Combination therapy for the treatment of mastocytosis

Abstract

To provide a method for the treatment of mastocytosis.SOLUTION: The present disclosure relates to the use of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or 1-(5-(7-amino-1-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)-4-bromo-2-fluorophenyl)-3-phenylurea, or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor such as a RAS, RAF, MEK or ERK inhibitor.SELECTED DRAWING: None

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 624,453, filed January 31, 2018, which is incorporated herein by reference in its entirety.

[0002] c-KIT (also known as KIT, CD117, and stem cell factor receptor) is a 145 kDa transmembrane tyrosine kinase protein that acts as a type III receptor. The c-KIT proto-oncogene, located on chromosome 4q11-21, encodes the c-KIT receptor, whose ligand is stem cell factor (SCF, hematopoietic stem cell factor, kit ligand, mast cell growth factor). The receptor possesses tyrosine protein kinase activity, and binding of the ligand SCF leads to the autophosphorylation of c-KIT and its association with substrates such as phosphatidylinositol 3-kinase (PI3K). Tyrosine phosphorylation by protein tyrosine kinases is particularly important in cell signaling and can mediate signals for key intracellular processes such as proliferation, survival, differentiation, apoptosis, adhesion, invasion, and migration.

[0003] The receptor tyrosine kinase c-KIT gene is important for mast cell proliferation, survival, differentiation, and homeostasis. Activating mutations or overexpression of the c-KIT gene enhance the ability of the c-KIT receptor to initiate intracellular pathways that result in abnormal mast cell proliferation.

[0004] Mastocytosis is a rare disorder characterized by the abnormal accumulation of mast cells (MCs) in the skin, bone marrow, and internal organs (e.g., liver, spleen, gastrointestinal tract, and lymph nodes). Cases beginning in adulthood tend to be chronic and involve bone marrow in addition to the skin, whereas in childhood, symptoms are often characterized by skin manifestations without internal organ involvement and often resolve during adolescence. In most adult patients, mastocytosis tends to be persistent, and in a minority of patients, it may progress to more advanced categories. Mastocytosis can be classified into specific types depending on the patient's symptoms and overall condition. Types of mastocytosis include cutaneous mastocytosis (e.g., maculopapular cutaneous mastocytosis, mastocytoma, and diffuse cutaneous mastocytosis) and systemic mastocytosis (e.g., indolent systemic mastocytosis (ISM), smoldering systemic mastocytosis (SSM), systemic mastocytosis with associated clonal hematologic non-mast cell lineage disease (SM-AHN), aggressive systemic mastocytosis (ASM), mast cell leukemia (MCL), and mast cell sarcoma). In rare cases, invasive neoplastic mast cell proliferation results in end-organ damage, significantly shortening patient lifespan and requiring cytoreductive therapy. The pathological accumulation of neoplastic mast cells caused by oncogenic mutations in c-KIT has been found to be the cause of systemic mastocytosis. The predominant activating KIT mutation is an aspartic acid to valine substitution at residue 816 (KIT D816V). Patients with systemic mastocytosis, whose mast cells frequently contain the activating D816V c-KIT mutation, may be inactive against invasive disease and may experience symptoms related to mast cell mediator release. Recurrent anaphylaxis or inactive systemic mastocytosis with vascular collapse in the absence of skin lesions is a specific subtype of inactive systemic mastocytosis, and this clonal MC activation disorder represents a significant proportion of all mast cell activation syndromes. The V560G KIT mutation is extremely rare in patients with systemic mastocytosis, and its biological and prognostic impact is unknown. Currently, most tyrosine kinase inhibitors only show modest efficacy in advanced disease states and are associated with significant side effects. Furthermore, the KIT D816V mutation is a rare condition that affects mastocytosis. Some aggressive KIT mutations, including those listed above, are resistant to classical ATP-competitive KIT inhibitors, such as imatinib, sunitinib, sorafenib, and regorafenib. An inhibitor of c-KIT D816V, midostaurin, was recently approved for the treatment of SM in 2017.

[0005] Although the c-KIT D816V mutation is a c-KIT primary mutation reported as a driver of systemic mastocytosis (SM), secondary c-KIT mutations conferring resistance to specific c-KIT inhibitors ("resistant c-KIT secondary mutations") have also been reported in mastocytosis patients, including, for example, point mutations, in-frame deletions or insertions, or missense mutations of Y269C, Y503_F504insAY, V560D, or K642E in the c-KIT gene.

[0006] Activating mutations or overexpression of the c-KIT gene lead to neoplastic mast cell proliferation. Given the complex functions of c-KIT and the potential utility of c-KIT inhibitors in the treatment of drug-resistant systemic mastocytosis, inhibitors and therapeutic treatments with beneficial therapeutic properties are needed. Summary of the Invention

[0007] The present disclosure relates in part to the use of c-KIT inhibitors, such as 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea (Compound A), and MAPKAP pathway inhibitors, such as MEK inhibitors, such as trametinib, or ERK inhibitors, such as ulixertinib, or RAF inhibitors, such as LY3009120, to induce apoptosis of mastocytosis cells.

[0008] Provided in the present disclosure is a method for treating mastocytosis in a patient in need thereof, comprising administering to the patient an effective amount of a c-KIT inhibitor, for example, 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea (Compound A described herein), and an effective amount of an inhibitor of mitogen-activated protein kinase (MEK inhibitor), and / or an effective amount of an extracellular signal-regulated kinase inhibitor (ERK inhibitor).

[0009] For example, a method for treating systemic mastocytosis in a patient in need thereof includes administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof, and an effective amount of an inhibitor of mitogen-activated protein kinase (MEK inhibitor) or an ERK inhibitor.

[0010] Also contemplated in this disclosure is a method of treating mastocytosis in a patient in need thereof, comprising administering to the patient an effective amount of a c-KIT inhibitor and an effective amount of a RAF inhibitor. [Brief explanation of the drawings]

[0011] [Figure 1-1] FIG. 1A shows a graphical representation of cell proliferation following treatment with the indicated agents together with Compound A compared to vehicle control in HMC1.1 V560G (left panel) and HMC1.2 V560G / D816V (right panel) cell lines.

[0012] [Figure 1-2] FIG. 1B shows a graphical representation of cell proliferation following treatment with the indicated agents together with Compound B compared to vehicle control in HMC1.1 V560G (left panel) and HMC1.2 V560G / D816V (right panel) cell lines.

[0013] [Figure 2-1] FIG. 2A shows a graphical representation of caspase activity in the HMC1.2 V560G / D816V cell line after various indicated treatments with Compound A and trametinib.

[0014] [Figure 2-2] FIG. 2B provides a matrix representation of synergy based on the combination index method for various treatments with Compound A and trametinib in the HMC1.2 V560G / D816V cell line.

[0015] [Figure 2-3] FIG. 2C provides the combination index plot for the combination of the MEK inhibitor trametinib with Compound A.

[0016] [Figure 3-1] FIG. 3A shows a graphical representation of caspase activity in the HMC1.2 V560G / D816V cell line after various indicated treatments with Compound B and trametinib.

[0017] [Figure 3-2] FIG. 3B provides a matrix diagram of synergy based on the combination index method for various treatments with Compound B and trametinib in the HMC1.2 V560G / D816V cell line.

[0018] [Figure 3-3] FIG. 3C provides the combination index plot for the combination of the MEK inhibitor trametinib with Compound B.

[0019] [Figure 4-1] FIG. 4A shows a graphical representation of caspase activity in the HMC1.2 V560G / D816V cell line after various indicated treatments with Compound A and binimetinib.

[0020] [Figure 4-2] FIG. 4B provides a matrix representation of synergy based on the combination index method for various treatments with Compound A and binimetinib in the HMC1.2 V560G / D816V cell line.

[0021] [Figure 4-3] FIG. 4C provides a combination index plot for the combination of the MEK inhibitor binimetinib with Compound A.

[0022] [Figure 5-1] FIG. 5A shows a graphical representation of caspase activity in the HMC1.2 V560G / D816V cell line after various indicated treatments with Compound B and binimetinib.

[0023] [Figure 5-2] FIG. 5B provides a matrix representation of synergy based on the combination index method for various treatments with Compound B and binimetinib in the HMC1.2 V560G / D816V cell line.

[0024] [Figure 5-3] FIG. 5C provides the combination index plot for the combination of the MEK inhibitor binimetinib with Compound B.

[0025] [Figure 6-1] FIG. 6A shows a graphical representation of caspase activity in the HMC1.2 V560G / D816V cell line after various indicated treatments with Compound A and cobimetinib.

[0026] [Figure 6-2] FIG. 6B provides a matrix diagram of synergy based on the combination index method for various treatments with Compound A and cobimetinib in the HMC1.2 V560G / D816V cell line.

[0027] [Figure 6-3] FIG. 6C provides a combination index plot for the combination of the MEK inhibitor cobimetinib with Compound A.

[0028] [Figure 7-1] FIG. 7A shows a graphical representation of caspase activity in the HMC1.2 V560G / D816V cell line after various indicated treatments with Compound B and cobimetinib.

[0029] [Figure 7-2] FIG. 7B provides a matrix representation of synergy based on the combination index method for various treatments with Compound B and cobimetinib in the HMC1.2 V560G / D816V cell line.

[0030] [Figure 7-3] FIG. 7C provides the combination index plot for the combination of the MEK inhibitor cobimetinib with Compound B.

[0031] [Figure 8-1] FIG. 8A shows a graphical representation of caspase activity in the HMC1.2 V560G / D816V cell line after various indicated treatments with Compound A and the ERK inhibitor ulixertinib.

[0032] [Figure 8-2] FIG. 8B provides a matrix representation of synergy based on the combination index method for various treatments with Compound A and the ERK inhibitor ulixertinib in the HMC1.2 V560G / D816V cell line.

[0033] [Figure 8-3] FIG. 8C provides a combination index plot for the combination of the ERK inhibitor ulixertinib with Compound A.

[0034] [Figure 9] FIG. 9A shows the inhibition of colony growth from treatment with single-agent Compound A, single-agent trametinib, and the combination of the MEK inhibitor trametinib with Compound A in HMC1.2 V560G / D816V cells.

[0035] Figure 9B shows a graphical representation of the inhibition of colony growth from treatment in HMC1.2 V560G / D816V cells with single-agent Compound A, single-agent trametinib, and the combination of the MEK inhibitor trametinib with Compound A. Arrows indicate the absence of colony growth.

[0036] [Figure 10] FIG. 10A shows the inhibition of colony growth from treatment with single-agent Compound B, single-agent trametinib, and the combination of the MEK inhibitor trametinib with Compound B in HMC1.2 V560G / D816V cells.

[0037] 10B shows a graphical representation of the inhibition of colony growth from treatment in HMC1.2 V560G / D816V cells with single-agent Compound B, single-agent trametinib, and the combination of the MEK inhibitor trametinib with Compound B. Arrows indicate the absence of colony growth.

[0038] [Figure 11] FIG. 11A shows the inhibition of colony growth from treatment with single-agent Compound A, single-agent binimetinib, and the combination of the MEK inhibitor binimetinib with Compound A in HMC1.2 V560G / D816V cells.

[0039] Figure 11B shows the effect of single agent Compound A, single agent Compound B, and single agent Compound C on the expression of ATP in HMC1.2 V560G / D816V cells. 1 shows a graphical representation of the inhibition of colony growth from treatment with the MEK inhibitor binimetinib, and the combination of the MEK inhibitor binimetinib with Compound A. Arrows indicate the absence of colony growth.

[0040] [Figure 12] FIG. 12A shows the inhibition of colony growth from treatment with single-agent Compound B, single-agent binimetinib, and the combination of the MEK inhibitor binimetinib with Compound B in HMC1.2 V560G / D816V cells.

[0041] 12B shows a graphical representation of the inhibition of colony growth from treatment in HMC1.2 V560G / D816V cells with single-agent Compound B, single-agent binimetinib, and the combination of the MEK inhibitor binimetinib with Compound B. Arrows indicate the absence of colony growth.

[0042] [Figure 13] FIG. 13A shows the inhibition of colony growth from treatment with single-agent Compound A, single-agent cobimetinib, and the combination of the MEK inhibitor cobinimetinib with Compound A in HMC1.2 V560G / D816V cells.

[0043] FIG. 13B shows a graphical representation of the inhibition of colony growth from treatment with single agent Compound A, single agent cobimetinib, and the combination of the MEK inhibitor cobimetinib with Compound A in HMC1.2 V560G / D816V cells.

[0044] [Figure 14] FIG. 14A shows the inhibition of colony growth from treatment with single agent Compound B, single agent cobimetinib, and the combination of the MEK inhibitor cobimetinib with Compound B in HMC1.2 V560G / D816V cells.

[0045] FIG. 14B shows a graphical representation of the inhibition of colony growth from treatment with single agent Compound B, single agent cobimetinib, and the combination of the MEK inhibitor cobimetinib with Compound B in HMC1.2 V560G / D816V cells.

[0046] [Figure 15] Figure 15A shows a graphical representation of caspase activity in HMC1.2 V560G / D816V cells transfected with empty vector (EV, left panel) or N-ras G12D (right panel) after various treatments with Compound A and trametinib at 24 hours.

[0047] Figure 15B shows a graphical representation of caspase activity in HMC1.2 V560G / D816V cells transfected with empty vector (left panel) or N-ras G12D (right panel) after various treatments with Compound A and trametinib at 48 hours.

[0048] [Figure 16] Figure 16A shows a graphical representation of caspase activity in HMC1.2 V560G / D816V cells transfected with empty vector (EV, left panel) or N-ras G12D (right panel) after various treatments with Compound A and cobimetinib at 24 hours.

[0049] Figure 16B shows a graphical representation of caspase activity in HMC1.2 V560G / D816V cells transfected with empty vector (left panel) or N-ras G12D (right panel) after various treatments with Compound A and cobimetinib at 48 hours.

[0050] [Figure 17-1] FIG. 17A shows the inhibition of colony growth from treatment with single-agent Compound A, single-agent trametinib, and the combination of the MEK inhibitor trametinib and Compound A in empty vector (EV)-transfected HMC1.2 V560G / D816V cells.

[0051] Figure 17B shows HMC1.2 V560G / D81 transfected with empty vector (EV). 1 shows a graphical representation of the inhibition of colony growth from treatment in 6V cells with single agent Compound A, single agent trametinib, and the combination of the MEK inhibitor trametinib with Compound A. Arrows indicate the absence of colony growth.

[0052] [Figure 17-2] FIG. 17C shows the inhibition of colony growth from treatment with single-agent Compound A, single-agent trametinib, and the combination of the MEK inhibitor trametinib and Compound A in N-ras G12D-transfected HMC1.2 V560G / D816V cells.

[0053] Figure 17D shows a graphical representation of the inhibition of colony growth from treatment of N-ras G12D-transfected HMC1.2 V560G / D816V cells with single-agent Compound A, single-agent trametinib, and the combination of the MEK inhibitor trametinib and Compound 1. Arrows indicate the absence of colony growth.

[0054] [Figure 18-1] FIG. 18A shows the inhibition of colony growth from treatment with single-agent Compound A, single-agent cobimetinib, and the combination of the MEK inhibitor cobimetinib and Compound A in empty vector (EV)-transfected HMC1.2 V560G / D816V cells.

[0055] Figure 18B shows the inhibition of colony growth from treatment in empty vector (EV)-transfected HMC1.2 V560G / D816V cells with single-agent Compound A, single-agent cobimetinib, and the combination of the MEK inhibitor cobimetinib and Compound A. Arrows indicate the absence of colony growth.

[0056] [Figure 18-2] FIG. 18C shows the inhibition of colony growth from treatment with single-agent Compound A, single-agent cobimetinib, and the combination of the MEK inhibitor cobimetinib and Compound A in N-ras G12D-transfected HMC1.2 V560G / D816V cells.

[0057] Figure 18D shows a graphical representation of the inhibition of colony growth from treatment of N-ras G12D-transfected HMC1.2V560G / D816V cells with single-agent Compound A, single-agent cobimetinib, and the combination of the MEK inhibitor cobimetinib and Compound A. Arrows indicate the absence of colony growth. DETAILED DESCRIPTION OF THE INVENTION

[0058] As shown in the accompanying Examples, the combination of a c-KIT inhibitor, e.g., 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea (Compound A), with a MAPKAP pathway inhibitor, e.g., the MEK inhibitor trametinib, or the ERK inhibitor ulixertinib, or the RAF inhibitor LY3009120, is unexpectedly found to synergize and induce apoptosis in mastocytosis cells. Additionally, the combination therapies disclosed herein are cytocidal, as opposed to merely cytostatic.

[0059] Without wishing to be bound by any particular theory, it is believed that many c-KIT inhibitors inhibit only specific mutant forms of c-KIT and do not effectively inhibit the c-KIT D816V mutation that causes SM. The present disclosure provides a method of treating c-KIT-mediated mastocytosis by inhibiting both c-KIT and MEK using a c-KIT inhibitor in combination with a MAPKAP pathway inhibitor. In certain embodiments, the c-KIT inhibitor, as referred to herein, is Compound A or a pharmaceutically acceptable salt thereof, Compound B or a pharmaceutically acceptable salt thereof, midostaurin or a pharmaceutically acceptable salt thereof, BLU-285 or a pharmaceutically acceptable salt thereof, PLX9486 or a pharmaceutically acceptable salt thereof, or crenolanib or Surprisingly, c-KIT inhibitors, such as Compound A and Compound B (and their pharmaceutically acceptable salts), act synergistically with inhibitors of MEK, ERK, or RAF to inhibit mastocytosis cell proliferation and induce apoptosis of mastocytosis cells.

[0060]

[0060] Accordingly, in certain embodiments, the present disclosure provides methods of inducing cytolethal mast cell death, inducing mast cell apoptosis, inhibiting mast cell growth or proliferation, inhibiting mast cell mediator release, reducing the amount of accumulated mast cells in a tissue or organ, reducing the volume of mastocytosis-associated tumors, e.g., mast cell leukemia or mast cell sarcoma, and / or inhibiting mast cell regrowth, by contacting mast cells, e.g., mast cells containing a c-KIT mutation, with a c-KIT inhibitor and a MAPKAP pathway inhibitor. In various embodiments, the mast cells are contacted in vitro, in vivo, or ex vivo. In certain embodiments, c-KIT inhibitors are disclosed herein as Compound A or a pharmaceutically acceptable salt thereof, Compound B or a pharmaceutically acceptable salt thereof, midostaurin or a pharmaceutically acceptable salt thereof, BLU-285 or a pharmaceutically acceptable salt thereof, PLX9486 or a pharmaceutically acceptable salt thereof, or crenolanib or a pharmaceutically acceptable salt thereof; MEK inhibitors are disclosed herein as trametinib, cobimetinib, selumetinib, or binimetinib; ERK inhibitors include but are not limited to ulixertinib, SCH772984, LY3214996, ravoxertinib, and VX-11e; and RAF inhibitors include but are not limited to LY3009120, vemurafenib, or dabrafenib.

[0061] In certain embodiments, the present disclosure includes methods of treating a subject having mastocytosis, mast cell leukemia, or acute myeloid leukemia, comprising administering to the subject effective amounts of (i) a KIT inhibitor, e.g., 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof, or 1-(5-(7-amino-1-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)-4-bromo-2-fluorophenyl)-3-phenylurea or a pharmaceutically acceptable salt thereof, and (ii) a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, or ulixertinib. In certain embodiments of any of the methods disclosed herein, the mastocytosis is systemic mastocytosis resulting from a D816V mutation in the c-KIT gene in mast cells. definition

[0062] As used herein, "Compound A and Compound B" refer to 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea and 1-(5-(7-amino-1-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)-4-bromo-2-fluorophenyl)-3-phenylurea, respectively. Pharmaceutically acceptable salts, tautomers, hydrates, and solvates of Compound A and Compound B are also contemplated in this disclosure. The structures of Compound A and Compound B are shown below: [ka] 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea (Compound A) [ka] 1-(5-(7-amino-1-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)-4-bromo-2-fluorophenyl)-3-phenylurea (Compound B)

[0063] Methods for preparing Compound A and Compound B are disclosed in US Pat. No. 8,461,179 B1, the contents of which are incorporated herein by reference.

[0064] Exemplary methods and materials are described herein. In the specification and the appended claims, unless the context clearly dictates otherwise, the singular also includes the plural. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0065]

[0065] Throughout this disclosure, various patents, patent applications, and publications are referenced. The disclosures of these patents, patent applications, and publications in their entireties are incorporated by reference into this disclosure to more fully describe the state of the art as known to those skilled in the art as of the date of this disclosure. In the event of any inconsistency between these patents, patent applications, and publications and this disclosure, the present disclosure shall control. For convenience, certain terms employed in the specification, examples, and claims are collected here. Unless otherwise specified, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The first definition provided for a group or word provided in this disclosure applies to that group or word individually or as part of another group throughout this disclosure, unless otherwise indicated.

[0066] A "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the United States Food and Drug Administration as acceptable for use in humans or veterinary medicine.

[0067] "Pharmaceutically acceptable salts" include acid addition salts.

[0068] "Pharmaceutically acceptable acid addition salts" refers to those salts which retain the biological effectiveness and properties of the free base, which are not biologically or otherwise undesirable, and which are not salts of inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; and salts of acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, acetic ... It is formed from organic acids such as galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0069] A "pharmaceutical composition" refers to a formulation of a compound described herein (e.g., Compound A or a pharmaceutically acceptable salt thereof) with a vehicle generally acceptable in the art for delivery of biologically active compounds to mammals, e.g., humans. Such vehicles include all pharmaceutically acceptable carriers, diluents, or excipients therefor.

[0070]

[0001] "MAPKAP pathway inhibitors" are inhibitors of the MAP kinase signaling pathway. Inhibitors of this pathway include RAS inhibitors, RAF inhibitors (e.g., vemurafenib, dabrafenib, LY3009120), MEK inhibitors (e.g., trametinib, binimetinib, cobimetinib), and ERK inhibitors (e.g., ulixertinib).

[0071]

[00070] For example, a c-KIT inhibitor in combination with a MAPKAP pathway inhibitor. Subjects or patients "in need of treatment" with the disclosed combination therapies include subjects with diseases and / or conditions that can be treated with the combinations disclosed herein to achieve beneficial therapeutic results, such as mastocytosis, mast cell leukemia, or acute myeloid leukemia. A beneficial outcome in the treatment of mastocytosis may include complete remission, partial response, clinical improvement, or stable disease as defined by the IWG-MRT-ECNM criteria (Gotlib et al., Blood 2013;121:2393-401). In certain embodiments, the subject in need of treatment is afflicted with cutaneous mastocytosis (e.g., maculopapular cutaneous mastocytosis, mastocytoma, and diffuse cutaneous mastocytosis) or systemic mastocytosis (e.g., indolent systemic mastocytosis, smoldering systemic mastocytosis, systemic mast cells with associated clonal hematologic non-mast cell lineage disorders, aggressive systemic mastocytosis, mast cell leukemia, and mast cell sarcoma). In certain embodiments, the subject is a mammal, for example, a human or other mammal.

[0072] When used in connection with a compound or other therapeutic agent disclosed herein, the term "effective amount" refers to an amount of a therapeutic agent, e.g., Compound A or a MAPKAP pathway inhibitor, alone or in combination, that is useful for treating or preventing a disease or disorder. An effective amount of a therapeutic agent used is an amount of each therapeutic agent that, when used in combination, is useful for treating or preventing a disease or disorder, even if the amount of one or both of the therapeutic agents in the absence of the other agent is not effective in treating or preventing the disease or disorder. In certain embodiments, an effective amount is an amount that results in inducing cytolethal mast cell death, inducing mast cell apoptosis, reducing the amount of accumulated mast cells in a tissue or organ, reducing mastocytosis symptoms, inhibiting mast cell mediator release, inhibiting mast cell proliferation, and / or inducing mast cell regression, where the mast cells contain an activating mutation in c-KIT kinase, including the activating c-KIT D816V mutation. An "effective amount" can vary depending on the form of administration, the specific location of the disease or disorder, and the age, weight, and overall health of the subject. The amount of compound administered will depend on the extent, severity, and type of disease or condition, the amount of treatment desired, and the release characteristics of the pharmaceutical formulation. It will also depend on the subject's health, size, weight, age, sex, and tolerance to the drug. Typically, the compounds are administered for a period of time sufficient to achieve the desired therapeutic effect.

[0073] The terms "treatment," "treat," and "treating" are meant to encompass the full range of interventions in the patient being treated, e.g., a patient suffering from mastocytosis or acute myeloid leukemia (AML). Treating may be curing, ameliorating, or at least partially alleviating the disorder. In certain embodiments, treatment is carried out with the intent of inducing cytolethal mast cell death, inducing mast cell apoptosis, reducing the amount of accumulated mast cells in a tissue or organ, reducing mastocytosis symptoms, inhibiting mast cell mediator release, inhibiting mast cell proliferation, and / or inducing mast cell regression in the subject being treated. In certain embodiments, treatment with the combination therapies disclosed herein alleviates, delays, or reverses one or more of the symptoms of mastocytosis and / or induces regression of mastocytosis, even if the mastocytosis is not actually eliminated. In some embodiments, treatment includes completely eliminating the disease or disorder, e.g., mastocytosis or AML. In other embodiments, treatment results in a complete response, partial response, clinical improvement, or stable disease as defined by the IWG-MRT-ECNM criteria (Gotlib et al, Blood 2013;121:2393-401).

[0074] As used herein, "mast cells" includes mast cells (also called mast cells) and CD34+ mast cell precursors.

[0075] As used herein, "neoplasm" refers to abnormal tissue that grows by more rapid than normal cell proliferation. Neoplasms exhibit a partial or complete lack of structural organization and functional coordination with normal tissue, and usually form a discrete mass of tissue that can be either benign (benign tumor) or malignant (cancer).

[0076]

[00075] As used herein, "tumor" refers to a mass. The term can refer to either a non-malignant (harmless) or malignant (cancerous) growth. Malignant growths can originate in solid organs or bone marrow. The latter are often called liquid tumors. "Tumor" encompasses mast cell leukemia and mast cell sarcoma, collectively referred to herein as "mastocytosis tumors."

[0077] In certain embodiments, the therapeutic effect of treating mastocytosis according to the methods disclosed herein can be measured using standard response criteria known in the art. For example, the "response criteria for complete remission (CR), partial remission (PR), clinical improvement (CI), or stable disease (SD)" used to quantify the effect of treatment on aggressive systemic mastocytosis, mast cell leukemia, and systemic mastocytosis associated with myeloid neoplasms are defined as follows: The criteria can be any of those defined by RT-ECNM (Gotlib et al, Blood 2013;121:2393-401). For example, a complete response (CR) requires all four criteria, a response duration of 12 weeks or more, and the absence of aggregated compact neoplastic mast cells in the BM or other biopsied organs other than the skin. A serum tryptase concentration of <20 ng / mL† and peripheral blood count remission, defined as ANC with a normal differential, of ≥1 × 10 9 / L, Hb level ≥ 11 g / dL, and platelet count ≥ 100 × 10 9Complete response is defined as complete remission of 1 / L with palpable hepatosplenomegaly and all biopsy-confirmed or suspected SM-related organ damage (CI findings); partial response (PR)* requires all three criteria, a response duration of ≥12 weeks, the absence of both CR and progressive disease (PD), a ≥50% reduction in neoplastic MC in bone marrow and / or in organs other than the skin with biopsy-confirmed SM-related organ damage, a ≥50% reduction in serum tryptase levels, and resolution of one or more biopsy-confirmed or suspected SM-related organ damage (CI findings); clinical improvement (CI) requires a response duration of ≥12 weeks, the fulfillment of one or more of the non-hematopoietic and / or hematopoietic response criteria (see Table 3), and the absence of both CR / PR and progressive disease (PD). Stable disease (SD) is the absence of CR, PR, CI, or PD criteria.

[0078] Guidelines for determining response include: (1) only disease-related Grade 2 or greater organ damage is evaluable as a primary endpoint in clinical trials; (2) the response assessments of CR, PR, SD, PD, and loss or response (LOR) should apply only to these Grade 2 or greater organ damage findings within the context of the trial; (3) the disease status at the time of patient removal from the trial is particularly relevant to the updated status of the initial Grade 2 or greater organ damage finding; and (4) drug-related toxicity and / or other clinical problems (e.g., gastrointestinal bleeding in the setting of worsening anemia or transfusion dependency) must be ruled out before assigning a designated PD or LOR in patients with worsening baseline Grade 2 or greater organ damage.

[0079] In certain embodiments, the therapeutic effect of treating AML according to the methods disclosed herein can be measured using standard response criteria known in the art. For example, the "response criteria for treatment of AML" used to quantify the effect of treatment on AML include minimal residual disease (CR), as defined in Blood. 2017 Jan 26;129(4):424-447. MRD- ) without complete remission (CR), complete remission (CR), CR with incomplete hematologic recovery (CR i ), morphologic leukemia-free state (MLFS), partial response (PR), stable disease (SD), or progressive disease (PD), and are summarized as follows: complete response without minimal residual disease (CR); MRD- ): If pretreatment testing is performed, CR with negative genetic markers by RT-qPCR or CR with negative MFC. Complete remission (CR): bone marrow blasts <5%; absence of blasts in the bloodstream and blasts with Auer rods; absence of extramedullary disease; absence of extramedullary disease; absolute neutrophil count (ANC) ≥ 1.0 x 10 9 / L (1000 / μL); platelet count ≥ 100 × 10 9 / L (100,000 / μL); CR with incomplete blood recovery (CR i ): Residual neutropenia (<1.0×10 9 / L [1000 / μL]) or thrombocytopenia (<100 × 10 9 All CR criteria except ≥ 100,000 / μL (≥ 100,000 / μL); Morphologic Leukemia-Free State (MLFS): < 5% bone marrow blasts; no blasts with Auer rods; no extramedullary disease; no need for hematologic recovery; Partial Response (PR): All hematologic criteria for CR; a reduction in bone marrow blast percentage to 5%-25%; and a reduction in pretreatment bone marrow blast percentage of at least 50%.

[0080] "Combination therapy" refers to the combination of two or more therapeutic agents, such as a c-KIT inhibitor (Compound A or its derivatives) Therapy involves administering to a patient a therapeutic agent (such as a pharmaceutical acceptable salt, midostaurin, BLU-285, PLX9486, or crenolanib) and a MAPKAP pathway inhibitor (including, but not limited to, trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib, and LY3009120). Two or more therapeutic agents may be delivered simultaneously, for example, in separate pharmaceutical compositions or in the same pharmaceutical composition, or two or more therapeutic agents may be delivered at different times. For example, two or more therapeutic agents may be delivered simultaneously or during overlapping periods, and / or one therapeutic agent may be delivered before or after the other therapeutic agent. Treatment involving a combination of a KIT inhibitor, such as Compound A, and a MAPKAP pathway inhibitor may involve treatment with either single agent preceded or followed by a period of simultaneous treatment with both agents. However, it is intended that effective amounts of two or more therapeutic agents will be present in the patient's body during some period of time. Treatment method

[0081]

[00080] In one embodiment, the present disclosure provides a method for treating obesity, e.g., systemic mastocytosis (SM). In one embodiment, the present disclosure provides a method for treating or preventing mastocytosis, optionally c-KIT-mediated mastocytosis, comprising providing or administering to a subject in need thereof an effective amount of a c-KIT inhibitor in combination with an effective amount of a MAPKAP pathway inhibitor, e.g., trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib, or LY3009120. In one embodiment, the present disclosure provides a method for treating or preventing mastocytosis, optionally c-KIT-mediated mastocytosis, e.g., systemic mastocytosis (SM), comprising providing or administering to a subject in need thereof an effective amount of Compound A (or a pharmaceutically acceptable salt thereof) or Compound B (or a pharmaceutically acceptable salt thereof) in combination with an effective amount of a MAPKAP pathway inhibitor, e.g., trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib, or LY3009120. In related embodiments, the present disclosure provides methods for treating or preventing mast cell tumors, e.g., mast cell leukemia or mast cell sarcoma, optionally c-KIT-mediated mastocytosis, comprising providing or administering to a subject in need thereof an effective amount of a c-KIT inhibitor in combination with an effective amount of a MAPKAP pathway inhibitor, e.g., trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib, or LY3009120. In related embodiments, the disclosure provides methods for treating or preventing mast cell tumors, optionally c-KIT-mediated mast cell tumors, e.g., mast cell leukemia or mast cell sarcoma, comprising providing or administering to a subject in need thereof an effective amount of Compound A (or a pharmaceutically acceptable salt thereof) or Compound B (or a pharmaceutically acceptable salt thereof), in combination with an effective amount of a MAPKAP pathway inhibitor, e.g., trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib, or LY3009120.

[0082] In another example, the present disclosure provides a method of treating mastocytosis in a patient in need thereof, comprising administering to the patient an effective amount of a c-KIT inhibitor and an effective amount of one or more MAPKAP pathway inhibitors, which may be selected from the group consisting of rapidly progressing fibrosarcoma (RAF) kinase inhibitors, inhibitors of mitogen-activated protein kinase (MEK inhibitors), and extracellular signal-regulated kinase inhibitors (ERK inhibitors).

[0083] In one embodiment of such disclosed methods, the mastocytosis is associated with a c-KIT mutation. In some embodiments, the c-KIT mutation is an activating mutation.

[0084] In another embodiment, mastocytosis can involve mast cells with a primary mutation in exon 17 of the c-KIT gene. In some embodiments, the primary mutation is a c-KIT D816 mutation. In some embodiments, the primary mutation is one of D816V, D816Y, D816F, D816H, F522C, K5091, V560G, V559G, and del419. In some embodiments, the primary mutation is D816V.

[0085] Mastocytosis may also include mast cells with a c-KIT secondary mutation. In some embodiments, the c-KIT secondary mutation is in one of exons 9, 11, 13, or 17. In some embodiments, the c-KIT secondary mutation is one of the following mutations: Y269C, Y503_F504insAY, V560D, or K642E.

[0086]

[00085] The disclosed methods can be used to determine whether mastocytosis is associated with a c-KIT primary mutation. For example, the method may further comprise determining whether the mastocytosis is associated with a c-KIT primary mutation or whether the mastocytosis is associated with a c-KIT secondary mutation. In some embodiments, determining whether the mastocytosis is associated with a c-KIT primary mutation or whether the mastocytosis is associated with a c-KIT secondary mutation comprises identifying a mutation in DNA extracted from a tumor sample. In yet other embodiments, determining whether the mastocytosis is associated with a c-KIT primary mutation or whether the mastocytosis is associated with a c-KIT secondary mutation comprises identifying a mutation in tumor DNA in blood or identifying a mutation in peripheral blood leukocytes in blood.

[0087] The mastocytosis can be systemic mastocytosis. In some embodiments, the systemic mastocytosis is selected from the group consisting of inactive systemic mastocytosis, smoldering systemic mastocytosis, systemic mastocytosis with associated clonal hematologic non-mast cell lineage disease, aggressive systemic mastocytosis, mast cell leukemia, and mast cell sarcoma. In some embodiments, the mastocytosis is inactive systemic mastocytosis, optionally with anaphylactic recurrences or systemic mastocytosis with vascular collapse in the absence of skin lesions. In some embodiments, the mastocytosis is smoldering systemic mastocytosis.

[0088] In some embodiments, the mastocytosis is systemic mastocytosis with an associated clonal hematologic non-mast cell lineage disorder. In some embodiments, the mastocytosis is aggressive systemic mastocytosis. In some embodiments, the mastocytosis is mast cell leukemia or mast cell sarcoma. In some embodiments, the mastocytosis is cutaneous mastocytosis. In some embodiments, the mastocytosis is selected from the group consisting of maculopapular cutaneous mastocytosis, mastocytoma, or diffuse cutaneous mastocytosis.

[0089] In such disclosed methods, the c-KIT inhibitor can be selected from the group consisting of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof, midostaurin or a pharmaceutically acceptable salt thereof, imatinib mesylate, sunitinab malate, midostaurin, regorafenib, crenolanib, PTX9486, or BLU-285 (avapritinib) or a pharmaceutically acceptable salt thereof.

[0090] Further, the MEK inhibitor can be selected from the group consisting of trametinib, selumetinib, cobimetinib, and binimetinib. In some embodiments, the MEK inhibitor is binimetinib. In some embodiments, the MEK inhibitor is trametinib. In some embodiments, the ERK inhibitor is selected from the group consisting of ulixertinib, SCH772984, and LY3214996. In some embodiments, the c-KIT inhibitor and the MEK and / or ERK inhibitor are substantially The two agents are administered simultaneously or sequentially.

[0091]

[00090] This method also includes the use of other targeted cancer therapeutics, targeted cancer biological agents, immune checkpoint inhibitors, This may involve administering an inhibitor, or a chemotherapeutic agent.

[0092] Furthermore, administration of an effective amount of a c-KIT inhibitor, and administration of an effective amount of an inhibitor of mitogen-activated protein kinase (MEK inhibitor), and / or an effective amount of an extracellular signal-regulated kinase inhibitor (ERK inhibitor) for two or more weeks according to the intended methods can result in a patient in at least partial remission.

[0093] Also provided is a method for treating systemic mastocytosis in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof and an effective amount of a MAPKAP pathway inhibitor. In these disclosed methods, the MAPKAP pathway inhibitor is selected from the group consisting of a rapidly progressing fibrosarcoma (RAF) kinase inhibitor, an inhibitor of mitogen-activated protein kinase (MEK inhibitor), and an extracellular signal-regulated kinase inhibitor (ERK inhibitor).

[0094] In one embodiment, systemic mastocytosis can be associated with a c-KIT mutation. For example, the mutation can be a c-KIT D816 mutation. In some embodiments, the mutation is one of D816V, D816Y, D816F, D816H, F522C, K5091, V560G, V559G, and del419. In some embodiments, the mutation is one of A553D, C433Y, D419Y, D572A, D816F, D816H, D816I, D816V, D816Y, D820G, del419, dup(501-502), E839K, F522C, I817V, InsFF419, InsV815-I816, K509I, N822I, R815K, T417V, V560G, V559I, or Y418Y. In some embodiments, the mutation is D816V.

[0095] Additionally, mastocytosis may be associated with an additional c-KIT mutation, which is one of the following mutations: Y269C, Y503_F504insAY, V560D, or K642E.

[0096]

[00095] In these disclosed methods, the MEK inhibitor is trametinib, selmetinib, or In some embodiments, the MEK inhibitor is binimetinib. In some embodiments, the MEK inhibitor is trametinib. The ERK inhibitor can be selected from the group consisting of ulixertinib, SCH772984, and LY3214996.

[0097] The present disclosure further provides a method of treating mastocytosis in a patient in need thereof, comprising administering to the patient an effective amount of a c-KIT inhibitor and an effective amount of a RAF inhibitor.

[0098] In such disclosed methods, the RAF inhibitor can be a pan-RAF inhibitor or a B-RAF inhibitor, including vemurafenib, dabrafenib, and LY3009120. Additionally, the c-KIT inhibitor can be 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharmaceutically acceptable salt thereof. It is possible to do this.

[0099] Certain embodiments of the methods disclosed herein include methods of treating mastocytosis or mast cell tumors, the methods comprising inducing cytolethal mast cell death, inducing mast cell apoptosis, reducing the amount of accumulated mast cells in a tissue or organ, reducing mastocytosis symptoms, inhibiting mast cell mediator release, inhibiting mast cell proliferation, and / or inducing mast cell regression, wherein the mast cells contain one or more activating mutations in c-KIT kinase, e.g., the activating KIT D816V mutation. In certain embodiments, the methods include methods of eradicating mastocytosis, e.g., mast cell tumors, in a subject. In some embodiments, treatment results in a complete remission, partial response, clinical improvement, or stable disease as defined by the IWG-MRT-ECNM criteria (Gotlib et al., Blood 2013;121:2393-401).

[0100] In another related embodiment, the present disclosure provides a method of treating or preventing acute myeloid leukemia (AML), optionally c-KIT-mediated (AML), comprising administering to a subject in need thereof an effective amount of a c-KIT inhibitor in combination with an effective amount of a MAPKAP pathway inhibitor, e.g., trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib, or LY3009120. In a related embodiment, the present disclosure provides a method of treating or preventing acute myeloid leukemia (AML), optionally c-KIT-mediated (AML), comprising administering to a subject in need thereof an effective amount of Compound A (or a pharmaceutically acceptable salt thereof) or Compound B (or a pharmaceutically acceptable salt thereof) in combination with an effective amount of a MAPKAP pathway inhibitor, e.g., trametinib, cobimetinib, selumetinib, binimetinib, ulixertinib, or LY3009120.

[0101] [000100] When the methods described herein refer to treatment with Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, it is meant that only one of Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, is required. However, it will be understood that these methods also encompass administering to a patient both Compound A or a pharmaceutically acceptable salt thereof, and Compound B or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor. The methods described herein also include administering Compound A and a MAPKAP pathway inhibitor to a subject, whereby Compound A is metabolized to Compound B in vivo, and the mixture of Compound A and Compound B in vivo effectively treats the subject in combination with the MAPKAP pathway inhibitor.

[0102] Certain embodiments of the disclosed methods and compositions are practiced using a combination of Compound A, or a pharmaceutically acceptable salt thereof, and trametinib; a combination of Compound A, or a pharmaceutically acceptable salt thereof, and selumetinib; a combination of Compound A, or a pharmaceutically acceptable salt thereof, and cobimetinib; or a combination of Compound A, or a pharmaceutically acceptable salt thereof, and binimetinib.

[0103] In one embodiment, Compound A, or a pharmaceutically acceptable salt thereof, and a MEK inhibitor, such as trametinib or binimetinib, are administered to a subject suffering from c-KIT-mediated mastocytosis. In another embodiment, Compound B, or a pharmaceutically acceptable salt thereof, and a MEK inhibitor, such as trametinib or binimetinib, are administered to a subject suffering from c-KIT-mediated mastocytosis.

[0104] In a related embodiment, Compound A or a pharmaceutically acceptable salt thereof may be combined with, for example, In another embodiment, compound B or a pharmaceutically acceptable salt thereof and a MEK inhibitor, e.g., trametinib or binimetinib, are administered to a patient suffering from a mast cell tumor, including, but not limited to, mast cell leukemia or mast cell sarcoma, where the growth or progression of the mast cell tumor is caused by an activating c-KIT mutation, e.g., a KIT D816V mutation. In certain embodiments, compound A or a pharmaceutically acceptable salt thereof or compound B or a pharmaceutically acceptable salt thereof and a MEK inhibitor, for example, trametinib or binimetinib, are administered to patients suffering from AML, optionally caused by an activating c-KIT primary mutation, for example, a c-KIT exon 8 activating mutation or a c-KIT exon 17 mutation, including, but not limited to, a mutation at D816 or a mutation at N822 (Journal of Clinical Oncology 2006 24:24,3904-3911).

[0105] Certain embodiments of the disclosed methods and compositions are practiced using a combination of Compound A, or a pharmaceutically acceptable salt thereof, and ulixertinib; a combination of Compound A, or a pharmaceutically acceptable salt thereof, and SCH772984,e; a combination of Compound A, or a pharmaceutically acceptable salt thereof, and LY3214996; a combination of Compound A, or a pharmaceutically acceptable salt thereof, and lavoxertinib; or a combination of Compound A, or a pharmaceutically acceptable salt thereof, and VX-11.

[0106] In one embodiment, Compound A, or a pharmaceutically acceptable salt thereof, and an ERK inhibitor, e.g., ulixertinib, are administered to a subject suffering from c-KIT-mediated mastocytosis. In another embodiment, Compound B, or a pharmaceutically acceptable salt thereof, and an ERK inhibitor, e.g., ulixertinib, are administered to a subject suffering from c-KIT-mediated mastocytosis.

[0107] In a related embodiment, Compound A, or a pharmaceutically acceptable salt thereof, and an ERK inhibitor, e.g., ulixertinib, are administered to a patient suffering from a mast cell tumor, including, but not limited to, mast cell leukemia or mast cell sarcoma, where mast cell tumor growth or progression is driven by a primary activating c-KIT mutation, e.g., a KIT D816V mutation. In another embodiment, Compound B, or a pharmaceutically acceptable salt thereof, and an ERK inhibitor, e.g., ulixertinib, are administered to a patient suffering from a mast cell tumor, including, but not limited to, mast cell leukemia or mast cell sarcoma, where mast cell tumor growth or progression is driven by a primary activating c-KIT mutation, e.g., a KIT D816V mutation. In certain embodiments, Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, and an ERK inhibitor, for example, ulixertinib, are administered to a patient suffering from AML, optionally caused by a primary activating c-KIT mutation, for example, a c-KIT exon 8 activating mutation or a c-KIT exon 17 mutation, including, but not limited to, a mutation at D816 or a mutation at N822 (Journal of Clinical Oncology 2006 24:24, 3904-3911).

[0108] Specific embodiments of the disclosed methods and compositions include the combination of Compound A, or a pharmaceutically acceptable salt thereof, with LY3009120; Compound A, or a pharmaceutically acceptable salt thereof, with dabrafenib; or Compound A, or a pharmaceutically acceptable salt thereof, with vemurafenib. This is carried out using a combination of

[0109] In one embodiment, compound A or a pharmaceutically acceptable salt thereof and a RAF inhibitor, e.g., LY3009120, dabrafenib, or vemurafenib, are administered to a subject suffering from c-KIT-mediated mastocytosis. In another embodiment, compound B or a pharmaceutically acceptable salt thereof and a RAF inhibitor, e.g., LY3009120, dabrafenib, or vemurafenib, are administered to a subject suffering from c-KIT-mediated mastocytosis. In a related embodiment, compound A or a pharmaceutically acceptable salt thereof and a RAF inhibitor, e.g., LY3009120, dabrafenib, or vemurafenib, are administered to a patient suffering from a mast cell tumor, including, but not limited to, mast cell leukemia or mast cell sarcoma, where mast cell tumor growth or progression is driven by a primary activating c-KIT mutation, e.g., the KIT D816V mutation. In another embodiment, compound B or a pharmaceutically acceptable salt thereof and a RAF inhibitor, e.g., LY3009120, dabrafenib, or vemurafenib, are administered to a patient suffering from a mast cell tumor, including, but not limited to, mast cell leukemia or mast cell sarcoma, where mast cell tumor growth or progression is driven by a primary activating c-KIT mutation, e.g., the KIT D816V mutation. In certain embodiments, compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, and a RAF inhibitor, for example, LY3009120, dabrafenib, or vemurafenib, are administered to patients suffering from AML, and optionally the AML is caused by a primary activating c-KIT mutation, for example, a c-KIT exon 8 activating mutation or a c-KIT exon 17 mutation, including, but not limited to, a mutation at D816 or a mutation at N822 (Journal of Clinical Oncology 2006 24:24,3904-3911).

[0110] Exemplary c-KIT inhibitors that can be used in accordance with the disclosed methods and compositions include, but are not limited to, Compound A or a pharmaceutically acceptable salt thereof, Compound B or a pharmaceutically acceptable salt thereof, midostaurin, BLU-285, PLX9486, and crenolanib. Exemplary MEK inhibitors that can be used in accordance with the disclosed methods and compositions include, but are not limited to, trametinib, selumetinib, cobimetinib, and binimetinib. Exemplary ERK inhibitors that can be used in accordance with the disclosed methods and compositions include, but are not limited to, ulixertinib, SCH772984, LY3214996, ravoxertinib, and VX-11e. Exemplary RAF inhibitors that can be used in accordance with the disclosed methods and compositions include, but are not limited to, LY3009120, dabrafenib, and vemurafenib.

[0111] [000110] Treatment with Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, includes administering Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, before, after, simultaneously with, or during a period of overlap with administration of the MAPKAP pathway inhibitor. It is understood that the effective amount of any of Compound A or a pharmaceutically acceptable salt thereof, Compound B or a pharmaceutically acceptable salt thereof, another c-KIT inhibitor, or a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, may be different when used in the combinations disclosed herein compared to when either of these agents is used by itself for the same purpose, e.g., to treat or prevent mastocytosis or mast cell tumors, e.g., mast cell leukemia or AML. In certain embodiments, the effective amount of Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, when administered in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, is less than when administered as a monotherapy, e.g., to treat or prevent mastocytosis or mast cell tumors. In certain embodiments, the effective amount of a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, is less when administered in combination with Compound A or a pharmaceutically acceptable salt thereof, or when administered in combination with Compound B or a pharmaceutically acceptable salt thereof, e.g., to treat or prevent mastocytosis or mast cell tumors.

[0112] Any of the methods disclosed herein may further include determining whether the mastocytosis cells, mast cell tumor, or AML to be treated is associated with one or more c-KIT gene mutations. Such a determination may be made by conventional methods for determining the presence of gene mutations in a biological sample obtained from a subject, such as a bone marrow sample, tissue sample, peripheral blood sample, or plasma sample. Furthermore, such a determination may be made by reviewing the results of performed tests to determine the presence of one or more c-KIT gene mutations in the biological sample obtained from the patient. In certain embodiments of any of the methods disclosed herein, the methods are performed on a subject whose mastocytosis, mast cell tumor, or AML has been identified as being associated with one or more c-KIT gene mutations. The c-KIT gene mutations include, but are not limited to, any of those specifically described herein. In certain embodiments of the methods disclosed herein, the methods are not performed on a subject whose mastocytosis, mast cell tumor, or AML has been identified as not being associated with one or more c-KIT gene mutations.

[0113] In various embodiments of any of the methods disclosed herein, treatment with either Compound A or a pharmaceutically acceptable salt thereof or Compound B or a pharmaceutically acceptable salt thereof in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, induces cytolethal mast cell death, induces mast cell apoptosis, reduces the amount of accumulated mast cells in a tissue or organ, reduces mastocytosis symptoms, inhibits mast cell mediator release, inhibits mast cell proliferation, and / or induces mast cell regression, wherein the mast cells express activated KIT Activating mutations in c-KIT kinase, including the D816V mutation, are encompassed. Methods for measuring or determining the amount of mast cell apoptosis, mast cell death, inhibition of mast cell proliferation and growth, inhibition of mast cell mediator release, mutant KIT allele burden in peripheral blood, eradication of mastocytosis and mast cell tumors, complete remission, partial response, clinical improvement, or stable disease are known in the art and include any of the methods described herein.

[0114] In certain embodiments, treatment with a c-KIT inhibitor, e.g., Compound A or a pharmaceutically acceptable salt thereof or Compound B or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, results in an increase in the amount of apoptosis of mastocytosis cells or mast cells compared to the amount of apoptosis of the same type of mastocytosis cells or mast cells untreated or treated with the MAPKAP pathway inhibitor alone or with a c-KIT inhibitor alone, e.g., Compound A or Compound B. For example, apoptosis can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or It may be increased by at least a factor of 20. In certain embodiments, the amount of apoptosis is determined by measuring caspase activity in KIT mutant mast cells or mast cell lines, including the HMC1.2 mast cell line containing the KIT D816V mutation.

[0115] In certain embodiments, treatment with a c-KIT inhibitor, e.g., Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, results in a decrease in the accumulation of mast cells in the skin or an internal organ, e.g., the liver, spleen, bone marrow, and / or small intestine, compared to the amount of mast cell accumulation in the same organ untreated or treated with the MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, alone or with a c-KIT inhibitor, e.g., midostaurin, BLU-285, Compound A, or Compound B, alone. For example, the amount or number of mast cells accumulated in an organ may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0116] [000115] In certain embodiments, treatment with a c-KIT inhibitor, e.g., Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, results in a complete remission as defined by the IWG-MRT-ECNM criteria (Gotlib et al., Blood 2013;121:2393-401).

[0117] In certain embodiments, treatment with a c-KIT inhibitor, e.g., Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, results in a partial response as defined by the IWG-MRT-ECNM criteria (Gotlib et al., Blood 2013;121:2393-401).

[0118] In certain embodiments, treatment with a c-KIT inhibitor, e.g., Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, results in clinical improvement as defined by the IWG-MRT-ECNM criteria (Gotlib et al., Blood 2013;121:2393-401).

[0119] In certain embodiments, treatment with a c-KIT inhibitor, e.g., Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, results in stable disease as defined by the IWG-MRT-ECNM criteria (Gotlib et al., Blood 2013;121:2393-401).

[0120] In certain embodiments, treatment with a c-KIT inhibitor, e.g., Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, induces apoptosis or inhibits proliferation of resistant mastocytosis cells that contain a KIT secondary mutation in addition to the KIT D816V primary mutation. KIT secondary mutations include, but are not limited to, point mutations, in-frame deletions or insertions, or missense mutations of Y269C, Y503_F504insAY, V560D, or K642E in the c-KIT gene (Lasho et al., Br J Haematol 173, 153-156).

[0121] [000120] In certain embodiments, treatment with a c-KIT inhibitor, e.g., Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, induces apoptosis or inhibits proliferation of resistant mastocytosis cells that contain mutations, e.g., resistance mutations, in other genes, including any of the genes disclosed herein. In certain embodiments, mutations in these other genes may include gain of function mutations in NRas (Haematologica 2011;96(03):459-463.doi:10.3324 / haematol.2010.031690) or loss of function mutations in TET2 (Leukemia 2009;23:900-04; Blood,6 December 2012;120(24):4846-49). The presence of other epigenetic or transcriptional regulatory factor mutations was detected in SM with mutations in DNMT3A, ASXL1, and CBL in 12%, 12%, and 4% of patients, respectively (PloS 1.2012;7:e43090). Furthermore, some mastocytosis patients also exhibit mutations in the spliceosome machinery. The spliceosome ensures the correct linear order of exons spliced ​​into mRNA. Hanssens et al. reported a 23.6% incidence of SRSF2, 5.6% SF3B1, and 2.7% U2AF1 mutations in a group of 72 mastocytosis patients (Haematologica 2014;99:830-35). Such mastocytosis cases with complex genomic drivers may benefit from the treatment disclosed herein with a combination of a c-KIT inhibitor and a MEK inhibitor compared to treatment with only a MEK inhibitor, e.g., trametinib, or only a c-KIT inhibitor, e.g., Compound A or Compound B, or no treatment.For example, apoptosis can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold. For example, the amount or number of proliferation of resistant mastocytosis cells can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0122] In certain embodiments, the resistant mastocytosis cells are resistant to apoptosis-mediated cell death or cytocidal activity by treatment with a c-KIT inhibitor, e.g., Compound A, Compound B, midostaurin, BLU-285, PLX9486, or crenolanib, and / or are resistant to apoptosis-mediated cell death or cytocidal activity by treatment with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, when used as monotherapy.

[0123] In certain embodiments, treatment with a combination of a c-KIT inhibitor, e.g., Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, results in eradication of mastocytosis, e.g., mast cell tumors. In certain embodiments, eradication of mastocytosis means that there is no longer any detectable mastocytosis in the patient. In certain embodiments, eradication of mastocytosis is achieved within at least 12 weeks, at least 24 weeks, at least 1 year, at least 2 years, or at least 3 years after initiation of treatment of mastocytosis with the combination therapy disclosed herein. The patient has been free of detectable mastocytosis for at least 5 years. Eradication of mastocytosis can be determined by the criteria of complete remission as defined by the IWG-MRT-ECNM criteria (Gotlib et al., Blood 2013;121:2393-401).

[0124] The present disclosure describes combination therapies involving the administration of a c-KIT inhibitor, e.g., Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120. The combination therapies described herein can be used by themselves or in combination with one or more additional therapeutic agents. For example, Compound A or a pharmaceutically acceptable salt thereof, or Compound B or a pharmaceutically acceptable salt thereof, and a MAPKAP pathway inhibitor can be administered together with a cancer targeted therapeutic agent, a cancer targeted biological agent, an immune checkpoint inhibitor, or a chemotherapeutic agent. In another embodiment, Compound A or Compound B and a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, are administered without any other therapeutic agent. The therapeutic agent can be administered together with another therapeutic agent described herein in the combination therapy or sequentially with the therapeutic agent.

[0125] Combination therapy can be achieved by administering two or more therapeutic agents, each formulated and administered separately, or by administering two or more therapeutic agents in a single formulation. Other combinations are also encompassed by combination therapy. In combination therapy, two or more therapeutic agents can be administered simultaneously, but this is not required. For example, administration of a first agent (or combination of agents) can precede administration of a second agent (or combination of agents) by minutes, hours, days, or weeks. Thus, two or more agents can be administered within minutes of each other, or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days of each other, or within 2, 3, 4, 5, 6, 7, 8, 9, or more weeks of each other. Longer intervals are possible in some cases. In many cases, it is desirable, but not necessary, for the two or more drugs used in combination therapy to be present in the patient's body at the same time.

[0126] [000125] Combination therapy can also include more than one administration of one or more drugs used in the combination, with the component drugs being administered in a different order. For example, when drug X and drug Y are used in combination, they can be administered one or more times sequentially in any combination, e.g., in the order XYX, XXY, YXY, Y-YX, XXYY, etc. Additionally, administration of two or more agents in a combination may be preceded or followed by a dosing interval in which at least one of the agents in the combination is omitted from treatment.

[0127] Additional therapeutic agents that may be administered in accordance with the present disclosure, for example for treating mastocytosis or mast cell tumors, include inhibitors of STAT5, including but not limited to ruxolitinib, tofacitinib, or fedratinib; inhibitors of BTK, including but not limited to ibrutinib, PCI29732, acalabrutinib, or AVL-292; idelalisib, dactolisib, pictilisib, LY294002, buparlisib, pilallisib, duvelisib, PF-04691502, voxtalisib, omitali inhibitors of PI3 kinase, including but not limited to parisib, gedatolisib, apitolisib, or wortmannin; inhibitors of AKT kinase, including but not limited to MK-2206, perifosine, GSK690693, GSK2141795, ipatasertib, AZD5363, afuresertib, or AT7867; DNA methylation inhibitors, including but not limited to 5-azacytidine or 5-aza-2'-deoxycytidine; bortezomib, carfilzomib, MLN9708, ONX 0912, interferon alpha (IFN-α), proteosome inhibitors, including but not limited to cladribine, and lysosomotropic agents, including but not limited to chloroquine, hydroxychloroquine, or quinacrine.

[0128] In certain embodiments, the additional therapeutic agent is selected from 5-azacytidine, 5-aza-2'-deoxycytidine, and cladribine.

[0129] In certain embodiments, a subject in need thereof is treated with a combination of one or two of Compound A or a pharmaceutically acceptable salt thereof, such as trametinib, binimetinib, ulixertinib, or LY3009120, and a MAPKAP pathway inhibitor that is 5-azacytidine. In certain embodiments, the MAPKAP pathway inhibitor is trametinib. In certain embodiments, the MAPKAP pathway inhibitor is binimetinib. In certain embodiments, the MAPKAP pathway inhibitor is ulixertinib. In certain embodiments, the MAPKAP pathway inhibitor is LY3009120, dabrafenib, or vemurafenib.

[0130] In certain embodiments, a subject in need thereof is treated with a combination of Compound A or a pharmaceutically acceptable salt thereof, such as trametinib, binimetinib, ulixertinib, or LY3009120, and a MAPKAP pathway inhibitor that is 5-aza-2'-deoxycytidine. In certain embodiments, the MAPKAP pathway inhibitor is trametinib. In certain embodiments, the MAPKAP pathway inhibitor is binimetinib. In certain embodiments, the MAPKAP pathway inhibitor is ulixertinib. In certain embodiments, the MAPKAP pathway inhibitor is LY3009120, dabrafenib, or vemurafenib.

[0131] [000130] In certain embodiments, a subject in need thereof is treated with a combination of Compound A or a pharmaceutically acceptable salt thereof, such as trametinib, binimetinib, ulixertinib, or LY3009120, and a MAPKAP pathway inhibitor that is cladribine. In certain embodiments, the MAPKAP pathway inhibitor is trametinib. In certain embodiments, the MAPKAP pathway inhibitor is binimetinib. In certain embodiments, the MAPKAP pathway inhibitor is ulixertinib. In certain embodiments, the MAPKAP pathway inhibitor is LY3009120, dabrafenib, or vemurafenib. Additional therapeutic agents that may be administered according to the present disclosure include, but are not limited to, arsenic trioxide, cyclophosphamide, cytarabine, daunorubicin, doxorubicin, enasidenib, idarubicin, quizartinib, mitoxantrone, thioguanine, or vincristine. In certain embodiments, a subject with AML is treated with Compound A, or a pharmaceutically acceptable salt thereof, in combination with arsenic trioxide, cyclophosphamide, cytarabine, daunorubicin, doxorubicin, enasidenib, idarubicin, quizartinib, mitoxantrone, thioguanine, or vincristine.

[0132] Pharmaceutical Composition Aspects of the present disclosure are directed to therapeutic methods comprising administering a combination of compounds disclosed herein, or one or more pharmaceutical compositions comprising such compounds and a pharmaceutically acceptable diluent, excipient, or carrier. In certain embodiments, the methods disclosed herein involve administering a first pharmaceutical composition comprising a c-KIT inhibitor, e.g., Compound A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, excipient, or carrier, and a second pharmaceutical composition comprising a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, and a pharmaceutically acceptable diluent, excipient, or carrier. In certain embodiments, the methods disclosed herein comprise: The method involves administering a first pharmaceutical composition comprising Compound B or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent, excipient, or carrier, and a second pharmaceutical composition comprising a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, and a pharmaceutically acceptable diluent, excipient, or carrier. In certain embodiments, the methods disclosed herein involve administering pharmaceutical compositions comprising a ciKIT inhibitor, e.g., Compound A or a pharmaceutically acceptable salt thereof, a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, and a pharmaceutically acceptable diluent, excipient, or carrier. In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising compound B, or a pharmaceutically acceptable salt thereof, a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, and a pharmaceutically acceptable diluent, excipient, or carrier.

[0133] In the pharmaceutical compositions of the compounds described herein, pharmaceutically acceptable carriers can be either solid or liquid. Solid forms include powders, tablets, dispersible granules, capsules, cachets, and suppositories. Powders and tablets may be comprised of about 5 to about 95 percent active ingredient. Suitable solid carriers are known in the art and include, for example, magnesium carbonate, magnesium stearate, talc, sugar, or lactose. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration. Examples of pharmaceutically acceptable carriers and methods for manufacturing various compositions can be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co., Easton, Pa., incorporated herein by reference in its entirety.

[0134] Liquid form preparations include solution, suspension and emulsion.For example, for parenteral injection, water or water-propylene glycol solution, or for oral solution, suspension and emulsion, sweetener and opacifier are added.Liquid form preparations also include the solution for intranasal administration.

[0135] Liquid compositions, particularly injectable ones, can be prepared, for example, by dissolving, dispersing, etc. For example, the disclosed compounds are dissolved or mixed with a pharmaceutically acceptable solvent, such as, for example, water, saline, aqueous dextrose, glycerin, ethanol, etc., to form an injectable isotonic solution or suspension. Proteins such as albumin, chylomicron particles, or serum proteins can be used to solubilize the disclosed compounds.

[0136] [000135] Parenteral injectable administration is generally used for subcutaneous, intramuscular, or intravenous injections and infusions. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, or as solid forms suitable for dissolving in liquid prior to injection.

[0137] Aerosol formulations suitable for inhalation may also be used. These formulations may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas, for example nitrogen.

[0138] Also contemplated for use are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions, and emulsions.

[0139] dose In some embodiments, when Compound A or Compound B (or a pharmaceutically acceptable salt thereof) is used in combination with a MAPKAP pathway inhibitor, e.g., trametinib, binimetinib, ulixertinib, or LY3009120, for a treatment protocol, the two therapeutic agents may be administered together or in a "dual regimen," in which the two therapeutic agents are dosed and administered separately. When Compound A or B (or a pharmaceutically acceptable salt thereof) and the MAPKAP pathway inhibitor are dosed separately, a typical dose of Compound A or Compound B (or a pharmaceutically acceptable salt thereof) administered to a subject in need of treatment is typically about 5 mg / day to about 5000 mg / day, and in other embodiments, about 50 mg / day to about 1000 mg / day. Other doses may be from about 10 mmol up to about 250 mmol / day, about 20 mmol to about 70 mmol / day, or about 30 mmol to about 60 mmol / day. Effective dosages of the disclosed compounds, when used in relation to the indicated effects, range from about 0.5 mg to about 5,000 mg of the disclosed compounds required to treat the condition. Compositions for in vivo or in vitro use can contain about 0.5, 5, 20, 50, 75, 100, 150, 250, 500, 750, 1,000, 1,250, 2,500, 3,500, or 5,000 mg of the disclosed compounds, or can contain a range of amounts from one to another listed in the dosage chart. For oral administration, a typical recommended daily dosage regimen can range from about 1 mg / day to about 500 mg / day, or 1 mg / day to 200 mg / day, in a single dose or in two to four divided doses. In one embodiment, a typical daily oral dosage regimen is 150 mg.

[0140] In certain embodiments, the dose of the MAPKAP pathway inhibitor is consistent with previously disclosed doses and / or doses approved for use by the Food and Drug Administration. In other embodiments, the dose of the MAPKAP pathway inhibitor is less than the previously approved dose, e.g., about 20%, about 50%, or about 80% of the approved dose. In certain embodiments, the dose of trametinib is about 0.5 mg to 20 mg orally daily, e.g., about 1 mg daily or 2 mg daily. In certain embodiments, the dose of cobimetinib is about 10 mg to 200 mg daily, e.g., about 30 mg or about 60 mg daily. In certain embodiments, the dose of binimetinib is about 10 mg to 200 mg twice daily, e.g., about 25 mg or about 45 mg twice daily. In certain embodiments, the dose of selumetinib is about 10 mg to 200 mg daily, for example, about 30 mg or about 75 mg twice daily.

[0141] [000140] The amount and frequency of administration of the compounds described herein and / or pharmaceutically acceptable salts thereof and other therapeutic agents will be regulated according to the judgment of the attending physician, taking into account factors such as the age, condition and size of the patient, and the severity of the condition being treated.

[0142] The compounds of the present disclosure (e.g., Compound A or Compound B (and pharmaceutically acceptable salts thereof), MAPKAP pathway inhibitors, and other therapeutic agents) can be administered by any suitable route. The compounds can be administered orally (e.g., with food) in capsules, suspensions, tablets, pills, dragees, liquids, gels, syrups, slurries, etc. Methods for encapsulating compositions (such as in hard gelatin or cyclodextran coatings) are known in the art (Baker, et al., "Controlled Release of Biological Active Agents," John Wiley and Sons, 1986, incorporated herein by reference in its entirety). The compounds can be administered to a subject in conjunction with an acceptable pharmaceutical carrier as part of a pharmaceutical composition. The dosage form of the pharmaceutical composition will vary according to the route of administration selected. A suitable pharmaceutical carrier can contain inactive ingredients that do not interact with the compound. The carrier is biocompatible, i.e., non-toxic, non-inflammatory, non-immunogenic, and does not produce other undesirable reactions at the site of administration.

[0143] An exemplary pharmaceutical composition comprises a compound described herein, e.g., Compound A, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, such as a) a diluent, e.g., purified water, a triglyceride oil, e.g., hydrogenated or partially hydrogenated vegetable oil or a mixture thereof, corn oil, olive oil, sunflower oil, safflower oil, a fish oil or an ester thereof, e.g., EPA or DHA, or a triglyceride or a mixture thereof, an omega-3 fatty acid or a derivative thereof, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose, and / or glycine; b) a diluent, e.g., silica, talc, stearic acid, a magnesium salt or a calcium salt thereof, sodium oleate, sodium stearate, magnesium stearate, benzoic acid c) lubricants, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, magnesium carbonate, natural sugars, such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, waxes and / or polyvinylpyrrolidone, if desired; d) disintegrants, such as starch, agar, methylcellulose, bentonite, xanthan gum, alginic acid or its sodium salt, or effervescent mixtures; e) absorbents, colorants, flavors and sweeteners; f) e.g. Tween 80, Labrasol, HPMC, DOSS, Caproyl and / or g) an emulsifier or dispersing agent, such as 909, Labrafac, Labrafil, Peceol, Transcutol, CAPMUL MCM, CAPMUL PG-12, Captex 355, Gelucire, Vitamin E TGPS or other acceptable emulsifier; and / or g) a carrier such as a cyclodextrin, hydroxypropyl cyclodextrin, PEG 400, PEG 200, or an agent that enhances absorption of the compound.

[0144] If prepared as a fixed dose, such combinations will employ the compounds described herein within the dosage ranges described herein or known to those of skill in the art.

[0145] Because the compounds described herein (e.g., Compound A and Compound B and MAPKAP pathway inhibitors) are intended for use in pharmaceutical compositions, those skilled in the art will appreciate that they can be provided in substantially pure form, e.g., at least 60% pure, at least 75% pure, at least 85% pure, and at least 98% pure (w / w). Pharmaceutical formulations may be in unit dosage form. In such formulations, Compound A or Compound B is prepared in suitably sized unit doses containing an appropriate amount, e.g., an effective amount to achieve a desired purpose as described herein. [Example]

[0146] [000145] It has been unexpectedly discovered that combination treatment of either Compound A or a pharmaceutically acceptable salt thereof with a MAPKAP pathway inhibitor synergistically induces apoptosis of mast cells driven by mutant c-KIT, which is responsible for systemic mastocytosis. Furthermore, this combination therapy inhibits the proliferation of mastocytosis cells. Furthermore, the combination therapy disclosed herein appears to have a cytotoxic effect on mastocytosis, as opposed to a merely cytostatic effect as determined by combination treatment induced caspase activation. This unexpected finding was identified in biochemical and cellular assays, including those described herein.

[0147] The present disclosure is further illustrated by the following examples, which are not to be construed as limiting the disclosure in scope or spirit to the specific procedures described therein. It should be understood that the examples are presented to illustrate particular embodiments and are not intended to limit the scope of the present disclosure. It should further be understood that various other embodiments, modifications, and equivalents may be made to those skilled in the art without departing from the spirit of the present disclosure and / or the scope of the appended claims.

[0148] Example 1. Treatment of mutant KIT mast cell lines with Compound A or Compound B inhibits cell proliferation and KIT phosphorylation in mastocytosis cell lines A study was conducted to demonstrate that treatment with Compound A or Compound B inhibits cell proliferation of mutant KIT HMC1.1 KIT V560G and HMC1.2 KIT V560G / D816V mast cell lines. Assays were performed in 96-well plates seeded with 10,000 cells per well. Cells were treated with vehicle control, Compound A, or Compound B at various concentrations, grown for 72 hours, and then cell proliferation was assessed.

[0149] Figure 1A is a graphical representation showing the relative rates of cell proliferation determined for various concentrations of Compound A. Treatment with Compound A inhibited cell proliferation in HMC1.1 V560G and HMC1.2 V560G / D816V mast cell lines with IC50 values ​​of 2.6 nM and 97 nM, respectively.

[0150] Figure 1B is a graphical representation showing the relative rates of cell proliferation determined for various concentrations of Compound B. Treatment with Compound B inhibited cell proliferation in HMC1.1 V560G and HMC1.2 V560G / D816V mast cell lines with IC50 values ​​of 2.3 nM and 61 nM, respectively.

[0151] Example 2. Combined treatment with Compound A and trametinib induces apoptosis in mastocytosis cell lines [000150] Combination treatment with Compound A and the MEK inhibitor trametinib improves HMC1.2 A study was conducted to demonstrate the induction of apoptosis in the KIT V560G / D816V mastocytosis cell line. Assays were performed in 96-well plates seeded with 10,000 cells per well. Cells were treated with vehicle control, Compound A, trametinib, or a combination thereof at various concentrations and allowed to grow for 24 hours. Apoptosis was assessed by measuring caspase 3 / 7 activity.

[0152] Figure 2A is a graphical representation showing the relative rates of cell apoptosis determined for various treatments. Figure 2B is a matrix diagram of synergy based on the combination index (CI) method described by Chou and Talalay (1984) and Chou and Martin (2005) computer software. CI<1 indicates synergy, CI=1 indicates additive effect, and CI>1 indicates antagonism. Unexpectedly, combined treatment with Compound A and the MEK inhibitor trametinib demonstrated strong synergy in inducing apoptosis of HMC1.2 KIT V560G / D816V cells. Figure 2C is a CI combination index plot demonstrating the strong synergy of the combination of Compound A and trametinib in inducing apoptosis of HMC1.2 KIT V560G / D816V mast cells.

[0153] Example 3. Combined treatment with Compound B and trametinib induces apoptosis in mastocytosis cell lines Studies were also conducted to demonstrate that combined treatment with Compound B and trametinib induces apoptosis in the HMC1.2 KIT V560G / D816V mastocytosis cell line. Assays were performed as described in Example 2. Apoptosis was assessed by measuring caspase 3 / 7 activity.

[0154] Figure 3A is a graphical representation showing the relative rates of cell apoptosis determined for various treatments. Figure 3B is a matrix diagram of synergy based on the combination index (CI) method described by Chou and Talalay (1984) and Chou and Martin (2005) computer software. CI<1 indicates synergy, CI=1 indicates additive effect, and CI>1 indicates antagonism. Unexpectedly, combined treatment with Compound B and the MEK inhibitor trametinib demonstrated strong synergy for inducing apoptosis of HMC1.2 KIT V560G / D816V cells. Figure 3C is a CI combination index plot demonstrating the strong synergy of the combination of Compound B and trametinib for inducing apoptosis of HMC1.2 KIT V560G / D816V mast cells.

[0155] Example 4. Combined treatment with Compound A and binimetinib induces apoptosis in mastocytosis cell lines Combined treatment with Compound A and binimetinib also induces apoptosis in the HMC1.2 KIT V560G / D816V mastocytosis cell line. Assays were performed as described in Example 2. Apoptosis was assessed by measuring caspase 3 / 7 activity.

[0156] [000155] Figure 4A is a graphical representation showing the relative percentage of cell apoptosis determined for various treatments. Figure 4B is a matrix diagram of synergy based on the combination index (CI) method described by Chou and Talalay (1984) and Chou and Martin's computer software (2005). Unexpectedly, combined treatment with Compound A and the MEK inhibitor binimetinib demonstrated strong synergy in inducing apoptosis of HMC1.2 KIT V560G / D816V cells. Figure 4C is a CI combination index plot demonstrating the strong synergy of the combination of Compound A and binimetinib in inducing apoptosis of HMC1.2 KIT V560G / D816V mast cells.

[0157] Example 5. Combined treatment with Compound B and binimetinib induces apoptosis in mastocytosis cell lines Figure 5A is a graphical representation showing the relative rates of cell apoptosis determined for various treatments with Compound B and binimetinib. Figure 5B is a matrix diagram of synergy based on the combination index (CI) method described by Chou and Talalay (1984) and Chou and Martin (2005) computer software. CI<1 indicates synergy, CI=1 indicates additive effect, and CI>1 indicates antagonism. Unexpectedly, combined treatment with Compound B and the MEK inhibitor binimetinib demonstrated strong synergy in inducing apoptosis of HMC1.2 KIT V560G / D816V cells. Figure 5C is a CI combination index plot demonstrating the strong synergy of the combination of Compound B and binimetinib in inducing apoptosis of HMC1.2 KIT V560G / D816V mast cells.

[0158] Example 6. Combined treatment with Compound A and cobimetinib induces apoptosis in mastocytosis cell lines Figure 6A is a graphical representation showing the relative percentage of cell apoptosis determined for various treatments of Compound A and cobimetinib. Figure 6B is a matrix diagram of synergy based on the combination index (CI) method, as described by Chou and Talalay (1984) and Chou and Martin (2005) computer software. Unexpectedly, combination therapy with Compound A and the MEK inhibitor cobimetinib demonstrated strong synergy in inducing apoptosis in HMC1.2 KIT V560G / D816V cells. Figure 6C is a combination index plot of CI, showing the relative percentage of cell apoptosis determined for various treatments of Compound A and cobimetinib. Figure 6B is a matrix diagram of synergy based on the combination index (CI) method, as described by Chou and Talalay (1984) and Chou and Martin (2005) computer software. Unexpectedly, combination therapy with Compound A and the MEK inhibitor cobimetinib demonstrated strong synergy in inducing apoptosis in HMC1.2 KIT V560G / D816V cells. We demonstrate potent synergy for the combination of Compound A and cobimetinib to induce apoptosis of 16V mast cells.

[0159] Example 7. Combined treatment with Compound B and cobimetinib induces apoptosis in mastocytosis cell lines Figure 7A is a graphical representation showing the relative rates of cell apoptosis determined for various treatments with Compound B and cobimetinib. Figure 7B is a matrix diagram of synergy based on the combination index (CI) method described by Chou and Talalay (1984) and Chou and Martin (2005) computer software. CI<1 indicates synergy, CI=1 indicates additive effect, and CI>1 indicates antagonism. Unexpectedly, combined treatment with Compound B and the MEK inhibitor cobimetinib demonstrated strong synergy for inducing apoptosis of HMC1.2 KIT V560G / D816V cells. Figure 7C is a CI combination index plot demonstrating the strong synergy of the combination of Compound B and cobimetinib for inducing apoptosis of HMC1.2 KIT V560G / D816V mast cells.

[0160] Example 8. Combined treatment with Compound A and ulixertinib induces apoptosis in mastocytosis cell lines Figure 8A is a graphical representation showing the relative rates of cell apoptosis determined for various treatments with Compound A and the ERK inhibitor ulixertinib. Figure 8B is a matrix diagram of synergy based on the combination index (CI) method, as described by Chou and Talalay (1984) and Chou and Martin (2005) computer software. Unexpectedly, combination therapy with Compound A and ulixertinib demonstrated strong synergy in inducing apoptosis of HMC1.2 KIT V560G / D816V cells. Figure 8C is a CI combination index plot demonstrating the strong synergy of the combination of Compound A and ulixertinib in inducing apoptosis of HMC1.2 KIT V560G / D816V mast cells.

[0161] Example 9. Combined treatment with Compound B and ulixertinib induces apoptosis in mastocytosis cell lines [000160] The combination treatment of Compound B and the ERK inhibitor ulixertinib can be evaluated for inducing apoptosis in HMC1.2 KIT V560G / D816V mast cells. A synergy matrix diagram based on the combination index (CI) method can be generated as described by Chou and Talalay (1984) and Chou and Martin's computer software (2005). The combination treatment of Compound B and ulixertinib can be used to demonstrate the synergistic effect of inducing apoptosis in HMC1.2 KIT V560G / D816V cells. The combination index plot of CI can be used to demonstrate the strong synergistic effect of the combination of Compound B and ulixertinib in inducing apoptosis in HMC1.2 KIT V560G / D816V mast cells.

[0162] Example 10. Combined treatment with Compound A and SCH772984 induces apoptosis in mastocytosis cell lines The combined treatment of Compound A and the ERK inhibitor SCH772984 can be evaluated for inducing apoptosis in HMC1.2 KIT V560G / D816V mast cells. A matrix diagram of synergy based on the combination index (CI) method can be generated as described by Chou and Talalay (1984) and Chou and Martin's computer software (2005). The combined treatment of Compound A and SCH772984 can be used to demonstrate the synergistic effect of inducing apoptosis in HMC1.2 KIT V560G / D816V cells. CI combination index plot can be used to demonstrate synergy for the combination of Compound A and SCH772984 in inducing apoptosis of HMC1.2 KIT V560G / D816V mast cells.

[0163] Example 11. Combined treatment with Compound B and SCH772984 induces apoptosis in mastocytosis cell lines The combined treatment of compound B and the ERK inhibitor SCH772984 can be evaluated for inducing apoptosis in HMC1.2 KIT V560G / D816V mast cells. A synergy matrix diagram based on the combination index (CI) method can be generated as described by Chou and Talalay (1984) and Chou and Martin's computer software (2005). The combined treatment of compound A and SCH772984 can be used to demonstrate the synergistic effect of inducing apoptosis in HMC1.2 KIT V560G / D816V cells. The combined index plot of CI can be used to demonstrate the synergistic effect of the combined use of compound B and SCH772984 in inducing apoptosis in HMC1.2 KIT V560G / D816V mast cells.

[0164] Example 12. Combined treatment with Compound A and LY3009120 induces apoptosis in mastocytosis cell lines The combined treatment of Compound A and the RAF inhibitor LY3009120 can be evaluated for inducing apoptosis in HMC1.2 KIT V560G / D816V mast cells. A synergy matrix diagram based on the combination index (CI) method can be generated as described by Chou and Talalay (1984) and Chou and Martin (2005) computer software. The combined treatment of Compound A and LY3009120 can be used to demonstrate the synergistic effect of inducing apoptosis in HMC1.2 KIT V560G / D816V cells. The combined index plot of CI can be used to demonstrate the strong synergistic effect of the combined use of Compound A and LY3009120 to induce apoptosis in HMC1.2 KIT V560G / D816V mast cells.

[0165] Example 13. Combined treatment with Compound B and LY3009120 induces apoptosis in mastocytosis cell lines The combined treatment of Compound B and the RAF inhibitor LY3009120 can be evaluated for inducing apoptosis in HMC1.2 KIT V560G / D816V mast cells. A synergy matrix diagram based on the combination index (CI) method can be generated as described by Chou and Talalay (1984) and Chou and Martin's computer software (2005). The combined treatment of Compound B and LY3009120 can be used to demonstrate the synergistic effect of inducing apoptosis in HMC1.2 KIT V560G / D816V cells. The combined index plot of CI can be used to demonstrate the synergistic effect of the combined use of Compound B and LY3009120 to induce apoptosis in HMC1.2 KIT V560G / D816V mast cells.

[0166] Example 14. Combined treatment with Compound A and trametinib leads to a synergistic decrease in colony growth of the HMC1.2 KIT D816V mast cell line [000165] A study was conducted to demonstrate that combined treatment with Compound A and the MEK inhibitor trametinib leads to reduced colony growth of HMC1.2 compared to treatment with either agent alone. 10,000 HMC1.2 cells were grown in soft agar and incubated for 10 days with various concentrations of Compound A, trametinib, or a combination of Compound A and trametinib. Drug treatment was removed, and viable colony growth was observed after an additional 5 days. Figure 9A shows representative photographs of colony growth of HMC1.2 mast cells after treatment with trametinib (0, 10, or 25 nM), compound A (0, 25, or 50 nM), either as single agents or as a matrix combining various concentrations of compound A with various concentrations of trametinib. While single-agent treatment with compound A or trametinib resulted in colony growth at all concentrations 5 days after drug removal, the combination of compound A and trametinib resulted in little colony growth 5 days after drug removal. The combination of compound A (25 nM) with trametinib (25 nM) completely eradicated colony growth 5 days after drug removal. Unexpectedly, the combination of Compound A (50 nM) with trametinib (10 or 25 nM) resulted in complete eradication of viable HMC1.2 cell growth to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification 5 days after drug removal, whereas no eradication was observed with treatment with single-agent Compound A or single-agent trametinib. Further extension of colony growth to an additional 8 days after drug removal (a total of 13 days) maintained eradication to the limit of detection for the combination of Compound A (50 nM) and trametinib (10 and 25 nM). However, incubation with the combination of Compound A (25 nM) and trametinib (25 nM) resulted in the growth of approximately 15–20 colonies after an additional 8 days (Figure 9A, right panel).

[0167] Figure 9B is a graphical representation quantifying colony growth of HMC1.2 mast cells after various treatments from Figure 9A. Combination treatment with 50 nM Compound A and either 10 nM or 25 nM trametinib unexpectedly resulted in eradication of colony growth to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification (Figure 9B, see arrow). Combination treatment with 25 nM Compound A and 25 nM trametinib unexpectedly resulted in eradication of colony growth to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification (Figure 9B, see arrow), eradication not observed with treatment with either single-agent Compound A or trametinib.

[0168] Example 15. Combined treatment with Compound B and trametinib leads to a synergistic decrease in colony growth of the HMC1.2 KIT D816V mast cell line Studies were also performed with Compound B and the MEK inhibitor trametinib to assess the reduction of colony growth in HMC1.2 cells as described in Example 14.

[0169] Figure 10A shows representative photographs of colony growth of HMC1.2 mast cells after treatment with trametinib (0, 10, or 25 nM), Compound B (0, 25, or 50 nM), either as single agents or as a matrix combining various concentrations of Compound B with various concentrations of trametinib. Single-agent treatment with Compound B or trametinib resulted in colony growth at all concentrations 5 days after drug removal, while the combination of Compound B and trametinib resulted in little colony growth 5 days after drug removal. The combination of Compound B (25 nM) with trametinib (25 nM) significantly reduced the proliferation of viable HMC1.2 cells 5 days after drug removal. The combination of Compound B (50 nM) with trametinib (10 or 25 nM) unexpectedly resulted in complete eradication of viable HMC1.2 cell proliferation to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification 5 days after drug removal, while no eradication was observed with treatment with single-agent Compound B or single-agent trametinib. Further extension of colony growth to an additional 8 days after drug removal (13 days total) maintained eradication to the limit of detection of colony growth with the combination of Compound B (50 nM) and trametinib (25 nM). Figure 10B is a graphical representation quantifying colony growth of HMC1.2 mast cells after various treatments from Figure 10A. Combination treatment with 50 nM Compound B and either 10 nM or 25 nM trametinib unexpectedly resulted in eradication of colony growth to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification, while eradication was not observed with treatment with either single-agent Compound B or trametinib (Figure 10B, arrows (See markings).

[0170] Example 16. Combined treatment with Compound A and binimetinib leads to a synergistic decrease in colony growth of the HMC1.2 KIT D816V mast cell line Figure 11A shows representative photographs of colony growth of HMC1.2 mast cells after treatment with binimetinib (0, 250, or 500 nM), Compound A (0, 25, or 50 nM), either as single agents or as a matrix in which various concentrations of Compound A were combined with various concentrations of binimetinib. Single-agent treatment with Compound A or binimetinib resulted in colony growth at all concentrations 5 days after drug withdrawal, while the combination of Compound A and binimetinib resulted in little colony growth 5 days after drug withdrawal. Combination of Compound A (25 nM) with binimetinib (250 nM) significantly reduced proliferation of viable HMC1.2 cells 5 days after drug withdrawal. Unexpectedly, the combination of Compound A (50 nM) with binimetinib (250 nM or 500 nM) resulted in complete eradication of viable HMC1.2 cell growth to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification 5 days after drug removal, whereas no eradication was observed with treatment with single-agent Compound A or single-agent binimetinib. Further extension of colony growth to an additional 8 days after drug removal (a total of 13 days) demonstrated that the combination of Compound A (50 nM) and binimetinib (500 nM) maintained eradication to the limit of detection of colony growth. However, the combination of Compound A (50 nM) and binimetinib (250 nM) resulted in the growth of approximately 10–15 colonies.

[0171] [000170] Figure 11B is a graphical representation quantifying colony growth of HMC1.2 mast cells after various treatments from Figure 11A. Combination treatment with 50 nM Compound A and 250 nM or 500 nM binimetinib unexpectedly resulted in eradication of colony growth to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification; eradication was not observed with treatment with either single-agent Compound A or binimetinib (see arrows in Figure 11B).

[0172] Example 17. Combined treatment with Compound B and binimetinib leads to a synergistic decrease in colony growth of the HMC1.2 KIT D816V mast cell line Figure 12A shows representative photographs of colony growth of HMC1.2 mast cells after treatment with binimetinib (0, 250, or 500 nM), Compound B (0, 25, or 50 nM), either as single agents or as a matrix in which various concentrations of Compound B were combined with various concentrations of binimetinib. Single-agent treatment with Compound B or binimetinib resulted in colony growth at all concentrations 5 days after drug withdrawal, while the combination of Compound B and binimetinib resulted in little colony growth 5 days after drug withdrawal. The combination of Compound B (25 nM) with binimetinib (250 nM) significantly reduced the proliferation of live HMC1.2 cells 5 days after drug withdrawal. Combination of Compound B (25 nM) with binimetinib (500 nM) resulted in complete eradication of viable HMC1.2 cell proliferation to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification 5 days after drug withdrawal. Unexpectedly, combination of Compound B (50 nM) with binimetinib (250 nM or 500 nM) resulted in complete eradication of viable HMC1.2 cell proliferation to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification 5 days after drug withdrawal, whereas no eradication was observed with treatment with single-agent Compound B or single-agent binimetinib. With colony growth further extended to an additional 8 days after drug removal (total of 13 days), eradication remained maintained down to the limit of detection for colony growth with the combination of Compound B (50 nM) with binimetinib (250 or 500 nM) and Compound B (25 nM) with binimetinib (500 nM).

[0173] Figure 12B is a graphical representation quantifying colony growth of HMC1.2 mast cells after various treatments from Figure 12A. Combination treatment with 25 nM Compound B and 500 nM binimetinib and combination treatment with 50 nM Compound B and either 250 nM or 500 nM binimetinib. Combination treatment with Compound B unexpectedly resulted in eradication of colony growth to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification, whereas eradication was not observed with treatment with either single-agent Compound B or binimetinib (Figure 12B, see arrow).

[0174] Example 18. Combination treatment with Compound A and cobimetinib leads to a synergistic decrease in colony growth of the HMC1.2 KIT D816V mast cell line Figure 13A shows representative photographs of colony growth of HMC1.2 mast cells after treatment with cobimetinib (0, 25, or 50 nM), Compound A (0, 25, or 50 nM), either as single agents or as a matrix in which various concentrations of Compound A were combined with various concentrations of cobimetinib. While single-agent treatment with Compound A or cobimetinib resulted in colony growth at all concentrations 5 days after drug withdrawal, the combination of Compound A and cobimetinib resulted in little colony growth 5 days after drug withdrawal compared to either single-agent treatment. Combination of Compound A (50 nM) with cobimetinib (25 or 50 nM) significantly reduced proliferation of viable HMC1.2 cells 5 days after drug withdrawal.

[0175] Figure 13B is a graphical representation quantifying colony growth of HMC1.2 mast cells after various treatments from Figure 13A. Combination treatment with 50 nM Compound A and either 25 nM or 50 nM cobimetinib resulted in a significant decrease in colony growth compared to either Compound A or cobimetinib alone.

[0176] Example 19. Combination treatment with Compound B and cobimetinib leads to a synergistic decrease in colony growth of the HMC1.2 KIT D816V mast cell line [000175] Figure 14A shows representative photographs of colony growth of HMC1.2 mast cells after treatment with cobimetinib (0, 25, or 50 nM), Compound B (0, 25, or 50 nM), either as single agents or as a matrix combining various concentrations of Compound B with various concentrations of cobimetinib. Single-agent treatment with Compound B or cobimetinib resulted in colony growth at all concentrations 5 days after drug withdrawal, while the combination of Compound B and cobimetinib resulted in little colony growth 5 days after drug withdrawal compared to either single-agent treatment. Combination of Compound B (50 nM) with cobimetinib (25 or 50 nM) significantly reduced proliferation of viable HMC1.2 cells 5 days after drug withdrawal.

[0177] Figure 14B is a graphical representation quantifying colony growth of HMC1.2 mast cells after various treatments from Figure 14A. Combination treatment with 50 nM Compound B and either 25 nM or 50 nM cobimetinib resulted in a significant decrease in colony growth compared to either Compound B or cobimetinib alone.

[0178] Example 20. Combined treatment with Compound A and the ERK inhibitor ulixertinib leads to a synergistic decrease in colony growth of the HMC1.2 KIT D816V mast cell line Combination treatment of Compound A and ulixertinib can be evaluated for inhibition of colony growth of the HMC1.2 KIT D816V mast cell line according to the method of Example 14.

[0179] Example 21. Combined treatment with Compound B and the ERK inhibitor ulixertinib leads to a synergistic decrease in colony growth of the HMC1.2 KIT D816V mast cell line Combination treatment of Compound B and ulixertinib can be evaluated for inhibition of colony growth of the HMC1.2 KIT D816V mast cell line according to the method of Example 14.

[0180] Example 22. Combined treatment with Compound A and the RAF inhibitor LY3009120 leads to a synergistic decrease in colony growth of the HMC1.2 KIT D816V mast cell line Combination treatment of Compound A and the RAF inhibitor LY3009120 can be evaluated for inhibition of colony growth of the HMC1.2 KIT D816V mast cell line according to the method of Example 14.

[0181] Example 23. Combined treatment with Compound B and LY3009120 leads to a synergistic decrease in colony growth of the HMC1.2 KIT D816V mast cell line [000180] Combined treatment of Compound B and the RAF inhibitor LY3009120 can be evaluated for inhibition of colony growth of the HMC1.2 KIT D816V mast cell line according to the method of Example 14.

[0182] Example 24. Combined treatment with Compound A and the MEK inhibitor trametinib induces apoptosis in the HMC1.2 KIT V560G / D816V mastocytosis cell line transfected with mutant N-ras G12D A study was conducted to demonstrate that combined treatment with Compound A and trametinib induces apoptosis in HMC1.2 V560G / D816V cells transfected with mutant N-ras G12D. Assays were performed in 96-well plates seeded with 10,000 cells per well. Cells were treated with vehicle control, Compound A, trametinib, or their combination at various concentrations, and the cells were grown for 24 and 48 hours. Apoptosis was assessed by measuring caspase 3 / 7 activity.

[0183] Figure 15A is a graphical representation showing the relative percentage of caspase activity determined for various treatments at 24 hours. Combined treatment with Compound A and trametinib for 24 hours induced apoptosis in HMC1.2 V560G / D816V cells transfected with empty vector (EV) (left panel) or mutant N-ras G12D (right panel). The combination of Compound A with trametinib led to a synergistic increase in apoptosis in HMC1.2 cells transfected with EV and N-ras G12D.

[0184] Figure 15B is a graphical representation showing the relative percentage of caspase activity determined for various treatments at 48 hours. Combined treatment with Compound A and trametinib for 48 hours induced apoptosis in HMC1.2 V560G / D816V cells transfected with empty vector (EV) (left panel) or mutant N-ras G12D (right panel). The combination of Compound A with trametinib led to a synergistic increase in apoptosis in HMC1.2 cells transfected with EV and N-ras G12D. Apoptosis was greater in N-ras transfected cells than in EV-transfected cells.

[0185] Example 25. Combined treatment with Compound A and the MEK inhibitor cobimetinib induces apoptosis in the HMC1.2 KIT V560G / D816V mastocytosis cell line transfected with mutant N-ras G12D Figure 16A is a graphical representation showing the relative percentage of caspase activity determined for various treatments at 24 hours. Combined treatment with Compound A and cobimetinib for 24 hours induced apoptosis in HMC1.2 V560G / D816V cells transfected with empty vector (EV) (left panel) or mutant N-ras G12D (right panel). The combination of Compound A with cobimetinib led to a synergistic increase in apoptosis in HMC1.2 cells transfected with EV and N-ras G12D. Apoptosis was greater in N-ras transfected cells than in EV-transfected cells.

[0186] [000185] Figure 16B shows caspase activity determined for various treatments at 48 hours. Figure 1 shows a graphical representation of the relative percentage of apoptosis in HMC1.2 V560G / D816V cells transfected with empty vector (EV) (left panel) or mutant N-ras G12D (right panel). Combination treatment with Compound A and cobimetinib for 48 hours induced apoptosis in HMC1.2 V560G / D816V cells transfected with empty vector (EV) (left panel) or mutant N-ras G12D (right panel). Combination of Compound A with cobimetinib led to a synergistic increase in apoptosis in EV-transfected HMC1.2 cells and N-ras G12D-transfected HMC1.2 cells. Apoptosis was greater in N-ras-transfected cells than in EV-transfected cells.

[0187] Example 26. Combination treatment with Compound A and the MEK inhibitor trametinib inhibits N-ras Transfection with G12D or empty vector leads to a synergistic decrease in colony growth of the HMC1.2 KIT D816V mast cell line A study was conducted to demonstrate that combined treatment with Compound A and trametinib leads to reduced colony growth of HMC1.2 cells transfected with empty vector (EV) or N-ras G12D compared with either single-agent treatment. HMC1.2 cells were incubated with various concentrations of Compound A, trametinib, or a combination of Compound A and trametinib for 10 days. Drug treatment was removed, and viable colony growth was observed after an additional 5 or 13 days.

[0188] Figure 17A shows representative photographs of colony growth of EV-transfected HMC1.2 mast cells after treatment with trametinib (0, 1, 10, or 25 nM), compound A (0, 25, or 50 nM), either as single agents or as a matrix in which various concentrations of compound A were combined with various concentrations of trametinib. While single-agent treatment with compound A or trametinib resulted in colony growth at all concentrations 5 days after drug removal, the combination of compound A and trametinib resulted in little colony growth 5 days after drug removal. The combination of compound A (50 nM) with trametinib (1 nM) resulted in a significant decrease in colony growth compared to single-agent compound A or single-agent trametinib, especially with 1 nM trametinib. The combination of Compound A (50 nM) with trametinib (10 or 25 nM) unexpectedly resulted in complete eradication of viable HMC1.2 cell proliferation to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification 5 days after drug removal, while no eradication was observed with treatment with single-agent Compound A or single-agent trametinib. Further extended colony growth for an additional 8 days after drug removal (a total of 13 days after drug removal) maintained eradication to the limit of detection of colony growth with the combination of Compound A (50 nM) and trametinib (25 nM).

[0189] Figure 17B is a graphical representation quantifying colony growth of HMC1.2 mast cells transfected with EVs after various treatments from Figure 17A. Combination treatment with 50 nM Compound A and either 10 nM or 25 nM trametinib unexpectedly resulted in eradication of colony growth to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification, whereas eradication was not observed with treatment with either single-agent Compound A or trametinib (Figure 17B, see arrow).

[0190] Figure 17C shows representative photographs of colony growth of N-ras G12D-transfected HMC1.2 cells after treatment with trametinib (0, 1, 10, or 25 nM), compound A (0, 25, or 50 nM), either as single agents or as a matrix combining various concentrations of compound A with various concentrations of trametinib. Single-agent treatment with compound A or trametinib resulted in colony growth at all concentrations 5 days after drug removal, while the combination of compound A and trametinib resulted in little colony growth 5 days after drug removal. Combination of compound A (25 nM) with trametinib (25 nM) resulted in a significant decrease in viable HMC1.2 cell growth 5 days after drug removal, and unexpectedly, combination of compound A (50 nM) with trametinib (10 or 25 nM) resulted in a 5-fold decrease in growth 5 days after drug removal. The combination of Compound A (50 nM) and trametinib (25 nM) resulted in complete eradication of growth of N-ras G12D-transfected HMC1.2 cells to the limit of detection as determined by visualization by stereomicroscopy at 17C, whereas eradication was not observed with treatment with either single-agent Compound A or trametinib. Further extension of colony growth to an additional 8 days after drug removal (a total of 13 days after drug removal), eradication of colony growth remained with the combination of Compound A (50 nM) and trametinib (25 nM) (Figure 17C).

[0191] [000190] Figure 17D is a graphical representation quantifying colony growth of various treatments from Figure 17C. Combination treatment with Compound A and trametinib resulted in superior inhibition of colony growth compared to treatment with either single agent. Unexpectedly, combination treatment with 50 nM Compound A and either 10 nM or 25 nM trametinib resulted in eradication of colony growth to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification 5 days after drug removal; eradication was not observed with treatment with either single agent Compound A or trametinib (see arrows in Figure 17D).

[0192] Example 27. Combination treatment with Compound A and cobimetinib leads to a synergistic decrease in colony growth of the HMC1.2 KIT D816V mast cell line transfected with N-ras G12D or empty vector A study was conducted to demonstrate that combined treatment with Compound A and cobimetinib leads to reduced colony growth of HMC1.2 cells transfected with empty vector (EV) or N-ras G12D compared with treatment with either agent alone. HMC1.2 cells were incubated with various concentrations of Compound A, cobimetinib, or a combination of Compound A and cobimetinib for 10 days. Drug treatment was removed, and viable colony growth was observed after an additional 5 or 10 days.

[0193] Figure 18A shows representative photographs of colony growth of EV-transfected HMC1.2 mast cells after treatment with cobimetinib (25 or 50 nM), compound A (0, 25, or 50 nM), either as single agents or as a matrix containing various concentrations of compound A combined with various concentrations of cobimetinib. While single-agent treatment with compound A or cobimetinib resulted in colony growth at all concentrations 5 days after drug removal, the combination of compound A and cobimetinib resulted in little colony growth 5 days after drug treatment was removed. Combination of compound A (25 nM) with cobimetinib (25 and 50 nM) significantly reduced the proliferation of viable HMC1.2 cells 5 days after drug treatment was removed. The combination of Compound A (50 nM) with cobimetinib (50 nM) unexpectedly resulted in complete eradication of viable HMC1.2 cell proliferation to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification 5 days after removal of drug treatment, while no eradication was observed with treatment with single-agent Compound A or single-agent cobimetinib. Further extension of colony growth to an additional 5 days after removal of drug treatment (a total of 10 days after drug removal) maintained eradication to the limit of detection of colony growth with the combination of Compound A (50 nM) and cobimetinib (50 nM).

[0194] Figure 18B is a graphical representation quantifying colony growth of HMC1.2 mast cells transfected with EVs after various treatments from Figure 18A. Combination treatment with 50 nM Compound A and 50 nM cobimetinib unexpectedly resulted in eradication of colony growth to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification, while eradication was not observed with treatment with either single-agent Compound A or cobimetinib (Figure 18B, see arrow).

[0195] Figure 18C shows the results of N-ras G12D transfected H cells after treatment with cobimetinib (0, 25, or 50 nM), Compound A (0, 25, or 50 nM), either as single agents or as a matrix combining various concentrations of Compound A with various concentrations of cobimetinib. Representative photographs of colony growth of HMC1.2 mast cells. Single-agent treatment with Compound A or cobimetinib resulted in colony growth at all concentrations 5 days after removal of drug treatment, while the combination of Compound A and cobimetinib resulted in little colony growth 5 days after removal of drug treatment. Combination of Compound A (25 nM) with cobimetinib (25 and 50 nM) significantly reduced viable HMC1.2 cell proliferation 5 days after removal of drug treatment. Unexpectedly, combination of Compound A (50 nM) with cobimetinib (25 or 50 nM) resulted in complete eradication of viable HMC1.2 cell proliferation to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification 5 days after removal of drug treatment, whereas no eradication was observed with treatment with single-agent Compound A or single-agent cobimetinib. Further extension of colony growth to an additional 10 days after removal of drug treatment showed that the combination of Compound A (50 nM) and cobimetinib (25 or 50 nM) still maintained eradication to the limit of detection of colony growth.

[0196] [000195] Figure 18D is a graphical representation quantifying colony growth after various treatments from Figure 18C. Combination treatment with Compound A and cobimetinib resulted in superior inhibition of colony growth compared to treatment with either single agent. Unexpectedly, combination treatment with 50 nM Compound A and 25 nM or 50 nM cobimetinib resulted in eradication of colony growth to the limit of detection as determined by visualization by stereomicroscopy at 5x magnification 5 days after drug removal, whereas eradication was not observed with treatment with either single agent Compound A or cobimetinib (see arrows in Figure 18D).

[0197] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described specifically in this disclosure which equivalents are intended to be encompassed by the following claims. Scope of claims at the time of international application [Item 1] A method for treating mastocytosis in a patient in need thereof, comprising administering to the patient an effective amount of a c-KIT inhibitor and an effective amount of one or more MAPKAP pathway inhibitors. [Item 2] The method of claim 1, wherein the MAPKAP pathway inhibitor is selected from the group consisting of a mitogen-activated protein kinase (MEK inhibitor), an extracellular signal-regulated kinase inhibitor (ERK inhibitor), and a rapidly progressing fibrosarcoma (RAF) kinase inhibitor. [Item 3] The method of claim 1 or 2, wherein the mastocytosis is associated with a c-KIT mutation. [Item 4] The method of any one of claims 1 to 3, wherein the c-KIT mutation is an activating mutation. [Item 5] The method according to any one of claims 1 to 4, wherein the mastocytosis involves mast cells with a primary mutation in exon 17 of the c-KIT gene. [Item 6] The method of claim 5, wherein the primary mutation is a c-KIT D816 mutation. 7. The method of claim 5 or 6, wherein the primary mutation is one of D816V, D816Y, D816F, D816H, F522C, K5091, V560G, V559G, and del419. [Item 8] The method of any one of claims 5 to 6, wherein the primary mutation is D816V. [Item 9] The method of any one of claims 1 to 8, wherein the mastocytosis involves mast cells with a c-KIT secondary mutation. 10. The method of claim 9, wherein the c-KIT secondary mutation is in one of exons 9, 11, 13, or 17. 11. The method of claim 9 or 10, wherein the c-KIT secondary mutation is one of the following mutations: Y269C, Y503_F504insAY, V560D, or K642E. [Item 12] The method of any one of claims 6 to 11, further comprising determining whether the mastocytosis is associated with a primary c-KIT mutation. [Item 13] The method of any one of claims 6 to 11, further comprising determining whether the mastocytosis is associated with a secondary c-KIT mutation. [Item 14] The method of claim 12 or 13, wherein determining whether the mastocytosis is associated with a primary c-KIT mutation or whether the mastocytosis is associated with a secondary c-KIT mutation comprises identifying mutations in DNA extracted from a tumor sample. 15. The method of claim 12 or 13, wherein determining whether the mastocytosis is associated with a primary c-KIT mutation or determining whether the mastocytosis is associated with a secondary c-KIT mutation comprises identifying a mutation in circulating tumor DNA or in circulating peripheral blood leukocytes. [Item 16] The method according to any one of claims 1 to 15, wherein the mastocytosis is systemic mastocytosis. [Item 17] The method of claim 16, wherein the systemic mastocytosis is selected from the group consisting of indolent systemic mastocytosis, smoldering systemic mastocytosis, systemic mastocytosis with associated clonal hematologic non-mast cell lineage disease, aggressive systemic mastocytosis, mast cell leukemia, and mast cell sarcoma. [Item 18] The method according to claim 16, wherein the mastocytosis is painless systemic mastocytosis, optionally systemic mastocytosis accompanied by recurrent anaphylactic episodes or vascular collapse without skin lesions. [Item 19] The method according to claim 16, wherein the mastocytosis is smoldering systemic mastocytosis. [Item 20] The method of claim 16, wherein the mastocytosis is systemic mastocytosis with an associated clonal hematologic non-mast cell lineage disorder. [Item 21] The method according to claim 16, wherein the mastocytosis is aggressive systemic mastocytosis. [Item 22] The method according to claim 16, wherein the mastocytosis is mast cell leukemia or mast cell sarcoma. [Item 23] The method according to any one of claims 1 to 15, wherein the mastocytosis is cutaneous mastocytosis. [Item 24] The method of claim 23, wherein the mastocytosis is selected from the group consisting of maculopapular cutaneous mastocytosis, mastocytoma, and diffuse cutaneous mastocytosis. 25. The method of any one of claims 1 to 24, wherein the c-KIT inhibitor is selected from the group consisting of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof, midostalin or a pharmaceutically acceptable salt thereof, imatinib mesylate, sunitinab malate, midostaurin, regorafenib, crenolanib, PTX9486, or BLU-285 or a pharmaceutically acceptable salt thereof. [Item 26] The method of any one of claims 2 to 25, wherein the MEK inhibitor is selected from the group consisting of trametinib, selumetinib, cobimetinib, and binimetinib. [Item 27] ​​The method of any one of claims 2 to 26, wherein the MEK inhibitor is binimetinib. [Item 28] The method of any one of claims 2 to 27, wherein the MEK inhibitor is trametinib. [Item 29] The method of any one of claims 2 to 25, wherein the ERK inhibitor is selected from the group consisting of ulixertinib, SCH772984, and LY3214996. [Item 30] The method according to any one of claims 2 to 29, wherein the c-KIT inhibitor and the MEK inhibitor and / or the ERK inhibitor are administered substantially simultaneously or sequentially. [Item 31] The method of any one of claims 1 to 30, further comprising administering another cancer-targeting therapeutic agent, cancer-targeting biological agent, immune checkpoint inhibitor, or chemotherapeutic agent. [Item 32] The method of any one of claims 1 to 31, wherein the patient is in at least partial remission two weeks or more after administration. [Item 33] A method for treating systemic mastocytosis in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharmaceutically acceptable salt thereof, and an effective amount of one or more MAPKAP pathway inhibitors. [Item 34] The method of claim 33, wherein the MAPKAP pathway inhibitor is selected from the group consisting of a mitogen-activated protein kinase (MEK inhibitor) and an extracellular signal-regulated kinase inhibitor (ERK inhibitor). [Item 35] The method of claim 33 or 34, wherein the systemic mastocytosis is associated with a c-KIT mutation. 36. The method of claim 33 or 34, wherein the mutation is a c-KIT D816 mutation. 37. The method of claim 36, wherein the mutation is one of D816V, D816Y, D816F, D816H, F522C, K5091, V560G, V559G, and del419. 38. The method of claim 37, wherein the mutation is one of A553D, C433Y, D419Y, D572A, D816F, D816H, D816I, D816V, D816Y, D820G, del419, dup(501-502), E839K, F522C, I817V, InsFF419, InsV815-I816, K509I, N822I, R815K, T417V, V560G, V559I, or Y418Y. 39. The method of any one of claims 34 to 38, wherein the mutation is D816V. 40. The method of any one of claims 34 to 38, wherein the mastocytosis is associated with an additional c-KIT mutation that is one of the following mutations: Y269C, Y503_F504insAY, V560D, or K642E. Item 41. The method of any one of claims 34 to 40, wherein the MEK inhibitor is selected from the group consisting of trametinib, selumetinib, cobimetinib, and binimetinib. Item 42. The method of any one of claims 34 to 40, wherein the MEK inhibitor is binimetinib. Item 43. The method of any one of claims 34 to 40, wherein the MEK inhibitor is trametinib. 44. The method of any one of claims 34 to 42, wherein the ERK inhibitor is selected from the group consisting of ulixertinib, SCH772984, and LY3214996. Item 45. A method for treating mastocytosis in a patient in need thereof, comprising administering to the patient an effective amount of a c-KIT inhibitor and an effective amount of a RAF inhibitor. [Item 46] The method of claim 44, wherein the RAF inhibitor is a pan-RAF inhibitor or a B-RAF inhibitor. 47. The method of claim 44 or 45, wherein the c-KIT inhibitor is 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharmaceutically acceptable salt thereof.

Claims

1. A medicament for use in the treatment of mastocytosis, comprising: Compound B: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof; and an effective amount of one or more MAPKAP pathway inhibitors; It is a combination of the MAPKAP pathway inhibitor is selected from the group consisting of a mitogen-activated protein kinase (MEK inhibitor) and an extracellular signal-regulated kinase inhibitor (ERK inhibitor); the mitogen-activated protein kinase (MEK inhibitor) is selected from the group consisting of trametinib, selumetinib, cobimetinib, and binimetinib; The extracellular signal-regulated kinase inhibitor (ERK inhibitor) is ulixertinib.

2. The pharmaceutical composition of claim 1, wherein the mastocytosis is accompanied by a c-KIT mutation.

3. The method of claim 1 or 2, wherein the c-KIT mutation is an activating mutation.

4. The method according to any one of claims 1 to 3, wherein the mastocytosis involves mast cells with a primary mutation in exon 17 of the c-KIT gene.

5. The method of claim 4, wherein the primary mutation is a c-KIT D816 mutation.

6. 6. The medicament of claim 4 or 5, wherein the primary mutation is one of D816V, D816Y, D816F, D816H, F522C, K5091, V560G, V559G, and del419.

7. The pharmaceutical of claim 4 or 5, wherein the primary mutation is D816V.

8. The pharmaceutical agent according to any one of claims 1 to 7, wherein the mastocytosis involves mast cells with a c-KIT secondary mutation.

9. The method of claim 8, wherein the c-KIT secondary mutation is in one of exons 9, 11, 13, or 17.

10. The method according to claim 8 or 9, wherein the c-KIT secondary mutation is one of the following mutations: Y269C, Y503_F504insAY, V560D, or K642E.

11. The method of any one of claims 5 to 10, wherein the treatment further comprises determining whether the mastocytosis is associated with a c-KIT primary mutation.

12. The method of any one of claims 5 to 10, wherein the treatment further comprises determining whether the mastocytosis is associated with a secondary c-KIT mutation.

13. The method of claim 11 or 12, wherein determining whether the mastocytosis is associated with a c-KIT primary or secondary mutation comprises identifying the mutation in DNA extracted from a tumor sample.

14. The method according to claim 11 or 12, wherein determining whether the mastocytosis is associated with a c-KIT primary mutation or a c-KIT secondary mutation comprises identifying the mutation in circulating tumor DNA or in circulating peripheral blood leukocytes.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the mastocytosis is systemic mastocytosis.

16. The pharmaceutical composition of claim 15, wherein the systemic mastocytosis is selected from the group consisting of indolent systemic mastocytosis, smoldering systemic mastocytosis, systemic mastocytosis with associated clonal hematologic non-mast cell lineage disease, aggressive systemic mastocytosis, mast cell leukemia, and mast cell sarcoma.

17. The pharmaceutical composition of claim 15, wherein the mastocytosis is indolent systemic mastocytosis, optionally systemic mastocytosis accompanied by anaphylactic recurrence or vascular collapse without skin lesions.

18. The pharmaceutical composition of claim 15, wherein the mastocytosis is smoldering systemic mastocytosis.

19. The pharmaceutical composition of claim 15, wherein the mastocytosis is systemic mastocytosis with an associated clonal hematologic non-mast cell lineage disorder.

20. The pharmaceutical composition of claim 15, wherein the mastocytosis is aggressive systemic mastocytosis.

21. The pharmaceutical composition of claim 15, wherein the mastocytosis is mast cell leukemia or mast cell sarcoma.

22. The pharmaceutical composition according to claim 1 , wherein the mastocytosis is cutaneous mastocytosis.

23. The method of claim 22, wherein the mastocytosis is selected from the group consisting of maculopapular cutaneous mastocytosis, mastocytoma, or diffuse cutaneous mastocytosis.

24. The pharmaceutical agent according to any one of claims 1 to 23, wherein the MEK inhibitor is binimetinib.

25. The pharmaceutical agent according to any one of claims 1 to 23, wherein the MEK inhibitor is trametinib.

26. The pharmaceutical agent according to any one of claims 1 to 25, wherein the c-KIT inhibitor and the MEK inhibitor and / or the ERK inhibitor are adapted to be administered simultaneously or sequentially.

27. 27. The pharmaceutical of any one of claims 1 to 26, wherein the treatment further comprises using another cancer targeted therapeutic agent, cancer targeted biological agent, immune checkpoint inhibitor, or chemotherapeutic agent.

28. 28. The method of any one of claims 1 to 27, wherein the patient is in at least partial remission after two or more weeks of use of the medicament.

29. A pharmaceutical for use in the treatment of systemic mastocytosis, comprising: Compound B: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof; and an effective amount of one or more MAPKAP pathway inhibitors selected from the group consisting of mitogen-activated protein kinase (MEK inhibitors), and extracellular signal-regulated kinase inhibitors (ERK inhibitors); It is a combination of the MEK inhibitor is selected from the group consisting of trametinib, selumetinib, cobimetinib, and binimetinib; The ERK inhibitor is ulixertinib. Medicine.

30. The pharmaceutical agent according to claim 29, wherein the systemic mastocytosis is associated with a c-KIT mutation.

31. The pharmaceutical agent according to claim 29, wherein the mutation is a c-KIT D816 mutation.

32. 32. The pharmaceutical of claim 31, wherein the mutation is one of D816V, D816Y, D816F, D816H, F522C, K5091, V560G, V559G, and del419.

33. 33. The pharmaceutical composition of claim 32, wherein the mutation is one of A553D, C433Y, D419Y, D572A, D816F, D816H, D816I, D816V, D816Y, D820G, del419, dup(501-502), E839K, F522C, I817V, InsFF419, InsV815-I816, K509I, N822I, R815K, T417V, V560G, V559I, or Y418Y.

34. The pharmaceutical of any one of claims 31 to 33, wherein the mutation is D816V.

35. The method of any one of claims 29 to 33, wherein the mastocytosis is accompanied by an additional c-KIT mutation, which is one of the following mutations: Y269C, Y503_F504insAY, V560D, or K642E.

36. The pharmaceutical agent of any one of claims 29 to 35, wherein the MAPKAP pathway inhibitor is a MEK inhibitor selected from the group consisting of trametinib, selumetinib, cobimetinib, and binimetinib.

37. The pharmaceutical agent according to any one of claims 29 to 33, wherein the MEK inhibitor is binimetinib.

38. The pharmaceutical agent according to any one of claims 29 to 33, wherein the MEK inhibitor is trametinib.

Images