 2 to about 1200 mg/m 2 , or from about 800 mg/m 2 to about 1200 mg/m 2 , or from about 800 mg/m 2 to about 1600 mg/m 2 .
  • the first agent and/or the second agent are administered to the patient or individual having or suffering from cancer in an amount of about 200 mg/m 2 , about 300
  • the amount of the first agent and the amount of the second agent are within the same range listed above. In some embodiments, the amount of the first agent and the amount of the second agent are in different ranges listed above.
  • the first agent comprises N- [5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H- pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof, and is administered to a patient or individual having or suffering from cancer in an amount such that the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)- 2-(tetrahydro-2H-pyran-4-ylamino)-benzamide the patient receives ranges from about 200 mg/m 2 to about 1600 mg/ m 2 , or from about 200 mg/ m 2 to about 1200 mg/m 2 , or from about 200 mg/m 2 to about 1000 mg/m 2 , or from about 400
  • the first agent comprises N- [5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H- pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof, and is administered to a patient or individual having or suffering from cancer in an amount such that the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)- 2-(tetrahydro-2H-pyran-4-ylamino)-benzamide the patient receives is about 200 mg/m 2 ,
  • the amount of N-[5-(3,5-difluorobenzyl)-lH- indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide the patient receives is about 200 mg/m 2 , about 300 mg/m 2 , about 400 mg/m 2 , about 500 mg/m 2 , or about 600 mg/m 2 .
  • the amount ofN-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide the patient receives is about 200 mg/m 2 . In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-2H-pyran-4-ylamino)-benzamide the patient receives is about 300 mg/m 2 .
  • the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide the patient receives is about 400 mg/m 2 . In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide the patient receives is about 500 mg/m 2 .
  • the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide the patient receives is about 600 mg/m 2 .
  • the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer in an amount such that the amount of the patient receives is about 0.5 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg, including increments therein.
  • the first agent comprises N-[5-(3,5-difluorobenzyl)-lH- indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof, and the amount of N-[5-(3,5-difluorobenzyl)- lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)- benzamide that the patient receives is about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg.
  • the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 200 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol- 3-yl] -4-(4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 300 mg.
  • the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives is about 400 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 500 mg.
  • the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 600 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol- 3-yl] -4-(4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 700 mg.
  • the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives is about 800 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 900 mg.
  • the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 1000 mg.
  • the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer in an amount such that the amount the first agent and/or second agent that the patient receives per day is about 0.5 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg, including increments therein.
  • the first agent comprises N- [5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H- pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof, and is administered to a patient or individual having or suffering from cancer in an amount such that the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)- 2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives per day is about 0.5 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about
  • the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives once per day is about 200 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives once per day is about 300 mg.
  • the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives once per day is about 400 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives once per day is about 500 mg.
  • the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives once per day is about 600 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives once per day is about 700 mg.
  • the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives once per day is about 800 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives once per day is about 900 mg.
  • the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives once per day is about 1000 mg.
  • the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer in multiple dosages for a treatment period of 2 to 50 days. In some embodiments, the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer in multiple dosages of about 50 to about 200 mg/kg per dose over a treatment period of 5 to 42 days. In some embodiments, the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer with an oral dosage of about 60 mg/kg twice a day (BID), seven times per week.
  • BID twice a day
  • the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer with an oral dosage of about 60 mg/kg twice a day (BID), seven times per week for six weeks, on alternate weekly basis (i.e., one week on and one week off).
  • BID twice a day
  • Some embodiments include any of the methods described herein, wherein the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer in an amount ranging from about 0.01 mg/kg to about 100 mg/kg, or from about 0.02 mg/kg to about 50 mg/kg, or from about 0.05 mg/kg to about 25 mg/kg, or from about 0.1 mg/kg to about 20 mg/kg, or from about 0.2 mg/kg to about 10 mg/kg, or from about 0.5 mg/kg to about 5 mg/kg, or from about 1 mg/kg to about 2 mg/kg.
  • the amount of the first agent and the amount of the second agent are within the same range listed above. In some embodiments, the amount of the first agent and the amount of the second agent are in different ranges listed above.
  • any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l -yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide further comprise a weakly basic organic compound and at least one acidulant.
  • the at least one acidulant is an organic acidulant.
  • the at least one acidulant is selected from tartaric acid, maleic acid, fumaric acid, citric acid, and betaine
  • the at least one acidulant is fumaric acid. In some embodiments, the at least one acidulant is tartaric acid. In some embodiments, the at least one acidulant is maleic acid. In some embodiments, the at least one acidulant is citric acid. In some embodiments, the at least one acidulant is betaine hydrochloride.
  • any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l -yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide further comprise at least one acidulant.
  • the at least one acidulant is an organic acidulant.
  • the at least one acidulant is selected from tartaric acid, maleic acid, fumaric acid, citric acid, and betaine hydrochloride.
  • the least one acidulant is fumaric acid.
  • the at least one acidulant is tartaric acid. In some embodiments, the at least one acidulant is maleic acid. In some embodiments, the at least one acidulant is citric acid. In some embodiments, the at least one acidulant is betaine hydrochloride.
  • compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and at least one acidulant are in the form of a tablet or capsule.
  • pharmaceutical compositions in the form of a tablet In some embodiments are provided pharmaceutical compositions in the form of a capsule.
  • compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and at least one acidulant comprises from about 10 mg to about 1000 mg of the N-[5-(3,5-difluorobenzyl)- lH-indazol-3-yl] -4-(4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-pyran-4-ylamino)-benzamide and at least one acidulant.
  • compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-2H-pyran-4-ylamino)-benzamide when
  • Tmax of the N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin- l-yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide in the plasma of the patient is between about 2 hours and 6 hours following the administration of the pharmaceutical composition to the patient.
  • compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-2H-pyran-4-ylamino)-benzamide when
  • the N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l -yl)-2-(tetrahydro-pyran-4- ylamino)-benzamide provides a pharmacokinetic profile in the patient wherein the Cmax of the N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro- pyran-4-ylamino)-benzamide in the plasma of the patient is between about 2080 nM and about 21 10 nM following the administration of the pharmaceutical composition to the patient.
  • compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-2H-pyran-4-ylamino)-benzamide when
  • the N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l -yl)-2-(tetrahydro-pyran-4- ylamino)-benzamide provides a pharmacokinetic profile in the patient wherein the Cmax of the N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro- pyran-4-ylamino)-benzamide in the plasma of the patient is between 80% to 125% of 2560 nM, based on a 90 percent confidence interval following the administration of the pharmaceutical composition to the patient.
  • compositions comprising N-[5-(3, 5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that provides an AUC(0 to 24) of the N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l- yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide in the plasma of a patient of between about 28,900 nM*hr and about 30,800 nM*hr following administration of the pharmaceutical composition to the patient in a fasted state, and wherein the composition comprises a total dose of about 800 mg of the N-[5-(3,5-difluorobenzyl)-l
  • any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3, 5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide provide a Tmax of the N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-pyran-4-ylamino)-benzamide in the plasma of a patient of between about 2 hours and about 6 hours following administration of the pharmaceutical composition to the patient in a fasted state.
  • This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the bioactive compositions and formulations provided herein (including activity, pharmacokinetics, pharmacodynamics, and bioavailability thereof), the physiological condition of the patient treated (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication) or cells, the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. Further, an effective or therapeutically effective amount may vary depending on whether the one or more bioactive compositions and formulations provided herein is administered alone or in combination with other drug(s), other therapy/therapies or other therapeutic method(s) or modality/modalities.
  • a typical dosage may range from about 0.1 mg/kg to about 100 mg/kg or more, depending on the factors mentioned above. In other alternatives, the dosage may range from about 0.1 mg/kg to about 100 mg/kg; or about 1 mg/kg to about 100 mg/kg; or about 5 mg/kg up to about 100 mg/kg.
  • suitable dosage may range from about 1 mg/kg to about 10 g/kg; or about 10 mg/kg to about 1 g/kg; or about 50 mg/kg up to about 10 g/kg. Additional guidance with regard to this aspect can be found in, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005, which is incorporated by reference in its entirety herein.
  • implementations of the methods according to this and other aspects of the present disclosure further include acquiring knowledge of a genetic alteration in the cancer of the patient from a second analytical assay prior to the administering step.
  • the second analytical assay can generally be any analytical assay known to those having ordinary skill in the art, and can be for example an antibody-based assay, a nucleotide-based assay, or an enzymatic activity assay.
  • Non-limiting examples of suitable second analytical assays include capillary electrophoresis, nucleic acid sequencing, polypeptide sequencing, restriction digestion, nucleic acid amplification-based assays, nucleic acid hybridization assay, comparative genomic hybridization, real-time PCR, quantitative reverse transcription PCR (qRT-PCR), PCR-RFLP assay, HPLC, mass-spectrometric genotyping, fluorescent in- situ hybridization (FISH), next generation sequencing (NGS), and a kinase activity assay.
  • Other examples of suitable second analytical assays include ELISA, immunohistochemistry, Western blotting, mass spectrometry, flow cytometry, protein-microarray,
  • FISH analysis is used to identify the chromosomal rearrangement resulting in the one or more molecular alterations such as the fusion genes or gene products as described herein.
  • at least a first probe tagged with a first detectable label can be designed to target a first gene of a fusion gene, such as in one or more exons of the gene and at least a second probe tagged with a second detectable label can be designed to target a second gene of the fusion gene, such as in one or more exons of the genes (for example, the exons containing the part of the protein that includes the tyrosine kinase domain).
  • the at least one first probe and the at least one second probe will be closer together in a patient who carries the fusion compared to a patient who does not carry the fusion gene or gene product.
  • a variation of a FISH assay for example, "break-apart FISH," is used to evaluate a patient selected by a method provided herein.
  • at least one probe targeting the fusion junction and at least one probe targeting an individual gene of the fusion are utilized.
  • FISH assays are performed using formalin-fixed, paraffin- embedded tissue sections that are placed on slides. The DNA from the tissue sample sections is denatured to single-stranded form and subsequently allowed to hybridize with the appropriate DNA probes that can be designed and prepared using methods and techniques known to those having ordinary skill in the art.
  • any unbound probe may be removed by a series of washes and the nuclei of the cells are counter-stained with DAPI (4',6 diamidino-2-phenylindole), a DNA-specific stain that fluoresces blue.
  • DAPI 4,',6 diamidino-2-phenylindole
  • Hybridization of the probe or probes are viewed using a fluorescence microscope equipped with appropriate excitation and emission filters, allowing visualization of the fluorescent signals.
  • a break-apart FISH assay may be used to detect multiple types of rearrangements involving the ALK gene locus.
  • tumor cells from some patients having non-small cell lung cancer (NSCLC) display an ALK-positive FISH pattern as detected using single interference filter sets comprising green (FITC), red (Texas red), and blue (4',6-diamidino-2-phenylindole) as well as dual (red/green) and triple (blue, red, green) band-pass filters.
  • FITC green
  • red Texas red
  • blue (4',6-diamidino-2-phenylindole
  • a fusion of the ALK gene is visualized as split orange and green signals, single orange signals, or single orange and single green signals.
  • the cancer is selected from the group consisting of anaplastic large-cell lymphoma (ALCL), colorectal cancer (CRC), cholangiocarcinoma, gastric, glioblastomas (GBM), leiomyosarcoma, melanoma, non-small cell lung cancer (NSCLC), squamous cell lung cancer, neuroblastoma (NB), ovarian cancer, pancreatic cancer, prostate cancer, medullary thyroid cancer, breast cancer, and papillary thyroid cancer.
  • ACL anaplastic large-cell lymphoma
  • CRC colorectal cancer
  • GBM glioblastomas
  • NSCLC non-small cell lung cancer
  • NB neuroblastoma
  • ovarian cancer pancreatic cancer
  • prostate cancer medullary thyroid cancer
  • breast cancer and papillary thyroid cancer.
  • the knowledge of the presence of the one or more molecular alterations is obtained from an assay performed simultaneously on a plurality of biological samples.
  • the plurality of biological samples may be assayed in a multitest platform.
  • multitest platform is intended to encompass any suitable means to contain one or more reaction mixtures, suspensions, or detection reactions. As such, the outcomes of a number of screening events can be assembled onto one surface, resulting in a “multitest platform” having, or consisting of multiple elements or parts to do more than one experiment simultaneously. It is intended that the term “multitest platform” encompasses protein chips, microtiter plates, multi-well plates, microcards, test tubes, petri plates, trays, slides, and the like. In some embodiments, multiplexing can further include simultaneously conducting a plurality of screening events in each of a plurality of separate biological samples.
  • the number of biological samples analyzed can be based on the number of spots on a slide and the number of tests conducted in each spot.
  • the number of biological samples analyzed can be based on the number of wells in a multi-well plate and the number of tests conducted in each well.
  • 6-well, 12- well, 24-well, 48-well, 96-well, 384-well, 1536-well or 3456-well microtiter plates can be useful in the presently disclosed methods, although it will be appreciated by those in the art, not each microtiter well need contain an individual biological sample.
  • very high numbers of tests can be run simultaneously.
  • the plurality of biological samples includes at least 6, 12, 24, 48, 96, 200, 384, 400, 500, 1000, 1250, 1500, or 3000 samples, including increments therein.
  • the one or more molecular alterations is selected from a genetic mutation, a gene amplification, a gene rearrangement, a single-nucleotide variation (SNV), a deletion, an insertion, an InDel mutation, one or more single nucleotide point mutations (SNPs), an epigenetic alteration, a splicing variant, an RNA/protein overexpression, and an aberrant RNA/protein expression.
  • the genetic alteration includes an insertion of a heterologous nucleic acid sequence within a coding sequence of a biomarker gene.
  • the insertion forms a chimeric nucleic acid sequence that encodes a fusion peptide.
  • the acquiring knowledge of the one or more molecular alterations further comprises determining a nucleic acid sequence and/or an amino acid sequence comprising the one or more molecular alterations.
  • the nucleic acid sequence comprising the one or more molecular alterations from a selected cancer patient tumor is sequenced.
  • the sequence is determined by a next generation sequencing method.
  • the assay includes one or more antibodies that bind to at least two, three, four, or all of ALK, ROS 1, TrkA, TrkB and TrkC biomarkers.
  • the one or more molecular alterations detected in the biological sample involve at least two, at least three, or at least four of the biomarkers.
  • the knowledge of the presence of the one or more molecular alterations in the biological sample is acquired from an assay that includes contacting the biological sample with one or more antibodies or fragments thereof that are specific for the biomarkers.
  • the specific antibodies are monoclonal antibodies.
  • the specific antibodies include at least one of D5F3®, D4D5®, C 17F1®, and combinations thereof.
  • the biological sample is contacted with one or more of the specific antibodies simultaneously.
  • the biological sample is sequentially contacted with the specific antibodies.
  • the one or more molecular alterations results in elevated expression of one or more of the ALK, ROS1, TrkA, TrkB, and TrkC biomarkers.
  • the knowledge of the one or more molecular alterations is acquired from an assay wherein determining whether the expression of one or more biomarker is elevated includes: (a) determining the expression level of the one or more biomarkers in the biological sample; and (b) comparing the determined expression level to a reference expression level.
  • the pharmaceutical compositions comprise a physical admixture of the various ingredients in solid, liquid, or gelcap form.
  • Other embodiments comprise at least two separated ingredients in a single dosage unit or dosage form, such as, for example, a two- or three-layer tablet in which at least two active ingredients are located in separate layers or regions of the tablet, optionally separated by a third material, such as, for example, a sugar layer or other inert barrier to prevent contact between the first two ingredients.
  • two or more active ingredients are separately formulated into individual dosage units, which are then packaged together for ease of administration.
  • One embodiment comprises a package containing a plurality of individual dosage units. This embodiment may, for example, comprise a blister package.
  • multiple blister-packed dosage units are present on a single sheet, and those units that are to be administered together are packaged in the same or adjacent blisters of the blister pack.
  • any other packaging can be used in which two active ingredients are packaged together for concurrent or sequential use.
  • the methods relate to the use of any of the compounds as described herein, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of abnormal cell growth in a mammal.
  • the present disclosure further relates to the use of any of the compounds as described herein, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of abnormal cell growth in a mammal wherein the abnormal cell growth is cancerous or non-cancerous.
  • the abnormal cell growth is cancerous.
  • the abnormal cell growth is non-cancerous.
  • a pharmaceutical composition comprising the first agent and/or the second agent further comprises at least one pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may comprise a conventional pharmaceutical carrier or excipient. Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents (such as hydrates and solvates).
  • the pharmaceutical compositions may, if desired, contain additional ingredients such as flavorings, binders, excipients and the like.
  • tablets containing various excipients, such as citric acid may be employed together with various disintegrants such as starch, alginic acid and certain complex silicates and with binding agents such as sucrose, gelatin and acacia.
  • lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes.
  • Solid compositions of a similar type may also be employed in soft and hard filled gelatin capsules.
  • Non-limiting examples of materials therefore, include lactose or milk sugar and high molecular weight polyethylene glycols.
  • the active compound therein may be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof.
  • the pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository.
  • Exemplary parenteral administration forms include solutions or suspensions of active compounds in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms may be suitably buffered, if desired.
  • the pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages.
  • compositions of the disclosure comprise a therapeutically effective amount of at least one compound provided herein and an inert, pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical carriers employed may be either solid or liquid.
  • Exemplary solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
  • Exemplary liquid carriers are syrup, peanut oil, olive oil, water and the like.
  • the inventive compositions may include time-delay or time-release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or the like.
  • a bioavailability enhancer such as Labrasol, Gelucire or the like, or formulator, such as CMC (carboxy-methylcellulose), PG (propyleneglycol), or PEG (poly ethylenegly col)
  • CMC carboxy-methylcellulose
  • PG propyleneglycol
  • PEG poly ethylenegly col
  • Gelucire® a semi-solid vehicle that protects active ingredients from light, moisture and oxidation, may be added, e.g. , when preparing a capsule formulation.
  • the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form, or formed into a troche or lozenge.
  • the amount of solid carrier may vary, but generally will be from about 25 mg to about 1 g.
  • the preparation may be in the form of syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or non-aqueous liquid suspension.
  • a semi-solid carrier is used, the preparation may be in the form of hard and soft gelatin capsule formulations.
  • the inventive compositions are prepared in unit-dosage form appropriate for the mode of administration, e.g., parenteral or oral administration.
  • a salt of a compound of the present disclosure may be dissolved in an aqueous solution of an organic or inorganic acid, such as a 0.3 M solution of succinic acid or citric acid. If a soluble salt form is not available, the agent may be dissolved in a suitable co-solvent or combinations of co-solvents. Examples of suitable co-solvents include alcohol, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin and the like in concentrations ranging from 0 to 60% of the total volume.
  • a compound of the present disclosure is dissolved in DMSO and diluted with water.
  • the composition may also be in the form of a solution of a salt form of the active ingredient in an appropriate aqueous vehicle such as water or isotonic saline or dextrose solution.
  • the agents of the compounds of the present disclosure may be formulated into aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers known in the art.
  • Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained using a solid excipient in admixture with the active ingredient (agent), optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients include: fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; and cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used, which may optionally contain gum arabic, polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agents.
  • compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present disclosure may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethan
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit-dosage form, e.g. , in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active agents may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds of the present disclosure may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion-exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical carrier for hydrophobic compounds is a co-solvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • the co-solvent system may be a VPD co-solvent system.
  • VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the non-polar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the VPD co-solvent system (VPD: 5 W) contains VPD diluted 1 : 1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
  • the proportions of a co-solvent system may be suitably varied without destroying its solubility and toxicity characteristics.
  • co-solvent components may be varied: for example, other low-toxicity non-polar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose.
  • hydrophobic pharmaceutical compounds may be employed.
  • Liposomes and emulsions are known examples of delivery vehicles or carriers for hydrophobic drugs.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity due to the toxic nature of DMSO.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • additional strategies for protein stabilization may be employed.
  • the pharmaceutical compositions also may comprise suitable solid- or gel-phase carriers or excipients.
  • These carriers and excipients may provide marked improvement in the bioavailability of poorly soluble drugs.
  • Examples of such carriers or excipients include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • additives or excipients such as Gelucire®, Capryol®, Labrafil®, Labrasol®, Lauroglycol®, Plurol®, Peceol®,
  • Transcutol® and the like may be used.
  • the pharmaceutical composition may be incorporated into a skin patch for delivery of the drug directly onto the skin.
  • an exemplary daily dose generally employed will be from about 0.001 to about 1000 mg/kg of body weight, with courses of treatment optionally repeated at appropriate intervals.
  • the pharmaceutically acceptable formulations of the present disclosure may contain a compound of the present disclosure, or a salt or solvate thereof, in an amount from about 0.5 w/w % to about 95 w/w %, or from about 1 w/w % to about 95 w/w %, or from about 1 w/w % to about 75 w/w %, or from about 5 w/w % to about 75 w/w %, or from about 10 w/w % to about 75 w/w %, or from about 10 w/w % to about 50 w/w %.
  • the compounds provided herein, or salts or solvates thereof may be administered to a mammal suffering from abnormal cell growth, such as a human, either alone or as part of a pharmaceutically acceptable formulation, once a week, once a day, twice a day, three times a day, or four times a day, or even more frequently.
  • Administration of the compounds provided herein may be effected by any method that enables delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral inj ection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal administration.
  • Bolus doses can be used, or infusions over a period of 1, 2, 3, 4, 5, 10, 15, 20, 30, 60, 90, 120 or more minutes, or any intermediate time period can also be used, as can infusions lasting 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 16, 20, 24 or more hours or lasting for 1 -7 days or more.
  • Infusions can be administered by drip, continuous infusion, infusion pump, metering pump, depot formulation, or any other suitable means.
  • Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian patients to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the dose and dosing regimen is adjusted in accordance with methods well- known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present disclosure.
  • dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular patient, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the present disclosure encompasses intra-patient dose- escalation as determined by the skilled artisan. Determining appropriate dosages and regimens for administration of the chemotherapeutic agent are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings provided herein.
  • the methods disclosed herein are useful for the treatment of cancers including but not limited to cancers of the: circulatory system, for example, heart (sarcoma [angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma], myxoma, rhabdomyoma, fibroma, lipoma and teratoma), mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue; respiratory tract, for example, nasal cavity and middle ear, accessory sinuses, larynx, trachea, bronchus and lung such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma
  • SCLC small cell lung cancer
  • reproductive system for example, gynecological, uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous
  • cystadenocarcinoma mucinous cystadenocarcinoma, unclassified carcinoma] granulosa- thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma) and other sites associated with female genital organs; placenta, penis, prostate, testis, and other sites associated with male genital organs; hematologic system, for example, blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, mye
  • the second agent used in combination with the first agent described herein comprises an MEK inhibitor or an ERK inhibitor.
  • the second agent comprises an MEK inhibitor.
  • MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS-703988.
  • the second agent comprises an ERK inhibitor.
  • ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • anti-angiogenesis agent e.g., an agent that stops tumors from developing new blood vessels.
  • anti-angiogenesis agents include for example VEGF inhibitors, VEGFR inhibitors, TIE-2 inhibitors, PDGFR inhibitors, angiopoetin inhibitors, PKC-beta inhibitors, COX-2 (cyclooxygenase II) inhibitors, integrins (alpha-v/beta-3), MMP-2 (matrix- metalloproteinase 2) inhibitors, and MMP-9 (matrix-metalloprotienase 9) inhibitors.
  • Preferred anti-angiogenesis agents include sunitinib (Sutent®), bevacizumab (Avastin®), axitinib (AG 13736), SU 14813 (Pfizer), and AG 13958 (Pfizer).
  • Additional anti-angiogenesis agents include vatalanib (CGP 79787), Sorafenib (Nexavar®), pegaptanib octasodium (Macugen®), vandetanib (Zactima®), PF-0337210 (Pfizer), SU 14843 (Pfizer), AZD 2171 (AstraZeneca), ranibizumab (Lucentis®),
  • Neovastat® (AE 941), tetrathiomolybdata (Coprexa®), AMG 706 (Amgen), VEGF Trap (AVE 0005), CEP 7055 (Sanofi-Aventis), XL 880 (Exelixis), telatinib (BAY 57-9352), and CP-868,596 (Pfizer).
  • anti-angiogenesis agents include enzastaurin (LY 317615), midostaurin (CGP 41251), perifosine (KRX 0401), teprenone (Selbex®) and UCN 01 (Kyowa Hakko).
  • anti-angiogenesis agents which can be used in conjunction with one or more pharmaceutical compositions described herein include celecoxib (Celebrex®), parecoxib (Dynastat®), deracoxib (SC 59046), lumiracoxib (Preige®), valdecoxib
  • anti-angiogenesis agents include exisulind (Aptosyn®), salsalate
  • anti-angiogenesis agents include ABT 510 (Abbott), apratastat (TMI 005), AZD 8955 (AstraZeneca), incyclinide (Metastat®), and PCK 3145 (Procyon).
  • anti-angiogenesis agents include acitretin (Neotigason®), plitidepsin (Aplidine®), cilengtide (EMD 121974), combretastatin A4 (CA4P), fenretinide (4 HPR), halofuginone (Tempostatin®), Panzem® (2-methoxy estradiol), PF-03446962 (Pfizer), rebimastat (BMS 275291), catumaxomab (Removab®), lenalidomide (Revlimid®) squalamine (EVIZON®), thalidomide (Thalomid®), Ukrain® (NSC 631570), Vitaxin® (MEDI 522), and zoledronic acid (Zometa®).
  • the second agent used in combination with the first agent described herein is a so-called signal transduction inhibitor (e.g., inhibiting the means by which regulatory molecules that govern the fundamental processes of cell growth, differentiation, and survival communicated within the cell).
  • Signal transduction inhibitors include small molecules, antibodies, and antisense molecules.
  • Signal transduction inhibitors include for example kinase inhibitors (e.g. , tyrosine kinase inhibitors or serine/threonine kinase inhibitors) and cell cycle inhibitors.
  • More specifically signal transduction inhibitors include, for example, ALK inhibitors, ROS 1 inhibitors, TrkA inhibitors, TrkB inhibitors, TrkC inhibitors, farnesyl protein transferase inhibitors, EGF inhibitor, ErbB-1 (EGFR) inhibitors, ErbB-2 inhibitors, pan erb inhibitors, IGF1R inhibitors, MEK inhibitors, c-Kit inhibitors, FLT-3 inhibitors, K-Ras inhibitors, PI3 kinase inhibitors, JAK inhibitors, STAT inhibitors, Raf kinase inhibitors, Akt inhibitors, mTOR inhibitor, P70S6 kinase inhibitors, inhibitors of the WNT pathway and so called multi-targeted kinase inhibitors.
  • ALK inhibitors for example, ALK inhibitors, ROS 1 inhibitors, TrkA inhibitors, TrkB inhibitors, TrkC inhibitors, farnesyl protein transferase inhibitors, EGF inhibitor, ErbB-1 (EGFR)
  • signal transduction inhibitors that may be used include gefitinib (Iressa®), cetuximab (Erbitux®), erlotinib (Tarceva®), trastuzumab (Herceptin®), sunitinib (Sutent®) imatinib (Gleevec®), and PD325901 (Pfizer).
  • Additional examples of signal transduction inhibitors which may be used include BMS 214662 (Bristol-Myers Squibb), lonafarnib (Sarasar®), pelitrexol (AG 2037), matuzumab (EMD 7200), nimotuzumab (TheraCIM h-R3®), panitumumab (Vectibix®), Vandetanib (Zactima®), pazopanib (SB 786034), ALT 110 (Alteris Therapeutics), BIBW 2992 (Boehringer Ingelheim), and Cervene® (TP 38).
  • BMS 214662 Bristol-Myers Squibb
  • lonafarnib Sarasar®
  • pelitrexol AG 2037
  • matuzumab EMD 7200
  • nimotuzumab TheraCIM h-R3®
  • panitumumab Vectibix®
  • signal transduction inhibitors include PF- 2341066 (Pfizer), PF-299804 (Pfizer), canertinib (CI 1033), pertuzumab (Omnitarg®), Lapatinib (Tycerb®), pelitinib (EKB 569), miltefosine (Miltefosin®), BMS 599626 (Bristol-Myers Squibb), Lapuleucel-T (Neuvenge®), NeuVax® (E75 cancer vaccine), Osidem® (IDM 1), mubritinib (TAK-165), CP-724,714 (Pfizer), panitumumab (Vectibix®), lapatinib (Tycerb®), PF-299804 (Pfizer), pelitinib (EKB 569), and pertuzumab
  • signal transduction inhibitors include ARRY 142886 (Array Biopharm), everolimus (Certican®), zotarolimus (Endeavor®), temsirolimus (Torisel®), AP 23573 (ARIAD), and VX 680 (Vertex).
  • signal transduction inhibitors that may be used include XL 647 (Exelixis), sorafenib (Nexavar®), LE-AON (Georgetown University), and GI-4000 (Globelmmune).
  • Other signal transduction inhibitors that may be used include ABT 751 (Abbott), alvocidib (flavopiridol), BMS 387032 (Bristol Myers), EM 1421 (Erimos), indisulam (E 7070), seliciclib (CYC 200), BIO 112 (One Bio), BMS 387032 (Bristol-Myers Squibb), PD 0332991 (Pfizer), AG 024322 (Pfizer), LOXO-101 (Loxo Oncology), crizotinib, ceritinib, trametinib, PD0325901, selumetinib, cobimetinib, refametinib, pimasertib,
  • PD0325901 selumetinib, cobimetinib, refametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E6201, GDC-0623, and TAK-733.
  • signal transduction inhibitors that may be used include trametinib, selumetinib, cobimetinib, refametinib, pimasertib, and binimetinib.
  • a signal transduction inhibitor that may be used includes trametinib.
  • a signal transduction inhibitor that may be used includes, selumetinib.
  • a signal transduction inhibitor that may be used includes cobimetinib.
  • a signal transduction inhibitor that may be used includes refametinib.
  • a signal transduction inhibitor that may be used includes pimasertib.
  • a signal transduction inhibitor that may be used includes binimetinib.
  • the second agent is selected from are classical
  • Classical antineoplastic agents include but are not limited to hormonal modulators such as hormonal, anti-hormonal, androgen agonist, androgen antagonist and anti-estrogen therapeutic agents, histone deacetylase (HDAC) inhibitors, gene silencing agents or gene activating agents, ribonucleases, proteosomics, Topoisomerase I inhibitors, Camptothecin derivatives, Topoisomerase II inhibitors, alkylating agents, antimetabolites, poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor, microtubulin inhibitors, antibiotics, plant derived spindle inhibitors, platinum-coordinated compounds, gene therapeutic agents, antisense oligonucleotides, vascular targeting agents (VTAs), and statins.
  • hormonal modulators such as hormonal, anti-hormonal, androgen agonist, androgen antagonist and anti-estrogen therapeutic agents
  • HDAC histone deacetylase
  • Gene silencing agents or gene activating agents ribonu
  • Examples of classical antineoplastic agents include, but are not limited to, glucocorticoids, such as dexamethasone, prednisone, prednisolone,
  • methylprednisolone hydrocortisone
  • progestins such as medroxyprogesterone, megestrol acetate (Megace), mifepristone (RU-486), Selective Estrogen Receptor
  • SERMs Selective Estrogen-Receptor Downregulators
  • SED's Selective Estrogen-Receptor Downregulators
  • fulvestrant exemestane
  • Anastrozole Arimidex
  • atamestane fadrozole
  • letrozole Femara
  • gonadotropin-releasing hormone GnRH; also commonly referred to as luteinizing hormone-releasing hormone [LHRH]
  • agonists such as buserelin (Suprefact), goserelin (Zoladex), leuprorelin (Lupron), and tripto
  • antineoplastic agents examples include, but are not limited to, suberolanilide hydroxamic acid (SAHA, Merck Inc./Aton Pharmaceuticals), depsipeptide (FR901228 or FK228), G2M-777, MS-275, pivaloyloxymethyl butyrate and PXD-101 ; Onconase (ranpirnase), PS-341 (MLN-341), Velcade (bortezomib), 9- aminocamptothecin, belotecan, BN-80915 (Roche), camptothecin, diflomotecan, edotecarin, exatecan (Daiichi), gimatecan, 10-hydroxy camptothecin, irinotecan HC1 (Camptosar), lurtotecan, Orathecin (rubitecan, Supergen), SN-38, topotecan, camptothecin, 10- hydroxy campto
  • nemorubicin novantrone (mitoxantrone), pirarubicin, pixantrone, procarbazine,
  • the second agent is selected from dihydrofolate reductase inhibitors.
  • dihydrofolate reductase inhibitors include, but are not limited to, methotrexate and NeuTrexin (trimetrexate), purine antagonists (such as 6- mercaptopurine riboside, mercaptopurine, 6-thioguanine, cladribine, clofarabine (Clolar), fludarabine, nelarabine, and raltitrexed), pyrimidine antagonists (such as 5-fluorouracil (5- FU), Alimta (premetrexed disodium, LY231514, MTA), capecitabine (Xeloda®), cytosine arabinoside, Gemzar® (gemcitabine, Eli Lilly), Tegafur (UFT Orzel or Uforal and including TS-1 combination of tegafur, gimestat and otostat), dox
  • antimetabolites such as eflornithine, hydroxyurea, leucovorin, nolatrexed (Thymitaq), triapine, trimetrexate, N-(5- N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl)-L-glutamic acid, AG-014699 (Pfizer Inc.), ABT-472 (Abbott Laboratories), INO-1001 (Inotek Pharmaceuticals), KU-0687 (KuDOS Pharmaceuticals) and GPI 18180 (Guilford Pharm Inc) and combinations thereof.
  • antineoplastic cytotoxic agents include, but are not limited to, Abraxane (Abraxis Bioscience, Inc.), Batabulin (Amgen), EPO 906 (Novartis), Vinflunine (Bristol-Myers Squibb Company), actinomycin D, bleomycin, mitomycin C, neocarzinostatin (Zinostatin), vinblastine, vincristine, vindesine, vinorelbine (Navelbine), docetaxel (Taxotere), Ortataxel, paclitaxel (including Taxoprexin a DHA/paclitaxel conjugate), cisplatin, carboplatin, Nedaplatin, oxaliplatin (Eloxatin), Satraplatin, Camptosar, capecitabine (Xeloda), oxaliplatin (Eloxatin), Taxotere alitretinoin, Canfosfamide (
  • antineoplastic agents include, but are not limited to, as Advexin (ING 201), TNFerade (GeneVec, one or more compounds which express TNF alpha in response to radiotherapy), RB94 (Baylor College of Medicine), Genasense (Oblimersen, Genta), Combretastatin A4P (CA4P), Oxi-4503, AVE-8062, ZD- 6126, TZT-1027, Atorvastatin (Lipitor, Pfizer Inc.), Provastatin (Pravachol, Bristol-Myers Squibb), Lovastatin (Mevacor, Merck Inc.), Simvastatin (Zocor, Merck Inc.), Fluvastatin (Lescol, Novartis), Cerivastatin (Bay col, Bayer), Rosuvastatin (Crestor, AstraZeneca), Lovostatin, Niacin (Advicor, Kos Pharmaceuticals), Caduet, Lip
  • the methods disclosed herein comprise administering to the patient a combination further comprising one or more additional agents.
  • the one or more additional agents may be independently selected from the anti-angiogenesis agents described herein, the so-called signal transduction inhibitors described herein, the classical antineoplastic agents described herein, and the dihydrofolate reductase inhibitors described herein.
  • the example herein is provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with using the present technology.
  • the example herein is also presented in order to more fully illustrate the preferred aspects of the present technology.
  • the example should in no way be construed as limiting the scope of the present technology, as defined by the appended claims.
  • the example can include or incorporate any of the variations, aspects or aspects of the present technology described above.
  • the variations, aspects or aspects described above may also further each include or incorporate the variations of any or all other variations, aspects or aspects of the present technology.
  • Example 1 Pharmaceutical composition comprising A-[5-(3,5-difluorobenzyl)-lH- indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)- benzamide.
  • a pharmaceutical composition comprising N-[5-(3,5-difluorobenzyl)-lH-indazol- 3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and betaine hydrochloride was prepared as follows.
  • N-[5-(3,5-Difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l -yl)-2- (tetrahydro-2H-pyran-4-ylamino)-benzamide was screened through a 60-mesh sieve and was transferred to the batch mixing container.
  • the betaine hydrochloride was ground with a mortar and pestle, screened through a 60-mesh sieve and then transferred to the batch mixing container.
  • the resulting mixture was filled into gelatin capsule shells, opaque white, size #00.
  • the body and cap of the capsules were separated, the capsule bodies were placed into a capsule device, ensuring the top of the capsule body was flush with the surface of the filling device by moving the spacer of the device.
  • the powder blend was poured onto the surface of the filling device, volumetrically filling the body of the capsules, and scraping the excess powder evenly until all capsule bodies are filled.
  • the powder was firmly tamped into the shells one time using a tamper. Additional powder blend was added to fill the remainder of the capsule and any excess powder was scraped off. The tamping, filling, and scraping procedures were repeated for each capsule until the desired capsule fill weight was achieved.
  • the filled capsules were collected in a 10-mesh sieve and were de-dusted by agitating them lightly.
  • the filled capsule weight range acceptance limits were set at 93% to 107%.
  • the average weight of the empty capsule shells was 119.4 mg.
  • capsules are used for concurrent or sequential administration with a separate pharmaceutical composition comprising a second agent.
  • the second agent may be a MEK inhibitor or an ERK inhibitor.
  • Example 2 Combination therapy for patients with non-small cell lung cancer.
  • One or more appropriate doses of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and a second agent are administered to a human patient for the treatment of non-small cell lung cancer.
  • Expected results from treatment with the combination include (1) reduction of the size of a cancer tumor, (2) inhibition (that is, slowing to some extent, preferably stopping) of cancer tumor metastasis, (3) inhibition to some extent (that is, slowing to some extent, preferably stopping) of cancer tumor growth, and/or, (4) relief to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer.
  • the second agent may be a MEK inhibitor or an ERK inhibitor.
  • MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988.
  • ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • Example 3 Combination therapy for patients with papillary thyroid cancer.
  • One or more appropriate doses of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and a second agent are administered to a human patient for the treatment of papillary thyroid cancer.
  • Expected results from treatment with the combination include (1) reduction of the size of a cancer tumor, (2) inhibition (that is, slowing to some extent, preferably stopping) of cancer tumor metastasis, (3) inhibition to some extent (that is, slowing to some extent, preferably stopping) of cancer tumor growth, and/or, (4) relief to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer.
  • the second agent may be a MEK inhibitor or an ERK inhibitor.
  • MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988.
  • ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • Example 4 Combination therapy for patients with neuroblastoma.
  • One or more appropriate doses of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and a second agent are administered to a human patient for the treatment of neuroblastoma.
  • Expected results from treatment with the combination include (1) reduction of the size of a cancer tumor, (2) inhibition (that is, slowing to some extent, preferably stopping) of cancer tumor metastasis, (3) inhibition to some extent (that is, slowing to some extent, preferably stopping) of cancer tumor growth, and/or, (4) relief to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer.
  • the second agent may be a MEK inhibitor or an ERK inhibitor.
  • MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988.
  • ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • Example 5 Combination therapy for patients with pancreatic cancer.
  • One or more appropriate doses of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and a second agent are administered to a human patient for the treatment of pancreatic cancer.
  • Expected results from treatment with the combination include (1) reduction of the size of a cancer tumor, (2) inhibition (that is, slowing to some extent, preferably stopping) of cancer tumor metastasis, (3) inhibition to some extent (that is, slowing to some extent, preferably stopping) of cancer tumor growth, and/or, (4) relief to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer.
  • the second agent may be a MEK inhibitor or an ERK inhibitor.
  • MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988.
  • ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • Example 6 Combination therapy for patients with melanoma.
  • One or more appropriate doses of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and a second agent are administered to a human patient for the treatment of melanoma.
  • Expected results from treatment with the combination include (1) reduction of the size of a cancer tumor, (2) inhibition (that is, slowing to some extent, preferably stopping) of cancer tumor metastasis, (3) inhibition to some extent (that is, slowing to some extent, preferably stopping) of cancer tumor growth, and/or, (4) relief to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer.
  • the second agent may be a MEK inhibitor or an ERK inhibitor.
  • MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS-703988.
  • ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS- 134, AEZS-136, and IDN-5491.
  • Example 7 Combination therapy for patients with colorectal cancer.
  • One or more appropriate doses of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and a second agent are administered to a human patient for the treatment of colorectal cancer.
  • Expected results from treatment with the combination include (1) reduction of the size of a cancer tumor, (2) inhibition (that is, slowing to some extent, preferably stopping) of cancer tumor metastasis, (3) inhibition to some extent (that is, slowing to some extent, preferably stopping) of cancer tumor growth, and/or, (4) relief to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer.
  • the second agent may be a MEK inhibitor or an ERK inhibitor.
  • MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988.
  • ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
  • This Example describes studies performed to generate transgenic Ba/F3 cells expressing a TPM3-TrkA-G595R fusion protein.
  • a cDNA encoding TPM3-TrkA-G595R fusion was cloned from entrectinib-resistant cells by a PCR-based technique and subsequently inserted into a lentiviral vector pVL-EFla-MCS-IRES-Puro (BioSettia, San Diego, CA).
  • vesicular stomatitis virus GP (VSVG)-pseudo-typed lentiviruses containing the TPM3-TrkA-G595R cDNA were transduced into the murine IL-3 dependent pro-B cell Ba/F3 at different multiplicity of infections (MOIs) with 8 ⁇ g/mL of polybrene (EMD Millipore).
  • MOIs multiplicity of infections
  • the transduced Ba/F3 cells were selected in the murine IL-3 containing RPMI media supplemented with 10% FBS and 1 ⁇ g/mL of puromycin for two weeks.
  • the stable cell pools were further selected in RPMI media (GIBCO®) supplemented with 10% FBS (fetal bovine serum) and without murine IL-3 for 4 weeks.
  • the resulting supernatants were saved for Western Blot analysis. 20-40 ⁇ g of the resulting proteins were separated by SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The resulting membranes were blotted with the following antibodies (obtained from Cell Signalling unless otherwise stated) according to the procedures described in the respective manuals: phospho-TrkA-Y490, phospho- TrkA-Y785, pan-Trk, PLCY1, phospho-PLCYl-Y783, MEK1/2, phospho-MEKl/2 (S217/S221), ⁇ -actin.
  • VDF polyvinylidene difluoride
  • Example 10 In vitro Activities of entrectinib (RXDX-101), trametinib, and the combination of entrectinib and trametinib on the inhibition of growth of Ba/F3 cell lines expressing TPM3-TrkA-G959R
  • DMSO dimethylsulfoxide
  • entrectinib RXDX-101
  • trametinib at a concentration of about 30 nM
  • a combination of entrectinib at a concentration of 300 nM and trametinib at a concentration of 30 nM.
  • the cells in each respective well were counted every 3 to 4 days using CountessTM cell counter (Invitrogen, Carlsbad, CA) in the presence of Trypan Blue (Invitrogen, Carlsbad, CA).
  • the media containing those cells were diluted at 1 : 10 with fresh growth media containing the same respective concentrations of entrectinib, trametinib or the combination of entrectinib and trametinib.
  • the wells that contained fewer than 1 million live cells on the day the cells in each well were counted went unchanged. The results of the study are shown in FIG.
  • Example 11 In vivo study of mice implanted with cells expressing TPM3-TrkA- G595R following administration of entrectinib (RXDX-101), trametinib, or a combination of entrectinib and trametinib.
  • RXDX-101 entrectinib
  • trametinib trametinib
  • trametinib or a combination of entrectinib and trametinib.
  • GMP Good manufacture practice
  • Ignyta Lignyta
  • the entrectinib in vivo oral dosing solution was made by suspending entrectinib in a solution of 0.5% methyl cellulose (Lot #: 147255, M352-500, Fisher) and 1% Tween 80 (Lot #: MKB58228V, P4780-100ML, Sigma) at a concentration of 5 mg/mL.
  • Trametinib in vivo oral dosing solution was made by dissolving trametinib in a solution of DMSO (Lot #: SHBG1596V, D8418-100ML, Sigma), PEG 400 (Lot #: MKBX3961V, 202398-500G, Sigma), Tween 80 (Lot #: MKB58228V, P4780-100ML, Sigma) and water.
  • the final concentration of trametinib was 0.1 mg/mL and the amounts of each component in the resulting solution were 3.3 % DMSO, 16.7% PEG 400, 15% Tween 80 and 65% water, with the water being added just prior to dosing.
  • Athymic nu/nu female mice between 6-7 weeks of age were ordered from Charles River. Animals were allowed to acclimate 3 days prior to the start of the study (e.g., cell implantation). Animals were housed in irradiated, individual HEPA ventilated cages (Innocage ® IVC, Innovive USA) on a 12-hour light-dark cycle at 68-79°F and 30-70% humidity. Animals were provided with irradiated chow (Teklad 2920X) and acidified drinking water (Innovive) ad libitum as per the animal care and use protocol established at Explora Biolabs (Ignyta ACUP# EB15-050). Animals were identified by uniquely numbered ear-tags.
  • KM 12 cells were cultured, harvested, and suspended in 1 : 1 FBS free culture media: marti gel at a concentration of 10 million cells/mL. Cells were implanted under the skin on the right flank of each mouse on day 0. The implantation volume was 200 ⁇ . Tumors grew until the mean tumor volume reached approximately 130 mm 3 . On day 8 mice were randomized into 6 treatment groups (10 mice/group) and treatment began. Animals were treated p.o. q.d.
  • Tumors measurements and body weights were collected 2x/week over the duration of the study. Tumor growth was assessed by caliper and tumor volumes were calculated using the equation (1). Animals with tumor volumes > 2,000 mm 3 were removed from study.
  • volume (mm 3 ) [ length (mm) x width (mm) x width (mm) ] / 2 (1)
  • Body weights were collected using a laboratory scale. Toxicity was evaluated based on body weight loss. Animals losing ⁇ 5% of their body weight were removed from the study.
  • %TGI percentage of tumor growth inhibition
  • %TGI [ ( TVyehicie - TVtreatment) / ( TVyehicie " TVinitiai ) ] X 100 [0246]
  • Table 1 A summary table of the average tumor volume for each group, for each measurement day is shown in Table 1.
  • the %TGI for each day was calculated as described above and these data are summarized in Table 2. Evaluation of significance by two-way ANOVA (Prism) was limited out to day 14 due to one death in the entrectinib + trametinib group. Body weight data for this study shows that this death is likely due to toxicity induced by trametinib. Analysis showed that by day 14 only the trametinib alone groups was significantly different from vehicle (p ⁇ 0.05, two-way ANOVA, Prism).
  • Table 1 Average tumor volume for each treatment group on each day measured.
  • TrkA human tropomyosin receptor kinase A
  • TrkB human tropomyosin receptor kinase B
  • TrkC human tropomyosin receptor kinase C
  • ALK human ALK tyrosine kinase receptor

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Abstract

Described herein are combinations comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROSl, TrkA, TrkB, or TrkC activity, or a combination thereof, and methods of using such combinations in the treatment of patients having cancer.

Description

COMBINATIONS FOR THE TREATMENT OF CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62/269,805, filed December 18, 2015, and U.S. Provisional Application No. 62/428,498, filed November 30, 2016, both of which are hereby incorporated by reference in their entirety.
FIELD
[0002] The present disclosure relates to methods of treating cancer in a patient, wherein the patient is known to possess at least one molecular alteration in ALK, ROS1, NTRK1, NTRK2, NTRK3, TrkA, TrkB, or TrkC, or a combination thereof, comprising
administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor or ALK, ROS1, TrkA, TrkB, or TrkC, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor.
BACKGROUND
[0003] The malfunctioning of protein kinases (PKs) is the hallmark of numerous diseases. A large share of the oncogenes and proto-oncogenes involved in human cancers encode for PKs. For a general reference to PKs malfunctioning or deregulation see, for instance, Current Opinion in Chemical Biology 1999, 3:459-465, which is incorporated by reference herein in its entirety.
[0004] Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor belonging to the insulin receptor subfamily of RTKs: the ALK gene is located on chromosome 2 and is expressed mainly in neuronal cells, especially during development. Many data from the literature have demonstrated that the ALK fusion proteins have strong oncogenic potentials.
[0005] ROS1 belongs to the insulin-receptor superfamily. Like other tyrosine kinase receptor molecules, it plays a role in relaying growth signals from the environment outside the cell into the cell's nucleus. It is 1 of 2 orphan receptor tyrosine kinase family members with no known binding ligand. Genetic changes in ROS1, such as gene rearrangements, mutations, or copy number increases, create oncogenes, which can lead to cancer. ROS1 was discovered in NSCLC patients in the form of a fusion protein (6 different partners for ROS 1) and is found in approximately 2% of patients with NSCLC. Two other ROS1 gene rearrangements have been detected in a variety of other cancers, including glioblastoma multiforme, cholangiocarcinoma, ovarian cancer, gastric adenocarcinoma, colorectal cancer, inflammatory myofibroblastic tumor, angiosarcoma, and epitheloid hemangioendothelioma.
[0006] Trks are the high affinity receptor tyrosine kinases activated by a group of soluble growth factors called neurotrophins (NT). The Trk receptor family has three members-- TrkA, TrkB and TrkC. Among the neurotrophins are (i) nerve growth factor (NGF) which activates TrkA, (ii) brain-derived neurotrophic factor (BDNF) and NT-4/5 which activate TrkB and (iii) NT3 which activates TrkC. Trks are widely expressed in neuronal tissue and are implicated in the maintenance, signaling and survival of neuronal cells (Patapoutian et al , Current Opinion in Neurobiology, 2001, 11, 272-280, incorporated by reference in its entirety herein). NTRK1 encodes the TrkA receptor tyrosine kinase. TrkA activates the PI3K/AKT, PKC and ERK1/2 pathways which promote cell growth and survival.
[0007] Recent literature has shown that overexpression, activation, amplification and/or mutation of Trks are associated with many cancers including neuroblastoma (Brodeur, G. M., Nat. Rev. Cancer 2003, 3, 203-216, incorporated by reference in its entirety herein), ovarian cancer (Davidson. B., et al , Clin. Cancer Res. 2003, 9, 2248-2259, incorporated by reference in its entirety herein), breast cancer (Kruettgen et al , Brain Pathology 2006, 16: 304-310, incorporated by reference in its entirety herein), prostate cancer (Dionne et al , Clin. Cancer Res. 1998, 4(8): 1887-1898, incorporated by reference in its entirety herein), pancreatic cancer (Dang et al, Journal of Gastroenterology and Hepatology 2006, 21(5): 850-858, incorporated by reference in its entirety herein), multiple myeloma (Hu et al , Cancer Genetics and Cytogenetics 2007, 178: 1-10, incorporated by reference in its entirety herein), astrocytoma and medulloblastoma (Kruettgen et al , Brain Pathology 2006, 16: 304-310, incorporated by reference in its entirety herein) glioma (Hansen et al, Journal of Neurochemistry 2007, 103: 259-275, incorporated by reference in its entirety herein), melanoma (Truzzi et al, Journal of Investigative Dermatology 2008, 128(8): 2031-2040, incorporated by reference in its entirety herein), thyroid carcinoma (Brzezianska et al. , Neuroendocrinology Letters 2007, 28(3), 221-229, incorporated by reference in its entirety herein), lung adenocarcinoma (Perez-Pinera et al, Molecular and Cellular Biochemistry 2007, 295(1&2), 19-26, incorporated by reference in its entirety herein), large cell neuroendocrine tumors (Marchetti et al , Human Mutation 2008, 29(5), 609-616, incorporated by reference in its entirety herein), and colorectal cancer (Bardelli, A., Science 2003, 300, 949, incorporated by reference in its entirety herein). In preclinical models of cancer, Trk inhibitors are efficacious in both inhibiting tumor growth and stopping tumor metastasis. In particular, non-selective small molecule inhibitors of Trk A, B and C and Trk/Fc chimeras were efficacious in both inhibiting tumor growth and stopping tumor metastasis (Nakagawara, A. Cancer Letters 2001, 169: 107-114; Meyer et al , Leukemia 2007, 1-10; Pierottia and Greco, Cancer Letters 2006, 232:90-98; Eric Adriaenssens et al, Cancer Res 2008, 68:(2) 346-351; Truzzi et al, Journal of Investigative Dermatology 2008, 128(8): 2031-2040, each of which is incorporated by reference in its entirety herein).
Therefore, an inhibitor of the Trk family of kinases is expected to have utility in the treatment of cancer.
SUMMARY
[0008] In one aspect, disclosed herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof; and the second agent is a MEK inhibitor or an ERK inhibitor.
[0009] In another aspect, disclosed herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in ALK.
[0010] In another aspect, disclosed herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the ALK receptor tyrosine kinase polypeptide. In some embodiments, the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G1202 or G1269 of the ALK polypeptide set forth in SEQ ID NO: 4. In some embodiments, the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G1202 of the ALK polypeptide set forth in SEQ ID NO: 4. In some embodiments, the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G1269 of the ALK polypeptide set forth in SEQ ID NO: 4.
[0011] In another aspect, disclosed herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a ROSl inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in ROSl.
[0012] In another aspect, disclosed herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a ROSl inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the ROSl receptor tyrosine kinase polypeptide. In some embodiments, the at least one mutation in the ROSl receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G2032 or G2101 of the ROSl polypeptide set forth in SEQ ID NO: 5. In some
embodiments, the at least one mutation in the ROSl receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G2032 of the ROSl polypeptide set forth in SEQ ID NO: 5. In some embodiments, the at least one mutation in the ROSl receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G2101 of the ROSl polypeptide set forth in SEQ ID NO: 5.
[0013] In another aspect, disclosed herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkA inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK1.
[0014] In another aspect, disclosed herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkA inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkA receptor tyrosine kinase polypeptide. In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G595 or G667 of the TrkA polypeptide set forth in SEQ ID NO: 1. In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G595 of the TrkA polypeptide set forth in SEQ ID NO:
1. In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G595R). In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G667 of the TrkA polypeptide set forth in SEQ ID NO: 1. In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G667C).
[0015] In another aspect, disclosed herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkB inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK2.
[0016] In another aspect, disclosed herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkB inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkB receptor tyrosine kinase polypeptide. In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G639 or G709 of the TrkB polypeptide set forth in SEQ ID NO: 2. In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G639 of the TrkB polypeptide set forth in SEQ ID NO:
2. In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G639R). In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G709 of the TrkB polypeptide set forth in SEQ ID NO: 2. In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G709C). [0017] In another aspect, disclosed herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkC inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK3.
[0018] In another aspect, disclosed herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkC inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkC receptor tyrosine kinase polypeptide. In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G623 or G696 of the TrkC polypeptide set forth in SEQ ID NO: 3. In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G623 of the TrkC polypeptide set forth in SEQ ID NO: 3. In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G623R). In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G696 of the TrkC polypeptide set forth in SEQ ID NO: 3. In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G696C).
[0019] In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0020] In some embodiments, the second agent is a MEK inhibitor. In some
embodiments, the MEK inhibitor is an inhibitor of MEKl , MEK2, or a combination thereof. In some embodiments, the MEK inhibitor is selected from the group consisting of
PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS-703988. In some embodiments, the MEK inhibitor is PD0325901. In some embodiments, the MEK inhibitor is selumetinib. In some embodiments, the MEK inhibitor is cobimetinib. In some embodiments, the MEK inhibitor is refametinib. In some embodiments, the MEK inhibitor is trametinib. In some embodiments, the MEK inhibitor is pimasertib. In some embodiments, the MEK inhibitor is binimetinib. In some embodiments, the MEK inhibitor is AZD8330. In some embodiments, the MEK inhibitor is R04987655. In some embodiments, the MEK inhibitor is R05126766. In some embodiments, the MEK inhibitor is WX-554. In some embodiments, the MEK inhibitor is E-6201. In some embodiments, the MEK inhibitor is GDC-0623. In some embodiments, the MEK inhibitor is TAK-733. In some embodiments, the MEK inhibitor is RG-7304. In some embodiments, the MEK inhibitor is CKBP-002. In some embodiments, the MEK inhibitor is RDEA-436. In some embodiments, the MEK inhibitor is sorafenib. In some embodiments, the MEK inhibitor is PD-184352. In some embodiments, the MEK inhibitor is GSK-2091976A. In some embodiments, the MEK inhibitor is AS-703988.
[0021] In some embodiments, the second agent is an ERK inhibitor. In some
embodiments, the ERK inhibitor is an inhibitor of ERK1, ERK2, or a combination thereof. In some embodiments, the ERK inhibitor is selected from the group consisting of TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491. In some embodiments, the ERK inhibitor is TG-02. In some embodiments, the ERK inhibitor is MK-8353. In some embodiments, the ERK inhibitor is ulixertinib. In some embodiments, the ERK inhibitor is HE-3235. In some embodiments, the ERK inhibitor is AEZS-134. In some embodiments, the ERK inhibitor is AEZS-136. In some embodiments, the ERK inhibitor is IDN-5491.
[0022] In some embodiments, the cancer is selected from non-small cell lung cancer, papillary thyroid cancer, neuroblastoma, pancreatic cancer, melanoma, and colorectal cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is papillary thyroid cancer. In some embodiments, the cancer is neuroblastoma. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is colorectal cancer.
[0023] In some embodiments, the combination is a pharmaceutical composition comprising the therapeutically effective amount of the first agent, the therapeutically effective amount of the second agent, and at least one pharmaceutically acceptable carrier.
[0024] In some embodiments, the combination is concurrent administration of a first pharmaceutical composition comprising the therapeutically effective amount of the first agent and a second pharmaceutical composition comprising the therapeutically effective amount of the second agent.
[0025] In some embodiments, the combination is sequential administration of a first pharmaceutical composition comprising the therapeutically effective amount of the first agent and a second pharmaceutical composition comprising the therapeutically effective amount of the second agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 is a Western Blot analysis from Example 9 demonstrating that p-MEKl/2 (Ser217/221) and total MEK1/2 are upregulated in the Ba/F3 cells expressing TPM3-TrkA- G595R versus Ba/F3 cells expressing wild-type TPM3-TrkA.
[0027] FIG. 2 is a plot of the growth of live cells from Example 10, demonstrating that Ba/F3 cells expressing TPM3-TrkA-G595R that are treated with the combination of entrectinib (300 nM) and trametinib (30 nM) displayed significant inhibition of growth (up to 40 days) compared to the cells that were untreated (DMSO), those treated with entrectinib (300 nM) alone and those treated with trametinib (30 nM) alone.
[0028] FIG. 3 is a plot of the results from Example 11, which demonstrates that the growth of tumors in mice implanted with cells expressing TPM3-TrkA-G595R was reduced in those mice to which a combination of entrectinib (RXDX-101) (60 mg/kg) and trametinib (1 mg/kg) was administered versus tumor growth in those mice in the control group (Vehicle), those mice treated with entrectinib (60 mg/kg) alone, and those mice treated with tremetinib (1 mg/kg) alone.
DETAILED DESCRIPTION
[0029] The term "N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l- yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide," as used herein, means the compound having the chemical structure,
H
Figure imgf000011_0001
N-[5 -(3 ,5 -Difluorobenzy 1)- 1 H-indazol-3-yl] -4-(4-methy 1-piperazin- 1 -y l)-2-(tetrahy dro- pyran-4-ylamino)-benzamide may be prepared as described in United States Patent No. 8,299,057, the disclosure of which is hereby incorporated by reference in its entirety. N-[5- (3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l -yl)-2-(tetrahydro-pyran-4- ylamino)-benzamide is associated with CAS Registry Number 1 108743-60-7. The N-[5- (3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l -yl)-2-(tetrahydro-pyran-4- ylamino)-benzamide compound may also be described as "entrectinib" and/or "RXDX- 101."
[0030] The singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes one or more cells, including mixtures thereof. "A and/or B" is used herein to include all of the following alternatives: "A," "B," "A or B," and "A and B".
[0031] The term "about," as used herein, means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. Where ranges are provided, they are inclusive of the boundary values.
[0032] The term "acidulant," as used herein, means a chemical compound that is acidic in nature. As used herein, the term "organic acidulant" means an acidulant the chemical composition of which contains carbon. As used herein, the term "inorganic acidulant" means an acidulant the composition of which does not contain carbon. [0033] The terms "administration" and "administering," as used herein, refer to the delivery of a bioactive composition or formulation by an administration route including, but not limited to, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, topically, or combinations thereof.
[0034] The term "AUC," as used herein, means the area under the curve of a plot of the concentration of a compound in the plasma of a patient versus time.
[0035] The terms "anaplastic lymphoma kinase" and "ALK," as used herein, mean the ALK tyrosine kinase receptor or CD246 (cluster of differentiation 246), which is an enzyme that in humans is encoded by the ALK gene and also has the UniProt identifier
ALK HUMAN.
[0036] The term "AZD8330," as used herein, means the compound having the chemical structure,
Figure imgf000012_0001
Alternative names for AZD8330 include AZD-8330 and 2-(2-fluoro-4-iodophenylamino)- N-(2-hydroxyethoxy)-l,5-dimethyl-6-oxo-l,6-dihydropyridine-3-carboxarnide. AZD8330 is associated with CAS Registry Number 869357-68-6.
[0037] The term "betaine hydrochloride," as used herein, means a compound having CAS Registry Number 590-46-5 and the common names l-carboxy-NNN- trimethylmethanaminium chloride and (carboxymethyl)trimethylammonium hydrochloride.
[0038] The term "binimetinib," as used herein, means the compound having the chemical structure,
Figure imgf000012_0002
Alternative names for binimetinib include MEK162, ARRY-162, ARRY-438162, and 5-((4- bromo-2-fluorophenyl)amino)-4-fluoro-N-(2-hydroxyethoxy)-l-methyl-lH- benzo[d]imidazole-6-carboxamide. Binimetinib is associated with CAS Registry Number 606143-89-9.
[0039] The term "biological sample," as used herein, encompasses a variety of sample types obtained from an organism and can be used in a diagnostic or monitoring assay. The sample may be of a healthy tissue, diseased tissue, or tissue suspected of being diseased tissue. The sample may be a biopsy taken, for example, during a surgical procedure. The sample may be collected via means of fine needle aspiration, scraping or washing a cavity to collects cells or tissue therefrom. The sample may be of a tumor such as, for example, solid and hematopoietic tumors as well as of neighboring healthy tissue. The sample may be a smear of individual cells or a tissue section. The term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components. The term encompasses clinical samples, and also includes cells in cell culture, cell supernatants, cell lysates, cell extracts, cell homogenates, subcellular components including synthesized proteins, serum, plasma, bodily and other biological fluids, and tissue samples. The biological sample can contain compounds that are not naturally intermixed with the cell or tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like. In one embodiment, the sample is preserved as a frozen sample or as formaldehyde- or paraformaldehy de-fixed paraffin-embedded (FFPE) tissue preparation. For example, the sample can be embedded in a matrix, e.g., an FFPE block or a frozen sample.
[0040] The term "biomarker," as used herein, means a molecule whose level of nucleic acid or protein product has a quantitatively differential concentration or level with respect to an aspect of a biological state of a patient. "Biomarker" is used interchangeably with "marker" herein. The level of the biomarker can be measured at both the nucleic acid level as well as the polypeptide level. At the nucleic acid level, a nucleic acid gene or a transcript which is transcribed from any part of the patient's chromosomal and extrachromosomal genome, including for example the mitochondrial genome, may be measured. Preferably an RNA transcript, more preferably an RNA transcript includes a primary transcript, a spliced transcript, an alternatively spliced transcript, or an mRNA of the biomarker is measured. At the polypeptide level, a pre-propeptide, a propeptide, a mature peptide or a secreted peptide of the biomarker may be measured. A biomarker can be used either solely or in conjunction with one or more other identified biomarkers so as to allow correlation to the biological state of interest as defined herein. Specific examples of biomarkers covered by the present disclosure include ALK, ROS1, TrkA, TrkB, and TrkC.
[0041] As used herein "cancer" refers to any malignant and/or invasive growth or tumor caused by abnormal cell growth. As used herein "cancer" refers to solid tumors named for the type of cells that form them, cancer of blood, bone marrow, or the lymphatic system. Examples of solid tumors include but are not limited to sarcomas and carcinomas. Examples of cancers of the blood include but are not limited to leukemias, lymphomas and myeloma. The term "cancer" includes but is not limited to a primary cancer that originates at a specific site in the body, a metastatic cancer that has spread from the place in which it started to other parts of the body, a recurrence from the original primary cancer after remission, and a second primary cancer that is a new primary cancer in a person with a history of previous cancer of different type from latter one. These terms also mean the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell, such as a leukemia cell. These terms include a solid tumor, a soft tissue tumor, or a metastatic lesion. As used herein, the term "cancer" includes premalignant, as well as malignant cancers. In certain embodiments, the cancer is a solid tumor, a soft tissue tumor, or a metastatic lesion.
[0042] The term "chemotherapeutic agent," as used herein, means a chemical substance, such as a cytotoxic or cytostatic agent, that is used to treat a condition, particularly cancer.
[0043] As used herein, the term "Cmax" means the peak concentration that a compound achieves in the plasma of a patient after the compound, or a pharmaceutical composition comprising the compound, has been administrated to the patient. In some embodiments, the compound, or a pharmaceutical composition comprising the compound, is administered orally to a patient to achieve a particular Cmax. [0044] The term "cobimetinib," as used herein, means the compound having the chemical structure,
Figure imgf000015_0001
Alternative names for cobimetinib include GDC-0973, XL-518, RG7420, Cotellic™, and (^[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl]3-hydroxy-3-(piperidin-2- yl]azetidin-l -yl)methanone. Cobimetinib is associated with CAS Registry Number 934660- 93-2.
[0045] The terms "combination" and "in combination with," as used herein, mean the administration of a compound provided herein together with an at least one additional pharmaceutical or medicinal agent (e.g., an anti-cancer agent), either sequentially or simultaneously. It includes dosing simultaneously, or within minutes or hours of each other, or on the same day, or on alternating days, or dosing the compound provided herein on a daily basis, or multiple days per week, or weekly basis, for example, while
administering another compound such as a chemotherapeutic agent on the same day or alternating days or weeks or on a periodic basis during a time simultaneous therewith or concurrent therewith, or at least a part of the time during which the compound provided herein is dosed. For example, N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l -yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof could be dosed every day or several days a week while the chemotherapeutic agent is dosed on alternating days or alternating weeks or other periods of time, such as every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1. 12, 13, 14 or more days.
[0046] The term "E6201 ," as used herein, means the compound having the chemical structure,
Figure imgf000015_0002
Alternative names for E6201 include (3S,4R, 5Z, 8S,9S, l l£)-14-(ethylamino)-8,9,16- trihydroxy-3,4-dimethyl-3,4,9,10-tetrahydro-lH-2-benzoxacyclotetradecine-l,7(8H)-dione. E6201 is associated with CAS Registry Number 603987-35-5.
[0047] The term "ERK," as used herein, means the family of extracellular signal- regulated kinases (ERKs), and are protein kinase intracellular signaling molecules. ERKs include, but are not limited to, ERKl and ERK2. As used herein, the term "ERKl" means extracellular signal-regulated kinase 1 having the UniProt identifier MK03_HUMAN and encoded by the MAPK3 gene. As used herein, the term "ERK2" means extracellular signal- regulated kinase 2 having the UniProt identifier MK01_HUMAN and encoded by the MAPKl gene. ERKl and ERK2 are also referred to by those having ordinary skill in the art as MAPK3 and MAPKl, respectively. A reference to ERKl is a reference to MAPK3. A reference to ERK2 is a reference to MAPKl. Unless otherwise indicated, use of the term "ERK" may refer to ERKl, ERK2, or a combination thereof.
[0048] The term "food effect," as used herein, means a change in the rate and/or extent of absorption of a compound in a patient when the compound is administered to the patient shortly after a meal (fed conditions) as compared to the rate and/or extent of absorption of the compound when the compound is administered to the patient under fasting conditions. As used herein, the term "no food effect" means that there is no significant difference in the rate and/or extent of absorption of a compound in a patient when the compound is administered to the patient in fed conditions compared to fasting conditions.
[0049] The term "GDC-0623," as used herein, means the compound having the chemical structure,
Figure imgf000016_0001
Alternative names for GDC-0623 include 5-[(2-fluoro-4-iodophenyl)amino]-N-(2- hydroxyethoxy)-imidazo[l,5-a]pyridine-6-carboxarnide. GDC-0623 is associated with CAS Registry Number 1168091-68-6. The term "HE-3235," as used herein, means the compound having the chemical
Figure imgf000017_0001
Alternative names for HE-3235 include HE3235, H E3235, Apotone®, and
(3R,5S, 8R,9S, 10S,13S,14S, 11R)- 17-ethyny 1- 10, 13 -dimethy lhexadecahy dro- 1 H- cyclopenta[a]phenanthrene-3,17-diol. HE-3235 is associated with CAS Registry Number 183387-50-0.
[0051] The term "IDN-5491," as used herein, means the compound having the chemical structure,
Figure imgf000017_0002
Alternative names for IDN-5491 include hyperforin triemthoxybenzoate and (Ι^^Λ,Τ^,δΛ)- l-isobutyryl-8-methyl-3,5,7-tris(3-methyl-2-buten-l-yl)-8-(4-methyl-3-penten-l-yl)-4,9- dioxobicyclo[3.3. l]non-2-en-2-yl 2,3,4-trimethoxybenzoate.
[0052] The term "MEK," as used herein, means the family of mitogen/extracellular signal-regulated kinases (MEKs), and are kinase enzymes which phosphorylate mitogen- activated protein kinase (MAPK). MEK is also known as MAP2K and MAPKK. Isoforms of MEK include, but are not limited to, MEK1 and MEK2. As used herein, the term "MEK1" means mitogen/extracellular signal-regulated kinase-1 having the UniProt identifier MP2K1_HUMAN and encoded by the MAP2K1 gene. As used herein, the term "MEK2" means mitogen/extracellular signal-regulated kinase-2 having the UniProt identifier MP2K2 HUMAN and encoded by the MAP2K2 gene. Unless otherwise indicated, use of the term "MEK" may refer to MEK1, MEK2, or a combination thereof. [0053] The term "microarray," as used herein, is an ordered arrangement of array elements (for example, small samples of a biological sample from a patient such as tissue samples) mounted on a solid support capable of binding other molecule species or antibodies. The array elements are arranged so that there are preferably at least one or more different array elements.
[0054] The terms "molecular alteration" and "genetic alteration," as used herein, mean any variation in the genetic or protein sequence in or more cells of a patient as compared to the corresponding wild-type genes or proteins. One or more molecular alterations include, but are not limited to, genetic mutations, gene amplifications, splice variants, deletions, gene rearrangements, single-nucleotide variations (SNVs), insertions, and aberrant RNA/protein expression. The terms "molecular alteration" and "genetic alteration" are used interchangeably herein.
[0055] The term "patient," as used herein, means a mammal, including, but not limited to, a human, a dog or a cat. In some embodiments, the patient is a human. In some
embodiments, the patient is a dog. In some embodiments, the patient is a cat.
[0056] The term "PD0325901," as used herein, means the compound having the chemical structure,
Figure imgf000018_0001
Alternative names for PD0325901 include PD 0325901, PD325901, and JV-[(2R)-2,3- dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide.
PD0325901 is associated with CAS Registry Number 391210-10-9.
[0057] The term "PD184352," as used herein, means the compound having the chemical structure,
Figure imgf000018_0002
Alternative names for PD 184352 include CI- 1040 and 2-(2-chloro-4-iodophenylamino)-N- cyclopropylmethoxy-3,4-difluorobenzamide. PD184352 is associated with CAS Registry Number 212631-79-3.
[0058] The term "pimasertib," as used herein, means the compound having the chemical structure,
Figure imgf000019_0001
Altemative names for pimasertib include AS-703026, AS 703026, AS703026,
MSC1936369B, N-[(25 -2,3-dihydroxypropyl]-3-(2-fluoro-4-iodoanilino)pyridine-4- carboxamide, and N-(2,3-dihydroxypropyl)-l-((2-fluoro-4- iodophenyl)amino)isonicotinamide. Pimasertib is associated with CAS Registry Number 1236699-92-5.
[0059] The term "refametinib," as used herein means the compound having the chemical structure,
Figure imgf000019_0002
Altemative names for refametinib include BAY-86-9766, RDEA119, and N-[3,4-difluoro- 2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl]-l-[(25)-2,3-dihydroxypropyl]- cyclopropanesulfonamide. Refametinib is associated with CAS Registry Number 923032-
37-5.
[0060] The term "R04987655," as used herein, means the compound having the chemical structure,
Figure imgf000019_0003
Alternative names for R04987655 include RO 4987655, RO-4987655, RG 7167, CH- 4987655, CH 4987655, CH4987655, and 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-N-(2- hydroxyethoxy)-5-[(3-oxooxazinan-2-yl)methyl]benzamide. R04987655 is associated with CAS Registry Number 874101-00-5.
[0061] The term "R05126766," as used herein, means the compound having the chemical structure,
Figure imgf000020_0001
Alternative names for R05126766 include RO-5126766 and CH5126766. R05126766 is associated with CAS Registry Number 946128-88-7.
[0062] The term "ROS1," as used herein, means the ROS 1 receptor tyrosine-protein kinase having the UniProt designation ROS 1 HUMAN and encoded by the ROS 1 gene.
[0063] The term "selumetinib," as used herein, means the compound having the chemical structure,
Figure imgf000020_0002
Alternative names for selumetinib include AZD6244, ARRY- 142886, and 6-(4-bromo-2- chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxarnide. Selumetinib is associated with CAS Registry Number 606143-52-6.
[0064] The term "solid support," as used herein, means the well-understood solid materials to which various components such as, for example, proteins and nucleic acids, are physically attached, thereby immobilizing the components. The term "solid support," as used herein, means a non-liquid substance. A solid support can be, but is not limited to, a membrane, sheet, gel, glass, plastic or metal. Immobilized components may be associated with a solid support by covalent bonds and/or via non-covalent attractive forces such as hydrogen bond interactions, hydrophobic attractive forces and ionic forces, for example [0065] The term "sorafenib," as used herein, means the compound having the chemical structure,
Figure imgf000021_0001
Alternative names for sorafenib include BAY 43-9006, Nexavar®, and 4-[4-[[4-chloro-3- (trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide. Sorafenib is associated with CAS Registry Number 284461-73-0.
[0066] The term "TAK-733" as used herein, means the compound having the chemical structure,
Figure imgf000021_0002
Altemative names for TAK-733 include (i?)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro- 4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione. TAK-733 is associated with CAS Registry Number 1035555-63-5.
[0067] The term "therapeutically effective amount," as used herein, means that amount of the compound or compounds being administered which will relieve to some extent one or more of the symptoms of the disorder being treated. In reference to the treatment of a cancer, a therapeutically effective amount refers to that amount which has the effect of (1) reducing the size of a cancer tumor, (2) inhibiting (that is, slowing to some extent, preferably stopping) cancer tumor metastasis, (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) cancer tumor growth, and/or, (4) relieving to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer. [0068] The term "trametinib" as used herein, means the compound having the chemical structure,
Figure imgf000022_0001
Alternative names for trametinib include GSK1 120212 and Mekinist®. Trametinib is associated with CAS Registry Number 871700-17-3.
[0069] The term "tropomyosin receptor kinase," as used herein, means the family of tropomyosin receptor kinases (Trks) that are activated by peptide hormones of the neurotrophin family and include, but are not limited to, TrkA, TrkB, and TrkC. As used herein, the term "TrkA" means wild-type tropomyosin receptor kinase A having the UniProt identifier NTRK1_HUMAN and encoded by the NTRK1 gene. As used herein, the term "TrkB" means wild-type tropomyosin receptor kinase B having the UniProt identifier NTRK2 HUMAN and encoded by the NTRK2 gene. As used herein, the term "TrkC" means wild-type tropomyosin receptor kinase C having the UniProt identifier
NTRK3 HUMAN and encoded by the NTRK3 gene. TrkA, TrkB and TrkC are also referred to by those having ordinary skill in the art as Trkl, Trk2 and Trk3, respectively. A reference to TrkA is a reference to Trkl . A reference to TrkB is a reference to Trk2. A reference to TrkC is a reference to Trk3.
[0070] The term "ulixertinib," as used herein, means the compound having the chemical structure,
Figure imgf000022_0002
Alternative names for ulixertinib include BVD-523, VRT752271, and 4-(5-chloro-2- (isopropylamino)pyridin-4-yl)-N-((5 - l -(3-chl orophenyl)-2-hydroxy ethyl)- lH-pyrrole-2- carboxamide. Ulixertinib is associated with CAS Registry Number 869886-67-9.
[0071] Provided herein, in one aspect, are methods of treating cancer in a patient in need thereof, the methods comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof.
[0072] In another aspect, provided herein are methods of treating cancer in a patient in need thereof, the methods comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof; and the second agent comprises a MEK inhibitor or an ERK inhibitor.
[0073] In another aspect, provided herein are methods of treating cancer in a patient in need thereof, the methods comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof; and the second agent is a MEK inhibitor or an ERK inhibitor.
[0074] In another aspect, provided herein are methods of treating cancer in a patient in need thereof, the methods comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in ALK.
[0075] In another aspect, provided herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the ALK receptor tyrosine kinase polypeptide.
[0076] In another aspect, provided herein are methods of treating cancer in a patient in need thereof, the methods comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a ROS1 inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in ROS1. [0077] In another aspect, provided herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a ROSl inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the ROSl receptor tyrosine kinase polypeptide.
[0078] In another aspect, provided herein are methods of treating cancer in a patient in need thereof, the methods comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkA inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK1.
[0079] In another aspect, provided herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkA inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkA receptor tyrosine kinase polypeptide.
[0080] In another aspect, provided herein are methods of treating cancer in a patient in need thereof, the methods comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkB inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK2.
[0081] In another aspect, provided herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkB inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkB receptor tyrosine kinase polypeptide.
[0082] In another aspect, provided herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkC inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK3.
[0083] In another aspect, provided herein are methods of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkC inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkC receptor tyrosine kinase polypeptide.
[0084] In some embodiments, knowledge of the at least one genetic alteration is acquired from an antibody-based assay. The antibody-based assay can generally be any antibody- based assay, and can be, for example, ELISA, immunohistochemistry, western blotting, mass spectrometry, flow cytometry, protein-microarray, immunofluorescence, and a multiplex detection assay. In some embodiments, the antibody-based assay includes an immunohistochemistry analysis.
[0085] In some embodiments, identifying a ALK, ROS 1 , TrkA, TrkB, or TrkC modulation defect such as an up-regulation defect or a down-regulation defect, for example a null mutation such as a ALK, ROS 1, TrkA, TrkB, or TrkC deletion or a ALK, ROS 1, TrkA, TrkB, or TrkC chimeric locus encoding a constitutively active ALK, ROS 1, TrkA, TrkB, or TrkC kinase in a cancer or precancerous pancreatic cell in an individual comprises assaying for ALK, ROS1, TrkA, TrkB, or TrkC activity in a cell extract from a pancreatic cancerous or precancerous cell population. In some embodiments, identifying a ALK, ROS 1, TrkA, TrkB, or TrkC modulation defect such as an up-regulation defect or a down- regulation defect, for example a null mutation such as a ALK, ROS 1, TrkA, TrkB, or TrkC deletion or a ALK, ROS1, TrkA, TrkB, or TrkC chimeric locus encoding a constitutively active ALK, ROS1, TrkA, TrkB, or TrkC kinase in a cancer or precancerous pancreatic cell in an individual comprises assaying for ALK, ROS1, TrkA, TrkB, or TrkC transcript accumulation in an RNA population from a pancreatic cancerous or precancerous cell population. In some embodiments, identifying a ALK, ROS 1, TrkA, TrkB, or TrkC modulation defect such as an up-regulation defect or a down-regulation defect, for example a null mutation such as a ALK, ROS 1, TrkA, TrkB, or TrkC deletion or a ALK, ROS 1, TrkA, TrkB, or TrkC chimeric locus encoding a constitutively active ALK, ROS 1, TrkA, TrkB, or TrkC kinase in a cancer or precancerous pancreatic cell in an individual comprises determining the nucleic acid sequence such as the genomic deoxyribonucleic acid sequence in a cell or cells or a cell population comprising a cell or cells from a pancreatic cancerous or precancerous cell population.
[0086] In some embodiments, methods of the present disclosure are to treat, reduce the symptoms of, ameliorate the symptoms of, delay the onset of, or otherwise
pharmaceutically address a condition in a patient selected from non-small cell lung cancer, papillary thyroid cancer, neuroblastoma, pancreatic cancer and colorectal cancer and possibly other indications in which a defect in the modulation of ALK, ROS l, TrkA, TrkB, or TrkC activity, or a combination thereof, or up-regulation, misregulation or deletion thereof might play a role by administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROSl, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)- lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)- benzamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof. In some embodiments, the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX- 554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD- 184352, GSK-2091976A, and AS-703988. In some embodiments, the ERK inhibitor is an ERK1 inhibitor, an ERK2 inhibitor, or a combination thereof. In some embodiments, the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS- 136, and IDN-5491.
[0087] In some embodiments, methods of the present disclosure are to treat, reduce the symptoms of, ameliorate the symptoms of, delay the onset of, or otherwise
pharmaceutically address a condition in a patient selected from non-small cell lung cancer, papillary thyroid cancer, neuroblastoma, pancreatic cancer and colorectal cancer associated with a ALK, ROSl, TrkA, TrkB, or TrkC down-regulation defect, for example a null mutation such as a ALK, ROSl, TrkA, TrkB, or TrkC deletion by identifying a ALK, ROS l, TrkA, TrkB, or TrkC down-regulation defect, for example a null mutation such as a ALK, ROS l, TrkA, TrkB, or TrkC deletion in a cancer or precancerous cell in the patient, and administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a
combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof. In some embodiments, the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK- 2091976A, and AS-703988. In some embodiments, the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof. In some embodiments, the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
[0088] In some embodiments are provided methods for treating cancer in a patient in need thereof, the method comprising: (a) acquiring knowledge of the presence of at least one genetic alteration in a biological sample from the patient, wherein the at least one genetic alteration is detected by an assay comprising one or more antibodies that bind to one or more of ALK, ROS1, TrkA, TrkB, and TrkC biomarkers; (b) and administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS 1, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof. In some embodiments, the MEK inhibitor is selected from
PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS-703988. In some embodiments, the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof. In some embodiments, the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491. [0089] In some embodiments are provided methods for treating cancer in a patient in need thereof, the method comprising: (1) testing one or more cells comprising a tumor from the patient for the presence of at least one of ALK, ROS1, TrkA, TrkB, or TrkC; and (2) if the one or more cells tests positive for at least one of ALK, ROS 1, TrkA, TrkB, or TrkC, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof. In some embodiments, the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988. In some embodiments, the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof. In some embodiments, the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
[0090] In some embodiments are provided methods for treating cancer in a patient in need thereof, the method comprising: (1) testing one or more cells comprising tumor tissue from the patient for the presence of at least one of ALK, ROS1, TrkA, TrkB, or TrkC; and (2) if the one or more cells tests positive for at least one of ALK, ROS1, TrkA, TrkB, or TrkC, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof. In some embodiments, the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988. In some embodiments, the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof. In some embodiments, the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
[0091] In some embodiments are provided methods for treating cancer in a patient in need thereof, the method comprising: (a) acquiring knowledge of the presence of at least one genetic alteration in at least one target gene in the cancer patient, wherein the at least one target gene is selected from ALKl, BDNF, NGF, NGFR, NTF3, NTF4, ROS 1, SORT1, NTRK1, NTRK2, and NTRK3; and (b) administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor based on the recognition that the combination is effective in treating cancer patients having the at least one genetic alteration in the at least one target gene. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH- indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide^ or a pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof. In some embodiments, the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK- 2091976A, and AS-703988. In some embodiments, the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof. In some embodiments, the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
[0092] In some embodiments are provided methods for treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor, and wherein prior to the administration of the combination, the patient is known to possess at least one genetic alteration in at least one target gene selected from ALKl, BDNF, NGF, NGFR, NTF3, NTF4, ROS 1, SORT1, NTRK1, NTRK2, and NTRK3. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof. In some embodiments, the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK- 2091976A, and AS-703988. In some embodiments, the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof. In some embodiments, the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
[0093] In some embodiments are provided methods for treating cancer in a patient in need thereof, comprising administering to the patient known to possess at least one genetic alteration in at least one target gene selected from ALK1, BDNF, NGF, NGFR, NTF3, NTF4, ROSl, SORTl, NTRKl, NTRK2, and NTRK3 a therapeutically effective amount of a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS l, TrkA, TrkB, or TrkC activity, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof. In some embodiments, the MEK inhibitor is selected from
PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS-703988. In some embodiments, the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof. In some embodiments, the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
[0094] In some embodiments are provided methods of treating a cancer patient, the method comprising administering to the patient a therapeutically effective amount of a combination comprising a therapeutically effective amount of a first agent and a
therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS l, TrkA, TrkB, or TrkC activity, or a combination thereof; the second agent is selected from a MEK inhibitor and an ERK inhibitor; the cancer patient is has at least one genetic alteration in at least one target gene; and the target gene is selected from ALK1, BDNF, NGF, NGFR, NTF3, NTF4, ROS1, SORT1, NTRK1, NTRK2, and NTRK3. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4- methyl-piperazin-1 -yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a
pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof. In some embodiments, the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK- 2091976A, and AS-703988. In some embodiments, the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof. In some embodiments, the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
[0095] In some embodiments are provided methods of treating a cancer patient, comprising (a) acquiring knowledge of the presence of at least one genetic alteration in at least one target gene selected from ALK1, BDNF, NGF, NGFR, NTF3, NTF4, ROS1, SORT1, NTRK1, NTRK2, and NTRK3 in the patient; and (b) administering to the patient a therapeutically effective amount of a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROS1, TrkA, TrkB, or TrkC activity, or a
combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the MEK inhibitor is a MEK1 inhibitor, a MEK2 inhibitor, or a combination thereof. In some embodiments, the MEK inhibitor is selected from PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK- 2091976A, and AS-703988. In some embodiments, the ERK inhibitor is an ERKl inhibitor, an ERK2 inhibitor, or a combination thereof. In some embodiments, the ERK inhibitor is selected from TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN- 5491. [0096] In some embodiments are provided methods wherein the tumors are caused by the presence of non-small cell lung cancer, papillary thyroid cancer, neuroblastoma, pancreatic cancer or colorectal cancer in the patient. In some embodiments are provided methods wherein one or more of the cells comprising the tumors in the patient test positive for the presence of a gene that expresses at least one of ALK, ROSl, TrkA, TrkB, or TrkC kinase or one or more of the cells comprising the tumors in the patient demonstrates at least one of ALK, ROSl, TrkA, TrkB, or TrkC kinase activity.
[0097] In some embodiments are provided methods wherein one or more of the cells comprising the tumors in the patient test positive for at least one gene rearrangement comprising a gene, or a fragment thereof, that expresses at least one of ALK, ROS l, TrkA, TrkB, or TrkC. In some embodiments are provided such methods wherein the cells test positive for at least one of ROSl, TrkA, TrkB, or TrkC. In some embodiments are provided methods wherein the cells test positive for ALK. In some embodiments are provided methods wherein the cells test positive for ROSl . In some embodiments are provided methods wherein the cells test positive for at least one of TrkA, TrkB and TrkC. In some embodiments are provided methods wherein the cells test positive for TrkA. In some embodiments are provided methods wherein the cells test positive for TrkB. In some embodiments are provided such methods wherein the cells test positive for TrkC.
[0098] In some embodiments are provided methods of treating cancer in a patient in need thereof, the method comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of at least one of ALK, ROSl, TrkA, TrkB, or TrkC; and (2) if the one or more cells tests positive for at least one of ALK, ROS l, TrkA, TrkB, or TrkC activity, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor, a ROS l inhibitor, a TrkA inhibitor, a TrkB inhibitor, or a TrkC inhibitor, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0099] In some embodiments are provided methods of treating cancer in a patient in need thereof, the method comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of ALK; and (2) if the one or more cells tests positive for ALK activity, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)- lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)- benzamide, or a pharmaceutically acceptable salt thereof.
[0100] In some embodiments are provided methods of treating cancer in a patient in need thereof, the method comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of ROS1 ; and (2) if the one or more cells tests positive for ROS1 activity, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ROS1 and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0101] In some embodiments are provided methods of treating cancer in a patient in need thereof, the method comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of TrkA, TrkB, or TrkC, or a combination thereof; and (2) if the one or more cells tests positive for TrkA, TrkB, or TrkC activity, or a combination thereof, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of TrkA, TrkB, or TrkC, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro- 2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0102] In some embodiments are provided methods of treating cancer in a patient in need thereof, the method comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of TrkA; and (2) if the one or more cells tests positive for TrkA activity, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of TrkA, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof. [0103] In some embodiments are provided methods of treating cancer in a patient, the method comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of TrkB; and (2) if the one or more cells tests positive for TrkB activity, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of TrkB, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0104] In some embodiments are provided methods of treating cancer in a patient, the method comprising: (1) testing one or more cells comprising the tumors in the patient for the presence of TrkC; and (2) if the one or more cells tests positive for TrkC activity, administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of TrkC, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments, the first agent is N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0105] In some embodiments of the methods provided herein, the patient has at least one mutation in the TrkA receptor tyrosine kinase polypeptide. For example, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide may be a genetic alteration of SEQ ID NO: 1. In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G595 or G667 of the TrkA polypeptide set forth in SEQ ID NO: 1. In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G595 of the TrkA polypeptide set forth in SEQ ID NO: 1. In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G595R). In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G667 of the TrkA polypeptide set forth in SEQ ID NO: 1. In some embodiments, the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G667C). [0106] In some embodiments of the methods provided herein, the patient at least one mutation in the TrkB receptor tyrosine kinase polypeptide. For example, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide may be a genetic alteration of SEQ ID NO: 2. In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G639 or G709 of the TrkB polypeptide set forth in SEQ ID NO: 2. In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G639 of the TrkB polypeptide set forth in SEQ ID NO:
2. In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G639R). In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G709 of the TrkB polypeptide set forth in SEQ ID NO: 2. In some embodiments, the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G709C).
[0107] In some embodiments of the methods provided herein, the patient has at least one mutation in the TrkC receptor tyrosine kinase polypeptide. For example, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide may be a genetic alteration of SEQ ID NO: 3. In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G623 or G696 of the TrkC polypeptide set forth in SEQ ID NO: 3. In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G623 of the TrkC polypeptide set forth in SEQ ID NO:
3. In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G623R). In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G696 of the TrkC polypeptide set forth in SEQ ID NO: 3. In some embodiments, the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G696C).
[0108] In some embodiments of the methods provided herein, the patient has at least one mutation in ALK receptor tyrosine kinase polypeptide. For example, the at least one mutation in the ALK receptor tyrosine kinase polypeptide may be a genetic alteration of SEQ ID NO: 4. In some embodiments, the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G1202 or G1269 of the ALK polypeptide set forth in SEQ ID NO: 4. In some embodiments, the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G1202 of the ALK polypeptide set forth in SEQ ID NO: 4. In some embodiments, the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G1269 of the ALK polypeptide set forth in SEQ ID NO: 4.
[0109] In some embodiments of the methods provided herein, the patient has at least one mutation in the ROS1 receptor tyrosine kinase polypeptide. For example, the at least one mutation in the ROS1 receptor tyrosine kinase polypeptide may be a genetic alteration of SEQ ID NO: 5. In some embodiments, the at least one mutation in the ROS1 receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G2032 or G2101 of the ROS1 polypeptide set forth in SEQ ID NO: 5. In some embodiments, the at least one mutation in the ROS1 receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G2032 of the ROS1 polypeptide set forth in SEQ ID NO: 5. In some embodiments, the at least one mutation in the ROS1 receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G2101 of the ROS 1 polypeptide set forth in SEQ ID NO: 5.
[0110] In some embodiments, the combination is a pharmaceutical composition comprising the therapeutically effective amount of the first agent, the therapeutically effective amount of the second agent, and at least one pharmaceutically acceptable carrier.
[0111] In some embodiments, the combination is concurrent administration of a first pharmaceutical composition comprising the therapeutically effective amount of the first agent and a second pharmaceutical composition comprising the therapeutically effective amount of the second agent
[0112] In some embodiments, the combination is sequential administration of a first pharmaceutical composition comprising the therapeutically effective amount of the first agent and a second pharmaceutical composition comprising the therapeutically effective amount of the second agent. In some embodiments, the first pharmaceutical composition is administered prior to the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition. [0113] In some embodiments are provided any of the methods described herein wherein the patient is suffering from cancer selected from non-small cell lung cancer, papillary thyroid cancer, neuroblastoma, melanoma, pancreatic cancer, and colorectal cancer.
[0114] In some embodiments are provided any of the methods described herein wherein the patient is suffering from cancer selected from non-small cell lung cancer,
neuroblastoma, melanoma, pancreatic cancer and colorectal cancer.
[0115] In some embodiments are provided any of the methods described herein wherein the patient is suffering from cancer selected from non-small cell lung cancer,
neuroblastoma, melanoma, and colorectal cancer.
[0116] In some embodiments are provided any of the methods described herein wherein the patient is suffering from cancer selected from non-small cell lung cancer,
neuroblastoma, and colorectal cancer.
[0117] In some embodiments are provided any of the methods described herein wherein the patient is suffering from non-small cell lung cancer.
[0118] In some embodiments are provided any of the methods described herein wherein the patient is suffering from papillary thyroid cancer.
[0119] In some embodiments are provided any of the methods described herein wherein the patient is suffering from neuroblastoma.
[0120] In some embodiments are provided any of the methods described herein wherein the patient is suffering from melanoma.
[0121] In some embodiments are provided any of the methods described herein wherein the patient is suffering from pancreatic cancer.
[0122] In some embodiments are provided any of the methods described herein wherein the patient is suffering from colorectal cancer.
[0123] In some embodiments are provided any of the methods described herein wherein the second agent comprises one or more chemotherapeutic agents or radiotherapy, such as radiotherapy as commonly administered to treat, ameliorate the symptoms of, or prevent or delay the onset of cancer. Such agents include, but are not limited to, antihormonal agents such as antiestrogens, antiandrogens and aromatase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, agents that target microtubules, platin-based agents, alkylating agents, DNA damaging or intercalating agents, antineoplastic antimetabolites, other kinase inhibitors, other anti-angiogenic agents, inhibitors of kinesins, therapeutic monoclonal antibodies, inhibitors of mTOR, histone deacetylase inhibitors, farnesyl transferase inhibitors, and inhibitors of hypoxic response.
[0124] In some embodiments are provided a product or kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy.
[0125] In some embodiments are provided a product or a kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy, wherein the first agent comprises an ALK inhibitor, a ROS 1 inhibitor, a TrkA inhibitor, a TrkB inhibitor, or a TrkC inhibitor, or a combination thereof, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments are provided a product or kit wherein the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0126] In some embodiments are provided a product or a kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy, wherein the first agent comprises an ALK inhibitor, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments are provided a product or kit wherein the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0127] In some embodiments are provided a product or a kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy, wherein the first agent comprises a ROS 1 inhibitor, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments are provided a product or kit wherein the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0128] In some embodiments are provided a product or a kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy, wherein the first agent comprises a TrkA inhibitor, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments are provided a product or kit wherein the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0129] In some embodiments are provided a product or a kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy, wherein the first agent comprises a TrkB inhibitor, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments are provided a product or kit wherein the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0130] In some embodiments are provided a product or a kit comprising a combination of a first agent and a second agent as a combined preparation for simultaneous, separate or sequential use in anticancer therapy, wherein the first agent comprises a TrkC inhibitor, and the second agent is selected from a MEK inhibitor and an ERK inhibitor. In some embodiments are provided a product or kit wherein the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0131] In some embodiments of the methods provided herein, the first agent comprises N- [5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H- pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
[0132] In some embodiments of the methods provided herein, the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer in an amount ranging from about 200 mg/m2 to about 1600 mg/m2, or from about 200 mg/m2 to about 1200 mg/m2, or from about 200 mg/m2 to about 1000 mg/m2, or from about 400 mg/m2 to about 1200 mg/m2, or from about 400 mg/m2 to about 1000 mg/m2, or from about 800 mg/m2 to about 1000 mg/m2, or from about 800 mg/m2 to about 1200 mg/m2, or from about 800 mg/m2 to about 1200 mg/m2, or from about 800 mg/m2 to about 1600 mg/m2. In some embodiments, the first agent and/or the second agent are administered to the patient or individual having or suffering from cancer in an amount of about 200 mg/m2, about 300
2 2 2 2 2 mg/m , about 400 mg/m , about 500 mg/m , about 600 mg/m , about 700 mg/m , about 800 mg/m , about 900 mg/m , about 1000 mg/m , about 1100 mg/m , about 1200 mg/m , about 1300 mg/m2, about 1400 mg/m2, about 1500 mg/m2, about 1600 mg/m2, about 1700 mg/m2, about 1800 mg/m2, about 1900 mg/m2, or about 2000 mg/m2. In some embodiments, the amount of the first agent and the amount of the second agent are within the same range listed above. In some embodiments, the amount of the first agent and the amount of the second agent are in different ranges listed above.
[0133] In some embodiments of the methods provided herein, the first agent comprises N- [5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H- pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof, and is administered to a patient or individual having or suffering from cancer in an amount such that the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)- 2-(tetrahydro-2H-pyran-4-ylamino)-benzamide the patient receives ranges from about 200 mg/m2 to about 1600 mg/ m2, or from about 200 mg/ m2 to about 1200 mg/m2, or from about 200 mg/m2 to about 1000 mg/m2, or from about 400 mg/m2 to about 1200 mg/m2, or from about 400 mg/m2 to about 1000 mg/m2, or from about 800 mg/m2 to about 1000 mg/m2, or from about 800 mg/m2 to about 1200 mg/m2, or from about 800 mg/m2 to about 1200 mg/m2, or from about 800 mg/m2 to about 1600 mg/m2.
[0134] In some embodiments of the methods provided herein, the first agent comprises N- [5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H- pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof, and is administered to a patient or individual having or suffering from cancer in an amount such that the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)- 2-(tetrahydro-2H-pyran-4-ylamino)-benzamide the patient receives is about 200 mg/m2,
2 2 2 2 2 about 300 mg/m , about 400 mg/m , about 500 mg/m , about 600 mg/m , about 700 mg/m , about 800 mg/m2, about 900 mg/m2, about 1000 mg/m2, about 1100 mg/m2, about 1200
2 2 2 2 2 mg/m , about 1300 mg/m , about 1400 mg/m , about 1500 mg/m , about 1600 mg/m , about 1700 mg/m2, about 1800 mg/m2, about 1900 mg/m2, or about 2000 mg/m2, including increments therein. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH- indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide the patient receives is about 200 mg/m2, about 300 mg/m2, about 400 mg/m2, about 500 mg/m2, or about 600 mg/m2. In some embodiments, the amount ofN-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide the patient receives is about 200 mg/m2. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-2H-pyran-4-ylamino)-benzamide the patient receives is about 300 mg/m2. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide the patient receives is about 400 mg/m2. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3- yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide the patient receives is about 500 mg/m2. In some embodiments, the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide the patient receives is about 600 mg/m2.
[0135] In some embodiments of the methods provided herein, the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer in an amount such that the amount of the patient receives is about 0.5 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg, including increments therein.
[0136] In some embodiments, the first agent comprises N-[5-(3,5-difluorobenzyl)-lH- indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof, and the amount of N-[5-(3,5-difluorobenzyl)- lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)- benzamide that the patient receives is about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 200 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol- 3-yl] -4-(4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 300 mg. In some embodiments, the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives is about 400 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 500 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 600 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol- 3-yl] -4-(4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 700 mg. In some embodiments, the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives is about 800 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 900 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives is about 1000 mg.
[0137] In some embodiments of the methods provided herein, the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer in an amount such that the amount the first agent and/or second agent that the patient receives per day is about 0.5 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg, including increments therein.
[0138] In some embodiments of the methods provided herein, the first agent comprises N- [5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H- pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof, and is administered to a patient or individual having or suffering from cancer in an amount such that the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)- 2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives per day is about 0.5 mg, about 1 mg, about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg, including increments therein. In some embodiments, the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives once per day is about 200 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives once per day is about 300 mg. In some embodiments, the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives once per day is about 400 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives once per day is about 500 mg. In some embodiments, the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives once per day is about 600 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives once per day is about 700 mg. In some embodiments, the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives once per day is about 800 mg. In some embodiments, the amount of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that the patient receives once per day is about 900 mg. In some embodiments, the amount of N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4- ylamino)-benzamide that the patient receives once per day is about 1000 mg.
[0139] In some embodiments, the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer in multiple dosages for a treatment period of 2 to 50 days. In some embodiments, the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer in multiple dosages of about 50 to about 200 mg/kg per dose over a treatment period of 5 to 42 days. In some embodiments, the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer with an oral dosage of about 60 mg/kg twice a day (BID), seven times per week. In some embodiments, the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer with an oral dosage of about 60 mg/kg twice a day (BID), seven times per week for six weeks, on alternate weekly basis (i.e., one week on and one week off).
[0140] Some embodiments include any of the methods described herein, wherein the first agent and/or the second agent are administered to a patient or individual having or suffering from cancer in an amount ranging from about 0.01 mg/kg to about 100 mg/kg, or from about 0.02 mg/kg to about 50 mg/kg, or from about 0.05 mg/kg to about 25 mg/kg, or from about 0.1 mg/kg to about 20 mg/kg, or from about 0.2 mg/kg to about 10 mg/kg, or from about 0.5 mg/kg to about 5 mg/kg, or from about 1 mg/kg to about 2 mg/kg. In some embodiments, the amount of the first agent and the amount of the second agent are within the same range listed above. In some embodiments, the amount of the first agent and the amount of the second agent are in different ranges listed above.
[0141] In some embodiments are provided any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l -yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide further comprise a weakly basic organic compound and at least one acidulant. In some embodiments, the at least one acidulant is an organic acidulant. In some embodiments, the at least one acidulant is selected from tartaric acid, maleic acid, fumaric acid, citric acid, and betaine
hydrochloride. In some embodiments, the at least one acidulant is fumaric acid. In some embodiments, the at least one acidulant is tartaric acid. In some embodiments, the at least one acidulant is maleic acid. In some embodiments, the at least one acidulant is citric acid. In some embodiments, the at least one acidulant is betaine hydrochloride.
[0142] In some embodiments are provided any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l -yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide further comprise at least one acidulant. In some embodiments, the at least one acidulant is an organic acidulant. In some embodiments, the at least one acidulant is selected from tartaric acid, maleic acid, fumaric acid, citric acid, and betaine hydrochloride. In some embodiments, the least one acidulant is fumaric acid. In some embodiments, the at least one acidulant is tartaric acid. In some embodiments, the at least one acidulant is maleic acid. In some embodiments, the at least one acidulant is citric acid. In some embodiments, the at least one acidulant is betaine hydrochloride.
[0143] In some embodiments are provided any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and at least one acidulant are in the form of a tablet or capsule. In some embodiments are provided pharmaceutical compositions in the form of a tablet. In some embodiments are provided pharmaceutical compositions in the form of a capsule.
[0144] In some embodiments are provided any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and at least one acidulant comprises from about 10 mg to about 1000 mg of the N-[5-(3,5-difluorobenzyl)- lH-indazol-3-yl] -4-(4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-pyran-4-ylamino)-benzamide and at least one acidulant.
[0145] In some embodiments are provided any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-2H-pyran-4-ylamino)-benzamide when
administered to a patient in a fasted state provides a pharmacokinetic profile in the patient wherein the Tmax of the N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin- l-yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide in the plasma of the patient is between about 2 hours and 6 hours following the administration of the pharmaceutical composition to the patient.
[0146] In some embodiments are provided any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-2H-pyran-4-ylamino)-benzamide when
administered to a patient in a fasted state at a total dose of about 800 mg of the N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l -yl)-2-(tetrahydro-pyran-4- ylamino)-benzamide provides a pharmacokinetic profile in the patient wherein the Cmax of the N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro- pyran-4-ylamino)-benzamide in the plasma of the patient is between about 2080 nM and about 21 10 nM following the administration of the pharmaceutical composition to the patient.
[0147] In some embodiments are provided any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-2H-pyran-4-ylamino)-benzamide when
administered to a patient in a fed state at a total dose of about 800 mg of the N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l -yl)-2-(tetrahydro-pyran-4- ylamino)-benzamide provides a pharmacokinetic profile in the patient wherein the Cmax of the N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro- pyran-4-ylamino)-benzamide in the plasma of the patient is between 80% to 125% of 2560 nM, based on a 90 percent confidence interval following the administration of the pharmaceutical composition to the patient. [0148] In some embodiments are provided any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin- 1 -yl)-2-(tetrahy dro-2H-pyran-4-ylamino)-benzamide when administered to a patient in a fasted state at a total dose of about 800 mg of the N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-pyran-4- ylamino)-benzamide provides a pharmacokinetic profile in the patient wherein the AUC(0 to 24) of the N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-pyran-4-ylamino)-benzamide in the plasma of the patient is between about 28,900 nM*hr and about 30,800 nM*hr following the administration of the pharmaceutical composition to the patient.
[0149] In some embodiments are provided any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3, 5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide that provides an AUC(0 to 24) of the N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l- yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide in the plasma of a patient of between about 28,900 nM*hr and about 30,800 nM*hr following administration of the pharmaceutical composition to the patient in a fasted state, and wherein the composition comprises a total dose of about 800 mg of the N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin- 1 -yl)-2-(tetrahy dro-pyran-4-ylamino)-benzamide.
[0150] In some embodiments are provided any of the methods described herein wherein the pharmaceutical compositions comprising N-[5-(3, 5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide provide a Tmax of the N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2- (tetrahydro-pyran-4-ylamino)-benzamide in the plasma of a patient of between about 2 hours and about 6 hours following administration of the pharmaceutical composition to the patient in a fasted state.
[0151] This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the bioactive compositions and formulations provided herein (including activity, pharmacokinetics, pharmacodynamics, and bioavailability thereof), the physiological condition of the patient treated (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication) or cells, the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. Further, an effective or therapeutically effective amount may vary depending on whether the one or more bioactive compositions and formulations provided herein is administered alone or in combination with other drug(s), other therapy/therapies or other therapeutic method(s) or modality/modalities. One skilled in the clinical and pharmacological arts will be able to determine an effective amount or therapeutically effective amount through routine experimentation, namely by monitoring a cell's or patient's response to administration of the one or more bioactive compositions and formulations provided herein and adjusting the dosage accordingly. A typical dosage may range from about 0.1 mg/kg to about 100 mg/kg or more, depending on the factors mentioned above. In other alternatives, the dosage may range from about 0.1 mg/kg to about 100 mg/kg; or about 1 mg/kg to about 100 mg/kg; or about 5 mg/kg up to about 100 mg/kg. For topical applications such as, for example, treatment of various hair conditions, according to some alternatives provided herein, suitable dosage may range from about 1 mg/kg to about 10 g/kg; or about 10 mg/kg to about 1 g/kg; or about 50 mg/kg up to about 10 g/kg. Additional guidance with regard to this aspect can be found in, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005, which is incorporated by reference in its entirety herein.
[0152] In some embodiments, implementations of the methods according to this and other aspects of the present disclosure further include acquiring knowledge of a genetic alteration in the cancer of the patient from a second analytical assay prior to the administering step. The second analytical assay can generally be any analytical assay known to those having ordinary skill in the art, and can be for example an antibody-based assay, a nucleotide-based assay, or an enzymatic activity assay. Non-limiting examples of suitable second analytical assays include capillary electrophoresis, nucleic acid sequencing, polypeptide sequencing, restriction digestion, nucleic acid amplification-based assays, nucleic acid hybridization assay, comparative genomic hybridization, real-time PCR, quantitative reverse transcription PCR (qRT-PCR), PCR-RFLP assay, HPLC, mass-spectrometric genotyping, fluorescent in- situ hybridization (FISH), next generation sequencing (NGS), and a kinase activity assay. Other examples of suitable second analytical assays include ELISA, immunohistochemistry, Western blotting, mass spectrometry, flow cytometry, protein-microarray,
immunofluorescence, and multiplex detection assay.
[0153] In some embodiments, FISH analysis is used to identify the chromosomal rearrangement resulting in the one or more molecular alterations such as the fusion genes or gene products as described herein. For example, to perform FISH, at least a first probe tagged with a first detectable label can be designed to target a first gene of a fusion gene, such as in one or more exons of the gene and at least a second probe tagged with a second detectable label can be designed to target a second gene of the fusion gene, such as in one or more exons of the genes (for example, the exons containing the part of the protein that includes the tyrosine kinase domain). The at least one first probe and the at least one second probe will be closer together in a patient who carries the fusion compared to a patient who does not carry the fusion gene or gene product. In some embodiments, a variation of a FISH assay, for example, "break-apart FISH," is used to evaluate a patient selected by a method provided herein. By this method, at least one probe targeting the fusion junction and at least one probe targeting an individual gene of the fusion, e.g. , at one or more exons and or introns of the gene, are utilized. In normal cells, both probes will be observed (or a secondary color will be observed due to the close proximity of the two genes of the gene fusion), and only the single gene probe will be observed when the translocation occurs or the probes, having differing colors, will be separated such that one of ordinary skill in the art observing the probes can determine that a relevant gene fusion or deletion is present in the sample. Generally, FISH assays are performed using formalin-fixed, paraffin- embedded tissue sections that are placed on slides. The DNA from the tissue sample sections is denatured to single-stranded form and subsequently allowed to hybridize with the appropriate DNA probes that can be designed and prepared using methods and techniques known to those having ordinary skill in the art. Following hybridization, any unbound probe may be removed by a series of washes and the nuclei of the cells are counter-stained with DAPI (4',6 diamidino-2-phenylindole), a DNA-specific stain that fluoresces blue. Hybridization of the probe or probes are viewed using a fluorescence microscope equipped with appropriate excitation and emission filters, allowing visualization of the fluorescent signals.
[0154] For example, a break-apart FISH assay may be used to detect multiple types of rearrangements involving the ALK gene locus. In the method, tumor cells from some patients having non-small cell lung cancer (NSCLC), display an ALK-positive FISH pattern as detected using single interference filter sets comprising green (FITC), red (Texas red), and blue (4',6-diamidino-2-phenylindole) as well as dual (red/green) and triple (blue, red, green) band-pass filters. A fusion of the ALK gene is visualized as split orange and green signals, single orange signals, or single orange and single green signals. [0155] Relevant molecular alterations with respect to ALK, ROS1, TrkA, TrkB and TrkC in biological samples derived from cancer patients may be examined and assessed using the same methods as described above, but by modifying the reagents, probes and other materials used in the assays in ways that are appropriate to the target molecular alteration and as can be readily determined by those having ordinary skill in the art.
[0156] Other variations of the FISH method known in the art are suitable for evaluating a patient selected in accordance with the methods provided herein.
[0157] In some embodiments of the methods provided herein, the cancer is selected from the group consisting of anaplastic large-cell lymphoma (ALCL), colorectal cancer (CRC), cholangiocarcinoma, gastric, glioblastomas (GBM), leiomyosarcoma, melanoma, non-small cell lung cancer (NSCLC), squamous cell lung cancer, neuroblastoma (NB), ovarian cancer, pancreatic cancer, prostate cancer, medullary thyroid cancer, breast cancer, and papillary thyroid cancer.
[0158] In some embodiments are provided such methods, wherein the knowledge of the presence of the one or more molecular alterations is obtained from an assay performed simultaneously on a plurality of biological samples. In some embodiments, the plurality of biological samples may be assayed in a multitest platform.
[0159] As used herein, the term "multitest platform" is intended to encompass any suitable means to contain one or more reaction mixtures, suspensions, or detection reactions. As such, the outcomes of a number of screening events can be assembled onto one surface, resulting in a "multitest platform" having, or consisting of multiple elements or parts to do more than one experiment simultaneously. It is intended that the term "multitest platform" encompasses protein chips, microtiter plates, multi-well plates, microcards, test tubes, petri plates, trays, slides, and the like. In some embodiments, multiplexing can further include simultaneously conducting a plurality of screening events in each of a plurality of separate biological samples. For example, the number of biological samples analyzed can be based on the number of spots on a slide and the number of tests conducted in each spot. In another example, the number of biological samples analyzed can be based on the number of wells in a multi-well plate and the number of tests conducted in each well. For example, 6-well, 12- well, 24-well, 48-well, 96-well, 384-well, 1536-well or 3456-well microtiter plates can be useful in the presently disclosed methods, although it will be appreciated by those in the art, not each microtiter well need contain an individual biological sample. Depending on the size of the microtiter plate and the number of the individual biological samples in each well, very high numbers of tests can be run simultaneously.
[0160] In some embodiments are provided such methods, wherein the plurality of biological samples includes at least 6, 12, 24, 48, 96, 200, 384, 400, 500, 1000, 1250, 1500, or 3000 samples, including increments therein.
[0161] In some embodiments are provided such methods, wherein the one or more molecular alterations is selected from a genetic mutation, a gene amplification, a gene rearrangement, a single-nucleotide variation (SNV), a deletion, an insertion, an InDel mutation, one or more single nucleotide point mutations (SNPs), an epigenetic alteration, a splicing variant, an RNA/protein overexpression, and an aberrant RNA/protein expression. In some embodiments are provided such methods, wherein the genetic alteration includes an insertion of a heterologous nucleic acid sequence within a coding sequence of a biomarker gene. In some embodiments are provided such methods, wherein the insertion forms a chimeric nucleic acid sequence that encodes a fusion peptide.
[0162] In some embodiments are provided such methods, wherein the acquiring knowledge of the one or more molecular alterations further comprises determining a nucleic acid sequence and/or an amino acid sequence comprising the one or more molecular alterations. In some embodiments, the nucleic acid sequence comprising the one or more molecular alterations from a selected cancer patient tumor is sequenced. In some embodiments, the sequence is determined by a next generation sequencing method.
[0163] Implementations of the methods according to this and other aspects of the present disclosure can include one or more of the following features. In some embodiments, the assay includes one or more antibodies that bind to at least two, three, four, or all of ALK, ROS 1, TrkA, TrkB and TrkC biomarkers. In some embodiments, the one or more molecular alterations detected in the biological sample involve at least two, at least three, or at least four of the biomarkers. In some embodiments, the knowledge of the presence of the one or more molecular alterations in the biological sample is acquired from an assay that includes contacting the biological sample with one or more antibodies or fragments thereof that are specific for the biomarkers. In some embodiments, the specific antibodies are monoclonal antibodies. In some embodiments, the specific antibodies include at least one of D5F3®, D4D5®, C 17F1®, and combinations thereof. In some embodiments, the biological sample is contacted with one or more of the specific antibodies simultaneously. In some embodiments, the biological sample is sequentially contacted with the specific antibodies. In some embodiments, the one or more molecular alterations results in elevated expression of one or more of the ALK, ROS1, TrkA, TrkB, and TrkC biomarkers. In some embodiments, the knowledge of the one or more molecular alterations is acquired from an assay wherein determining whether the expression of one or more biomarker is elevated includes: (a) determining the expression level of the one or more biomarkers in the biological sample; and (b) comparing the determined expression level to a reference expression level.
[0164] In some embodiments, the pharmaceutical compositions comprise a physical admixture of the various ingredients in solid, liquid, or gelcap form. Other embodiments comprise at least two separated ingredients in a single dosage unit or dosage form, such as, for example, a two- or three-layer tablet in which at least two active ingredients are located in separate layers or regions of the tablet, optionally separated by a third material, such as, for example, a sugar layer or other inert barrier to prevent contact between the first two ingredients. In other embodiments, two or more active ingredients are separately formulated into individual dosage units, which are then packaged together for ease of administration. One embodiment comprises a package containing a plurality of individual dosage units. This embodiment may, for example, comprise a blister package. In one embodiment of a blister package, multiple blister-packed dosage units are present on a single sheet, and those units that are to be administered together are packaged in the same or adjacent blisters of the blister pack. Alternatively, any other packaging can be used in which two active ingredients are packaged together for concurrent or sequential use.
[0165] In some embodiments, the methods relate to the use of any of the compounds as described herein, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of abnormal cell growth in a mammal. The present disclosure further relates to the use of any of the compounds as described herein, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of abnormal cell growth in a mammal wherein the abnormal cell growth is cancerous or non-cancerous. In some embodiments, the abnormal cell growth is cancerous. In another embodiment, the abnormal cell growth is non-cancerous.
[0166] In some embodiments, a pharmaceutical composition comprising the first agent and/or the second agent further comprises at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may comprise a conventional pharmaceutical carrier or excipient. Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents (such as hydrates and solvates). The pharmaceutical compositions may, if desired, contain additional ingredients such as flavorings, binders, excipients and the like. Thus for oral administration, tablets containing various excipients, such as citric acid may be employed together with various disintegrants such as starch, alginic acid and certain complex silicates and with binding agents such as sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes. Solid compositions of a similar type may also be employed in soft and hard filled gelatin capsules. Non-limiting examples of materials, therefore, include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration the active compound therein may be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof.
[0167] The pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository.
[0168] Exemplary parenteral administration forms include solutions or suspensions of active compounds in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms may be suitably buffered, if desired.
[0169] The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages.
[0170] The agents of the present disclosure may be formulated into pharmaceutical compositions as described below in any pharmaceutical form recognizable to the skilled artisan as being suitable. Pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of at least one compound provided herein and an inert, pharmaceutically acceptable carrier or diluent.
[0171] The pharmaceutical carriers employed may be either solid or liquid. Exemplary solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary liquid carriers are syrup, peanut oil, olive oil, water and the like. Similarly, the inventive compositions may include time-delay or time-release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or the like.
Further additives or excipients may be added to achieve the desired formulation properties. For example, a bioavailability enhancer, such as Labrasol, Gelucire or the like, or formulator, such as CMC (carboxy-methylcellulose), PG (propyleneglycol), or PEG (poly ethylenegly col), may be added. Gelucire®, a semi-solid vehicle that protects active ingredients from light, moisture and oxidation, may be added, e.g. , when preparing a capsule formulation.
[0172] If a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form, or formed into a troche or lozenge. The amount of solid carrier may vary, but generally will be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation may be in the form of syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or non-aqueous liquid suspension. If a semi-solid carrier is used, the preparation may be in the form of hard and soft gelatin capsule formulations. The inventive compositions are prepared in unit-dosage form appropriate for the mode of administration, e.g., parenteral or oral administration.
[0173] To obtain a stable water-soluble dose form, a salt of a compound of the present disclosure may be dissolved in an aqueous solution of an organic or inorganic acid, such as a 0.3 M solution of succinic acid or citric acid. If a soluble salt form is not available, the agent may be dissolved in a suitable co-solvent or combinations of co-solvents. Examples of suitable co-solvents include alcohol, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin and the like in concentrations ranging from 0 to 60% of the total volume. In an exemplary embodiment, a compound of the present disclosure is dissolved in DMSO and diluted with water. The composition may also be in the form of a solution of a salt form of the active ingredient in an appropriate aqueous vehicle such as water or isotonic saline or dextrose solution.
[0174] Proper formulation is dependent upon the route of administration selected. For injection, the agents of the compounds of the present disclosure may be formulated into aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. [0175] For oral administration, the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers known in the art. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained using a solid excipient in admixture with the active ingredient (agent), optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include: fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; and cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
[0176] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agents.
[0177] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
[0178] For administration intranasally or by inhalation, the compounds for use according to the present disclosure may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator and the like may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[0179] The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit-dosage form, e.g. , in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
[0180] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active agents may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
[0181] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0182] In addition to the formulations described above, the compounds of the present disclosure may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion-exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. A pharmaceutical carrier for hydrophobic compounds is a co-solvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be a VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the non-polar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD: 5 W) contains VPD diluted 1 : 1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. The proportions of a co-solvent system may be suitably varied without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity non-polar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose.
[0183] Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity due to the toxic nature of DMSO. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
[0184] The pharmaceutical compositions also may comprise suitable solid- or gel-phase carriers or excipients. These carriers and excipients may provide marked improvement in the bioavailability of poorly soluble drugs. Examples of such carriers or excipients include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Furthermore, additives or excipients such as Gelucire®, Capryol®, Labrafil®, Labrasol®, Lauroglycol®, Plurol®, Peceol®,
Transcutol® and the like may be used.
[0185] Further, the pharmaceutical composition may be incorporated into a skin patch for delivery of the drug directly onto the skin.
[0186] It will be appreciated that the actual dosages of the agents of this disclosure will vary according to the particular agent being used, the particular composition formulated, the mode of administration, and the particular site, host, and disease being treated. Those skilled in the art using conventional dosage-determination tests in view of the experimental data for a given compound may ascertain optimal dosages for a given set of conditions. For oral administration, an exemplary daily dose generally employed will be from about 0.001 to about 1000 mg/kg of body weight, with courses of treatment optionally repeated at appropriate intervals.
[0187] Additionally, the pharmaceutically acceptable formulations of the present disclosure may contain a compound of the present disclosure, or a salt or solvate thereof, in an amount from about 0.5 w/w % to about 95 w/w %, or from about 1 w/w % to about 95 w/w %, or from about 1 w/w % to about 75 w/w %, or from about 5 w/w % to about 75 w/w %, or from about 10 w/w % to about 75 w/w %, or from about 10 w/w % to about 50 w/w %.
[0188] The compounds provided herein, or salts or solvates thereof, may be administered to a mammal suffering from abnormal cell growth, such as a human, either alone or as part of a pharmaceutically acceptable formulation, once a week, once a day, twice a day, three times a day, or four times a day, or even more frequently.
[0189] Those of ordinary skill in the art will understand that with respect to the compounds of the present disclosure, the particular pharmaceutical formulation, the dosage, and the number of doses given per day to a mammal requiring such treatment, are all choices within the knowledge of one of ordinary skill in the art and can be determined without undue experimentation.
[0190] Administration of the compounds provided herein may be effected by any method that enables delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral inj ection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal administration. Bolus doses can be used, or infusions over a period of 1, 2, 3, 4, 5, 10, 15, 20, 30, 60, 90, 120 or more minutes, or any intermediate time period can also be used, as can infusions lasting 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 16, 20, 24 or more hours or lasting for 1 -7 days or more. Infusions can be administered by drip, continuous infusion, infusion pump, metering pump, depot formulation, or any other suitable means.
[0191] Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the mammalian patients to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present disclosure are dictated by and directly dependent on (a) the unique characteristics of the active agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
[0192] Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well- known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a patient may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a patient in practicing the present disclosure.
[0193] It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is to be further understood that for any particular patient, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the present disclosure encompasses intra-patient dose- escalation as determined by the skilled artisan. Determining appropriate dosages and regimens for administration of the chemotherapeutic agent are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings provided herein.
[0194] In some embodiments, the methods disclosed herein are useful for the treatment of cancers including but not limited to cancers of the: circulatory system, for example, heart (sarcoma [angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma], myxoma, rhabdomyoma, fibroma, lipoma and teratoma), mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue; respiratory tract, for example, nasal cavity and middle ear, accessory sinuses, larynx, trachea, bronchus and lung such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; gastrointestinal system, for example, esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), gastric, pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary tract, for example, kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and/or urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); liver, for example, hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor and glucagonoma); bone, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; nervous system, for example, neoplasms of the central nervous system (CNS), primary CNS lymphoma, skull cancer (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma
[pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); reproductive system, for example, gynecological, uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous
cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa- thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma) and other sites associated with female genital organs; placenta, penis, prostate, testis, and other sites associated with male genital organs; hematologic system, for example, blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; oral cavity, for example, lip, tongue, gum, floor of mouth, palate, and other parts of mouth, parotid gland, and other parts of the salivary glands, tonsil, oropharynx, nasopharynx, pyriform sinus, hypopharynx, and other sites in the lip, oral cavity and pharynx; skin, for example, malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids; adrenal glands: neuroblastoma; and other tissues including connective and soft tissue, retroperitoneum and peritoneum, eye, intraocular melanoma, and adnexa, breast, head or/and neck, anal region, thyroid, parathyroid, adrenal gland and other endocrine glands and related structures, secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites.
[0195] In some embodiments are provided methods disclosed herein wherein the second agent used in combination with the first agent described herein comprises an MEK inhibitor or an ERK inhibitor. In some embodiments, the second agent comprises an MEK inhibitor. Illustrative MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS-703988. In some embodiments, the second agent comprises an ERK inhibitor. Illustrative ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
[0196] In some embodiments are provided methods disclosed herein wherein the second agent used in combination with the first agent described herein is an anti-angiogenesis agent (e.g., an agent that stops tumors from developing new blood vessels). Examples of anti- angiogenesis agents include for example VEGF inhibitors, VEGFR inhibitors, TIE-2 inhibitors, PDGFR inhibitors, angiopoetin inhibitors, PKC-beta inhibitors, COX-2 (cyclooxygenase II) inhibitors, integrins (alpha-v/beta-3), MMP-2 (matrix- metalloproteinase 2) inhibitors, and MMP-9 (matrix-metalloprotienase 9) inhibitors.
Preferred anti-angiogenesis agents include sunitinib (Sutent®), bevacizumab (Avastin®), axitinib (AG 13736), SU 14813 (Pfizer), and AG 13958 (Pfizer).
[0197] Additional anti-angiogenesis agents include vatalanib (CGP 79787), Sorafenib (Nexavar®), pegaptanib octasodium (Macugen®), vandetanib (Zactima®), PF-0337210 (Pfizer), SU 14843 (Pfizer), AZD 2171 (AstraZeneca), ranibizumab (Lucentis®),
Neovastat® (AE 941), tetrathiomolybdata (Coprexa®), AMG 706 (Amgen), VEGF Trap (AVE 0005), CEP 7055 (Sanofi-Aventis), XL 880 (Exelixis), telatinib (BAY 57-9352), and CP-868,596 (Pfizer).
[0198] Other anti-angiogenesis agents include enzastaurin (LY 317615), midostaurin (CGP 41251), perifosine (KRX 0401), teprenone (Selbex®) and UCN 01 (Kyowa Hakko).
[0199] Other examples of anti-angiogenesis agents which can be used in conjunction with one or more pharmaceutical compositions described herein include celecoxib (Celebrex®), parecoxib (Dynastat®), deracoxib (SC 59046), lumiracoxib (Preige®), valdecoxib
(Bextra®), rofecoxib (Vioxx®), iguratimod (Careram®), IP 751 (Invedus), SC-58125 (Pharmacia) and etoricoxib (Arcoxia®).
[0200] Other anti-angiogenesis agents include exisulind (Aptosyn®), salsalate
(Amigesic®), diflunisal (Dolobid®), ibuprofen (Motrin®), ketoprofen (Orudis®) nabumetone (Relafen®), piroxicam (Feldene®), naproxen (Aleve®, Naprosyn®) diclofenac (Voltaren®), indomethacin (Indocin®), sulindac (Clinoril®), tolmetin (Tolectin®), etodolac (Lodine®), ketorolac (Toradol®), and oxaprozin (Daypro®).
[0201] Other anti-angiogenesis agents include ABT 510 (Abbott), apratastat (TMI 005), AZD 8955 (AstraZeneca), incyclinide (Metastat®), and PCK 3145 (Procyon).
[0202] Other anti-angiogenesis agents include acitretin (Neotigason®), plitidepsin (Aplidine®), cilengtide (EMD 121974), combretastatin A4 (CA4P), fenretinide (4 HPR), halofuginone (Tempostatin®), Panzem® (2-methoxy estradiol), PF-03446962 (Pfizer), rebimastat (BMS 275291), catumaxomab (Removab®), lenalidomide (Revlimid®) squalamine (EVIZON®), thalidomide (Thalomid®), Ukrain® (NSC 631570), Vitaxin® (MEDI 522), and zoledronic acid (Zometa®).
[0203] In some embodiments are provided methods disclosed herein wherein the second agent used in combination with the first agent described herein is a so-called signal transduction inhibitor (e.g., inhibiting the means by which regulatory molecules that govern the fundamental processes of cell growth, differentiation, and survival communicated within the cell). Signal transduction inhibitors include small molecules, antibodies, and antisense molecules. Signal transduction inhibitors include for example kinase inhibitors (e.g. , tyrosine kinase inhibitors or serine/threonine kinase inhibitors) and cell cycle inhibitors. More specifically signal transduction inhibitors include, for example, ALK inhibitors, ROS 1 inhibitors, TrkA inhibitors, TrkB inhibitors, TrkC inhibitors, farnesyl protein transferase inhibitors, EGF inhibitor, ErbB-1 (EGFR) inhibitors, ErbB-2 inhibitors, pan erb inhibitors, IGF1R inhibitors, MEK inhibitors, c-Kit inhibitors, FLT-3 inhibitors, K-Ras inhibitors, PI3 kinase inhibitors, JAK inhibitors, STAT inhibitors, Raf kinase inhibitors, Akt inhibitors, mTOR inhibitor, P70S6 kinase inhibitors, inhibitors of the WNT pathway and so called multi-targeted kinase inhibitors.
[0204] Among signal transduction inhibitors that may be used include gefitinib (Iressa®), cetuximab (Erbitux®), erlotinib (Tarceva®), trastuzumab (Herceptin®), sunitinib (Sutent®) imatinib (Gleevec®), and PD325901 (Pfizer).
[0205] Additional examples of signal transduction inhibitors which may be used include BMS 214662 (Bristol-Myers Squibb), lonafarnib (Sarasar®), pelitrexol (AG 2037), matuzumab (EMD 7200), nimotuzumab (TheraCIM h-R3®), panitumumab (Vectibix®), Vandetanib (Zactima®), pazopanib (SB 786034), ALT 110 (Alteris Therapeutics), BIBW 2992 (Boehringer Ingelheim), and Cervene® (TP 38).
[0206] Other examples of signal transduction inhibitors that may be used include PF- 2341066 (Pfizer), PF-299804 (Pfizer), canertinib (CI 1033), pertuzumab (Omnitarg®), Lapatinib (Tycerb®), pelitinib (EKB 569), miltefosine (Miltefosin®), BMS 599626 (Bristol-Myers Squibb), Lapuleucel-T (Neuvenge®), NeuVax® (E75 cancer vaccine), Osidem® (IDM 1), mubritinib (TAK-165), CP-724,714 (Pfizer), panitumumab (Vectibix®), lapatinib (Tycerb®), PF-299804 (Pfizer), pelitinib (EKB 569), and pertuzumab
(Omnitarg®).
[0207] Other examples of signal transduction inhibitors that may be used include ARRY 142886 (Array Biopharm), everolimus (Certican®), zotarolimus (Endeavor®), temsirolimus (Torisel®), AP 23573 (ARIAD), and VX 680 (Vertex).
[0208] Other signal transduction inhibitors that may be used include XL 647 (Exelixis), sorafenib (Nexavar®), LE-AON (Georgetown University), and GI-4000 (Globelmmune). [0209] Other signal transduction inhibitors that may be used include ABT 751 (Abbott), alvocidib (flavopiridol), BMS 387032 (Bristol Myers), EM 1421 (Erimos), indisulam (E 7070), seliciclib (CYC 200), BIO 112 (One Bio), BMS 387032 (Bristol-Myers Squibb), PD 0332991 (Pfizer), AG 024322 (Pfizer), LOXO-101 (Loxo Oncology), crizotinib, ceritinib, trametinib, PD0325901, selumetinib, cobimetinib, refametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E6201, GDC-0623, and TAK-733.
[0210] Other signal transduction inhibitors that may be used include trametinib,
PD0325901, selumetinib, cobimetinib, refametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E6201, GDC-0623, and TAK-733.
[0211] Other signal transduction inhibitors that may be used include trametinib, selumetinib, cobimetinib, refametinib, pimasertib, and binimetinib. A signal transduction inhibitor that may be used includes trametinib. A signal transduction inhibitor that may be used includes, selumetinib. A signal transduction inhibitor that may be used includes cobimetinib. A signal transduction inhibitor that may be used includes refametinib. A signal transduction inhibitor that may be used includes pimasertib. A signal transduction inhibitor that may be used includes binimetinib.
[0212] In some embodiments, the second agent is selected from are classical
antineoplastic agents. Classical antineoplastic agents include but are not limited to hormonal modulators such as hormonal, anti-hormonal, androgen agonist, androgen antagonist and anti-estrogen therapeutic agents, histone deacetylase (HDAC) inhibitors, gene silencing agents or gene activating agents, ribonucleases, proteosomics, Topoisomerase I inhibitors, Camptothecin derivatives, Topoisomerase II inhibitors, alkylating agents, antimetabolites, poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor, microtubulin inhibitors, antibiotics, plant derived spindle inhibitors, platinum-coordinated compounds, gene therapeutic agents, antisense oligonucleotides, vascular targeting agents (VTAs), and statins.
[0213] Examples of classical antineoplastic agents that may be used include, but are not limited to, glucocorticoids, such as dexamethasone, prednisone, prednisolone,
methylprednisolone, hydrocortisone, and progestins such as medroxyprogesterone, megestrol acetate (Megace), mifepristone (RU-486), Selective Estrogen Receptor
Modulators (SERMs; such as tamoxifen, raloxifene, lasofoxifene, afimoxifene, arzoxifene, bazedoxifene, fispemifene, ormeloxifene, ospemifene, tesmilifene, toremifene, trilostane and CHF 4227 (Cheisi)), Selective Estrogen-Receptor Downregulators (SERD's; such as fulvestrant), exemestane (Aromasin), anastrozole (Arimidex), atamestane, fadrozole, letrozole (Femara), gonadotropin-releasing hormone (GnRH; also commonly referred to as luteinizing hormone-releasing hormone [LHRH]) agonists such as buserelin (Suprefact), goserelin (Zoladex), leuprorelin (Lupron), and triptorelin (Trelstar), abarelix (Plenaxis), bicalutamide (Casodex), cyproterone, flutamide (Eulexin), megestrol, nilutamide
(Nilandron), and osaterone, dutasteride, epristeride, finasteride, Serenoa repens, PHL 00801, abarelix, goserelin, leuprorelin, triptorelin, bicalutamide, tamoxifen, exemestane, anastrozole, fadrozole, formestane, letrozole, and combinations thereof.
[0214] Examples of classical antineoplastic agents that may be used include, but are not limited to, suberolanilide hydroxamic acid (SAHA, Merck Inc./Aton Pharmaceuticals), depsipeptide (FR901228 or FK228), G2M-777, MS-275, pivaloyloxymethyl butyrate and PXD-101 ; Onconase (ranpirnase), PS-341 (MLN-341), Velcade (bortezomib), 9- aminocamptothecin, belotecan, BN-80915 (Roche), camptothecin, diflomotecan, edotecarin, exatecan (Daiichi), gimatecan, 10-hydroxy camptothecin, irinotecan HC1 (Camptosar), lurtotecan, Orathecin (rubitecan, Supergen), SN-38, topotecan, camptothecin, 10- hydroxy camptothecin, 9-aminocamptothecin, irinotecan, SN-38, edotecarin, topotecan, aclarubicin, paclitaxel, amonafide, amrubicin, annamycin, daunorubicin, doxorubicin, elsamitrucin, epirubicin, etoposide, idarubicin, galarubicin, hydroxy carbamide,
nemorubicin, novantrone (mitoxantrone), pirarubicin, pixantrone, procarbazine,
rebeccamycin, sobuzoxane, tafluposide, valrubicin, Zinecard (dexrazoxane), nitrogen mustard N-oxide, cyclophosphamide, AMD-473, altretamine, AP-5280, apaziquone, brostallicin, bendamustine, busulfan, carboquone, carmustine, chlorambucil, dacarbazine, estramustine, fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine, mafosfamide, mechlorethamine, melphalan, mitobronitol, mitolactol, mitomycin C, mitoxatrone, nimustine, ranimustine, temozolomide, thiotepa, and platinum-coordinated alkylating compounds such as cisplatin, Paraplatin (carboplatin), eptaplatin, lobaplatin, nedaplatin, Eloxatin (oxaliplatin, Sanofi), streptozocin, satrplatin, and combinations thereof.
[0215] In some embodiments, the second agent is selected from dihydrofolate reductase inhibitors. Examples of dihydrofolate reductase inhibitors that may be used include, but are not limited to, methotrexate and NeuTrexin (trimetrexate), purine antagonists (such as 6- mercaptopurine riboside, mercaptopurine, 6-thioguanine, cladribine, clofarabine (Clolar), fludarabine, nelarabine, and raltitrexed), pyrimidine antagonists (such as 5-fluorouracil (5- FU), Alimta (premetrexed disodium, LY231514, MTA), capecitabine (Xeloda®), cytosine arabinoside, Gemzar® (gemcitabine, Eli Lilly), Tegafur (UFT Orzel or Uforal and including TS-1 combination of tegafur, gimestat and otostat), doxifluridine, carmofur, cytarabine (including ocfosfate, phosphate stearate, sustained release and liposomal forms), enocitabine, 5-azacitidine (Vidaza), decitabine, and ethyny Icy ti dine) and other
antimetabolites such as eflornithine, hydroxyurea, leucovorin, nolatrexed (Thymitaq), triapine, trimetrexate, N-(5- N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl)-L-glutamic acid, AG-014699 (Pfizer Inc.), ABT-472 (Abbott Laboratories), INO-1001 (Inotek Pharmaceuticals), KU-0687 (KuDOS Pharmaceuticals) and GPI 18180 (Guilford Pharm Inc) and combinations thereof.
[0216] Other examples of classical antineoplastic cytotoxic agents that may be used include, but are not limited to, Abraxane (Abraxis Bioscience, Inc.), Batabulin (Amgen), EPO 906 (Novartis), Vinflunine (Bristol-Myers Squibb Company), actinomycin D, bleomycin, mitomycin C, neocarzinostatin (Zinostatin), vinblastine, vincristine, vindesine, vinorelbine (Navelbine), docetaxel (Taxotere), Ortataxel, paclitaxel (including Taxoprexin a DHA/paclitaxel conjugate), cisplatin, carboplatin, Nedaplatin, oxaliplatin (Eloxatin), Satraplatin, Camptosar, capecitabine (Xeloda), oxaliplatin (Eloxatin), Taxotere alitretinoin, Canfosfamide (Telcyta®), DMXAA (Antisoma), ibandronic acid, L-asparaginase, pegaspargase (Oncaspar®), Efaproxiral (Efaproxyn®~radiation therapy)), bexarotene (Targretin®), Tesmilifene (DPPE— enhances efficacy of cytotoxics)), Theratope®
(Biomira), Tretinoin (Vesanoid®), tirapazamine (Trizaone®), motexafin gadolinium (Xcytrin®) Cotara® (mAb), and NBI-3001 (Protox Therapeutics), polyglutamate-paclitaxel (Xyotax®) and combinations thereof.
[0217] Further examples of classical antineoplastic agents that may be used include, but are not limited to, as Advexin (ING 201), TNFerade (GeneVec, one or more compounds which express TNF alpha in response to radiotherapy), RB94 (Baylor College of Medicine), Genasense (Oblimersen, Genta), Combretastatin A4P (CA4P), Oxi-4503, AVE-8062, ZD- 6126, TZT-1027, Atorvastatin (Lipitor, Pfizer Inc.), Provastatin (Pravachol, Bristol-Myers Squibb), Lovastatin (Mevacor, Merck Inc.), Simvastatin (Zocor, Merck Inc.), Fluvastatin (Lescol, Novartis), Cerivastatin (Bay col, Bayer), Rosuvastatin (Crestor, AstraZeneca), Lovostatin, Niacin (Advicor, Kos Pharmaceuticals), Caduet, Lipitor, torcetrapib, and combinations thereof.
[0218] In some embodiments, the methods disclosed herein comprise administering to the patient a combination further comprising one or more additional agents. The one or more additional agents may be independently selected from the anti-angiogenesis agents described herein, the so-called signal transduction inhibitors described herein, the classical antineoplastic agents described herein, and the dihydrofolate reductase inhibitors described herein.
[0219] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges provided herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1 -5 articles refers to groups having 1 , 2, 3, 4, or 5 articles, and so forth.
[0220] The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase "consisting essentially of will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase "consisting of excludes any element not specified.
[0221] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
[0222] Headings, e.g. , (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.
[0223] The example herein is provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with using the present technology. The example herein is also presented in order to more fully illustrate the preferred aspects of the present technology. The example should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The example can include or incorporate any of the variations, aspects or aspects of the present technology described above. The variations, aspects or aspects described above may also further each include or incorporate the variations of any or all other variations, aspects or aspects of the present technology.
EXAMPLES
[0224] All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.
Example 1: Pharmaceutical composition comprising A-[5-(3,5-difluorobenzyl)-lH- indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)- benzamide.
[0225] A pharmaceutical composition comprising N-[5-(3,5-difluorobenzyl)-lH-indazol- 3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and betaine hydrochloride was prepared as follows.
Figure imgf000068_0001
Total 450.00 500.00 500.01
[0226] N-[5-(3,5-Difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l -yl)-2- (tetrahydro-2H-pyran-4-ylamino)-benzamide was screened through a 60-mesh sieve and was transferred to the batch mixing container. The betaine hydrochloride was ground with a mortar and pestle, screened through a 60-mesh sieve and then transferred to the batch mixing container. The mixture of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl- piperazin-l -yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and betaine hydrochloride were mixed by hand for about one minute, after which time the pre-gelatinized starch, colloidal silicon dioxide and one-half of the amount of required isomalt were added to the batch container. The resulting mixture was mixed by hand for about one minute. The magnesium stearate and the remaining isomalt were pre-blended through a 40-mesh sieve and then combined with materials in the batch mixing container. The final mixture was blended by hand for approximately 5 minutes.
[0227] The resulting mixture was filled into gelatin capsule shells, opaque white, size #00. The body and cap of the capsules were separated, the capsule bodies were placed into a capsule device, ensuring the top of the capsule body was flush with the surface of the filling device by moving the spacer of the device. The powder blend was poured onto the surface of the filling device, volumetrically filling the body of the capsules, and scraping the excess powder evenly until all capsule bodies are filled. The powder was firmly tamped into the shells one time using a tamper. Additional powder blend was added to fill the remainder of the capsule and any excess powder was scraped off. The tamping, filling, and scraping procedures were repeated for each capsule until the desired capsule fill weight was achieved. The filled capsules were collected in a 10-mesh sieve and were de-dusted by agitating them lightly.
[0228] The filled capsule weight range acceptance limits were set at 93% to 107%. The average weight of the empty capsule shells was 119.4 mg. The low capsule weight limit was set at (0.93 x 450 mg) + 119.4 mg = 538 mg. The high capsule weight limit was set at (1.07 x 450 mg) + 119.4 mg = 601 mg. Only those capsules meeting the weight limits were used in subsequent studies.
[0229] These capsules are used for concurrent or sequential administration with a separate pharmaceutical composition comprising a second agent. The second agent may be a MEK inhibitor or an ERK inhibitor.
Example 2. Combination therapy for patients with non-small cell lung cancer.
[0230] One or more appropriate doses of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and a second agent are administered to a human patient for the treatment of non-small cell lung cancer. Expected results from treatment with the combination, include (1) reduction of the size of a cancer tumor, (2) inhibition (that is, slowing to some extent, preferably stopping) of cancer tumor metastasis, (3) inhibition to some extent (that is, slowing to some extent, preferably stopping) of cancer tumor growth, and/or, (4) relief to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer. The second agent may be a MEK inhibitor or an ERK inhibitor. Examples of MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988. Examples of ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
Example 3. Combination therapy for patients with papillary thyroid cancer.
[0231] One or more appropriate doses of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and a second agent are administered to a human patient for the treatment of papillary thyroid cancer. Expected results from treatment with the combination, include (1) reduction of the size of a cancer tumor, (2) inhibition (that is, slowing to some extent, preferably stopping) of cancer tumor metastasis, (3) inhibition to some extent (that is, slowing to some extent, preferably stopping) of cancer tumor growth, and/or, (4) relief to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer. The second agent may be a MEK inhibitor or an ERK inhibitor. Examples of MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988. Examples of ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
Example 4. Combination therapy for patients with neuroblastoma.
[0232] One or more appropriate doses of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and a second agent are administered to a human patient for the treatment of neuroblastoma. Expected results from treatment with the combination, include (1) reduction of the size of a cancer tumor, (2) inhibition (that is, slowing to some extent, preferably stopping) of cancer tumor metastasis, (3) inhibition to some extent (that is, slowing to some extent, preferably stopping) of cancer tumor growth, and/or, (4) relief to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer. The second agent may be a MEK inhibitor or an ERK inhibitor. Examples of MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988. Examples of ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
Example 5. Combination therapy for patients with pancreatic cancer.
[0233] One or more appropriate doses of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and a second agent are administered to a human patient for the treatment of pancreatic cancer. Expected results from treatment with the combination, include (1) reduction of the size of a cancer tumor, (2) inhibition (that is, slowing to some extent, preferably stopping) of cancer tumor metastasis, (3) inhibition to some extent (that is, slowing to some extent, preferably stopping) of cancer tumor growth, and/or, (4) relief to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer. The second agent may be a MEK inhibitor or an ERK inhibitor. Examples of MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988. Examples of ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
Example 6. Combination therapy for patients with melanoma.
[0234] One or more appropriate doses of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and a second agent are administered to a human patient for the treatment of melanoma. Expected results from treatment with the combination, include (1) reduction of the size of a cancer tumor, (2) inhibition (that is, slowing to some extent, preferably stopping) of cancer tumor metastasis, (3) inhibition to some extent (that is, slowing to some extent, preferably stopping) of cancer tumor growth, and/or, (4) relief to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer. The second agent may be a MEK inhibitor or an ERK inhibitor. Examples of MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS-703988. Examples of ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS- 134, AEZS-136, and IDN-5491.
Example 7. Combination therapy for patients with colorectal cancer.
[0235] One or more appropriate doses of N-[5-(3,5-difluorobenzyl)-lH-indazol-3-yl]-4- (4-methyl-piperazin-l-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-benzamide and a second agent are administered to a human patient for the treatment of colorectal cancer. Expected results from treatment with the combination, include (1) reduction of the size of a cancer tumor, (2) inhibition (that is, slowing to some extent, preferably stopping) of cancer tumor metastasis, (3) inhibition to some extent (that is, slowing to some extent, preferably stopping) of cancer tumor growth, and/or, (4) relief to some extent (or, preferably, eliminating) one or more symptoms associated with the cancer. The second agent may be a MEK inhibitor or an ERK inhibitor. Examples of MEK inhibitors include, but are not limited to, PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS- 703988. Examples of ERK inhibitors include, but are not limited to, TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
Example 8. Generation of Ba/F3-TPM3-TrkA-G959R Cell Lines
[0232] This Example describes studies performed to generate transgenic Ba/F3 cells expressing a TPM3-TrkA-G595R fusion protein. A cDNA encoding TPM3-TrkA-G595R fusion was cloned from entrectinib-resistant cells by a PCR-based technique and subsequently inserted into a lentiviral vector pVL-EFla-MCS-IRES-Puro (BioSettia, San Diego, CA). After confirmation of the cDNA inserts by direct sequencing, vesicular stomatitis virus GP (VSVG)-pseudo-typed lentiviruses containing the TPM3-TrkA-G595R cDNA were transduced into the murine IL-3 dependent pro-B cell Ba/F3 at different multiplicity of infections (MOIs) with 8 μg/mL of polybrene (EMD Millipore). The transduced Ba/F3 cells were selected in the murine IL-3 containing RPMI media supplemented with 10% FBS and 1 μg/mL of puromycin for two weeks. The stable cell pools were further selected in RPMI media (GIBCO®) supplemented with 10% FBS (fetal bovine serum) and without murine IL-3 for 4 weeks.
Example 9. Effect of Treatment with Entrectinib (RXDX-101) on Ba/F3 Cells
Expressing TPM3-TrkA or TPM3-TrkA-G595R Mutation
[0236] Five to 10 million Ba/F3 cells expressing either TPM3-TrkA or TPM3-TrkA- G595R were incubated with different concentrations of entrectinib (RXDX-101) for 2 hours in a 5% CO2 incubator. The cells were washed twice with cold phosphate-buffered saline (PBS) (lx) and were then resuspended in lxRIPA buffer with protease and phosphatase inhibitor cocktails (EMDMillipore) and the resulting mixture was rocked for 30 minutes at 4 °C. The resulting lysates were clarified by centrifuging (10,000 x g) at 4 °C for 10 minutes. The resulting supernatants were saved for Western Blot analysis. 20-40 μg of the resulting proteins were separated by SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The resulting membranes were blotted with the following antibodies (obtained from Cell Signalling unless otherwise stated) according to the procedures described in the respective manuals: phospho-TrkA-Y490, phospho- TrkA-Y785, pan-Trk, PLCY1, phospho-PLCYl-Y783, MEK1/2, phospho-MEKl/2 (S217/S221), β-actin. The resulting bands were developed with ECL reagents (Amersham) and the images were captured using ChemiDoc imager (Bio-Rad). The results of the Western Blot analysis are shown in FIG. 1 and demonstrate that p-MEKl/2 (Ser217/221) and total MEKl/2 are upregulated in the Ba/F3 cells expressing TPM3-TrkA-G595R versus Ba/F3 cells expressing wild-type TPM3-TrkA.
Example 10. In vitro Activities of entrectinib (RXDX-101), trametinib, and the combination of entrectinib and trametinib on the inhibition of growth of Ba/F3 cell lines expressing TPM3-TrkA-G959R
[0237] The effects on the growth of a Ba/F3 cell line expressing TPM3-TrkA-G959R resulting from treatment with entrectinib (RXDX-101), trametinib, and the combination of entrectinib and trametinib were measured. Approximately one million Ba/F3 cells expressing TPM3-TrkA-G959R per well were seeded in 6-well plates in 5 mL media. To the wells were added one of the following from lOOOx stocks in DMSO: (a) no inhibitor (dimethylsulfoxide (DMSO) alone as control), (b) entrectinib (RXDX-101) at a concentration of about 300 nM, (c) trametinib at a concentration of about 30 nM, or (d) a combination of entrectinib (RXDX-101) at a concentration of 300 nM and trametinib at a concentration of 30 nM. The cells in each respective well were counted every 3 to 4 days using Countess™ cell counter (Invitrogen, Carlsbad, CA) in the presence of Trypan Blue (Invitrogen, Carlsbad, CA). For those wells that contained at least 1 million live cells on the day the cells in each well were counter, the media containing those cells were diluted at 1 : 10 with fresh growth media containing the same respective concentrations of entrectinib, trametinib or the combination of entrectinib and trametinib. The wells that contained fewer than 1 million live cells on the day the cells in each well were counted went unchanged. The results of the study are shown in FIG. 2, which demonstrates that the cells treated with the combination of entrectinib (300 nM) and trametinib (30 nM) displayed significant growth inhibition (up to 40 days) compared to the cells in wells containing no compound (DMSO control) and those containing entrectinib (300 nM) alone and trametinib (30 nM) alone.
Example 11. In vivo study of mice implanted with cells expressing TPM3-TrkA- G595R following administration of entrectinib (RXDX-101), trametinib, or a combination of entrectinib and trametinib. [0238] Athymic nu/nu mice, 6-7 weeks old, female mice were implanted subcutaneously (right flank) with 2 million KM-12 TPM3-NTRK1 G595R cells/mouse. Animals were randomized once the mean tumor volume reached -150 mm3. Animals received a total of 10 doses of Vehicle, 60 mg/kg entrectinib, 1 mg/kg trametinib, and combination of entrectinib (60 mg/kg) + trametinib (1 mg/kg), p.o. q.d. All treatments were tolerable. The results are shown in FIG. 3, which demonstrates that the growth of tumors in mice implanted with cells expressing TPM3-TrkA-G595R was reduced in those mice to which a combination of entrectinib (RXDX-101) (60 mg/kg) and trametinib (1 mg/kg) were administered versus tumor growth in those mice in the control group (Vehicle), those mice treated with entrectinib (60 mg/kg) alone, and those mice treated with trametinib (1 mg/kg) alone.
[0239] Good manufacture practice (GMP)-quality entrectinib (RXDX-101), was synthesized at Ignyta (Lot #: CA15-919, fill date 12/13/15). The entrectinib in vivo oral dosing solution was made by suspending entrectinib in a solution of 0.5% methyl cellulose (Lot #: 147255, M352-500, Fisher) and 1% Tween 80 (Lot #: MKB58228V, P4780-100ML, Sigma) at a concentration of 5 mg/mL. Trametinib in vivo oral dosing solution was made by dissolving trametinib in a solution of DMSO (Lot #: SHBG1596V, D8418-100ML, Sigma), PEG 400 (Lot #: MKBX3961V, 202398-500G, Sigma), Tween 80 (Lot #: MKB58228V, P4780-100ML, Sigma) and water. The final concentration of trametinib was 0.1 mg/mL and the amounts of each component in the resulting solution were 3.3 % DMSO, 16.7% PEG 400, 15% Tween 80 and 65% water, with the water being added just prior to dosing.
[0240] After randomization, all animals were dosed p.o. q.d. with solutions of (a) entrectinib vehicle, (b) entrectinib (5 mg/mL), (c) trametinib (0.1 mg/mL), or (d) a combination of entrectinib (5 mg/mL) and trametinib (0.1 mg/mL), at a dosing volume of about 10 mL/kg body weight.
[0241] Athymic nu/nu female mice between 6-7 weeks of age were ordered from Charles River. Animals were allowed to acclimate 3 days prior to the start of the study (e.g., cell implantation). Animals were housed in irradiated, individual HEPA ventilated cages (Innocage® IVC, Innovive USA) on a 12-hour light-dark cycle at 68-79°F and 30-70% humidity. Animals were provided with irradiated chow (Teklad 2920X) and acidified drinking water (Innovive) ad libitum as per the animal care and use protocol established at Explora Biolabs (Ignyta ACUP# EB15-050). Animals were identified by uniquely numbered ear-tags.
[0242] KM 12 cells were cultured, harvested, and suspended in 1 : 1 FBS free culture media: marti gel at a concentration of 10 million cells/mL. Cells were implanted under the skin on the right flank of each mouse on day 0. The implantation volume was 200 μΐνιηουββ. Tumors grew until the mean tumor volume reached approximately 130 mm3. On day 8 mice were randomized into 6 treatment groups (10 mice/group) and treatment began. Animals were treated p.o. q.d. with (a) vehicle (0.5% methyl cellulose and 1% Tween 80), (b) entrectinib (60 mg/kg), (c) 1 mg/kg trametinib, or (d) a combination of entrectinib (60 mg/kg) and trametinib (1 mg/kg). The final treatment for all groups was administered on day 17, for a total of 10 doses. The final tumor and body weight measurements were taken on the final day of dosing, day 17.
[0243] Tumors measurements and body weights were collected 2x/week over the duration of the study. Tumor growth was assessed by caliper and tumor volumes were calculated using the equation (1). Animals with tumor volumes > 2,000 mm3 were removed from study.
Volume (mm3) = [ length (mm) x width (mm) x width (mm) ] / 2 (1)
[0244] Body weights were collected using a laboratory scale. Toxicity was evaluated based on body weight loss. Animals losing <\ 5% of their body weight were removed from the study.
[0245] The percentage of tumor growth inhibition (%TGI) was calculated for each treatment group at each tumor measurement. The %TGI values were calculated using equation (2) below, where TVyehicie is the tumor volume for the vehicle-treated animals at a specified time point, TVinitiai is the initial tumor volume at the start of the treatment and TVtreatment is the tumor volume of the treatment group at a specified end-point time.
Differences in tumor volumes were assessed by ANOVA using GraphPad Prism (version 6.07, GraphPad Software, Inc.).
%TGI = [ ( TVyehicie - TVtreatment) / ( TVyehicie " TVinitiai ) ] X 100 [0246] A summary table of the average tumor volume for each group, for each measurement day is shown in Table 1. The %TGI for each day was calculated as described above and these data are summarized in Table 2. Evaluation of significance by two-way ANOVA (Prism) was limited out to day 14 due to one death in the entrectinib + trametinib group. Body weight data for this study shows that this death is likely due to toxicity induced by trametinib. Analysis showed that by day 14 only the trametinib alone groups was significantly different from vehicle (p<0.05, two-way ANOVA, Prism). The same was found if the entrectinib + trametinib group was omitted and the analysis was carried out to day 17. Statistical evaluation of each treatment group versus vehicle for each measurement day (i.e., day 11, 14 and 17) was also evaluated by one-way ANOVA. Differences were found between vehicle and trametinib alone and between vehicle and entrectinib + trametinib for days 14 and 17. All groups were found to be different from vehicle on day 17 (p<0.05, one-way ANOVA, Prism). Body weights remained stable throughout the study for the vehicle and entrectinib alone groups. Toxicity was observed for any animals receiving trametinib. One death was observed in the entrectinib + trametinib group and Body weight data for this animal showed a decline is weight prior to death.
Table 1. Average tumor volume for each treatment group on each day measured.
Figure imgf000077_0001
Table 2. Percent tumor growth inhibition (%TGI) for each treatment group on each day measured.
Figure imgf000077_0002
Sequence Listings
[0247] human tropomyosin receptor kinase A (TrkA)
Met Leu Arg Gly Gly Arg Arg Gly Gin Leu Gly Trp His Ser Trp Ala
Ala Gly Pro Gly Ser Leu Leu Ala Trp Leu He Leu Ala Ser Ala Gly
Ala Ala Pro Cys Pro Asp Ala Cys Cys Pro His Gly Ser Ser Gly Leu
Arg Cys Thr Arg Asp Gly Ala Leu Asp Ser Leu His His Leu Pro Gly
Ala Glu Asn Leu Thr Glu Leu Tyr He Glu Asn Gin Gin His Leu Gin
His Leu Glu Leu Arg Asp Leu Arg Gly Leu Gly Glu Leu Arg Asn Leu
Thr He Val Lys Ser Gly Leu Arg Phe Val Ala Pro Asp Ala Phe His
Phe Thr Pro Arg Leu Ser Arg Leu Asn Leu Ser Phe Asn Ala Leu Glu
Ser Leu Ser Trp Lys Thr Val Gin Gly Leu Ser Leu Gin Glu Leu Val
Leu Ser Gly Asn Pro Leu His Cys Ser Cys Ala Leu Arg Trp Leu Gin
Arg Trp Glu Glu Glu Gly Leu Gly Gly Val Pro Glu Gin Lys Leu Gin
Cys His Gly Gin Gly Pro Leu Ala His Met Pro Asn Ala Ser Cys Gly
Val Pro Thr Leu Lys Val Gin Val Pro Asn Ala Ser Val Asp Val Gly Asp Asp Val Leu Leu Arg Cys Gin Val Glu Gly Arg Gly Leu Glu Gin Ala Gly Trp He Leu Thr Glu Leu Glu Gin Ser Ala Thr Val Met Lys Ser Gly Gly Leu Pro Ser Leu Gly Leu Thr Leu Ala Asn Val Thr Ser Asp Leu Asn Arg Lys Asn Val Thr Cys Trp Ala Glu Asn Asp Val Gly Arg Ala Glu Val Ser Val Gin Val Asn Val Ser Phe Pro Ala Ser Val Gin Leu His Thr Ala Val Glu Met His His Trp Cys He Pro Phe Ser Val Asp Gly Gin Pro Ala Pro Ser Leu Arg Trp Leu Phe Asn Gly Ser Val Leu Asn Glu Thr Ser Phe lie Phe Thr Glu Phe Leu Glu Pro Ala Ala Asn Glu Thr Val Arg His Gly Cys Leu Arg Leu Asn Gin Pro Thr His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala Asn Pro Phe Gly Gin Ala Ser Ala Ser lie Met Ala Ala Phe Met Asp Asn Pro Phe Glu Phe Asn Pro Glu Asp Pro lie Pro Val Ser Phe Ser Pro Val Asp Thr Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr Pro Phe Gly Val Ser Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu Phe Leu Ser Thr Leu Leu Leu Val Leu Asn Lys Cys Gly Arg Arg Asn Lys Phe Gly He Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala Met Ser Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr Glu Gly Lys Gly Ser Gly Leu Gin Gly His He He Glu Asn Pro Gin Tyr Phe Ser Asp Ala Cys Val His His He Lys Arg Arg Asp He Val Leu Lys Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys His Asn Leu Leu Pro Glu Gin Asp Lys Met Leu Val Ala Val Lys Ala Leu Lys Glu Ala Ser Glu Ser Ala Arg Gin Asp Phe Gin Arg Glu Ala Glu Leu Leu Thr Met Leu Gin His Gin His He Val Arg Phe Phe Gly Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val Phe Glu Tyr Met Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly Pro Asp Ala Lys Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro Leu Gly Leu Gly Gin Leu Leu Ala Val Ala Ser Gin Val Ala Ala Gly Met Val Tyr Leu Ala Gly Leu His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Gin Gly Leu Val Val Lys He Gly Asp Phe Gly Met Ser Arg Asp lie Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met Leu Pro He Arg Trp Met Pro Pro Glu Ser He Leu Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu He Phe Thr Tyr Gly Lys Gin Pro Trp Tyr Gin Leu Ser Asn Thr Glu Ala He Asp Cys He Thr Gin Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys Pro Pro Glu Val Tyr Ala He Met Arg Gly Cys Trp Gin Arg Glu Pro Gin Gin Arg His Ser He Lys Asp Val His Ala Arg Leu Gin Ala Leu Ala Gin Ala Pro Pro Val Tyr Leu Asp Val Leu Gly (SEQ ID NO: 1)
[0248] human tropomyosin receptor kinase B (TrkB)
Met Ser Ser Trp He Arg Trp His Gly Pro Ala Met Ala Arg Leu Trp Gly Phe Cys Trp Leu Val Val Gly Phe Trp Arg Ala Ala Phe Ala Cys Pro Thr Ser Cys Lys Cys Ser Ala Ser Arg He Trp Cys Ser Asp Pro Ser Pro Gly He Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Val Asp Pro Glu Asn He Thr Glu He Phe He Ala Asn Gin Lys Arg Leu Glu He He Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Arg Asn Leu Thr He Val Asp Ser Gly Leu Lys Phe Val Ala His Lys Ala Phe Leu Lys Asn Ser Asn Leu Gin His He Asn Phe Thr Arg Asn Lys Leu Thr Ser Leu Ser Arg Lys His Phe Arg His Leu Asp Leu Ser Glu Leu He Leu Val Gly Asn Pro Phe Thr Cys Ser Cys Asp He Met Trp He Lys Thr Leu Gin Glu Ala Lys Ser Ser Pro Asp Thr Gin Asp Leu Tyr Cys Leu Asn Glu Ser Ser Lys Asn He Pro Leu Ala Asn Leu Gin He Pro Asn Cys Gly Leu Pro Ser Ala Asn Leu Ala Ala Pro Asn Leu Thr Val Glu Glu Gly Lys Ser He Thr Leu Ser Cys Ser Val Ala Gly Asp Pro Val Pro Asn Met Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His Met Asn Glu Thr Ser His Thr Gin Gly Ser Leu Arg He Thr Asn He Ser Ser Asp Asp Ser Gly Lys Gin He Ser Cys Val Ala Glu Asn Leu Val Gly Glu Asp Gin Asp Ser Val Asn Leu Thr Val His Phe Ala Pro Thr He Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys He Pro Phe Thr Val Lys Gly Asn Pro Lys Pro Ala Leu Gin Trp Phe Tyr Asn Gly Ala He Leu Asn Glu Ser Lys Tyr He Cys Thr Lys He His Val Thr Asn His Thr Glu Tyr His Gly Cys Leu Gin Leu Asp Asn Pro Thr His Met Asn Asn Gly Asp Tyr Thr Leu He Ala Lys Asn Glu Tyr Gly Lys Asp Glu Lys Gin He Ser Ala His Phe Met Gly Trp Pro Gly He Asp Asp Gly Ala Asn Pro Asn Tyr Pro Asp Val He Tyr Glu Asp Tyr Gly Thr Ala Ala Asn Asp He Gly Asp Thr Thr Asn Arg Ser Asn Glu He Pro Ser Thr Asp Val Thr Asp Lys Thr Gly Arg Glu His Leu Ser Val Tyr Ala Val Val Val He Ala Ser Val Val Gly Phe Cys Leu Leu Val Met Leu Phe Leu Leu Lys Leu Ala Arg His Ser Lys Phe Gly Met Lys Asp Phe Ser Trp Phe Gly Phe Gly Lys Val Lys Ser Arg Gin Gly Val Gly Pro Ala Ser Val He Ser Asn Asp Asp Asp Ser Ala Ser Pro Leu His His He Ser Asn Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly Gly Pro Asp Ala Val He He Gly Met Thr Lys He Pro Val He Glu Asn Pro Gin Tyr Phe Gly He Thr Asn Ser Gin Leu Lys Pro Asp Thr Phe Val Gin His He Lys Arg His Asn He Val Leu Lys Arg Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu Cys Pro Glu Gin Asp Lys He Leu Val Ala Val Lys Thr Leu Lys Asp Ala Ser Asp Asn Ala Arg Lys Asp Phe His Arg Glu Ala Glu Leu Leu Thr Asn Leu Gin His Glu His He Val Lys Phe Tyr Gly Val Cys Val Glu Gly Asp Pro Leu He Met Val Phe Glu Tyr Met Lys His Gly Asp Leu Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Val Leu Met Ala Glu Gly Asn Pro Pro Thr Glu Leu Thr Gin Ser Gin Met Leu His He Ala Gin Gin He Ala Ala Gly Met Val Tyr Leu Ala Ser Gin His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Asn Leu Leu Val Lys He Gly Asp Phe Gly Met Ser Arg Asp Val Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly His Thr Met Leu Pro lie Arg Trp Met Pro Pro Glu Ser He Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp Ser Leu Gly Val Val Leu Trp Glu He Phe Thr Tyr Gly Lys Gin Pro Trp Tyr Gin Leu Ser Asn Asn Glu Val He Glu Cys He Thr Gin Gly Arg Val Leu Gin Arg Pro Arg Thr Cys Pro Gin Glu Val Tyr Glu Leu Met Leu Gly Cys Trp Gin Arg Glu Pro His Met Arg Lys Asn He Lys Gly He His Thr Leu Leu Gin Asn Leu Ala Lys Ala Ser Pro Val Tyr Leu Asp He Leu Gly (SEQ ID NO: 2)
[0249] human tropomyosin receptor kinase C (TrkC)
Met Asp Val Ser Leu Cys Pro Ala Lys Cys Ser Phe Trp Arg He Phe Leu Leu Gly Ser Val Trp Leu Asp Tyr Val Gly Ser Val Leu Ala Cys Pro Ala Asn Cys Val Cys Ser Lys Thr Glu He Asn Cys Arg Arg Pro Asp Asp Gly Asn Leu Phe Pro Leu Leu Glu Gly Gin Asp Ser Gly Asn Ser Asn Gly Asn Ala Ser He Asn He Thr Asp He Ser Arg Asn He Thr Ser He His He Glu Asn Trp Arg Ser Leu His Thr Leu Asn Ala Val Asp Met Glu Leu Tyr Thr Gly Leu Gin Lys Leu Thr He Lys Asn Ser Gly Leu Arg Ser He Gin Pro Arg Ala Phe Ala Lys Asn Pro His Leu Arg Tyr He Asn Leu Ser Ser Asn Arg Leu Thr Thr Leu Ser Trp Gin Leu Phe Gin Thr Leu Ser Leu Arg Glu Leu Gin Leu Glu Gin Asn Phe Phe Asn Cys Ser Cys Asp He Arg Trp Met Gin Leu Trp Gin Glu Gin Gly Glu Ala Lys Leu Asn Ser Gin Asn Leu Tyr Cys He Asn Ala Asp Gly Ser Gin Leu Pro Leu Phe Arg Met Asn He Ser Gin Cys Asp Leu Pro Glu He Ser Val Ser His Val Asn Leu Thr Val Arg Glu Gly Asp Asn Ala Val He Thr Cys Asn Gly Ser Gly Ser Pro Leu Pro Asp Val Asp Trp He Val Thr Gly Leu Gin Ser He Asn Thr His Gin Thr Asn Leu Asn Trp Thr Asn Val His Ala He Asn Leu Thr Leu Val Asn Val Thr Ser Glu Asp Asn Gly Phe Thr Leu Thr Cys He Ala Glu Asn Val Val Gly Met Ser Asn Ala Ser Val Ala Leu Thr Val Tyr Tyr Pro Pro Arg Val Val Ser Leu Glu Glu Pro Glu Leu Arg Leu Glu His Cys He Glu Phe Val Val Arg Gly Asn Pro Pro Pro Thr Leu His Trp Leu His Asn Gly Gin Pro Leu Arg Glu Ser Lys He He His Val Glu Tyr Tyr Gin Glu Gly Glu He Ser Glu Gly Cys Leu Leu Phe Asn Lys Pro Thr His Tyr Asn Asn Gly Asn Tyr Thr Leu He Ala Lys Asn Pro Leu Gly Thr Ala Asn Gin Thr He Asn Gly His Phe Leu Lys Glu Pro Phe Pro Glu Ser Thr Asp Asn Phe He Leu Phe Asp Glu Val Ser Pro Thr Pro Pro He Thr Val Thr His Lys Pro Glu Glu Asp Thr Phe Gly Val Ser He Ala Val Gly Leu Ala Ala Phe Ala Cys Val Leu Leu Val Val Leu Phe Val Met He Asn Lys Tyr Gly Arg Arg Ser Lys Phe Gly Met Lys Gly Pro Val Ala Val He Ser Gly Glu Glu Asp Ser Ala Ser Pro Leu His His He Asn His Gly He Thr Thr Pro Ser Ser Leu Asp Ala Gly Pro Asp Thr Val Val He Gly Met Thr Arg He Pro Val He Glu Asn Pro Gin Tyr Phe Arg Gin Gly His Asn Cys His Lys Pro Asp Thr Tyr Val Gin His He Lys Arg Arg Asp He Val Leu Lys Arg Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu Ser Pro Thr Lys Asp Lys Met Leu Val Ala Val Lys Ala Leu Lys Asp Pro Thr Leu Ala Ala Arg Lys Asp Phe Gin Arg Glu Ala Glu Leu Leu Thr Asn Leu Gin His Glu His He Val Lys Phe Tyr Gly Val Cys Gly Asp Gly Asp Pro Leu lie Met Val Phe Glu Tyr Met Lys His Gly Asp Leu Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Met He Leu Val Asp Gly Gin Pro Arg Gin Ala Lys Gly Glu Leu Gly Leu Ser Gin Met Leu His He Ala Ser Gin He Ala Ser Gly Met Val Tyr Leu Ala Ser Gin His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Ala Asn Leu Leu Val Lys He Gly Asp Phe Gly Met Ser Arg Asp Val Tyr Ser Thr Asp Tyr Tyr Arg Leu Phe Asn Pro Ser Gly Asn Asp Phe Cys He Trp Cys Glu Val Gly Gly His Thr Met Leu Pro He Arg Trp Met Pro Pro Glu Ser He Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp Ser Phe Gly Val He Leu Trp Glu He Phe Thr Tyr Gly Lys Gin Pro Trp Phe Gin Leu Ser Asn Thr Glu Val He Glu Cys He Thr Gin Gly Arg Val Leu Glu Arg Pro Arg Val Cys Pro Lys Glu Val Tyr Asp Val Met Leu Gly Cys Trp Gin Arg Glu Pro Gin Gin Arg Leu Asn He Lys Glu He Tyr Lys He Leu His Ala Leu Gly Lys Ala Thr Pro He Tyr Leu Asp He Leu Gly (SEQ ID NO: 3)
[0250] human ALK tyrosine kinase receptor (ALK)
Met Gly Ala He Gly Leu Leu Trp Leu Leu Pro Leu Leu Leu Ser Thr Ala Ala Val Gly Ser Gly Met Gly Thr Gly Gin Arg Ala Gly Ser Pro Ala Ala Gly Pro Pro Leu Gin Pro Arg Glu Pro Leu Ser Tyr Ser Arg Leu Gin Arg Lys Ser Leu Ala Val Asp Phe Val Val Pro Ser Leu Phe Arg Val Tyr Ala Arg Asp Leu Leu Leu Pro Pro Ser Ser Ser Glu Leu Lys Ala Gly Arg Pro Glu Ala Arg Gly Ser Leu Ala Leu Asp Cys Ala Pro Leu Leu Arg Leu Leu Gly Pro Ala Pro Gly Val Ser Trp Thr Ala Gly Ser Pro Ala Pro Ala Glu Ala Arg Thr Leu Ser Arg Val Leu Lys Gly Gly Ser Val Arg Lys Leu Arg Arg Ala Lys Gin Leu Val Leu Glu Leu Gly Glu Glu Ala He Leu Glu Gly Cys Val Gly Pro Pro Gly Glu Ala Ala Val Gly Leu Leu Gin Phe Asn Leu Ser Glu Leu Phe Ser Trp Trp He Arg Gin Gly Glu Gly Arg Leu Arg He Arg Leu Met Pro Glu Lys Lys Ala Ser Glu Val Gly Arg Glu Gly Arg Leu Ser Ala Ala He Arg Ala Ser Gin Pro Arg Leu Leu Phe Gin He Phe Gly Thr Gly His Ser Ser Leu Glu Ser Pro Thr Asn Met Pro Ser Pro Ser Pro Asp Tyr Phe Thr Trp Asn Leu Thr Trp He Met Lys Asp Ser Phe Pro Phe Leu Ser His Arg Ser Arg Tyr Gly Leu Glu Cys Ser Phe Asp Phe Pro Cys Glu Leu Glu Tyr Ser Pro Pro Leu His Asp Leu Arg Asn Gin Ser Trp Ser Trp Arg Arg He Pro Ser Glu Glu Ala Ser Gin Met Asp Leu Leu Asp Gly Pro Gly Ala Glu Arg Ser Lys Glu Met Pro Arg Gly Ser Phe Leu Leu Leu Asn Thr Ser Ala Asp Ser Lys His Thr He Leu Ser Pro Trp Met Arg Ser Ser Ser Glu His Cys Thr Leu Ala Val Ser Val His Arg His Leu Gin Pro Ser Gly Arg Tyr He Ala Gin Leu Leu Pro His Asn Glu Ala Ala Arg Glu He Leu Leu Met Pro Thr Pro Gly Lys His Gly Trp Thr Val Leu Gin Gly Arg He Gly Arg Pro Asp Asn Pro Phe Arg Val Ala Leu Glu Tyr He Ser Ser Gly Asn Arg Ser Leu Ser Ala Val Asp Phe Phe Ala Leu Lys Asn Cys Ser Glu Gly Thr Ser Pro Gly Ser Lys Met Ala Leu Gin Ser Ser Phe Thr Cys Trp Asn Gly Thr Val Leu Gin Leu Gly Gin Ala Cys Asp Phe His Gin Asp Cys Ala Gin Gly Glu Asp Glu Ser Gin Met Cys Arg Lys Leu Pro Val Gly Phe Tyr Cys Asn Phe Glu Asp Gly Phe Cys Gly Trp Thr Gin Gly Thr Leu Ser Pro His Thr Pro Gin Trp Gin Val Arg Thr Leu Lys Asp Ala Arg Phe Gin Asp His Gin Asp His Ala Leu Leu Leu Ser Thr Thr Asp Val Pro Ala Ser Glu Ser Ala Thr Val Thr Ser Ala Thr Phe Pro Ala Pro He Lys Ser Ser Pro Cys Glu Leu Arg Met Ser Trp Leu He Arg Gly Val Leu Arg Gly Asn Val Ser Leu Val Leu Val Glu Asn Lys Thr Gly Lys Glu Gin Gly Arg Met Val Trp His Val Ala Ala Tyr Glu Gly Leu Ser Leu Trp Gin Trp Met Val Leu Pro Leu Leu Asp Val Ser Asp Arg Phe Trp Leu Gin Met Val Ala Trp Trp Gly Gin Gly Ser Arg Ala He Val Ala Phe Asp Asn He Ser He Ser Leu Asp Cys Tyr Leu Thr He Ser Gly Glu Asp Lys He Leu Gin Asn Thr Ala Pro Lys Ser Arg Asn Leu Phe Glu Arg Asn Pro Asn Lys Glu Leu Lys Pro Gly Glu Asn Ser Pro Arg Gin Thr Pro He Phe Asp Pro Thr Val His Trp Leu Phe Thr Thr Cys Gly Ala Ser Gly Pro His Gly Pro Thr Gin Ala Gin Cys Asn Asn Ala Tyr Gin Asn Ser Asn Leu Ser Val Glu Val Gly Ser Glu Gly Pro Leu Lys Gly He Gin He Trp Lys Val Pro Ala Thr Asp Thr Tyr Ser He Ser Gly Tyr Gly Ala Ala Gly Gly Lys Gly Gly Lys Asn Thr Met Met Arg Ser His Gly Val Ser Val Leu Gly He Phe Asn Leu Glu Lys Asp Asp Met Leu Tyr He Leu Val Gly Gin Gin Gly Glu Asp Ala Cys Pro Ser Thr Asn Gin Leu He Gin Lys Val Cys He Gly Glu Asn Asn Val He Glu Glu Glu He Arg Val Asn Arg Ser Val His Glu Trp Ala Gly Gly Gly Gly Gly Gly Gly Gly Ala Thr Tyr Val Phe Lys Met Lys Asp Gly Val Pro Val Pro Leu He He Ala Ala Gly Gly Gly Gly Arg Ala Tyr Gly Ala Lys Thr Asp Thr Phe His Pro Glu Arg Leu Glu Asn Asn Ser Ser Val Leu Gly Leu Asn Gly Asn Ser Gly Ala Ala Gly Gly Gly Gly Gly Trp Asn Asp Asn Thr Ser Leu Leu Trp Ala Gly Lys Ser Leu Gin Glu Gly Ala Thr Gly Gly His Ser Cys Pro Gin Ala Met Lys Lys Trp Gly Trp Glu Thr Arg Gly Gly Phe Gly Gly Gly Gly Gly Gly Cys Ser Ser Gly Gly Gly Gly Gly Gly Tyr He Gly Gly Asn Ala Ala Ser Asn Asn Asp Pro Glu Met Asp Gly Glu Asp Gly Val Ser Phe He Ser Pro Leu Gly He Leu Tyr Thr Pro Ala Leu Lys Val Met Glu Gly His Gly Glu Val Asn He Lys His Tyr Leu Asn Cys Ser His Cys Glu Val Asp Glu Cys His Met Asp Pro Glu Ser His Lys Val lie Cys Phe Cys Asp His Gly Thr Val Leu Ala Glu Asp Gly Val Ser Cys He Val Ser Pro Thr Pro Glu Pro His Leu Pro Leu Ser Leu He Leu Ser Val Val Thr Ser Ala Leu Val Ala Ala Leu Val Leu Ala Phe Ser Gly He Met He Val Tyr Arg Arg Lys His Gin Glu Leu Gin Ala Met Gin Met Glu Leu Gin Ser Pro Glu Tyr Lys Leu Ser Lys Leu Arg Thr Ser Thr He Met Thr Asp Tyr Asn Pro Asn Tyr Cys Phe Ala Gly Lys Thr Ser Ser He Ser Asp Leu Lys Glu Val Pro Arg Lys Asn He Thr Leu He Arg Gly Leu Gly His Gly Ala Phe Gly Glu Val Tyr Glu Gly Gin Val Ser Gly Met Pro Asn Asp Pro Ser Pro Leu Gin Val Ala Val Lys Thr Leu Pro Glu Val Cys Ser Glu Gin Asp Glu Leu Asp Phe Leu Met Glu Ala Leu He He Ser Lys Phe Asn His Gin Asn He Val Arg Cys He Gly Val Ser Leu Gin Ser Leu Pro Arg Phe He Leu Leu Glu Leu Met Ala Gly Gly Asp Leu Lys Ser Phe Leu Arg Glu Thr Arg Pro Arg Pro Ser Gin Pro Ser Ser Leu Ala Met Leu Asp Leu Leu His Val Ala Arg Asp He Ala Cys Gly Cys Gin Tyr Leu Glu Glu Asn His Phe He His Arg Asp He Ala Ala Arg Asn Cys Leu Leu Thr Cys Pro Gly Pro Gly Arg Val Ala Lys He Gly Asp Phe Gly Met Ala Arg Asp He Tyr Arg Ala Ser Tyr Tyr Arg Lys Gly Gly Cys Ala Met Leu Pro Val Lys Trp Met Pro Pro Glu Ala Phe Met Glu Gly He Phe Thr Ser Lys Thr Asp Thr Trp Ser Phe Gly Val Leu Leu Trp Glu He Phe Ser Leu Gly Tyr Met Pro Tyr Pro Ser Lys Ser Asn Gin Glu Val Leu Glu Phe Val Thr Ser Gly Gly Arg Met Asp Pro Pro Lys Asn Cys Pro Gly Pro Val Tyr Arg He Met Thr Gin Cys Trp Gin His Gin Pro Glu Asp Arg Pro Asn Phe Ala He He Leu Glu Arg He Glu Tyr Cys Thr Gin Asp Pro Asp Val He Asn Thr Ala Leu Pro He Glu Tyr Gly Pro Leu Val Glu Glu Glu Glu Lys Val Pro Val Arg Pro Lys Asp Pro Glu Gly Val Pro Pro Leu Leu Val Ser Gin Gin Ala Lys Arg Glu Glu Glu Arg Ser Pro Ala Ala Pro Pro Pro Leu Pro Thr Thr Ser Ser Gly Lys Ala Ala Lys Lys Pro Thr Ala Ala Glu He Ser Val Arg Val Pro Arg Gly Pro Ala Val Glu Gly Gly His Val Asn Met Ala Phe Ser Gin Ser Asn Pro Pro Ser Glu Leu His Lys Val His Gly Ser Arg Asn Lys Pro Thr Ser Leu Trp Asn Pro Thr Tyr Gly Ser Trp Phe Thr Glu Lys Pro Thr Lys Lys Asn Asn Pro He Ala Lys Lys Glu Pro His Asp Arg Gly Asn Leu Gly Leu Glu Gly Ser Cys Thr Val Pro Pro Asn Val Ala Thr Gly Arg Leu Pro Gly Ala Ser Leu Leu Leu Glu Pro Ser Ser Leu Thr Ala Asn Met Lys Glu Val Pro Leu Phe Arg Leu Arg His Phe Pro Cys Gly Asn Val Asn Tyr Gly Tyr Gin Gin Gin Gly Leu Pro Leu Glu Ala Ala Thr Ala Pro Gly Ala Gly His Tyr Glu Asp Thr He Leu Lys Ser Lys Asn Ser Met Asn Gin Pro Gly Pro (SEQ ID NO: 4)
[0251] human proto-oncogene tyrosine-protein kinase ROS, ROS 1 Met Lys Asn He Tyr Cys Leu He Pro Lys Leu Val Asn Phe Ala Thr Leu Gly Cys Leu Trp He Ser Val Val Gin Cys Thr Val Leu Asn Ser Cys Leu Lys Ser Cys Val Thr Asn Leu Gly Gin Gin Leu Asp Leu Gly Thr Pro His Asn Leu Ser Glu Pro Cys He Gin Gly Cys His Phe Trp Asn Ser Val Asp Gin Lys Asn Cys Ala Leu Lys Cys Arg Glu Ser Cys Glu Val Gly Cys Ser Ser Ala Glu Gly Ala Tyr Glu Glu Glu Val Leu Glu Asn Ala Asp Leu Pro Thr Ala Pro Phe Ala Ser Ser He Gly Ser His Asn Met Thr Leu Arg Trp Lys Ser Ala Asn Phe Ser Gly Val Lys Tyr He He Gin Trp Lys Tyr Ala Gin Leu Leu Gly Ser Trp Thr Tyr Thr Lys Thr Val Ser Arg Pro Ser Tyr Val Val Lys Pro Leu His Pro Phe Thr Glu Tyr He Phe Arg Val Val Trp He Phe Thr Ala Gin Leu Gin Leu Tyr Ser Pro Pro Ser Pro Ser Tyr Arg Thr His Pro His Gly Val Pro Glu Thr Ala Pro Leu He Arg Asn He Glu Ser Ser Ser Pro Asp Thr Val Glu Val Ser Trp Asp Pro Pro Gin Phe Pro Gly Gly Pro He Leu Gly Tyr Asn Leu Arg Leu He Ser Lys Asn Gin Lys Leu Asp Ala Gly Thr Gin Arg Thr Ser Phe Gin Phe Tyr Ser Thr Leu Pro Asn Thr He Tyr Arg Phe Ser He Ala Ala Val Asn Glu Val Gly Glu Gly Pro Glu Ala Glu Ser Ser He Thr Thr Ser Ser Ser Ala Val Gin Gin Glu Glu Gin Trp Leu Phe Leu Ser Arg Lys Thr Ser Leu Arg Lys Arg Ser Leu Lys His Leu Val Asp Glu Ala His Cys Leu Arg Leu Asp Ala He Tyr His Asn He Thr Gly He Ser Val Asp Val His Gin Gin He Val Tyr Phe Ser Glu Gly Thr Leu He Trp Ala Lys Lys Ala Ala Asn Met Ser Asp Val Ser Asp Leu Arg He Phe Tyr Arg Gly Ser Gly Leu He Ser Ser He Ser He Asp Trp Leu Tyr Gin Arg Met Tyr Phe He Met Asp Glu Leu Val Cys Val Cys Asp Leu Glu Asn Cys Ser Asn He Glu Glu He Thr Pro Pro Ser He Ser Ala Pro Gin Lys He Val Ala Asp Ser Tyr Asn Gly Tyr Val Phe Tyr Leu Leu Arg Asp Gly He Tyr Arg Ala Asp Leu Pro Val Pro Ser Gly Arg Cys Ala Glu Ala Val Arg He Val Glu Ser Cys Thr Leu Lys Asp Phe Ala He Lys Pro Gin Ala Lys Arg He He Tyr Phe Asn Asp Thr Ala Gin Val Phe Met Ser Thr Phe Leu Asp Gly Ser Ala Ser His Leu He Leu Pro Arg He Pro Phe Ala Asp Val Lys Ser Phe Ala Cys Glu Asn Asn Asp Phe Leu Val Thr Asp Gly Lys Val He Phe Gin Gin Asp Ala Leu Ser Phe Asn Glu Phe He Val Gly Cys Asp Leu Ser His He Glu Glu Phe Gly Phe Gly Asn Leu Val He Phe Gly Ser Ser Ser Gin Leu His Pro Leu Pro Gly Arg Pro Gin Glu Leu Ser Val Leu Phe Gly Ser His Gin Ala Leu Val Gin Trp Lys Pro Pro Ala Leu Ala He Gly Ala Asn Val He Leu He Ser Asp He He Glu Leu Phe Glu Leu Gly Pro Ser Ala Trp Gin Asn Trp Thr Tyr Glu Val Lys Val Ser Thr Gin Asp Pro Pro Glu Val Thr His He Phe Leu Asn He Ser Gly Thr Met Leu Asn Val Pro Glu Leu Gin Ser Ala Met Lys Tyr Lys Val Ser Val Arg Ala Ser Ser Pro Lys Arg Pro Gly Pro Trp Ser Glu Pro Ser Val Gly Thr Thr Leu Val Pro Ala Ser Glu Pro Pro Phe He Met Ala Val Lys Glu Asp Gly Leu Trp Ser Lys Pro Leu Asn Ser Phe Gly Pro Gly Glu Phe Leu Ser Ser Asp He Gly Asn Val Ser Asp Met Asp Trp Tyr Asn Asn Ser Leu Tyr Tyr Ser Asp Thr Lys Gly Asp Val Phe Val Trp Leu Leu Asn Gly Thr Asp He Ser Glu Asn Tyr His Leu Pro Ser He Ala Gly Ala Gly Ala Leu Ala Phe Glu Trp Leu Gly His Phe Leu Tyr Trp Ala Gly Lys Thr Tyr Val He Gin Arg Gin Ser Val Leu Thr Gly His Thr Asp He Val Thr His Val Lys Leu Leu Val Asn Asp Met Val Val Asp Ser Val Gly Gly Tyr Leu Tyr Trp Thr Thr Leu Tyr Ser Val Glu Ser Thr Arg Leu Asn Gly Glu Ser Ser Leu Val Leu Gin Thr Gin Pro Trp Phe Ser Gly Lys Lys Val He Ala Leu Thr Leu Asp Leu Ser Asp Gly Leu Leu Tyr Trp Leu Val Gin Asp Ser Gin Cys He His Leu Tyr Thr Ala Val Leu Arg Gly Gin Ser Thr Gly Asp Thr Thr He Thr Glu Phe Ala Ala Trp Ser Thr Ser Glu He Ser Gin Asn Ala Leu Met Tyr Tyr Ser Gly Arg Leu Phe Trp He Asn Gly Phe Arg He He Thr Thr Gin Glu He Gly Gin Lys Thr Ser Val Ser Val Leu Glu Pro Ala Arg Phe Asn Gin Phe Thr He He Gin Thr Ser Leu Lys Pro Leu Pro Gly Asn Phe Ser Phe Thr Pro Lys Val He Pro Asp Ser Val Gin Glu Ser Ser Phe Arg He Glu Gly Asn Ala Ser Ser Phe Gin He Leu Trp Asn Gly Pro Pro Ala Val Asp Τ Gly Val Val Phe Tyr Ser Val Glu Phe Ser Ala His Ser Lys Phe Leu Ala Ser Glu Gin His Ser Leu Pro Val Phe Thr Val Glu Gly Leu Glu Pro Tyr Ala Leu Phe Asn Leu Ser Val Thr Pro Tyr Thr Tyr Trp Gly Lys Gly Pro Lys Thr Ser Leu Ser Leu Arg Ala Pro Glu Thr Val Pro Ser Ala Pro Glu Asn Pro Arg He Phe lie Leu Pro Ser Gly Lys Cys Cys Asn Lys Asn Glu Val Val Val Glu Phe Arg Trp Asn Lys Pro Lys His Glu Asn Gly Val Leu Thr Lys Phe Glu He Phe Tyr Asn He Ser Asn Gin Ser He Thr Asn Lys Thr Cys Glu Asp Trp He Ala Val Asn Val Thr Pro Ser Val Met Ser Phe Gin Leu Glu Gly Met Ser Pro Arg Cys Phe He Ala Phe Gin Val Arg Ala Phe Thr Ser Lys Gly Pro Gly Pro Tyr Ala Asp Val Val Lys Ser Thr Thr Ser Glu He Asn Pro Phe Pro His Leu He Thr Leu Leu Gly Asn Lys He Val Phe Leu Asp Met Asp Gin Asn Gin Val Val Trp Thr Phe Ser Ala Glu Arg Val He Ser Ala Val Cys Tyr Thr Ala Asp Asn Glu Met Gly Tyr Tyr Ala Glu Gly Asp Ser Leu Phe Leu Leu His Leu His Asn Arg Ser Ser Ser Glu Leu Phe Gin Asp Ser Leu Val Phe Asp He Thr Val He Thr He Asp Trp He Ser Arg His Leu Tyr Phe Ala Leu Lys Glu Ser Gin Asn Gly Met Gin Val Phe Asp Val Asp Leu Glu His Lys Val Lys Tyr Pro Arg Glu Val Lys He His Asn Arg Asn Ser Thr He He Ser Phe Ser Val Tyr Pro Leu Leu Ser Arg Leu Tyr Trp Thr Glu Val Ser Asn Phe Gly Tyr Gin Met Phe Tyr Tyr Ser He He Ser His Thr Leu His Arg He Leu Gin Pro Thr Ala Thr Asn Gin Gin Asn Lys Arg Asn Gin Cys Ser Cys Asn Val Thr Glu Phe Glu Leu Ser Gly Ala Met Ala He Asp Thr Ser Asn Leu Glu Lys Pro Leu He Tyr Phe Ala Lys Ala Gin Glu He Trp Ala Met Asp Leu Glu Gly Cys Gin Cys Trp Arg Val He Thr Val Pro Ala Met Leu Ala Gly Lys Thr Leu Val Ser Leu Thr Val Asp Gly Asp Leu He Tyr Trp He He Thr Ala Lys Asp Ser Thr Gin He Tyr Gin Ala Lys Lys Gly Asn Gly Ala He Val Ser Gin Val Lys Ala Leu Arg Ser Arg His lie Leu Ala Tyr Ser Ser Val Met Gin Pro Phe Pro Asp Lys Ala Phe Leu Ser Leu Ala Ser Asp Thr Val Glu Pro Thr He Leu Asn Ala Thr Asn Thr Ser Leu Thr He Arg Leu Pro Leu Ala Lys Thr Asn Leu Thr Trp Tyr Gly He Thr Ser Pro Thr Pro Thr Tyr Leu Val Tyr Tyr Ala Glu Val Asn Asp Arg Lys Asn Ser Ser Asp Leu Lys Tyr Arg He Leu Glu Phe Gin Asp Ser He Ala Leu He Glu Asp Leu Gin Pro Phe Ser Thr Tyr Met lie Gin He Ala Val Lys Asn Tyr Tyr Ser Asp Pro Leu Glu His Leu Pro Pro Gly Lys Glu He Trp Gly Lys Thr Lys Asn Gly Val Pro Glu Ala Val Gin Leu He Asn Thr Thr Val Arg Ser Asp Thr Ser Leu He He Ser Trp Arg Glu Ser His Lys Pro Asn Gly Pro Lys Glu Ser Val Arg Tyr Gin Leu Ala He Ser His Leu Ala Leu He Pro Glu Thr Pro Leu Arg Gln Ser Glu Phe Pro Asn Gly Arg Leu Thr Leu Leu Val Thr Arg Leu Ser Gly Gly Asn He Tyr Val Leu Lys Val Leu Ala Cys His Ser Glu Glu Met Trp Cys Thr Glu Ser His Pro Val Thr Val Glu Met Phe Asn Thr Pro Glu Lys Pro Tyr Ser Leu Val Pro Glu Asn Thr Ser Leu Gin Phe Asn Trp Lys Ala Pro Leu Asn Val Asn Leu He Arg Phe Trp Val Glu Leu Gin Lys Trp Lys Tyr Asn Glu Phe Tyr His Val Lys Thr Ser Cys Ser Gin Gly Pro Ala Tyr Val Cys Asn He Thr Asn Leu Gin Pro Tyr Thr Ser Tyr Asn Val Arg Val Val Val Val Tyr Lys Thr Gly Glu Asn Ser Thr Ser Leu Pro Glu Ser Phe Lys Thr Lys Ala Gly Val Pro Asn Lys Pro Gly He Pro Lys Leu Leu Glu Gly Ser Lys Asn Ser He Gin Trp Glu Lys Ala Glu Asp Asn Gly Cys Arg He Thr Tyr Tyr He Leu Glu He Arg Lys Ser Thr Ser Asn Asn Leu Gin Asn Gin Asn Leu Arg Trp Lys Met Thr Phe Asn Gly Ser Cys Ser Ser Val Cys Thr Trp Lys Ser Lys Asn Leu Lys Gly He Phe Gin Phe Arg Val Val Ala Ala Asn Asn Leu Gly Phe Gly Glu Tyr Ser Gly He Ser Glu Asn He He Leu Val Gly Asp Asp Phe Trp He Pro Glu Thr Ser Phe He Leu Thr He He Val Gly He Phe Leu Val Val Thr He Pro Leu Thr Phe Val Trp His Arg Arg Leu Lys Asn Gin Lys Ser Ala Lys Glu Gly Val Thr Val Leu He Asn Glu Asp Lys Glu Leu Ala Glu Leu Arg Gly Leu Ala Ala Gly Val Gly Leu Ala Asn Ala Cys Tyr Ala lie His Thr Leu Pro Thr Gin Glu Glu lie Glu Asn Leu Pro Ala Phe Pro Arg Glu Lys Leu Thr Leu Arg Leu Leu Leu Gly Ser Gly Ala Phe Gly Glu Val Tyr Glu Gly Thr Ala Val Asp He Leu Gly Val Gly Ser Gly Glu He Lys Val Ala Val Lys Thr Leu Lys Lys Gly Ser Thr Asp Gin Glu Lys He Glu Phe Leu Lys Glu Ala His Leu Met Ser Lys Phe Asn His Pro Asn He Leu Lys Gin Leu Gly Val Cys Leu Leu Asn Glu Pro Gin Tyr He He Leu Glu Leu Met Glu Gly Gly Asp Leu Leu Thr Tyr Leu Arg Lys Ala Arg Met Ala Thr Phe Tyr Gly Pro Leu Leu Thr Leu Val Asp Leu Val Asp Leu Cys Val Asp He Ser Lys Gly Cys Val Tyr Leu Glu Arg Met His Phe He His Arg Asp Leu Ala Ala Arg Asn Cys Leu Val Ser Val Lys Asp Tyr Thr Ser Pro Arg He Val Lys He Gly Asp Phe Gly Leu Ala Arg Asp He Tyr Lys Asn Asp Tyr Tyr Arg Lys Arg Gly Glu Gly Leu Leu Pro Val Arg Trp Met Ala Pro Glu Ser Leu Met Asp Gly He Phe Thr Thr Gin Ser Asp Val Trp Ser Phe Gly He Leu He Trp Glu He Leu Thr Leu Gly His Gin Pro Tyr Pro
Ala His Ser Asn Leu Asp Val Leu Asn Tyr Val Gin Thr Gly Gly
Arg Leu Glu Pro Pro Arg Asn Cys Pro Asp Asp Leu Trp Asn Leu
Met Thr Gin Cys Trp Ala Gin Glu Pro Asp Gin Arg Pro Thr Phe
His Arg He Gin Asp Gin Leu Gin Leu Phe Arg Asn Phe Phe Leu
Asn Ser He Tyr Lys Ser Arg Asp Glu Ala Asn Asn Ser Gly Val
He Asn Glu Ser Phe Glu Gly Glu Asp Gly Asp Val He Cys Leu
Asn Ser Asp Asp He Met Pro Val Ala Leu Met Glu Thr Lys Asn
Arg Glu Gly Leu Asn Tyr Met Val Leu Ala Thr Glu Cys Gly Gin
Gly Glu Glu Lys Ser Glu Gly Pro Leu Gly Ser Gin Glu Ser Glu
Ser Cys Gly Leu Arg Lys Glu Glu Lys Glu Pro His Ala Asp Lys
Asp Phe Cys Gin Glu Lys Gin Val Ala Tyr Cys Pro Ser Gly Lys
Pro Glu Gly Leu Asn Tyr Ala Cys Leu Thr His Ser Gly Tyr Gly
Asp Gly Ser Asp (SEQ ID NO: 5)
[0252] Other embodiments are set forth in the following claims, along with the full scope of equivalents to which such claims are entitled.

Claims

WHAT IS CLAIMED IS:
1. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an inhibitor of ALK, ROSl, TrkA, TrkB, or TrkC activity, or a combination thereof; and the second agent is a MEK inhibitor or an ERK inhibitor.
2. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in ALK.
3. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is an ALK inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the ALK receptor tyrosine kinase polypeptide.
4. The method of claim 3, wherein the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G1202 or G1269 of the ALK polypeptide set forth in SEQ ID NO: 4.
5. The method of claim 4, wherein the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G1202 of the ALK polypeptide set forth in SEQ ID NO: 4.
6. The method of claim 4, wherein the at least one mutation in the ALK receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G1269 of the ALK polypeptide set forth in SEQ ID NO: 4.
7. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a ROSl inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in ROS l.
8. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a ROSl inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the ROSl receptor tyrosine kinase polypeptide.
9. The method of claim 8, wherein the at least one mutation in the ROSl receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G2032 or G2101 of the ROSl polypeptide set forth in SEQ ID NO: 5.
10. The method of claim 9, wherein the at least one mutation in the ROSl receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G2032 of the ROSl polypeptide set forth in SEQ ID NO: 5.
11. The method of claim 9, wherein the at least one mutation in the ROS 1 receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G2101 of the ROSl polypeptide set forth in SEQ ID NO: 5.
12. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkA inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRKl .
13. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkA inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkA receptor tyrosine kinase polypeptide.
14. The method of claim 13, wherein the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G595 or G667 of the TrkA polypeptide set forth in SEQ ID NO: 1.
15. The method of claim 14, wherein the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G595 of the TrkA polypeptide set forth in SEQ ID NO: 1.
16. The method of claim 15, wherein the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G595R).
17. The method of claim 14, wherein the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G667 of the TrkA polypeptide set forth in SEQ ID NO: 1.
18. The method of claim 17, wherein the at least one mutation in the TrkA receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G667C).
19. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkB inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK2.
20. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkB inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkB receptor tyrosine kinase polypeptide.
21. The method of claim 20, wherein the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G639 or G709 of the TrkB polypeptide set forth in SEQ ID NO: 2.
22. The method of claim 21, wherein the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G639 of the TrkB polypeptide set forth in SEQ ID NO: 2.
23. The method of claim 22, wherein the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G639R).
24. The method of claim 21, wherein the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G709 of the TrkB polypeptide set forth in SEQ ID NO: 2.
25. The method of claim 24, wherein the at least one mutation in the TrkB receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G709C).
26. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkC inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one genetic alteration in NTRK3.
27. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a combination comprising a therapeutically effective amount of a first agent and a therapeutically effective amount of a second agent, wherein the first agent is a TrkC inhibitor; the second agent is a MEK inhibitor or an ERK inhibitor; and the patient has at least one mutation in the TrkC receptor tyrosine kinase polypeptide.
28. The method of claim 27, wherein the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at a position corresponding to amino acid residue G623 or G696 of the TrkC polypeptide set forth in SEQ ID NO: 3.
29. The method of claim 28, wherein the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G623 of the TrkC polypeptide set forth in SEQ ID NO: 3.
30. The method of claim 29, wherein the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is Glu-to-Arg substitution (G623R).
31. The method of claim 28, wherein the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is at the position corresponding to amino acid residue G696 of the TrkC polypeptide set forth in SEQ ID NO: 3.
32. The method of claim 31, wherein the at least one mutation in the TrkC receptor tyrosine kinase polypeptide is Glu-to-Cys substitution (G696C).
33. The method of any one of claims 1-32, wherein the first agent is N-[5-(3,5- difluorobenzyl)-lH-indazol-3-yl]-4-(4-methyl-piperazin-l-yl)-2-(tetrahydro-2H- pyran-4-ylamino)-benzamide, or a pharmaceutically acceptable salt thereof.
34. The method of any one of claims 1-33, wherein the second agent is a MEK inhibitor.
35. The method of claim 34, wherein the MEK inhibitor is an inhibitor of MEK1,
MEK2, or a combination thereof.
36. The method of claim 34, wherein the MEK inhibitor is selected from the group
consisting of PD0325901, selumetinib, cobimetinib, refametinib, trametinib, pimasertib, binimetinib, AZD8330, R04987655, R05126766, WX-554, E-6201, GDC-0623, TAK-733, RG-7304, CKBP-002, RDEA-436, sorafenib, PD-184352, GSK-2091976A, and AS-703988.
37. The method of claim 36, wherein the MEK inhibitor is PD0325901.
38. The method of claim 36, wherein the MEK inhibitor is selumetinib.
39. The method of claim 36, wherein the MEK inhibitor is cobimetinib.
The method of claim 36, wherein the MEK inhibitor is refametinib.
The method of claim 36, wherein the MEK inhibitor is trametinib.
The method of claim 36, wherein the MEK inhibitor is pimasertib.
The method of claim 36, wherein the MEK inhibitor is binimetinib.
The method of claim 36, wherein the MEK inhibitor is AZD8330.
The method of claim 36, wherein the MEK inhibitor is R04987655.
The method of claim 36, wherein the MEK inhibitor is R05126766.
The method of claim 36, wherein the MEK inhibitor is WX-554.
The method of claim 36, wherein the MEK inhibitor is E-6201.
The method of claim 36, wherein the MEK inhibitor is GDC-0623.
The method of claim 36, wherein the MEK inhibitor is TAK-733.
The method of claim 36, wherein the MEK inhibitor is RG-7304.
The method of claim 36, wherein the MEK inhibitor is CKBP-002.
The method of claim 36, wherein the MEK inhibitor is RDEA-436.
The method of claim 36, wherein the MEK inhibitor is sorafenib.
The method of claim 36, wherein the MEK inhibitor is PD-184352.
The method of claim 36, wherein the MEK inhibitor is GSK-2091976A.
The method of claim 36, wherein the MEK inhibitor is AS-703988.
58. The method of any one of claims 1-33, wherein the second agent is an ERK inhibitor.
59. The method of claim 58, wherein the ERK inhibitor is an inhibitor of ERKl, ERK2, or a combination thereof.
60. The method of claim 58, wherein the ERK inhibitor is selected from the group consisting of TG-02, MK-8353, ulixertinib, HE-3235, AEZS-134, AEZS-136, and IDN-5491.
61. The method of claim 60, wherein the ERK inhibitor is TG-02.
62. The method of claim 60, wherein the ERK inhibitor is MK-8353.
63. The method of claim 60, wherein the ERK inhibitor is ulixertinib.
64. The method of claim 60, wherein the ERK inhibitor is HE-3235.
65. The method of claim 60, wherein the ERK inhibitor is AEZS-134.
66. The method of claim 60, wherein the ERK inhibitor is AEZS-136.
67. The method of claim 60, wherein the ERK inhibitor is IDN-5491.
68. The method of any one of claims 1-67, wherein the cancer is selected from non- small cell lung cancer, papillary thyroid cancer, neuroblastoma, pancreatic cancer, melanoma, and colorectal cancer.
69. The method of claim 68, wherein the cancer is non-small cell lung cancer.
70. The method of claim 68, wherein the cancer is papillary thyroid cancer.
71. The method of claim 68, wherein the cancer is neuroblastoma.
72. The method of claim 68, wherein the cancer is pancreatic cancer.
73. The method of claim 68, wherein the cancer is melanoma.
74. The method of claim 68, wherein the cancer is colorectal cancer.
75. The method of any one of claims 1 -74, wherein the combination is a pharmaceutical composition comprising the therapeutically effective amount of the first agent, the therapeutically effective amount of the second agent, and at least one
pharmaceutically acceptable carrier.
76. The method of any one of claims 1 -74 wherein the combination is concurrent
administration of a first pharmaceutical composition comprising the therapeutically effective amount of the first agent and a second pharmaceutical composition comprising the therapeutically effective amount of the second agent.
77. The method of any one of claims 1-74, wherein the combination is sequential
administration of a first pharmaceutical composition comprising the therapeutically effective amount of the first agent and a second pharmaceutical composition comprising the therapeutically effective amount of the second agent.
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